Midwest Islet Club 2025 Program

12:00 p.m. - 2:00 p.m. — Registration Opens / Poster Set Up / Lunch

• Registration and meeting check-in outside of the Tower Ballroom in Hine Hall
• Poster set up at Hine Hall
• Lunch in the Tower Ballroom

2:00 p.m. - 2:15 p.m. — Welcome Remarks

Location: Hine Hall Auditorium

• Welcome remarks presented by 2025 MIC Meeting Co-Chairs Amelia Linnemann, PhD, associate professor of pediatrics at the IU School of Medicine, and Andrew Templin, PhD, assistant professor of medicine at the IU School of Medicine. 

2:15 p.m. - 3:45 p.m. — Session 1: Metabolism within the Beta Cell

Location: Hine Hall Auditorium
​Moderator: Danielle Dean, PhD, Vanderbilt University

2:15 p.m.	Ava Stendahl, University of Michigan  Loss of the mitochondrial inorganic phosphate transporter impairs β cell glucose-stimulated insulin secretion despite a maintenance of ATP levels
2:30 p.m.	Shih-Ming (Annie) Huang, University of Wisconsin-Madison  β-cell primary cilia generate local ATP via glycolysis and communicate with mitochondria via the phosphoenolpyruvate cycle
2:45 p.m.	Simin Liu, University of Iowa The regulation of lipolysis by glucose in pancreatic islets
3:00 p.m.	Lu Liang, University of Wisconsin-Madison Deletion of PKM1 and PKM2 reveals their essential roles in regulating β-cell KATP channels, Ca2+ activity, and insulin secretion in mice
3:15 p.m.	Coffee/Snack break (30 minutes)

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4:00 p.m. - 5:15 p.m. — Session 2: Technological Advancements in Islet Research

Location: Hine Hall Auditorium
Moderator: Erica Cai, PhD, Indiana Biosciences Research Institute

3:45 p.m.	Kira Slepchenko, Ohio University Synchrotron X-ray fluorescence in single pancreatic beta-cells reveals novel stress-associated iron-regulating structures
4:00 p.m.	Ananya Bharath, Mayo Clinic Microfluidic Culture Enhances Long-Term Survival and Function of Pancreatic Islets
4:15 p.m.	Xinhang Dong, Washington University St. Louis Ultrastructure expansion microscopy of axonemal dynein in islet primary cilia
4:30 p.m.	Palwasha Khan, Ohio University Investigating the protective mechanism of a potential Type 1 diabetes therapeutic compound, in pancreatic beta cells: findings from transcriptome profiling
4:45 p.m.	Kelly Vazquez, Wheaton College Modulation of the Biomechanical Environment alters β-cell Function and Maturity
5:00 p.m.	Coffee/Snack break (15 minutes)

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5:15 p.m. - 6:30 p.m. — Paul Lacy Medal Award Presentation and Lecture  

Location: Hine Hall Auditorium

5:15 p.m. - 5:30 p.m.	Paul Lacy Medal Award Introduction - Carmella Evans-Molina, MD, PhD
5:30 p.m. - 6:30 p.m.	Paul Lacy Medal Award Lecture – Al Powers, MD

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6:30 p.m. - 8:30 p.m. — Cocktail Hour and Poster Session

Location:  Hine Hall Auditorium lobby, 1st floor hall outside of auditorium lobby, Hine Hall 2nd floor lobby and Hine Hall 2nd floor hall outside of 2nd floor lobby

• Heavy hors d’oeuvres will be provided during the poster session and cocktail hour. Dinner will be on one's own plans following the event.

9:00 a.m. - 10:30 a.m. — Session 3: Mitochondria: The Powerhouse of the Beta Cell

Location: Hine Hall Auditorium
Moderator: Sam Stephens, PhD, University of Iowa

9:00 a.m.	Emily Walker, University of Michigan Retrograde mitochondrial signaling governs the identity and maturity of metabolic tissues
9:15 a.m.	Kazuno Omori, Mayo Clinic Investigating effects of modulating glucokinase activity on human islet and mitochondrial function under diabetogenic conditions
9:30 a.m.	Debjyoti Kundu, Indiana Biosciences Research Institute Emerging Role of Ndufaf8: A mitochondrial Factor Shaping Beta Cell Metabolism, Stress Adaptation, and Immune Tolerance
9:45 a.m.	Becca Davidson, University of Michigan Mitochondrial DNA Integrity is Required for β-Cell Health In Vivo
10:00 a.m.	Coffee/Snack Break (30 minutes)

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10:30 a.m. - 12:00 p.m. — Session 4: Islet Inflammation and Immune Interactions 

Location: Hine Hall Auditorium
Moderator: Jon Piganelli, PhD, IU School of Medicine 

10:30 a.m.	Michael Schleh, Vanderbilt University Late-life caloric restriction reduces insulin secretory demand and alters islet immune composition by limiting antigen presentation and T-lymphocyte infiltration
10:45 a.m.	Jerry Xu, IU School of Medicine Proinflammatory stress activates neutral sphingomyelinase 2 based generation of a ceramide-enriched β-cell EV subpopulation
11:00 a.m.	Leslie Wagner, IU School of Medicine β-Cell Heterogeneity in the Acute IFN-α Response
11:15 a.m.	Charanya Muralidharan, University of Chicago Interferon Signaling in Type 1 Diabetes
11:30 a.m.	Sandra Blom, University of Iowa Proinflammatory cytokines mediate pancreatic β-cell specific alterations to Golgi integrity via iNOS-dependent mitochondrial inhibition
11:45 a.m.	Jacob Bartosiak, Medical College of Wisconsin  Evidence that innate immune activation controls gene expression in islet endocrine cells

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• Lunch will be provided in the Tower Ballroom.

1:00 p.m. - 3:00 p.m. — Session 5: Islet Physiology and Function

Location: Hine Hall Auditorium
Moderator: Emily Walker, PhD, University of Michigan

1:00 p.m.	Luhui Zhang, Mayo Clinic Time-restricted feeding prevents deleterious effects of diet-induced obesity on circadian regulation of β-cell function and transcription
1:15 p.m.	Snehasish Nag, IU School of Medicine  Decoding CHD5: Unveiling Its Role in Pancreatic Beta Cell Health and Function
1:30 p.m.	Temitayo Bamgbose, University of Alabama Birmingham Prokineticin 2 Promotes Beta-Cell Proliferation
1:45 p.m.	Isabella Melena, Washington University St. Louis Primary Cilia Regulate GLP-1 Signaling in Pancreatic Beta Cells
2:00 p.m.	Wenzhen Zhu, University of Michigan IRE1α Signaling is Required for Islet Alpha Cell Function
2:15 p.m.	Matthew Dickerson, Vanderbilt University Sodium/potassium ATPases promote alpha-cell glucagon secretion in response to hypoglycemia
2:30 p.m.	Coffee/Snack Break (20 minutes)

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2:50 p.m. - 4:45 p.m. — Session 6: Beta cell development, maturation and adaptation

Location: Hine Hall Auditorium
Moderator: Anoop Arunagiri, PhD, East Tennessee State University

2:50 p.m.	Avinil Das Sharma, IU School of Medicine Delineating the role of CHD4 in pancreatic islet development
3:05 p.m.	Matthew Varney, University of Pennsylvania The RNA binding protein hnRNPK regulates beta-cell insulin secretion and endocrine cell mass in pancreatic islets
3:20 p.m.	Hsuan Yeh, Children’s Hospital of Pittsburgh Glucocorticoid receptor deficiency impairs gestational β-cell compensation and contributes to gestational diabetes
3:35 p.m.	Katherine Perez, University of Alabama at Birmingham Tomosyn-2 Regulates β-Cell Proliferation and Functional Maturity in Neonatal Islets
3:50 p.m.	Jin Li, University of Michigan The mitochondrial chaperone GRPEL1 promotes β-cell development and maturation
4:05 p.m.	Catherina BP Villaca. Indiana University Indianapolis Beta cell translation capacity drives beta cell function
4:20 p.m.	Coffee/Snack Break (25 minutes)

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4:45 p.m. - 5:20 p.m. — Sorenson Young Investigator Award Presentation and Lecture

4:45 p.m. – 4:50 p.m.	Sorenson Young Investigator Award Introduction  - Amelia Linnemann, PhD
4:50 p.m. – 5:20 p.m.	Sorenson Young Investigator Award Lecture - Jing Hughes, MD, PhD

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5:20 p.m. - 6:00 p.m. — Oral and Poster Presentation Awards & Closing Remarks
Location: Hine Hall Auditorium

5:20 p.m. - 5:50 p.m.	Midwest Islet Club Oral and Poster Presentation Awards
5:50 p.m. - 6:00 p.m.	Concluding Remarks - MIC Organizers

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• Attendees can take this time to return to their hotel rooms, refresh and then travel or walk to Punch Bowl Social for dinner and entertainment.

Please join us for food, networking, games and entertainment at Punch Bowl Social in downtown Indianapolis! 

• MIC-organized food, beverage and entertainment access will be available until 10 p.m. However, attendees are welcome to continue socializing beyond this time. Punch Bowl Social closes at 11 p.m.

Location: Tower Ballroom

• Attending this optional breakfast for trainees and faculty requires advanced notice.

Diras2: a novel regulator of glucose metabolism and pancreatic islet survival

 

Nida Ajmal¹, Kathryn L. Corbin¹, Nico Cunningham², Palwasha Khan¹, Michael A. Kalwat³, and Craig S. Nunemaker¹

 

¹Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, 45701

²Honors Tutorial College (HTC), Ohio University, Athens, OH, 45701

³Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, IN, 46202

 

Diras2 is a small Ras-related GTPase predominantly expressed in the brain and has been linked to neurological conditions such as ADHD. Interestingly, our lab recently identified Diras2 expression in pancreatic islets, particularly in response to stress, suggesting it might play an unrecognized role in islet function. To elucidate the novel role of Diras2 in pancreatic islets and its physiological significance, we characterized global Diras2^-/- knockout male mice on C57B6N/J background compared to C57B6N/J wildtype (WT) mice with functional Diras2. The glucose tolerance test (GTT) and insulin tolerance test (ITT) under chow and high fat diet revealed enhanced glucose tolerance in Diras2^-/- mice, while body weight showed no significant differences compared to WT controls. Functional studies on islets isolated through collagenase digestion showed no significant difference in insulin secretion and content between both groups under 11mM and 28mM glucose concentrations, as quantified by ELISA. Stress response studies using beta-cell-specific stressors such as proinflammatory cytokines (IL-1β-5ng/mL, TNF-α 10ng/mL and IFN-ϒ, 100ng/mL) demonstrated that Diras2^-/- mice were significantly protected against cytokine-induced apoptosis after overnight treatment when measured through cell death markers such as propidium iodide and annexinV. These findings suggest that improved glucose tolerance in the absence of Diras2 may originate from the alterations in neural signals, given the high expression of Diras2 in the brain. Furthermore, Diras2 may possess a proapoptotic role in islets and reducing Diras2 expression may reduce cytokine-induced damage which further offer insights into its potential contribution to metabolic homeostasis, and its relevance as a therapeutic target for diabetes.

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A Chemical Approach to Improve Proinsulin Folding and Insulin Production

 

Maroof Alam¹, Nikita Daniel¹, Bhaskar K. Chatterjee², Shannon M. Lacy², Matthias C. Truttmann², Peter Arvan¹

 

¹Division of Metabolism, Endocrinology & Diabetes University of Michigan Medical Center, Ann Arbor, Michigan

²Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, Michigan

 

To synthesize biologically active insulin, proinsulin must fold in the endoplasmic reticulum (ER), including the intramolecular formation of B7-A7, A6-A11, and B19-A20 disulfide bonds. Two of these bonds (B7-A7 and B19-A20) are absolutely required for forward-trafficking of proinsulin from the ER. Non-native folding of proinsulin monomers leads to the mispairing of disulfide bonds, including the formation of aberrant intermolecular disulfide-linked complexes —  especially disulfide linked dimers. The ER chaperone BiP/GRP78 is thought to limit proinsulin misfolding. Beta cell treatments that inactivate BiP (such as the toxin SubAB) result in massive formation of aberrant intermolecular disulfide-linked complexes of proinsulin. The enzyme encoded by FICD is responsible for both BiP AMPylation (inactivation) and BiP deAMPylation (activation). Using a chemical screen to search for inhibitors of FICD-mediated BiP AMPylation (in preference to deAMPylation), the compound Closantel (C22) and its sodium salt (C73) were identified. We find these compounds preferentially inhibit FICD-mediated AMPylation in intact cells. Notably, both FICD AMPylation inhibitors enhance net proinsulin folding and forward-trafficking in INS1E cells. Our preliminary data suggest that AMPylation-inhibition also increases insulin production. Two murine models of diabetes: db/db mice, and Ins2-proinsulin-R(B22)E heterozygotes, develop insulin deficiency with significant proinsulin misfolding. Islets from these animals offer a suitable test system to establish whether FICD inhibitors can improve proinsulin folding and physiological insulin production. If so, FICD may be the first druggable target to enhance proinsulin folding, trafficking, and insulin production in rare patients with monogenic INS mutations, and in patients with common forms of type 2 diabetes.

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Gɑ_z Regulates Islet Hormone Secretion in a Mouse Model of Type 1 Diabetes

 

Amanda E. Allender^1,2, Rachel J. Fenske^1,2, Darby C. Peter^1,2, Mark T. Cadena^1,2, Haley N. Wienkes]^1,2, Kathryn A. Carbajal^1,2, Liam D. Hurley^1,2, Michelle E. Kimple^1,2

 

¹William S. Middleton Memorial Veterans Hospital

²Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA

 

Type 1 diabetes (T1D) is a disease of decreased functional beta-cell mass and impaired hormone secretion following immune infiltration to the pancreatic islet. Beta-cell proliferation, survival, and insulin secretion are regulated by G protein coupled receptors (GPCRs) that act through specific G proteins to regulate cellular second messenger levels like cyclic adenosine monophosphate (cAMP). Beta-cell cAMP potentiates insulin secretion and enhances proliferation and survival. Beta-cell cAMP production is stimulated by Glucagon-like peptide-1 receptor (GLP-1R) via G_s activity and inhibited by Prostaglandin EP3 receptor (EP3) via G_z activity. Previous work with the non-obese diabetic (NOD) mouse model of T1D revealed G_z contributes to disease progression, as mice lacking the catalytic alpha subunit Gɑ_z are protected from developing diabetes. Presence or absence of Gɑ_z does not impact glucose-stimulated insulin secretion (GSIS), but in female NOD mice Gɑ_z loss partially restores a defect in the potentiating effect of a GLP-1R agonist on GSIS. Delta-cells within the islet express GLP-1R, and GLP-1R activity potentiates glucose-stimulated somatostatin secretion (GSSS). Notably, islets from Gɑ_z-null mice exhibit greater GLP-1R agonist potentiation of GSSS compared to WT controls. This finding may be due to increased delta-cell GLP-1R expression in Gɑ_z-null mice compared to WT: a hypothesis we are testing by immunofluorescence experiments. Alternatively, Gɑ_z may be expressed in the delta-cell and have direct effects on somatostatin secretion: a hypothesis supported by previous immunohistochemical analyses. Regardless of the underlying mechanisms our experiments have revealed a critical role for Gɑ_z in regulating islet hormone secretion and susceptibility to T1D.

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Retinoid signaling and GATA4 cooperate to regulate pancreas development and function

 

Andrea Alvarez-Maldonado^1,2, Daria Podgorski², Annalisa Deguzman², Paul Gadue³, Lori Sussel⁴, and David S Lorberbaum^1,2,5

 

¹Cellular and Molecular Biology, University of Michigan, Ann Arbor

²Caswell Diabetes Institute, University of Michigan, Ann Arbor

³Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia

⁴Barbara Davis Center for Diabetes, University of Colorado, Denver, Anschutz Medical Campus ⁵Department of Pharmacology, University of Michigan, Ann Arbor

 

Understanding pancreatic development is crucial as many diseases that impact pancreas function stem from defects arising during organ specification. Among the many signals important for directing pancreas development are retinoids and the transcription factors (TFs) GATA4 and GATA6, which are both known to direct progenitor specification and differentiation. More than 80 mutations in GATA4 and GATA6 have been associated with pancreas agenesis, insufficiency, and diabetes. Interestingly, limiting retinoid signaling is necessary to accurately model these mutations in hPSC-derived islet-like cells, reinforcing the idea of RA-GATA cooperation. Despite advances like these in defining pancreatic developmental signals, critical knowledge gaps remain. To address these gaps, my project investigates how retinoic acid (RA), a component of the retinoid signaling pathway, interacts with GATA4 during pancreatic cell specification. We have confirmed a functional synergy between RA signaling and GATA4 that occurs during pancreas development and impacts adult function murine models of pancreatic progenitor specification. Our CRE-LOX system enables simultaneous RA inhibition and GATA4 knockout revealing striking phenotypes across pancreatic cells. Preliminary data show those compound mutant mice are hyperglycemic, exhibit disrupted islet architecture, develop cysts, and display abnormal exocrine tissue formation. These findings highlight RA-GATA4 synergy that occurs during pancreatic progenitor specification and establish a framework for understanding their roles in pancreas development, function, and disease. Insights from this research could help explain patient defects and inform novel therapeutic interventions for pancreatic disorders.

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Role of JAK/STAT Signaling Pathway in β-cell Extracellular Vesicle PD-L1 Shuttling and Regulation

 

Irene Amalaraj¹, Chaitra Rao¹, Saptarshi Roy², Jon D. Piganelli², Emily K. Sims¹

 

¹Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA

²Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

 

Type 1 Diabetes (T1D) progression is influenced by intercellular communication between immune cells and β-cells using extracellular vesicle (EV) cargo. We have previously demonstrated that treatment of β-cells with IFN-α or IFN-γ enriches expression of the immune checkpoint PD-L1 on EVs without an increase in EV number and that β-cell EV PD-L1 binds PD1 to inactivate CD8+T cells. However, mechanistic regulators of EV PD-L1 expression are not fully understood. Based on regulation of intracellular PD-L1 we hypothesized that the JAK/STAT signaling pathway enhances PD-L1 incorporation into β-cell EVs.

 

To test this, NIT-1 β-cell lines were transfected with siRNA for STAT1+2 or treated for 24 hours with JAK1/2 inhibitor (Barcitinib, SelleckChem) and TYK2 inhibitor (Deucravacitinib, MedChemExpress) before stimulation with 100 ng/mL IFN-α. EVs were isolated and analyzed for PD-L1 content and function using tetraspanin-based beads and an Exoview imaging system.

 

IFN-α treatment doubled EV PD-L1 cargo without increasing EV numbers. Combined STAT1/STAT2 inhibition abrogated IFN-α induced increases in EV PD-L1. Our findings indicate that STAT pathway activation is required for IFN-induced β cell EV PD-L1 shuttling, suggesting a role in paracrine signaling between β cells and the immune system in T1D.

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Dysfunctional β-cell Autophagy Stimulates Lysosomal Exocytosis, Promoting Alterations in the Secretome and Antigen Presentation Pathways

 

Matthew C. Austin¹, Saptarshi Roy², Jon D. Piganelli², Amelia K. Linnemann^3,4*

 

¹Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, United States

²Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States

³Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, United States

⁴Indiana Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, United States

 

We recently discovered that β-cell autophagy is impaired in NOD mice and humans prior to the onset of T1D, implicating this pathway in T1D pathogenesis. We also showed that impaired β-cell autophagy leads to ER stress, increased HLA-I expression, and enhanced β-cell immunogenicity. Based on these observations, here we sought to characterize the mechanisms by which defective autophagy increases β-cell HLA-I expression and enhanced immunogenicity. We used flow cytometry to assess how impaired autophagy affects Endo-C βH1 LAMP1 surface expression —a readout for lysosomal exocytosis —under basal and IFNα stimulated conditions. Under the same conditions, we performed flow cytometry on intracellular organelles to assess HLA-I co-localization with LAMP1+ organelles. Next, we performed Olink proteomics to assess the β-cell secretome under conditions of impaired autophagy. We found that β-cells treated with lysosome acidification inhibitor, Bafilomycin A1, showed a rapid increase of LAMP1 surface expression. This corresponded to alterations in the β-cell secretome under conditions of impaired autophagy combined with IFNα treatment. As our previous studies showed enhanced HLA-I expression when β-cell autophagy is impaired, we hypothesized that recycling of HLA-I complexes may contribute to this phenomenon. In support of this hypothesis, we detected LAMP1+/HLA-I+ double positive subcellular events, indicative of HLA-I molecule presence on lysosomes. Overall, we found that impaired β-cell autophagy induces lysosome exocytosis. This leads to changes in the β-cell secretome, thus likely altering the β-cell-immune cell dialogue. Impaired β-cell autophagy also enhances surface HLA-I expression due to reduced degradation and subsequent recycling, which may increase β-cell immune visibility.

 

Funding support from the Riley Children’s Foundation (AKL), R01DK124380 (AKL), DK064466 (MCA), and F30DK142522 (MCA).

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Determining the Role of the Activating Transcription Factor 5 in Pancreatic Beta-Cell Homeostasis

 

Romie Azor^1,2, Christine Juliana², Doris Stoffers² 

 

¹University of Pennsylvania Perelman School of Medicine Department of Biochemistry, Biophysics and Chemical Biology and Molecular Biophysics

²University of Institute of Diabetes, Obesity and Metabolism, Pennsylvania

 

The unfolded protein response (UPR) plays a critical role in fine-tuning the ability of the pancreatic β-cell to adapt insulin processing and secretion to regulate glucose homeostasis. While dysregulation of the UPR is a key driver of β-cell dysfunction and apoptosis, the mechanisms that regulate the endoplasmic reticulum (ER)  stress response remain incompletely defined. Our lab has identified Activating Transcription Factor 5 (ATF5), a stress-induced transcription factor, as a potential player in modulating β-cell ER stress responses. ATF5 exhibits tissue-specific expression and function, including localizing to mitochondria and participating in the mitochondrial UPR.  Here, we generated an Atf5 conditional allele (Atf5^fl/fl) and crossed to Tg(Ins2-Cre)Herr to obtain heterozygous and homozygous ATF5 β-cell deficient mice (Atf5^HETbKO and Atf5^bKO, respectively). Both male and female Atf5^HETbKO and Atf5^bKOmice exhibit normal body weight and ad libitum blood glucose levels. Male Atf5^HETbKO mice had impaired glucose tolerance with aging but not Atf5^bKO mice.  Interestingly, female Atf5^HETbKO and Atf5^bKO had impaired oral but not intraperitoneal glucose tolerance irrespective of age,  suggesting a role for Atf5 in the incretin  response in female mice. In parallel to these observations, we find that ATF5 is induced  by ER stress (thapsigargin) and glucolipotoxic conditions in mouse islets, by leveraging an epitope tagged endogenous ATF5 mouse model.  Our findings raise the possibility that ATF5 plays a role in the incretin response and ER stress.  Thus, we anticipate the findings of these studies will provide potential avenues for therapeutic targets to treat diabetes.

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Prokineticin 2 Promotes Beta-Cell Proliferation

 

Temitayo T. Bamgbose¹, Weidong Wang¹

 

¹Department of Genetics, Heersink School of Medicine, UAB Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, United States

Diabetes is characterized by progressive β-cell loss, death, dedifferentiation, and exhaustion. Thus, there is a need to discover novel therapeutic agents that can induce β-cell proliferation and regeneration to counteract β-cell death and exhaustion. We recently performed RNA-seq and discovered that high glucose increases the expression of PROK2 in β-cells. PROK2 is a secreted protein that plays diverse physiological roles, such as circadian rhythm regulation, inflammation, angiogenesis, and neuroprotection. However, there is a paucity of knowledge on the contribution of PROK2 to β-cell function. Through immunofluorescence staining, proliferation, and Glucose-stimulated insulin secretion (GSIS) assays, we report that PROK2 induces the proliferation of islet β-cells while preserving their insulin production and secretion function. Immunofluorescence staining also demonstrates that PROK2 expression is augmented in the pancreatic islets of physiological mouse models (pregnant and obese) that induce β-cell regeneration. Importantly, β-cell-specific deletion of PROK2 leads to a reduction in pancreatic β-cell mass. Consistently, we observed that in high-fat diet (HFD) fed mice, β-cell-specific deletion of PROK2 results in a reduction of blood insulin levels in the fed state. Collectively, our findings establish that PROK2 plays an important role in β-cell proliferation.

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Regionally defined stem cell derived islet differentiation

 

Manuj Bandral¹ and David Lorberbaum¹

 

¹University of Michigan

 

Type 1 diabetes (T1D) is an autoimmune disease resulting from destruction of insulin-producing β-cells in pancreatic islets. While exogenous insulin is the gold standard treatment, islet or whole pancreas transplantation remains an ideal cure, but limited by organ scarcity from donors. Alternatively, human pluripotent stem cells (hPSCs) hold great promise as a cell replacement therapy, but even the best in vitro generated stem cell-derived islets (SC-islets) possess compromised efficiency, specificity, and maturity. Since these protocols primarily rely on developmental cues discovered in rodent models, we propose using in vivo mouse models alongside in vitro hPSCs to address pitfalls in differentiation protocols and improve SC-islet maturation and function.

 

We are interested in the phase of pancreas development during which the dorsal and ventral pancreatic buds receive distinct signals before fusing into an integrated, functional pancreas. Such regional cues are not accounted for in hPSC derived SC-islets. We hypothesize that these regional signaling differences are essential for generating SC-islets and can improve differentiation of mature endocrine cells in vitro.

 

To address our hypothesis, we adapted published protocols to establish SC-islet differentiations and developed a mouse model allowing the isolation and separate cultivation of fetal dorsal and ventral pancreatic tissues. By combining these models with transcriptomics and proteomic analyses we will refine our differentiations to incorporate region-specific signaling cues to test our hypothesis. Ultimately, this work will contribute to the optimization of stem cell-based therapies for T1D, bringing us closer to a scalable cell replacement strategy to treat this disease.

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Evidence that innate immune activation controls gene expression in islet endocrine cells

 

Jacob T. Bartosiak¹, Katherine Harty¹, Elaine Schumacher¹, Polly A. Hansen¹, John A. Corbett¹

 

¹Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin

 

Cytokine signaling in islets has been suggested as a potential cause of type 1 diabetes due to the inhibition insulin secretion and islet degeneration. Our recent work suggests that the primary effects of cytokine signaling in islets may be protective and work through the upregulation of antipathogen and antioxidant genes in β-cells. Given that these studies have been predominantly performed in vitro using isolated islets and purified populations of endocrine cells, the goal of this study was to evaluate the effects of endogenously produced cytokines on gene expression in endocrine cells.

 

We explored the response of islets to endogenously produced cytokines following exposure to the well characterized bacterial and viral pathogen-associated molecular patterns (PAMP) lipopolysaccharide (gram^- bacteria) and poly(I:C) (dsRNA). Proinflammatory cytokines, such as IL-1β, are produced and circulated hematogenously within hours of PAMP exposure resulting in the upregulation of antiviral, antibacterial, and antioxidant genes and repression of identity factors in β-cells identified by scRNA-sequencing and confirmed in islets by qRT-PCR. Importantly, following PAMP clearance and reduction in cytokine levels, transcriptional responses return to baseline levels ~24 h after PAMP exposure. Using mice with β-cell specific deletion of the IL-1 signaling receptor, IL1r1, we have identified IL-1 as a key mediator of early islet responses to systemic innate immune activation. Our findings provide direct evidence for cross talk between the innate immune and endocrine systems and support our hypothesis that β-cells respond with the induction of protective cellular responses to reduce their susceptibility to virus-mediated damage.

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Characterizing the Role of β-cell Endocytic Processes in Autophagy

 

Yashaswini Battina¹, Alissa Muncy¹, Matthew Austin², and Amelia K. Linnemann^1,3

 

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN

²Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

³Indiana Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN

 

Type 1 Diabetes (T1D) is an autoimmune disease that results from the dysfunction and loss of insulin producing β-cells. Despite rising incidence rates, the mechanisms underlying disease pathogenesis remain poorly understood. Recent evidence suggests that early impairments in β-cell function may contribute to disease development. In line with these data, we recently observed that the cell degradative process of autophagy is impaired in human T1D, which may exacerbate immune-mediated β-cell destruction. The goal of this project is to better understand the mechanism by which impaired β-cell autophagy and related endocytic processes may contribute to early T1D pathogenesis. We performed bulk proteomics in islets from mice with defective β-cell autophagy and observed downregulation of several proteins involved in clathrin-mediated endocytosis. Notably, Rab11b, a key regulator of clathrin-mediated endocytosis of insulin granules and lysosome formation was reduced in autophagy deficient islets. Therefore, we set out to evaluate the functional role of Rab11b in β-cell autophagy. Using immunofluorescence staining of autophagy deficient and control mouse islets, the downregulation of the Rab11b protein was confirmed. We then stimulated Rab11b in a cultured β-like rat cell line using the lipophilic statin, simvastatin, in the absence and presence of the autophagy inhibitor, Bafilomycin A to evaluate autophagy regulation by Rab11b. Results indicated that autophagosome accumulation was occurring in absence of Rab11b, suggesting that Rab11b plays a critical role in β-cell autophagy. Understanding Rab11b downregulation could therefore provide insights into β-cell dysfunction during T1D pathogenesis.

 

Funding: R01DK124380

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Cysteine promotes islet α-cell glucagon secretion by limiting oxidative inhibition of sodium channels

 

Soma Behera¹, Matthew T. Dickerson¹, Prasanna K. Dadi¹, Jordyn R. Dobson¹, Spencer Peachee¹, Shannon E. Gibson¹, David A. Jacobson¹

 

¹ Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.

 

Recent studies highlight the essential role of amino acids in α-cell function, with cysteine (Cys) showing the strongest stimulation of glucagon secretion. Diabetic conditions not only elevate glucagon secretion but also alter plasma Cys levels. However, knowledge about the underlying mechanism(s) of α-cell Cys action is limited. As α-cells are high in taurine, which has been shown to inhibit K_ATP channels, we deprived taurine production by removing extracellular Cys. To our surprise, brief removal of extracellular Cys (4-12hrs) significantly decreased glucagon secretion under hypoglycemic conditions from both human and mouse islets. The defect in α-cell secretion was accompanied by reduced total Ca²⁺ influx and increased whole-cell K_ATP currents. L-cysteine supplementation rapidly restored islet glucagon secretion and enhanced α-cell Ca²⁺ influx. Furthermore, voltage-gated sodium channel amplitude and α-cell electrical excitability were reduced in Cys-deprived mouse and human α-cells. Moreover, treatment with reducing agents (dithiothreitol and 3-mercaptopyruvate (3-MP), generates H₂S) partially restored voltage-gated sodium channel currents; 3-MP was also able to restore Ca²⁺ influx in Cys-deprived mouse islets. Taken together, the data indicates that Cys deficiency leads to oxidative suppression of islet α-cell sodium channels, which impairs action potential firing, Ca²⁺ influx, and glucagon secretion. This work suggests that Cys plays an important role maintaining α-cell redox potential, which promotes electrical activity and glucagon secretion.

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Microfluidic Culture Enhances Long-Term Survival and Function of Pancreatic Islets

 

Ananya Bharath¹; José M. de Hoyos Vega, PhD, MS¹; Alan M. Gonzalez-Suarez, PhD, MS¹; Satish K. Sen, PhD¹; Thanh Nguyen¹; Kuntol Rakshit, PhD¹; Gulnaz Stybayeva, MD, PhD¹; Alexander Revzin, PhD^1,3; Aleksey Matveyenko, PhD^1,2

 

¹Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA; ²Division of Gastroenterology and Hepatology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA; ³Division of Endocrinology, Diabetes, Metabolism and Nutrition, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA

 

Human islet studies are constrained by the limited viability and functionality of islets in ex vivo culture. Extending this culture window can facilitate studies on long-term islet function and enhance islet viability prior to transplantation. Previously, we have demonstrated that small-volume confinement in microfluidic devices promotes the local accumulation of secreted factors, supporting phenotypic maintenance in specialized epithelial cell types. Here, we applied this principle to develop a polydimethylsiloxane (PDMS) microfluidic (µF) device optimized for long-term islet culture. The µF device features an array of microwells designed to support islet viability and promote long-term functionality. To validate the utility of the device, isolated mice (C57BL/6) and human islets were seeded into the microfluidic device and maintained for up to three weeks. Both mouse and human islets cultured in the device exhibited higher viability than those in 24-well plates when cultured in identical media volumes, as assessed by calcein-AM and ethidium homodimer-1 staining. Functional assessment revealed a 20-fold increase (p = 0.025) in the glucose-stimulated insulin secretion (GSIS) stimulation index in microfluidic-cultured mouse/human islets compared to well-plate cultures over 21 days. Fluorescence microscopy confirmed the preservation of islet cytoarchitecture and cellular composition throughout the culture period in microfluidic-cultured mouse/human islets. This platform is a valuable tool for preclinical research, enabling extended human islet studies and improving islet quality before transplantation.

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Proinflammatory cytokines mediate pancreatic β-cell specific alterations to Golgi integrity via iNOS-dependent mitochondrial inhibition

 

Sandra E. Blom^1,2, Riley M. Behan-Bush^1,3, James A. Ankrum^1,3, Ling Yang^1,4, and Samuel B. Stephens^1,2,4

 

¹Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA, USA

²Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Iowa, Iowa City, IA, USA

³Roy J. Carver Department of Biomedical Engineering, University of Iowa, Iowa City, IA, USA

⁴Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USA

 

Type 1 Diabetes (T1D) results from autoimmune-mediated destruction of pancreatic β-cells. Emerging evidence highlights the β-cell as an active participant in T1D development, yet how the β-cell contributes to its own demise remains a critical knowledge gap in the field. Our studies have uncovered a novel role for proinflammatory cytokines in dismantling the β-cell’s secretory program that may directly address this knowledge gap. Early in disease pathology, immune cell release of proinflammatory cytokines, interleukin-1β (IL-1β), interferon-γ (IFN-γ), and tumor necrosis factor (TNF-α), leads to β-cell mitochondrial dysfunction, loss of insulin secretion, ER stress, and decreased β-cell viability. Our recent work demonstrates that proinflammatory cytokines, IL-1β, TNF-α, and IFN-γ, also disrupt β-cell Golgi structure and function in mouse, rat, and human β-cells, which may further explain early defects in β-cell secretory function important for T1D development. The structural modifications include Golgi compaction and, in some instances, loss of the inter-connecting ribbon leading to Golgi fragmentation. We further show that Golgi structural alterations coincide with altered cell-surface glycoprotein composition. Our data demonstrate that iNOS generated nitric oxide (NO) is necessary and sufficient for β-cell Golgi re-structuring. Moreover, the unique sensitivity of the β-cell to NO-dependent mitochondrial inhibition results in β-cell specific Golgi alterations that are absent in other cell types, including α-cells. Collectively, our studies provide critical clues as to how β-cell secretory functions are specifically impacted by cytokines and NO that may contribute to the development of β-cell autoantigens relevant to T1D.

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RIPK1 regulates nucleic acid sensor expression in IFNγ+dsRNA-induced β-cell demise

 

Renato C.S. Branco^1,4,5, Christopher J. Contreras^2,4,5, Noyonika Mukherjee^3,4,5, Egan G. Mather⁴, Li Lin⁴, Kaitlyn A. Colglazier⁴, Erica P. Cai^4,5, Andrew T. Templin^2,3,4,5

 

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202

²Department of Medicine, Roudebush VA Medical Center, Indiana University School of Medicine, Indianapolis, IN 46202

³Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

⁴Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, IN 46202

⁵Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN 46202

 

Type 1 diabetes (T1D) is characterized by immune-mediated β-cell destruction that is associated with activation of antiviral signaling pathways. Prior studies established that IFNγ+poly I:C (a synthetic dsRNA) elicit β-cell cytotoxicity, and we previously identified receptor interacting protein kinase 1 (RIPK1) as a mediator of IFNγ+poly I:C (IPIC)-induced β-cell death. Here, we hypothesized that RIPK1 promotes IPIC-induced β-cell demise in part via effects on nucleic acid sensor expression. We treated NOD-derived NIT-1 β-cells with IPIC for 24 hours and assessed cell death and nucleic acid sensor gene expression. We compared control (CTL, non-targeting gRNA) and Ripk1 gene-edited (Ripk1Δ, gRNA targeting exons 2-3) NIT-1 cells, and evaluated small molecule RIPK1 (SZM’679) and JAK (ruxolitinib) inhibitors. Treatment with IFNγ or poly I:C alone did not increase cell death in NIT-1 CTL cells. In contrast, treatment with IPIC significantly increased cell death concomitant with upregulation of nucleic acid sensors including Tlr3, Sting1, cGas, Zbp1, Adar1, and Ifih1. Blockade of RIPK1 in NIT-1 Ripk1Δ cells or with SZM’679 protected from IPIC-induced cell death, and IPIC-induced Tlr3, Sting1, cGas, Zbp1, Adar1, and Ifih1 expression was reduced in Ripk1Δ cells. IPIC-induced cell death and nucleic acid sensor expression were also mitigated by JAK inhibition. Our findings indicate that RIPK1 promotes IFNγ+dsRNA-induced β-cell death in part via upregulation of nucleic acid sensor expression downstream of JAK signaling. Future studies will interrogate mechanisms of nucleic acid sensor and dsRNA-induced β-cell death in greater detail and examine our findings in primary islets and human β-cells.

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Targeting the opposing roles of PGE₂ receptors, EP3 and EP4, in the pathogenesis of T1D

 

Juliann Burkett¹, Jennifer Fuhr³, Audrey Lucerne⁵, Victoria Gaeth⁴, and Maureen Gannon^1,2,3,4

 

¹Vanderbilt University Department of Molecular Physiology & Biophysics

²Vanderbilt University Department of Cell and Developmental Biology

³Vanderbit University Medical Center Department of Medicine

⁴Vanderbilt University Interdisciplinary Graduate Program

⁵Boston University

 

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by the destruction of insulin-producing beta cells in the pancreas. As a disease of both the immune system and the islets, targeting beta-cell health and autoimmunity may have the greatest potential for prevention/reversal of T1D. Prostaglandin E₂ (PGE₂) can modulate both the immune system and beta cells via two receptors, EP3 and EP4. Manipulating these receptors promotes beta-cell health. Additionally, it is well established that PGE₂ plays critical, context-dependent roles in inflammation. We hypothesize that EP3 and EP4 have opposing roles in maintaining functional beta cells in T1D through beta-cell-intrinsic and immune-dependent mechanisms. To test this hypothesis, female nonobese diabetic (NOD) mice were treated with the disease-accelerating agent, cyclophosphamide, at 10 weeks, followed by four weeks of treatment with an EP3 antagonist, DG-041, and an EP4 agonist, Rivenprost, or corresponding vehicle compounds. Mice receiving the dual-targeting therapy were significantly protected from T1D onset, showed lower levels of insulitis, maintained greater beta-cell mass, and preserved MafA expression. There was also a trend toward increased beta-cell antioxidant responses. Furthermore, while systemic T cell populations remained unchanged, dual-targeting treatment induced an expansion of myeloid-derived suppressor cells and shifted macrophages to an anti-inflammatory M2-like state. These results support the hypothesis that EP3 and EP4 modulation affects both beta cells and immune cells in T1D progression.

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The Effects of Calorie restriction on the Onset of Type 1 Diabetes

 

Amanda Cambraia¹, Melanie Cutler¹, Shristi Shrestha², Cristiane dos Santos¹, Jean-

Philippe Cartailler², Rafael Arrojo e Drigo¹

 

¹Vanderbilt University, Department of Molecular Physiology and Biophysics, Nashville,

TN, USA.

²Creative Data Solutions, Vanderbilt Center for Stem Cell Biology, Nashville, TN, USA.

 

Type 1 diabetes (T1D) is an autoimmune disorder where immune T cells attack and destroy the insulin-producing beta cells in the pancreatic islets, leading to reduced insulin production and persistent hyperglycemia. Diet and nutrition play a crucial role in supporting immune system function and maintaining homeostasis. Caloric restriction (CR) has been shown to extend lifespan across various organisms and may also slow the aging process. Our lab has identified CR as a promising nutrition-based strategy for delaying the aging of beta cells and enhancing glucose metabolism at both the cellular and systemic levels. In the context of T1D, CR has demonstrated potential in enhancing glucose sensitivity, normalizing insulin levels, and promoting anti-inflammatory mediators. However, the underlying mechanisms of CR's impact on T1D and its ability to modulate the disease phenotype and autoimmunity remain unclear. To investigate the effects of CR on the onset and progression of T1D, we exposed 8-week-old female NOD/ShiLtJ mice to CR for 2 months. Our findings showed that while CR did not improve glucose homeostasis or reduce beta cell insulin release in vivo, it effectively delayed the onset of hyperglycemia. Additionally, CR mice exhibited substantially less immune cell infiltration and inflammation in the pancreas. To further understand these observations, we applied single-cell RNA sequencing and confocal imaging, which revealed reduced signaling pathways associated with the extracellular matrix and cell adhesion molecules in the CR pancreases. Future experiments will investigate the mechanisms underlying CR's effects on cell migration, metabolism, and immune system interactions.

 

Early Dysregulation of Autophagy and Immune-Mediated b-Cell Dysfunction contribute to Type 1 Diabetes pathogenesis in NOD mice

 

Esmeralda Castelblanco¹, Marguerite Mrad¹, Irving Ramirez-Sotero¹, Monika Bambouskova¹ and Maria S. Remedi¹

 

¹Department of Medicine, Division of Endocrinology, Metabolism and lipid Research,

Washington University School of Medicine in St Louis

 

Type 1 diabetes (T1D) is characterized by autoimmune destruction of pancreatic b-cells, leading to insulin deficiency. While genetic predisposition plays a significant role in T1D susceptibility, environmental factors are crucial in triggering β-cell autoimmunity. We used Non-Obese Diabetic (NOD) to investigate the temporal dynamic interplay between endoplasmic reticulum (ER) stress, pro-inflammatory cytokines, and autophagy in T1D pathogenesis. At eight weeks of age, when NOD mice have normal blood glucose levels, TFEB, the master regulator of autophagy and lysosomal function, exhibited perinuclear localization in β-cells and LC3II protein levels were significantly elevated. Despite the early activation of autophagy pathway, autophagic process was dysregulated. By 10 weeks of age, islets from NOD mice exhibited increased CHOP gene expression (ER stress marker) and high levels of proinflammatory cytokines, including IL-1b and IL-6. These changes precede the dramatic increase in immune cell infiltration, the elevated blood glucose levels, and the impaired glucose tolerance observed in 15-week-old NOD mice. At 18 weeks of age, islets from NOD mice exhibited a decrease in α-, β- and d-cell proportions, and interestingly, β-cells upregulated TNF-α protein. These results correlated with reduced insulin production, impaired autophagy and β-cell dysfunction. Together, these findings demonstrate significantly impaired autophagic flux and immune-mediated β-cell dysfunction in the NOD mice, highlighting the complex interplay between environmental triggers, autophagy dynamics, ER stress, inflammation, and β-cell destruction in T1 diabetes pathogenesis. Understanding these mechanisms provides valuable insights into potential therapeutic strategies aimed at preserving β-cell function and delaying T1D progression in susceptible individuals.

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Txnrd1 is required for normal glucose-stimulated insulin secretion in pancreatic β-cells

 

Erin E. Fahey¹, Alex M. Clifford¹, Kendall Cashion¹, John A. Corbett², and Jennifer S. Stancill¹

 

¹Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA

²Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA

 

Reactive oxygen species (ROS), generated during glucose metabolism, are essential mediators of glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, excessive ROS can lead to β-cell damage and dysfunction, contributing to the pathogenesis of Type 1 and 2 diabetes. β-cells express low levels of antioxidants catalase and glutathione peroxidase. However, we have previously demonstrated that thioredoxin reductase, particularly cytoplasmic Txnrd1, along with thioredoxin and peroxiredoxin, protect β-cells from oxidative damage. Given that Txnrd1 is a major regulator of ROS levels in β-cells, we hypothesized that it may also play a role in GSIS. In this study, we explored the role of Txnrd1 in GSIS by depleting or inhibiting Txnrd1 in INS-1(832/13) cells and in mouse and human islets. Under stimulatory conditions, both Txnrd1 depletion and inhibition blunted insulin secretion. However, glucose uptake was only reduced in cells treated with pharmacological inhibitors (auranofin and Txnrd1 inhibitor 1) but not in cells depleted of Txnrd1 suggesting divergent mechanisms for the blunting of insulin secretion. Interestingly, treatment with antioxidants (PEG-Catalase and Trolox) following Txnrd1 inhibition or depletion did not restore insulin secretion. These results suggest that impaired insulin secretion is not due to excessive ROS and that Txnrd1 may play a broader role in β-cell function, through redox signaling or other processes involved in insulin release. In future studies, we will focus on identifying the specific step in glucose-stimulated insulin secretion affected by Txnrd1 depletion and inhibition.

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Loss of RIPK1 protects from autoimmune-associated β-cell destruction in vitro and in vivo

 

Christopher J Contreras^1,3,5, Noyonika Mukherjee^2,3,5, Egan G. Mather³, Renato C.S. Branco^{3 4,5}, Nansa Amarsaikhan³, Erica P. Cai^3,5, Andrew T. Templin^1,2,3,5

 

¹Department of Medicine, Roudebush VA Medical Center and Indiana University School of Medicine, Indianapolis, IN

²Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

³Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, IN

⁴Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN

⁵Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN

 

Type 1 diabetes (T1D) is characterized by autoimmune-associated β-cell loss and insulin insufficiency. Receptor interacting protein kinase 1 (RIPK1) is a multifunctional protein that regulates survival and death signaling in non-islet cell types. We previously found that Ripk1 is upregulated in NOD islets and β-cells from T1D donors, and that Ripk1 deficiency protects from cytokine-induced β-cell death. Here, we hypothesized that RIPK1 contributes to autoimmune-mediated β-cell destruction. We performed in vitro coculture studies using splenocytes from diabetic NOD mice with either NIT-1 control (CTL, non-targeting gRNA) or NIT-1 Ripk1 gene-edited (Ripk1Δ, gRNA targeting exons 2-3) β-cells derived from NOD mice. We also performed in vivo transplantation of luciferase-expressing NIT-1 CTL and NIT-1 Ripk1Δ  cells on opposite flanks of non-diabetic NSG mice before administration of splenocytes isolated from diabetic NOD mice. Following in vitro coculture, splenocyte IFNg production was not different when exposed to CTL versus Ripk1Δ cells, and the percent of CD69+CD8+ splenocytes was also unchanged. However, β-cell death was significantly lower in NIT-1 Ripk1Δ versus NIT-1 CTL cells after coculture. For in vivo transplantation studies, graft luminescence was not different between NIT-1 CTL and NIT-1 Ripk1Δ cells at baseline or 7 days after autoreactive splenocyte injection. However, at days 19 and 22 post splenocyte administration graft luminescence was significantly higher in NIT-1 Ripk1Δ versus NIT-1 CTL cells. Together, our data indicate that β-cell RIPK1 promotes autoimmune-mediated demise separate from effects on immune cell activation. Additional studies are needed to understand the mechanisms by which RIPK1 protects from autoimmune-mediated β-cell killing in vitro and in vivo.

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Assessing Islet Function and Gene Expression Changes During T1D Onset in Diabetes Resistant NOD Mice

 

Kristen V Coutinho¹, Iztiba M Deeba¹, Samuel O Poole¹, Harshraj Shinde², Hubert M. Tse², and Chad S. Hunter¹

 

¹Department of Internal Medicine - Division of Endocrinology, Diabetes and Clinical Pharmacology, University of Kansas Medical Center, 1000 Hixon, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA

²Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, 1012 Cates Hall West, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA

 

Introduction and Objective: Type 1 diabetes (T1D) is an autoimmune disease affecting insulin-producing pancreatic β-cells. In T1D, excess reactive oxygen species (ROS) produced by islet-infiltrating leukocytes mediate β-cell death. The goal of this study is to investigate how decreasing ROS impacts β-cell function and T1D onset.

 

Methods: We compared the T1D NOD mouse model with the NOD.Ncf1^m1J strain, which is a NOD mouse with decreased NADPH oxidase-derived ROS generation and delayed T1D incidence. To address β-cell function and gene expression during the T1D onset, we assessed mice from 6-24 weeks (W) of age for glucose tolerance, plasma insulin, glucose-stimulated insulin secretion, key islet markers, and whole islet gene expression changes.

 

Results: Surprisingly, NOD.Ncf1^m1J mice had decreased glucose tolerance at 10W with reduced insulin secretion, as compared to NOD, even though NOD.Ncf1^m1J mice are T1D resistant up to 24W. This glucose intolerance in NOD.Ncf1^m1J mice prior to T1D suggests ROS also contributes to β-cell function. While no overt differences were observed for cell-specific islet markers at any age (e.g., insulin, somatostatin, and glucagon), whole islet transcriptomics revealed modulated transcription in 10W NOD.Ncf1^m1J mice, with an upregulation of Reg3a (encoding a cell survival factor) and downregulation of Foxa1 (encoding an islet transcription factor). This differential gene expression may contribute to β-cell survival and mitigate subsequent ROS-mediated cellular destruction.

 

Conclusion: Taken together, while ROS reduction in NOD.Ncf1^m1J mice delays T1D incidence, the resulting islet gene expression changes may also alter β-cell function.

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Inhibition of Mitochondrial Metabolism and NO Mediated Activation of the UPR

 

Charles Danaher¹, Jeff Pietroske¹, John Corbett¹

 

¹The Medical College of Wisconsin

 

Despite being implicated in β-cell destruction in type 1 diabetes, nitric oxide (NO) selectively activates multiple protective mechanisms in β-cells, including the unfolded protein response (UPR). NO exerts many of its protective effects through its reversible inhibition of mitochondrial metabolism. β-cells are metabolically “inflexible,” and cannot increase glycolytic flux in response to mitochondrial inhibition. Because of this, micromolar NO decreases ATP over eight-fold in β-cells. The mechanism by which NO activates the UPR is unknown. This study tests the hypothesis that it is the inhibition of mitochondrial oxidative metabolism and reduction in ATP that leads to UPR activation in β cells exposed to nitric oxide. To test this hypothesis, UPR activation was measured through eif2a phosphorylation and XBP1 mRNA splicing after exposure to mitochondrial inhibitors (complexes I, III, IV) and nitric oxide in INS 832/13 cells. Mouse embryonic fibroblasts (MEFs) cultured in glucose were used as a metabolically flexible non-β cell control. Nitric oxide and mitochondrial poisons selectively activate the UPR in INS 832/13 cells but do not MEF cells. MEFs were then cultured in glucose-free, 10mM galactose containing media. When MEF cells are forced to generate ATP through the mitochondrial oxidation of glutamine, they activate the UPR in a β-cell like manner. Nitric oxide’s activation of the UPR in beta cells is likely due to its inhibition of oxidative metabolism and the subsequent drop in cellular ATP. These findings suggest another protective action of NO (at iNOS derived levels) facilitated by β-cell’s unique lack of metabolic flexibility.

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Delineating the role of CHD4 in pancreatic islet development

 

Avinil Das Sharma^1,2,3,4, Rajani George^1,2,3, Abigail Taylor^1,2,3, Sukrati Kanojia^1,2,3,4, Rebecca K. Davidson^1,2,3,4, Kayla Huter^1,2,3, Kassandra Sandoval^1,2,3, Meredith Osmulski^1,2,3, Jason Spaeth^1,2,3,4*

 

¹Center for Diabetes & Metabolic Diseases, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

²Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

³Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America

⁴Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

 

Strategies to increase β-cell mass by directing stem cells toward a β-cell fate are a putative therapy for diabetes. Thus, understanding transcriptional programs governing endocrine progenitor differentiation is crucial for these efforts. Previously, we demonstrated that PDX1 interacts with the CHD4 subunit of the Nucleosome Remodelling and Deacetylase complex to regulate glucose homeostasis and insulin secretion in mature mouse β-cells. Here, we reveal that PDX1-CHD4 interactions also occur in NEUROG3-expressing mouse endocrine progenitor cells. We hypothesize CHD4 controls chromatin accessibility and gene expression essential for endocrine cell development. To test this hypothesis, we generated endocrine-progenitor-specific Chd4-deficient mice (Chd4^Δislet) using Neurog3-Cre. Following confirmation of CHD4 knockout throughout the entire islet, we discovered Chd4^Δislet mutant mice are glucose intolerant at 4 weeks of age and severely hyperglycemic by 6 weeks. The hyperglycemia is accompanied with reduced serum insulin and trending increase in serum glucagon levels. Immunofluorescence analyses shows reduced MAFA and insulin/glucagon co-expressing cells in 6-week-old Chd4^Δislet islets. By 8 weeks of age, Chd4^Δislet mutants exhibit altered islet morphology with reduced PDX1, MAFA, UCN3, and insulin levels and apparent increases in glucagon-positive cells per islet. These data suggest CHD4 loss from endocrine progenitor cells significantly impacts postnatal glucose homeostasis. We predict these deficiencies derive from the failed allocation of islet cells during development. To delineate the mechanistic action of CHD4 in modulating the chromatin landscape to permit proper islet formation, single-cell RNA and ATAC-seq will be performed on endocrine committed cells from Chd4^Δislet mutant embryos. 

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Mitochondrial DNA Integrity is Required for β-Cell Health In Vivo

 

Becca Davidson¹, Jie Zhu¹, Emma Reck¹, Ava Stendahl¹, Rudolf J. Wiesner², Scott Soleimanpour^1,3

 

¹Division of Metabolism, Endocrinology, & Diabetes and Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

²Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.

³VA Ann Arbor Healthcare System, Ann Arbor, Michigan, United States of America.

 

Type 2 diabetes (T2D) is a metainflammatory disease characterized by impairments in mitochondrial function and ultrastructure that contribute to the overall disruption of β-cell function. Mitochondria rely on both the nuclear genome as well as their own 16.6 kilobase-pair circular genome to generate the machinery required for oxidative phosphorylation (OXPHOS). Recently, our group identified a reduction in mitochondrial DNA (mtDNA) copy number in islets from donors with T2D, indicating a disruption in mitochondrial genome stability. While mtDNA genome instability is implicated in several diseases, its impact on β-cell dysfunction in diabetes has yet to be explored.

 

Here, we generated a mouse model prone to increased β-cell mtDNA deletions by selective expression of a dominant negative Twinkle (Twnk^K320E) helicase mutant, which is essential for mtDNA maintenance. Beginning at 5 weeks, β-Twnk^K320E mice exhibited impaired glucose tolerance, which progressively worsened with age. Circulating insulin concentrations following glucose stimulation were also reduced in β-Twnk^K320E mice by 5 weeks of age, yet there was no difference in β-cell mass, suggestive of a β cell functional defect. Further, we observed a complete loss of MafA, a key regulator of β-cell maturity, from a subset of β cells in β-Twnk^K320E mice. While mitochondrial mass was unchanged between groups, β‑Twnk^K320E islets have altered expression of subunits of all OXPHOS complexes, as well as reduced glucose-stimulated oxygen consumption, indicative of impaired mitochondrial function. Together, these data suggest that the accumulation of mtDNA deletions diminishes β-cell function and support the importance of mitochondrial genome integrity to β-cell health.

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Beta cell MicroRNA-21 (miR-21) transcription is induced by HIF1a in diferent diabetes models

 

Andre De Oliveira¹, Chaitra Rao¹, Jerry Xu¹ and Emily Sims¹

 

¹Division of Pediatric Endocrinology and Diabetology, Herman B Wells Center for Pediatric Research; Center for Diabetes and Metabolic Diseases; Indiana University School of Medicine, Indianapolis, IN 46202.

 

Proinflammatory cytokines, such as IL-1β, TNF-α and IFN-γ play a crucial role in the development of diabetes. We previously showed that cytokines also increase β-cell miR-21, leading to increased expression of dedifferentiation markers, loss of β-cell identity, and reduced glucose-stimulated insulin secretion. HIF1a has been suggested as a miR-21 putative transcriptional regulator in other systems. Besides its well-characterized activation by low oxygen tension, other factors such as reactive oxygen species and cytokines can also increase HIF1a activity. The aim of this work was to understand if HIF1a may play a role in miR-21 transcriptional regulation in diabetogenic models such as cytokine exposure. To address this question, INS1 cells were treated for 24h with 5 ng/mL IL-1β, while EndoC-βH1 cells and human islets were treated with IL-1β + 100 ng/mL IFN-γ + 10 ng/mL TNF-α. Gene expression was analyzed by RT-qPCR and protein abundance and distribution were analyzed by immunofluorescence. Cytokine treatment led to an increase in HIF1a message and protein, as well as increased miR-21 expression in INS1 and EndoC-βH1 cells as well as in cultured human islets. HIF1a knockdown reduced miR-21 expression at baseline and in response to cytokine treatment, suggesting that HIF1a may play a role in miR-21 transcriptional upregulation in the context of cytokines. These data suggest a HIF1a-miR-21 axis may be responsible for the impacts of miR-21 on beta cell dysfunction in the context of proinflammatory islet stress.

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Adaptations of offspring islets to maternal WSD-feeding and metformin use in a non-human primate model

 

Alexa DelBene¹, Darian Carroll¹, Jennifer Fuhr^3,4, Rene Lindsley⁵, Melissa Kirigiti⁵, Tyler Dean⁵, Stephanie Wesolowski^6,7, Carrie McCurdy⁸, Jed Friedman⁹, Kjersti Aagaard^10,11, Paul Kievet⁵, Maureen Gannon^1,3,4,12

 

 ¹Department of Molecular Physiology and Biophysics, Vanderbilt University

²Western Kentucky University

³Department of Veterans Affairs Tennessee Valley

⁴Department of Medicine, Vanderbilt University Medical Center

⁵Division of Cardiometabolic Health, Oregon National Primate Research Center

⁶Department of Pediatrics, University of Colorado School of Medicine

⁷Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine

⁸Department of Human Physiology, University of Oregon

⁹Harold Hamm Diabetes Center at the University of Oklahoma

¹⁰Department of Obstetrics and Gynecology, Baylor College of Medicine

¹¹Division of Maternal-Fetal Medicine, Baylor College of Medicine

¹²Department of Cell and Developmental Biology, Vanderbilt University

 

The Development Origins of Health and Disease Hypothesis states that events in early life, particularly in utero, have lasting impacts on offspring health and disease risk. As prevalence of diabetes and obesity increase amongst pregnant individuals, we must understand how diet and medications that control glycemic levels impact the developing offspring. Using a non-human primate model (NHP) of maternal overnutrition our group has demonstrated that fetal exposure to a Western-style diet (WSD) leads to post-natal insulin hypersecretion and alterations in expression of ion-channels involved in insulin secretion. Use of metformin (Met), a common oral glycemic agent, in pregnancy has been severely understudied and possible effects on the offspring need to be evaluated as Met is not metabolized by the maternal liver and is actively transported across the placenta. We hypothesize that Met exposure in utero disrupts beta-cell maturity, function, and differentiation in offspring islets regardless of diet. Utilizing an NHP model of maternal overnutrition with or without Met during pregnancy, we are analyzing offspring pancreas at fetal and juvenile time points. Our data suggests that fetuses exposed to Met have decreased total insulin protein content and decreased insulin granules by electron microscopy.

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Sodium/potassium ATPases promote alpha-cell glucagon secretion in response to hypoglycemia

 

M.T. Dickerson, P.K. Dadi, J.R. Dobson, S. Behera, S. Peachee, S. E. Gibson, and D.A. Jacobson

 

Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.

 

The catalytic (NKAα1) and auxiliary (NKAβ1) subunits of the Na⁺/K⁺ ATPase (NKA) protein complex are highly expressed in α-cells, yet their function remains unclear. Given the importance of NKA in cation homeostasis and membrane potential regulation, we investigated its role in tuning α-cell Ca²⁺ handling and glucagon secretion. Using electrophysiology, fluorescence imaging, and hormone secretion assays, we examined NKA subunit-specific regulation of α-cell function. This was accomplished utilizing transgenic mouse lines with α-cell-specific NKAβ1 knockout (αNKAβ1KO) or conditional NKAα1 ablation (αNKAα1KO) and a genetically encoded Ca²⁺ sensor (Salsa6f). Human pseudoislets were also employed that contained α-cell-specific NKAβ1 knockdown (αNKAβ1KD). Under hyperglycemic conditions (11 mM glucose (11G)), α-cell Ca²⁺ was elevated 210±31% in αNKAβ1KO islets compared to controls (P<0.05). During hypoglycemic stimulation (1 mM glucose (1G)), Ca²⁺ increased 274±30% in control α-cells (P<0.01), but was unchanged in NKAβ1KO α-cells. In response to 1G, glucagon secretion decreased 40±15% (P<0.01) in αNKAβ1KO mouse islets and 23±11% (P<0.05) in αNKAβ1KD human pseudoislets. αNKAβ1KO mice were glucose intolerant (37±14% AUC increase; P<0.05), with 71±12% lower plasma glucagon during an insulin tolerance test (ITT; P<0.001). Plasma glucagon was decreased 62±13% (P<0.01) during an ITT seven days post-tamoxifen-induced α-cell NKAα1KO. Isolated islets from these mice contained Salsa6f-positive α-cells, indicating NKAα1 ablation does not cause rapid or complete α-cell destruction. Glucagon secretion from αNKAα1KO islets in response to 1G decreased 70±12% (P<0.01). These findings establish NKAα1 and NKAβ1 as key regulators of α-cell Ca²⁺ handling and suggest glucose regulation of NKA activity modulates glycemic control of glucagon secretion.

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The Diabetes-Associated K⁺ channel TALK-2 promotes human β-cell endoplasmic reticulum Ca²⁺ leak resulting in reduced glucose-stimulated insulin secretion

 

Jordyn R. Dobson¹, Prasanna K. Dadi¹, Arya Y. Nakhe¹, Matthew T. Dickerson¹, Soma Bahera¹, Shannon E. Gibson¹, Spencer J. Peacheé¹ & David A. Jacobson¹

 

¹Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee

 

Polymorphisms in or near KCNK17, which encodes TALK-2, result in elevated islet KCNK17 expression and are associated with an increased predisposition for developing type 2 diabetes (T2D). KCNK17 is one of the most abundant β-cell K⁺ channel transcripts with a high islet expression specificity index. Yet the β-cell function(s) of TALK-2 have not been determined, thus, we examined how TALK-2 modulates β-cell Ca²⁺ handling and insulin secretion. TALK-2 was found to localize to the endoplasmic reticulum (ER) and plasma membrane. Heterologous TALK-2 expression resulted in accelerated Ca²⁺_ER release, reduced Ca²⁺_ER storage, and increased basal cytosolic Ca²⁺. ER membrane single channel recordings found that TALK-2 formed functional channels on the ER membrane. Furthermore, TALK-2 depolarized the ER membrane potential following Ca²⁺_ER release. Human islet TALK-2 function was also examined utilizing magnetically sorted β-cells with shRNA-mediated TALK-2 knockdown (TALK-2-KD). In dispersed β-cells, TALK-2-KD increased Ca²⁺_ER stores compared to Scramble shRNA controls. Additionally, cytosolic Ca²⁺ entry was reduced in TALK-2-KD β-cells under euglycemic conditions and increased under hyperglycemic conditions. Moreover, insulin secretion measured from pseudoislets with β-cell specific TALK-2-KD showed a reduction under euglycemic conditions and elevation under hyperglycemic conditions. These data suggest that TALK-2 promotes β-cell Ca²⁺_ER release, which increases basal insulin release and reduces glucose-stimulated insulin secretion (GSIS). Therefore, T2D-associated polymorphisms in KCNK17 are predicted to diminish β-cell Ca²⁺_ER stores, limit GSIS, elevate basal insulin release and thus enhance insulin resistance.    

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Ultrastructure expansion microscopy of axonemal dynein in islet primary cilia

 

Xinhang Dong^{1, 2}, Jeong Hun Jo², Jing Hughes^2*

 

¹Department of Biomedical Engineering, McKelvey School of Engineering, Washington University, One Brookings Drive, Saint Louis, MO

²Department of Medicine, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, 660 South Euclid Ave, Saint Louis, MO

 

^*Correspondence: Jing Hughes (jing.hughes@wustl.edu)

 

Primary cilia are vital sensory organelles whose structures are challenging to study due to their solitary nature and intricate cytoskeleton. Current imaging modalities are limited in their ability to visualize structural details that are important for understanding primary cilia function. Ultrastructure expansion microscopy (U-ExM) is a recent super-resolution imaging technique that physically expands biological specimens using a swellable hydrogel, allowing structural interrogation of small cellular components such as cilia. In this study, we apply U-ExM to mouse and human pancreatic islets to visualize the axonemal cytoskeleton and associated proteins in primary cilia. Our study reveals the expression of axonemal dynein in islet primary cilia and centrioles, with DNAI1 being a principal subunit which we validate using targeted shRNA knockdown. We conclude that U-ExM is suitable for localizing protein expression in pancreatic islet cilia which contain axonemal dynein.

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Defining dependencies between key proteins regulating islet function

 

Christopher H. Emfinger¹, Elyse C. Freiberger¹, Mary E. Rabaglia¹, Jelena Kolic², Shane P. Simonett¹, Michael Shortreed³, Lauren E. Clark¹, Donnie S. Stapleton¹, Kathryn L. Schueler¹, Kelly A. Mitok¹, Tara Price¹, Joshua J. Coon^3,4, Lloyd Smith³, Matthew J. Merrins^5,6, James D. Johnson², Mark P. Keller¹, Mark Craven⁷, and Alan D. Attie^1,3

 

¹Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706 USA

²Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada

³Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706 USA ⁴Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI, 53706 USA

⁵Department of Medicine, University of Wisconsin-Madison, Madison, WI, 53706 USA

⁶William S. Middleton Memorial Veterans Hospital, Madison, WI 53705 USA

⁷Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI, 53706 USA

 

Only ~20% of type 2 diabetes heritability is explained by high-risk single-nucleotide polymorphisms (SNPs), suggesting islet genes may strongly influence one another to alter diabetes risk. Machine learning (ML) algorithms can potentially identify such dependencies between key proteins regulating complex traits like islet function. We asked whether islet traits (e.g. secretion) could be accurately predicted from islet protein abundance. We trained models predicting such traits in two datasets: human islets from ~140 donors and islets from 374 genetically diverse mice fed a high-fat/high-sucrose diet.

 

Models derived from mouse proteins predicted well (R > 0.5 vs R ~0 for permuted data) for many traits (e.g. GLP-1 driven secretion, plasma insulin). Of the ~ 5000 detected proteins, < 50 proteins typically explained most of any given model’s prediction. Key model proteins enriched for pathways relevant for islet function and included unstudied proteins having orthologues with glycemia-related SNPs. The models also successfully predicted related traits using data from other mouse studies showing the predictions are reproducible. Importantly, the models successfully predicted the direction of effect on insulin secretion for some proteins (e.g. DPP8) where correlation alone failed.

 

Models learned from human data also predicted well for several traits (e.g. fatty-acid-driven secretion). Interestingly, many traits predicted well in human data were less well predicted using mouse models. Several traits well-predicted in mice (e.g. plasma insulin) are also signatures of diet responsivity. Uniformity of diet and other factors in the mice contrasts with the human donors’ diversity and may explain the predictive discrepancy.

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Txnrd1 Protects Against Glucose Intolerance After High-Fat Diet

 

Erin Fahey¹, Edward E. Schmidt², and Jennifer S. Stancill¹

 

¹Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA

²Department of Microbiology and Cell Biology, Montana State University, Bozeman, Montana, 59717, USA

 

β-cells of the pancreas are responsible for secreting insulin in response to elevated blood glucose levels. However, damage, death, or dysfunction to β-cells results in insufficient insulin, and therefore, prolonged, elevated blood glucose levels as seen in diabetes.  Our goal is to study the protective mechanisms of β-cells that maintain viability and function. Thioredoxin reductase 1 (Txnrd1) is part of an antioxidant pathway β-cells use to protect against damaging reactive oxygen species (ROS). Inhibition and deletion of Txnrd1 impairs β-cell function through decreased insulin secretion. This study examines the role of Txnrd1 in glucose homeostasis and maintenance of β-cell mass in vivo by using a mouse model with a β-cell specific knockout of Txnrd1 (Txnrd1^fl/fl;Ins1^Cre/+), hereafter called βTxnrd1 KO. By stimulating weight gain and insulin resistance with high-fat diet (HFD) in our βTxnrd1 KO mouse model, we can study the role of Txnrd1 in diabetes susceptibility and β-cell function and protection.  We found that male βTxnrd1 KO mice are more glucose intolerant than WT littermates, despite having normal total pancreatic insulin content, increased β-cell dysfunction or decreased insulin sensitivity. Interestingly, our data also suggests a sex difference for the role of Txnrd1 in glucose homeostasis as KO females are more tolerant of glucose challenges and effects of HFD and maintain normal total pancreatic insulin content.  Future studies aim to directly assess insulin sensitivity in this model to determine if this is contributing to the male phenotype we observed, shedding further light on the role of Txnrd1 in glucose homeostasis.

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Arginine vasopressin receptor 1b control of pancreatic alpha cell function and glucagon secretion

 

Shannon E. Gibson¹, Prasanna K. Dadi¹, Spencer J. Peacheé¹ & David A. Jacobson¹

 

¹Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee

 

During the pathogenesis of diabetes, pancreatic α-cell calcium (Ca²⁺) entry is increased leading to hyperglucagonemia and increased hepatic glucose output. Despite its importance, the precise mechanisms controlling α-cell glucose sensing remain poorly understood. Hypoglycemia has been shown to stimulate vasopressin (AVP) secretion, which becomes elevated under euglycemic conditions in diabetic patients. AVP activates V1bR, a Gq-coupled receptor encoded by Avpr1b, that is highly and selectively expressed in α-cells within islets. We hypothesize that activation of α-cell V1bR raises the threshold for hypoglycemia-stimulated glucagon secretion. To test this hypothesis, we developed a mouse model with α-cell selective Avpr1b knockdown (αV1bR-KD). To mimic hypoglycemia, glycolysis was blocked with 2-deoxy-D-glucose (2DG). αV1bR-KD mice showed reduced 2DG-stimulated glucagon secretion compared to controls. We further investigated the effects of V1bR on Ca²⁺ handling in α-cells expressing a ratio metric genetically encoded Ca²⁺ indicator (Salsa6f, tdTomato-GCaMP6f). Activation of V1bR resulted in a significant increase in Ca²⁺ influx in control islet α-cells, which was absent in islets from αV1bR-KD mice. Additionally, V1bR activation showed glucose sensitivity with a larger α-cell Ca²⁺ response under hypoglycemic (1 mM glucose) compared to hyperglycemic conditions (9 mM glucose). V1bR induced Ca²⁺ influx occurred in the absence of extracellular Ca²⁺, suggesting V1bR Gq-signaling activates IP₃-mediated endoplasmic reticulum Ca²⁺ release. These findings demonstrate that V1bR activation under hypoglycemic conditions plays a crucial role in regulating α-cell glucagon secretion. Future studies will uncover whether elevated AVP contributes to hyperglucagonemia in diabetes. 

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Acute Regulation of Islet Proinsulin Synthesis in Nondiabetic and Diabetic states

 

Noah Gleason PhD¹ , Peter Arvan MD PhD¹

 

¹Division of Metabolism, Endocrinology & Diabetes, University of Michigan, Ann Arbor

 

Nutrients control acute insulin release (studied extensively) and insulin synthesis (studied less extensively).  Proinsulin biosynthesis is regulated by various nutrients including glucose, but the molecular mechanisms remain unclear.  Newly-translated preproinsulin is delivered to the endoplasmic reticulum via the Sec61 translocon for conversion to proinsulin.  In the classical 30-min amino acid pulse-radiolabeling protocol, proinsulin biosynthesis is normalized to total TCA-precipitable CPM.  Using radiolabeling, Itoh and Okamoto (Nature 1980 283:100-102) famously reported a ~10-fold increase in isolated rat islets when acutely increasing glucose from 2.8 mM to 25 mM.  However: 1) the normalizing denominator can itself be affected by nutrients (and various stress conditions), and 2) one or more nonradioactive amino acids are typically depleted (“starved”) in the medium to promote incorporation of label.  We recently found that without depletion of any amino acids, a 30-min co-treatment with inhibitors (of Sec61alpha and proteasome) allows for quantitative measurement of proinsulin biosynthesis, detected by immunoblot as preproinsulin normalized to an islet loading control.  In preliminary experiments, pancreatic islets isolated from WT C57BL/6 mice (initially recovered overnight in RPMI with 11.1 mM glucose) were transferred for 2h to media containing glucose concentrations ranging from 2.8 - 20 mM.  In the last 30 min of incubation, islets co-treated with inhibitors 10 µM TL033-plus-MG132 exhibited an 8-fold increase in proinsulin biosynthesis.  This methodology enables for the first time the study of the contribution of amino acids, individually or in combination, to the biosynthesis of proinsulin in rodent islets or human islets from nondiabetic or diabetic individuals.

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Effect of temperature and preservative selection on C-peptide degradation in randomly voided urine

 

Eli Hagedorn^1,2,4,5, Cameron Rostron^1,2,4,5, Robert V. Considine^2,3,5, Anthony Acton^2,5, Mallory Oswalt^2,5, and Carmella Evans-Molina^1-7 

 

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202

²Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

³Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202

⁴Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

⁵Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN 46202

⁶Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

⁷Richard L. Roudebush VA Medical Center, Indianapolis, IN 46202

 

Type 1 diabetes (T1D) is characterized by loss of endogenous production of insulin and C-peptide, which are co-secreted by β cells. Evaluation of serum C-peptide after a mixed-meal challenge is the gold standard T1D diagnostic; however, urine C-peptide (UCP) has emerged as an alternative that avoids venipuncture and allows for home collection. Our objective was to establish best practices for remote monitoring of urine C-peptide in clinical cohorts.

 

Randomly voided urine was collected from 18 non-diabetic individuals (mean age = 46.6 years, range 22-63 years, 94.4% female). Samples were treated with boric acid (BA, [215mM]), sodium carbonate (SC, [8.0mM]), or no preservative (NP), incubated for 12, 24, 48, or 72 hours at 4°C or 22°C, and frozen at -80°C. UCP was quantified with TOSOH electrochemiluminescence immunoassays and normalized to creatinine. UCP degradation was expressed as percent change in UCP:Creatinine ratio (%UCP:Cr) from baseline.

 

In NP groups, incubation at 22°C decreased %UCP:Cr at 48 hours (-16%, p=0.0309) and 72 hours (-21%, p =0.0068) compared to 4°C. In BA groups, incubation at 22°C reduced %UCP:Cr at 72 hours (-16%; p=0.0053) compared to 4°C. SC samples showed no significant change in %UCP:Cr between 4°C and 22°C incubation. Assessing %UCP:Cr change from 22°C to 4°C, SC treatment showed a trend of improvement over BA at 48 hours (+10.80%) and 72 hours (+10.87%).

 

These findings indicate that C-peptide is stable in randomly voided urine and UCP:Cr may be a valuable tool for clinical studies. UCP stability is improved with refrigeration and SC as a preservative.

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SERCA2 modulates the lipidome and insulin release in pancreatic β-cells

   

Maryalice Hartley^1,5,8, Mackenzie Pearson⁸, Hai Hoang Bui⁷, Farooq Syed¹⁰, Kenneth D Roth⁷, Tatsuyoshi Kono^2,5, and Carmella Evans-Molina^1-7

 

¹Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

²Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202

³Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

⁴Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202

⁵Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN 46202

⁶Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

⁷Richard L. Roudebush VA Medical Center, Indianapolis, IN 46202

⁸Diabetes and Metabolic Research

⁹Molecular Pharmacology, Lilly Corporate Center, Eli Lilly & Company, Indianapolis, IN, 46285 U.S.A.

¹⁰Department of Diabetes and Immunology, Beckman Research Institute at the City of Hope, Duarte, CA 91010

 

The sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA2) pump plays a critical role in regulating calcium levels within the endoplasmic reticulum (ER) of pancreatic β-cells. Previously, we demonstrated reduced SERCA2 expression in islets from human organ donors with type 2 diabetes (T2D) compared to islets from non-diabetic organ donors. Furthermore, we showed that heterozygous deletion of SERCA2 in mice (S2HET) leads to reduced whole body glucose tolerance and impaired islet glucose stimulated insulin secretion (GSIS) in response to diet-induced obesity. Given that the ER is the major hub for lipid synthesis, we hypothesized that SERCA2 deficiency in the pancreatic β-cell may be associated with changes in lipid composition, potentially affecting insulin production and secretion. To test this hypothesis, islets isolated from wildtype (WT) and S2HET mice were treated with glucolipotoxic stress (GLT, 25mM glucose and 0.5mM palmitate) for 72 hours. GSIS was measured by ELISA and lipidomics were performed using LC-MS/MS. GSIS assays showed reduced insulin secretion and an unexpected increase in insulin and proinsulin content in GLT-treated S2HET islets compared to WT islets. Analysis of lipid subclasses revealed reduced cholesterol, sphingomyelin, and glucosylceramide in GLT-treated S2HET islets along with significant alterations to sphingolipid acyl chain lengths generated by dihydroceramide synthases (CerS) 2 and 4. Collectively, our findings suggest that decreased SERCA2 expression during T2D conditions induces lipid remodeling and impairs insulin secretion. Understanding this novel mechanism of β-cell dysfunction may provide valuable insight into the pathophysiology and treatment of T2D.

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Physiological β-cell responses to PAMP-stimulated endogenous cytokines

 

Katherine R. Harty¹, Jacob T. Bartosiak¹, Polly A. Hansen¹, John A. Corbett¹

 

¹Medical College of Wisconsin, Department of Biochemistry, Milwaukee, WI

 

It has been suggested that cytokines produced in response to infection contribute to the destruction of β-cells and development of T1D, yet following infection, most individuals do not develop diabetes. These and other observations suggest that there are physiological roles of cytokine signaling in the endocrine system. We recently identified several mechanisms by which cytokines function to protect β-cells. This study tests the hypothesis that cytokines released via PAMP-stimulated innate immune activation induce protective pathways in β-cells.

 

C57BL/6J mice and Sprague Dawley rats received IP injections of 0.33mg/kg or 3mg/kg LPS, or 8mg/kg or 12mg/kg Poly(I:C). Islets were harvested 3-24 hours post injection, and the gene responses of these islets were evaluated via qRT-PCR and scRNAseq. Protein expression was evaluated via immunofluorescence. In response to the in vivo administration of the molecular PAMPs, LPS and Poly(I:C), in mice and rats, there is a transient increase in the expression of antipathogen genes in islets that are detected as early as 3 hours which return to baseline by 24 hours post injection. Immunofluorescent imaging determined the level and endocrine cell types in which there were increases in the protein expression of antipathogen genes. This response is conserved between LPS and Poly(I:C) and are similar when comparing rats and mice.

 

PAMP-stimulated cytokine production in two different species induces protective and antipathogen responses in β-cells supporting our hypothesis that there are physiological roles for cytokine signaling in endocrine cells that function to protect β-cells from damage and destruction associated with invading pathogens.

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β-cell primary cilia generate local ATP via glycolysis and communicate with mitochondria via the phosphoenolpyruvate cycle

 

Shih Ming Huang¹, Hannah R. Foster ¹, Jing W. Hughes², and Matthew J. Merrins¹

 

¹Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, Madison, WI

²Department of Medicine, Division of Endocrinology, Metabolism & Lipid Research, Washington University School of Medicine, St. Louis, MO

 

Primary cilia are specialized antenna-like sensory and signaling organelles present in pancreatic β-cells that play important roles in mediating insulin secretion. However, it remains unclear how cilia are provided with ATP. Here, β-cell deletion of the ATP-generating enzyme pyruvate kinase (PK) was used to investigate autonomous ciliary signaling, while β-cell deletion of mitochondrial Pck2, which supplies PK with mitochondrial phosphoenolpyruvate, was used to assess extraciliary signaling from the mitochondria. The dynamics of plasma membrane and ciliary metabolism were examined in intact islets using live-cell 3D imaging using subcellularly targeted biosensors. At elevated glucose, plasma membrane ATP/ADP, pyruvate, and fructose 1,6-bisphosphate (FBP), exhibit regular oscillatory patterns. Glucose elevation elicited an initial rise followed by a workload-dependent fall in plasma membrane and ciliary ATP/ADP as shown in the presence of diazoxide. The initial glucose-dependent rise in ciliary ATP/ADP and pyruvate was reduced by β-cell deletion of PKm1 or PCK2, but not PKm2. Ciliary FBP, an upstream glycolytic intermediate of the PK reaction, shows a greater initial rise in PKm1-βKO, a reduction in PCK2-βKO, and no difference in PKm2-βKO. Experiments performed at low glucose with leucine stimulation were used to isolate retrograde PEP signaling from mitochondria to cilia. The leucine-dependent rise in ciliary ATP/ADP, pyruvate, and FBP observed in control cells was strongly reduced by β-cell deletion of PCK2 or PKm1, but not PKm2. Taken together, our findings indicate distinct signaling dynamics in the plasma membrane and ciliary domains, and demonstrate the importance of PKm1 and an extraciliary phosphoenolpyruvate cycle for control of ciliary bioenergetics.

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Gɑ_z Regulates β-cell Cholecystokinin Expression by Direct and Indirect Mechanisms

 

Liam D. Hurley^1,2, Rodsy Modhurima^1,2, Dawn Belt Davis^1,2, and Michelle E. Kimple^1,2,3

 

¹Research Service, William S. Middleton Memorial Veterans Hospital, Madison, WI

²Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin School of Medicine and Public Health, Madison, WI

³Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI

 

The gut-derived peptide hormone cholecystokinin (CCK) is produced in pancreatic β-cells during stress conditions and acts in an autocrine manner to promote β-cell survival. While several transcription factors may promote Cck expression, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) is known to occupy the Cck promoter and promote Cck transcription. The prostaglandin EP3 receptor (EP3)-coupled Gɑ_z protein has been well-characterized as a negative regulator of β-cell cAMP production, limiting the ability of the β-cell to survive in response to metabolic stress. Previous work from our lab revealed the EP3 agonist, sulprostone, reduces INS-1E β-cell cAMP production and Cck expression, suggesting a link between β-cell CCK and Gɑ_z signaling. To test this, we developed and validated an INS-1 (832/13) cell line stably overexpressing human Gɑ_z. We treated parental or Gɑ_z-overexpressing INS-1 (832/13) cells with sulprostone or vehicle control for 24 h, collecting mRNA and protein samples for downstream analysis. We observed Gɑ_z expression treatment significantly altered INS-1 cell expression of Cck. When Gɑ_z is overexpressed, Cck and Cckar expression is significantly increased as compared to parental INS-1 (832/13) cells, with preliminary findings in streptozotocin-treated cells suggesting a link with cell stress pathways. To study the impact of loss of Gɑ_z on Cck expression in the context of β-cell stress, we turned to the non-diabetic obese C57BL/6J Leptin^Ob (Ob) mouse model. As expected, none of the Ob mice from either genotype were diabetic, and Gɑ_z loss had no impact on fasting blood glucose levels. During obesity, islet Cck expression was increased 60-fold in wild-type Ob mice as compared to lean controls, consistent with previous studies linking obesity-driven metabolic stress to increased Cck gene expression. Gɑ_z loss affected Cck expression depending on metabolic state. In lean mouse islets, where Cck expression is already low/undetectable, Gɑ_z loss did not have an effect. However, compensatory upregulation of Cck transcription was significantly blunted in islets from Gɑ_z-null Ob mice as compared to wild-type Ob mice. Combined, our results from cell lines and mouse models suggest that Gɑ_z regulates Cck expression through both direct (i.e., cAMP-mediated) and indirect (i.e., cell stress-mediated) mechanisms.

 

This work was supported in part by US Department of Veterans Affairs awards I01 BX005804 (to M.E.K.), IK6 BX006804 (to M.E.K.) and I01 BX006389 (to D.B.D.). This work was also supported in part by National Institutes of Health award R01 DK137505 (to M.E.K.).

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Investigating the role of ß-cell specific Robo2 expression in the maintenance of islet architecture

 

Rachel Kasper¹, Lindsay Propst¹, Katelyn Kosfeld¹, and Barak Blum¹

 

¹Department of Cell and Regenerative Biology, University of Wisconsin-Madison

 

Islets of Langerhans in mice have a distinct cellular architecture with a beta cell core surrounded by a mantle of alpha and delta cells. Our lab identified Robo2 as a gene involved in the establishment and maintenance of this architecture. Robo2 is a cell surface receptor which we hypothesize is responsible for this cellular organization as well as the expansion capabilities of islets. This expansion capability is important because islets need to expand in response to an increased demand for insulin, specifically in events of obesity and pregnancy. A previous student in the lab showed that an inducible, ß-cell specific Robo2 knock-out (KO) mouse model developed disorganized islets as well as issues regulating blood glucose. We have now developed a constitutive Robo2 KO mouse which allows us to investigate Robo2’s role in development and maturation of islet architecture. I am continuing to characterize the ß-cell specific knock out of Robo2 as well as looking at ß-cell specific overexpression of Robo2. To do this, we have developed an AAV that induces expression of Robo2 tagged with eGFP. This model will be combined with compensatory islet expansion models, obesity and pregnancy, to give us more insights into Robo2’s involvement with islet expansion and organization.

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Investigating the protective mechanism of a potential Type 1 diabetes therapeutic compound, in pancreatic beta cells: findings from transcriptome profiling

 

Palwasha Khan^1,2, Nida Ajmal^1,2, Kathryn L. Corbin¹, Stephen C. Bergmeier³, Xin Tong⁴, Guoqiang Gu⁴, Craig S. Nunemaker^1,2

 

¹Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, 45701.

²Translational Biomedical Sciences (TBS) Graduate Program, Graduate College, Ohio University.

³Department of Chemistry, College of Arts and Sciences, Ohio University, Athens, OH, 45701.

⁴Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, 37232

 

Type 1 diabetes (T1D) is a multifactorial disorder marked by the destruction of pancreatic beta cells, which leads to insulin deficiency. Consequently, patients with T1D require lifelong insulin therapy to achieve normal glycemic control. Effective treatment demands protecting beta cells from cytokine-induced cell death and restoring insulin secretion, two essential functions lacking in current therapies. MSB-61 is our leading compound for T1D therapy, as it enhances insulin secretion within approximately 4 hours and protects islets from cytokine-induced cell death.

 

This study employs RNA sequencing to identify genes related to MSB-61’s effects and to explore its potential mechanism of action. To achieve this goal, CD-1 mouse islets were treated with 10 μM MSB-61, 50 μM MSB-61, or vehicle control for one hour (several hours before insulin effects can be observed), followed by RNA isolation for sequencing. Integrated differential expression and pathway analysis (iDEP) identified 91 differentially expressed genes (DEGs) from 57,010 transcripts, including non-coding RNAs. By filtering genes upregulated more than 5-fold, we narrowed the set down to 19 genes and 3 microRNAs. Notably, 12 genes and 2 microRNAs were strongly associated with Creb (cAMP response element binding). Among Creb’s core targets, we identified Fos, FosB, Fosl2, and the Nr4a family (Nr4a1, Nr4a2, Nr4a3), suggesting that MSB-61 influences Creb-related transcriptional networks, particularly those involving the AP-1 complex. Our model proposes that the Creb and AP-1 pathways regulate the function of MSB-61. These findings underscore the importance of RNA sequencing in identifying crucial molecular targets, which may facilitate the development of targeted therapies for T1D.

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STIM1 Deficiency Impairs Glucose regulation by Altering Insulin Granules, Mitochondria, and ER Ultrastructure in β cell

 

Tatsuyoshi  Kono^1,6,7,8 , Madeline R. McLaughlin^1,9, Paul Sohn^4,6,7, Preethi Krishnan^1,6,10, Wenting Wu^5,6, Chih-Chun Lee^1,6,7, Fang Huang⁹, Toshiya Senda¹³, Marjan Slak Rupnik^11,12, and *Carmella Evans-Molina^{1,2,3,4,6,7,8}

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202

²Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

³Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

⁴Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202

⁵Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202

⁶Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN 46202

⁷Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

⁸Roudebush VA Medical Center, Indianapolis, IN 46202

⁹Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907

¹⁰Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, Canada

¹¹Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

¹²Institute of Physiology, Faculty of Medicine, University of Maribor, Slovenia

¹³Structural Biology Research Center (SBRC), Institute of Materials Structure Science, High Energy Accelerator Research Organization, Ibaraki, Japan.

Impairments in endoplasmic reticulum (ER) calcium (Ca²⁺) homeostasis are closely associated with β cell dysfunction and the development of diabetes. Store-operated Ca²⁺ entry (SOCE) replenishes ER Ca²⁺ stores via plasma membrane Ca²⁺ channels, regulated by the ER Ca²⁺ sensor stromal interaction molecule 1 (STIM1). Female mice with β cell-specific STIM1 deletion exhibited reduced β cell mass, increased α cell mass, and diminished β cell maturity markers.

RNAseq analysis revealed 55 significantly modulated pathways, including “mitochondrial dysfunction,” “endocytosis,” and “estrogen receptor signaling.”  To confirm the effect on mitochondria, we performed morphology analysis and Ca²⁺ imaging using the RIP-promotor controlled-Mito CEPIA, which revealed the abnormal morphology and impaired mitochondrial Ca²⁺ level in STIM1 KO cell lines. Furthermore, the analysis using the fluorescent time construct, syncollin-dsRedE5TIMER, resulted in accumulation of old insulin granules in STIM1 KO cells. To survey granule morphology within β cells, TEM was performed using isolated islets. This analysis revealed a significant increase in the ratio of immature to mature granules, and a significant reduction in the granule halo size in islets from STIM1Δβ female mice compared to their littermate controls. To analyze ER volume and tubule diameter, we used Expansion Microscopy (ExM) and Single Molecule Localization Microscopy (SMLM). These analyses revealed a significant increase of ER volume and reduced tubule diameter in STIM1 KO cells under ER stress. Notably, STIM1 KO cells exhibited further decreases in tubule diameter in ER regions distal to the nucleus compared to WT cells, suggesting tubule location influences function and ultrastructural architecture under ER stress.

Our findings demonstrate a sexually dimorphic role for STIM1 in glucose metabolism regulation, linking STIM1 loss to insulin granule, mitochondrial and ER ultrastructural changes that impair β cell function.

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Emerging Role of Ndufaf8: A mitochondrial Factor Shaping Beta Cell Metabolism, Stress Adaptation, and Immune Tolerance

 

Debjyoti Kundu^1,2;Nansa Amarsaikhan ^1,2; Kyu Been Lee^1,2; Tanasha Lertjanyarak^1,2; Chialing Wu^1,2; Erica P. Cai^1,2,3.

 

¹Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute

²Center for Diabetes and Metabolism Diseases, Indiana University School of Medicine

³Department of Biochemistry and Molecular Biology, Indiana University School of Medicine

 

Pancreatic beta cells primarily rely on oxidative phosphorylation (OXPHOS) for ATP production to fuel the insulin secretory process. However, a byproduct of OXPHOS, reactive oxygen species (ROS), can accumulate rapidly, rendering beta cells particularly sensitive to oxidative stress. This vulnerability limits their capacity to manage additional stressors, such as ER stress and inflammation. To identify genetic targets that enhance beta cell resilience to ER stress, we conducted a genome-wide CRISPR-Cas9 screen. Among the significantly enriched guide-RNAs (gRNAs), those gRNAs targeting NADH: ubiquinone oxidoreductase assembly factor 8 (Ndufaf8), an assembly factor of mitochondrial electron transport chain complex I, were the most abundant. Using  a screen-identified gRNA, we generated NIT-1 beta cells from non-obese diabetic (NOD) mice carrying a loss-of-function mutation in Ndufaf8 (DNdufaf8), alongside control NIT-1 beta cells with a non-targeting gRNA. DNdufaf8 mouse beta cells exhibited decreased ROS accumulation, enhanced glucose-stimulated insulin secretion, and increased mitochondrial membrane potential in response to OXPHOS uncoupling. Bulk RNA sequencing further revealed that DNdufaf8 beta cells displayed upregulated glycolysis, HIF-1 signaling, and amino acid metabolism pathways. Notably, in an autoimmune mouse model of T1D, DNdufaf8 mouse beta cells exhibited significantly delayed immune-mediated killing compared to controls. These findings suggest that metabolic rewiring in beta cells may serve as a potential strategy to alleviate cellular stress, sustain energy production and enhance immune tolerance.

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Novel approaches to suppressing hypersecretion and protecting β-cell function for type 2 diabetes

 

Rachel Kuntz¹, Ini Aduroshakin¹, Andrea Ordóñez¹, Michael A. Kalwat^1,2*

 

¹Indiana Biosciences Research Institute, Indianapolis, IN

²Center for Diabetes and Metabolic Disease, Indiana University, Indianapolis, IN. *Corresponding author

 

Glucose-stimulated insulin release by pancreatic β-cells is dysfunctional in both type 2 diabetes (T2D) and congenital hyperinsulinism (HI). In pre-T2D, β-cells have elevated insulin production to combat peripheral insulin resistance. Conversely, in HI, genetic mutations cause β-cells to release insulin inappropriately, leading to hypoglycemia. Insulin hypersecretion in these conditions triggers a stress response, ultimately leading to β-cell failure. One approach to selectively target β-cells is to use the glucagon-like peptide 1 (GLP1) receptor, whose activation enhances insulin secretion and β-cell survival under stress. Additionally, inhibiting Ca²⁺ influx can prevent insulin release and protect β-cells from stress. We hypothesize that GLP1 signaling and Ca²⁺ influx inhibition can be used to synergistically suppress β-cell function while promoting long-term β-cell health. To identify new biology and therapeutic avenues, we studied insulin hypersecretion using human islets and mouse MIN6 β-cells modified to secrete a luciferase-linked insulin as a proxy reporter for insulin secretion. In control conditions, GLP1 enhanced insulin secretion as expected, nifedipine suppressed insulin secretion due to its inhibition of Ca²⁺ influx, and avexitide suppressed insulin secretion due to blocking the GLP1 receptor. We plan to expand our studies on the combinations of drugs directed to β-cells to determine their ability to preserve β-cell function and health, as well as synergistic properties associated with inhibiting Ca²⁺ influx. The conjugation of drugs like nifedipine to GLP1 could also improve its delivery to β-cells. We anticipate creating a method of drug delivery that could lead to innovations in therapeutic treatments for both HI and T2D.

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Exploiting SDF2L1 to protect against beta cell death and dysfunction in T1D

 

Tanasha Lertjanyarak¹, Nansalmaa Amarsaikhan¹, Andrea Ordóñez¹, Chialing Wu¹, Debjyoti Kundu¹, Kyu Been Lee¹, Michael A. Kalwat^1,2, Erica P. Cai^1,2

 

¹Indiana Biosciences Research Institute, Indianapolis, IN, USA

²Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA

 

Type 1 diabetes (T1D) is a life-threatening disease caused by the autoimmune destruction of insulin-secreting pancreatic β-cells, leading to impaired blood glucose regulation. In the United States, T1D affects over 1.6 million individuals. While insulin therapy is essential, it does not fully restore β-cell function, highlighting the need for new strategies to protect β-cells and prevent disease progression. The endoplasmic reticulum (ER) is critical for insulin production, but chronic ER stress can lead to β-cell dysfunction, apoptosis, and immune attack. Understanding how ER stress contributes to β-cell loss is crucial for developing effective interventions. This project identifies Stromal Derived Factor 2 Like 1 (SDF2L1) as a novel target for β-cell protection in diabetes. By cross-referencing genome-wide CRISPR knockout screening datasets with secretory stress transcriptomics, we hypothesize that SDF2L1 plays a maladaptive role in β-cell susceptibility to T1D-associated stressors. Our research demonstrates that CRISPR-mediated knockout of Sdf2l1 in non-obese diabetic (NOD) mouse-derived NIT-1 β-cells significantly improves resistance to ER stressors such as thapsigargin (TG) and inflammatory cytokines, enhancing β-cell survival and reducing surface MHC class I expression under cytokine treatment. In contrast, the overexpression of SDF2L1 in MIN6 β-cells stimulated with glucose was found to reduce insulin secretion. In the future, we aim to develop targeted therapeutics to deplete maladaptive factors like SDF2L1 in human β-cells, improving β-cell resilience and advancing understanding of stress adaptation in T1D. 

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The mitochondrial chaperone GRPEL1 promotes β-cell development and maturation

 

Jin Li¹; Jie Zhu¹; Ava Stendahl¹; Campbell Miller¹; Tanvi Mandadi¹; Scott A. Soleimanpour¹

 

¹Division of Metabolism, Endocrinology and Diabetes and Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA.

 

Recent findings demonstrate that defects in mitochondrial proteostasis contribute to β-cell failure and the development of diabetes. GrpE-like nucleotide exchange factor 1 (GRPEL1), a co-chaperone in the mtHSP70 mitochondrial protein import complex, promotes newly synthesized polypeptides to import and properly fold in the mitochondrial matrix which are critical for maintaining mitochondrial proteostasis. The role of GRPEL1 in β-cells is unknown. We generated mice bearing β-cell specific deletion of GRPEL1 (Grpel1^loxp/loxp; Ins1-Cre, hereafter known as β-Grpel1^KO) and found that GRPEL1 deletion led to impaired glucose intolerance rapidly after weaning and decreased glucose-stimulated insulin release. GRPEL1 deficiency resulted in increases in β-cell dedifferentiation markers, decreases in β-cell maturity markers, and reduced β-cell replication, which eventually caused the loss of β-cell mass. Further, we also observed a decrease in the ratio of β-cell/pancreatic area accompanied by reductions in β-cell replication and maturity markers after GRPEL1 deletion at postnatal day 1. A similar metabolic phenotype was observed following inducible β-cell specific GRPEL1 deficiency in adult islets. However, GRPEL1 deletion led to reductions in β-cell maturity markers, increases in β-cell immaturity markers, and increased apoptosis without effects on β-cell replication after inducible β-cell specific GRPEL1 deletion in adult islets. In the mitochondria, β-Grpel1^KO mice developed impaired respiration, abnormal mitochondrial ultrastructure, and reduced mitochondrial mass. Interestingly, the mitochondrial unfolded protein response was upregulated after GRPEL1 deficiency. Taken together, we observe that the mitochondrial chaperone GRPEL1 is required for β-cell development and maturation likely through the maintenance of mitochondrial proteostasis.

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Role of primary cilia in mediating ER stress in Pancreatic islet cells

 

Zipeng A. Li¹, Jung H. Cho¹, Jiayang Chen², Diana Hernandez¹, Samantha E. Adamson¹, Isabella Melena¹, Shannon E. Townsend¹, Fumihiko Urano¹, Joseph Dougherty², Jing W. Hughes¹*

 

¹Department of Medicine, Division of Endocrinology, Metabolism and Lipids Research, Washington University School of Medicine, 660 South Euclid Ave, Saint Louis, MO, USA ²Department of Neuroscience, Washington University School of Medicine, 660 South Euclid Ave, Saint Louis, MO, USA

 

*Corresponding author. Email: jing.hughes@wustl.edu

 

Primary cilia, integral cellular structures, are implicated in diabetes- related metabolic disorders. This study examined the role of primary cilia under acute ER stress using Thapsigargin (TG) on wild type (WT) and β-cell primary cilia KO islet cells (βCKO). Findings indicated increased cellular turnover rates, reminiscent of primary cilia dysfunction. Calcium signaling displayed advanced peak onset with reduced intensity and amplitude upon treatment. RNA profiling revealed significant differences in insulin processing and cellular stress pathways between WT and βCKO, with proteomics data suggesting an alternative βCKO islet mechanism in mediating ER stress in the absence of primary cilia. Transcriptomic analyses highlighted distinct differentially expressed genes (DEGs) clusters and gene ontology (GO) programs, pointing to potential functional & mechanistic implications. In conclusion, acute ER stress induction by TG showcases the pivotal role(s) of primary cilia in diabetes, with unique cellular responses in βCKO islets, highlighting potential therapeutic avenues.

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Deletion of PKM1 and PKM2 reveals their essential roles in regulating β-cell K_ATP channels, Ca²⁺ activity, and insulin secretion in mice

 

Lu Liang, Hannah R. Foster, Evgeniy Potapenko, and Matthew J. Merrins

 

Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, Madison, WI

 

Prior work has shown that the PKM1 and PKM2 isoforms of pyruvate kinase play a redundant role in K_ATP channel regulation, Ca²⁺ influx, and glucose-stimulated insulin secretion. Here, we used CRISPR to generate a double β-cell knockout of PKM1 and PKM2 (PKM1/2-βKO, Pkm^f/f:Ucn3-Cre), leaving only the allosteric PKL isoform intact. In vivo, PKM1/2-βKO mice exhibit hallmarks of diabetes, including fasting hyperglycemia and impaired glucose and meal tolerance due to reduced insulin secretion. While residual PKL was found to be capable of closing K_ATP channels in β-cells lacking PKM1/2, it was less effective than the physiological K_ATP regulators PKM1 and fructose-1,6-bisphosphate (FBP)-activated PKM2. Correspondingly, PKM1/2-βKO islets exhibited dysregulated Ca²⁺ oscillations in response to glucose, and were virtually unresponsive to leucine. Insulin secretion in response to glucose and tirzepatide was reduced by 75% in PKM1/2-βKO islets. Interestingly, pharmacologic activation of PKL with TEPP-46 had no impact on the Ca²⁺ phenotypes of PKM1/2-βKO islets, but fully rescued K_ATP closure and insulin secretion. These findings suggest that a pool of PKL is compartmentalized, and recruitable by TEPP-46, but not by the FBP produced endogenously by glycolysis. Our studies provide further evidence for metabolic compartmentation in the β-cells, and demonstrate an essential role for pyruvate kinase in the regulation of K_ATP channels and insulin secretion. They further reveal a novel role for pyruvate kinase in controlling β-cell Ca²⁺ that is independent of its action at the K_ATP channel.

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Using Novel Methods to Identify the Effect of Endogenous Pulsatile Insulin Secretion on Beta Cell Health

 

Brian List¹, Nicholas B. Whitticar¹, Kathryn Corbin¹, and Craig S. Nunemaker^1,2

 

¹Dept. of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University

²Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University

 

Beta cells secrete insulin in a pulsatile manner. An impairment in pulsatile insulin release is one of the earliest markers of developing type 2 diabetes and is connected to the development of insulin resistance. At the physiological level, insulin-responsive organs respond more effectively to pulsatile insulin.

 

A previous study into the function of pulsatile and non-pulsatile phenotypes of islets in the same mouse showed ER stress and signs of dysfunction connected with the non-pulsatile group. Which brings the question, are non-pulsatile islets dysfunctional because they are non-pulsatile, or are they non-pulsatile because they are dysfunctional? 

 

To provide causation to this link, our lab designed a custom microfluidic platform to deliver stimuli in media intermittently. In this study, islets were exposed to hyperglycemic conditions (20mM glucose) for >24hrs. We then used this system to impose pulsatile insulin secretion in beta cells with 3 minutes on and off delivery of the glucokinase inhibitor D-mannoheptulose. Glucokinase catalyzes glucose metabolism, which is the first step of GSIS, so inhibiting glucokinase would inhibit all downstream steps of glucose-stimulated insulin secretion. Administering this inhibitor on islets with hyperglycemia induced adaptive dysfunctions has shown restoration of function. Islets given intermittent MH were compared with continuous delivery of MH at a full (2.5mM) or half (1.25mM) dose. These treatments in the forced oscillatory system reversed the effects of hyperglycemia and restored glucose sensing to that of untreated islets, but continuous MH did not, suggesting pulsatile insulin secretion indeed has a role in the health of beta cells.

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The regulation of lipolysis by glucose in pancreatic islets

 

Siming Liu*, Lucy Kim*, Anamika Vikram, and Yumi Imai

 

*co-first authors

 

University of Iowa, Iowa City VA Medical Center

 

Lipolysis by adipose triglyceride lipase (ATGL) increases with glucose and produces metabolites that support insulin secretion. Upregulation of lipolysis by glucose is reduced in human islets affected by type 2 diabetes (T2D). Here, we aim to determine how glucose regulates lipolysis in beta cells and whether the regulation of lipolysis differs between beta and alpha cells.

First, we focused on alpha/beta hydrolase domain containing 5 (ABHD5), a co-activator of ATGL whose mutation is associated with T2D. Downregulation of ABHD5 increased lipid droplets (LDs) and triglyceride (TG) content in INS-1 cells and human beta cells indicating that ABHD5 regulates lipolysis. Moreover, 12 mM glucose stimulated lipolysis was blunted in ABHD5 downregulated INS-1 cells. Additionally, glucose-stimulated insulin secretion was impaired in ABHD5 downregulated INS-1 and human islets, as in ATGL downregulated beta cells.

Furthermore, 16.8 mM glucose promoted ABHD5 recruitment to the LD surface in INS-1 cells whereas the addition of H-89 reduced recruitment indicating partial cAMP-dependence. Thus, ABHD5 plays an important role in conferring glucose responsiveness of lipolysis in beta cells.

We then examined the regulation of lipolysis with the human specific ATGL-inhibitor, NG497, in beta and alpha cells. LDs increased at 5 mM and 11 mM glucose for NG497-treated beta cells but not alpha cells, which instead showed active lipolysis at 2.8 mM glucose with fatty acids. Furthermore, NG497 suppressed glucagon secretion in response to 1 mM glucose with amino acids in alpha cells. Thus, lipolysis is regulated differently in beta and alpha cells but supports glucagon secretion as well.

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Novel signaling intersections to improve human stem cell derived islet differentiation

 

Manuj Bandral^1,2, Andrea Àlvarez^1,2,3, Daria Podgorski^1,2, Paul Gadue⁴, Lori Sussel⁵, and David S Lorberbaum^1,2

 

¹Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA

²Elizabeth Weiser Caswell Diabetes Institute, University of Michigan, Ann Arbor, MI, USA

³Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA

⁴Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, PA, USA

⁵Barbara Davis Center for Diabetes, University of Colorado, Denver, Anschutz Medical Campus, CO, USA

 

Despite decades of research, critical knowledge gaps remain in pancreatic islet development. This is perhaps best exemplified by the inability to produce a full complement of mature beta cells within a stem cell derived islet (SC-islet) using even the best human pluripotent stem cell (hPSC) stepwise differentiation protocols. To address current knowledge gaps limiting these protocols, my newly established laboratory focuses on unraveling the complexities of intersecting signaling pathways and transcription factors that drive islet formation during embryogenesis and maintain islet function in adulthood. We have two major projects in our group that leverage the strengths of murine and hPSC models and are each related to our long term goals. First, we have identified a novel synergy between retinoic acid signaling and GATA transcription factors in mice, which significantly alters islet differentiation during fetal organ development and impairs endocrine function in adulthood. We are currently defining mechanisms contributing to these phenotypes. Second, we have successfully established an in house in vitro hPSC derived SC-islet differentiation platform allowing us to further refine these RA/GATA interactions in a human context. Furthermore, we are examining context specific signals from dorsal and ventral patterning events that are important for in vivo mouse and human development, but not fully explored in hPSC derived SC-islets. A better understanding of the molecular mechanisms underlying islet development is essential for improving therapeutic treatments for pancreas disease.

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Establishing a Luciferase Reporter Line for Quantitative Assessment of Stem Cell-Derived Islet Survival Following Transplantation

 

James Lu¹; Marlie M. Maestas¹; Sarah E. Gale¹; Jeffrey R. Millman¹

 

¹Washington University School of Medicine, St. Louis, MO, USA

 

Current treatment methodologies of Type 1 Diabetes (T1D) primarily aim to control symptoms rather than addressing the underlying autoimmune destruction of pancreatic beta cells. However, recent advancements in stem cell-based therapies present a potential path towards a functional cure for T1D through the transplantation of stem cell-derived islets (SC-islets). In this study, we detail the quantitative assessment of islet survival post-transplantation using functional insulin-producing islet-like clusters generated from human pluripotent stem cells (hPSCs) consisting of a luciferase reporter. First, hPSCs were transduced with a lentivirus containing the luciferase transgene to establish a reporter line to facilitate the quantitative assessment of islet engraftment post-transplantation. Using the 6-stage, planar differentiation protocol developed in the Millman Lab, hPSCs cells were then differentiated into SC-islet clusters. The functionality of the luciferase enzyme was verified in vitro by measuring luciferin bioluminescence in hPSCs prior to differentiation and in SC-islets after differentiation. Next, SC-islets were transplanted at a concentration of 10 million cells into muscle, kidney, or subcutaneous regions of immunodeficient mice. To quantitatively assess the survival of the islet engraftments, mice were injected with luciferin, and bioluminescence was measured for each transplantation location at various timepoints post-transplantation. The results demonstrate that luciferase-infected cells provided a reliable, quantifiable method to track SC-islet engraftments in vivo, with muscle and subcutaneous transplantations yielding the best imaging results. This method not only enhances our ability to assess islet survivability within research but also holds significant implications for improving stem cell therapies aimed at curing T1D.

 

Keywords: SC-islets. Type 1 diabetes, islet transplantation

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β-cell specific miR-483 deficiency in mice induces dedifferentiation and alteration of islet cell content

 

Aaron MacLeod, Katy Matson, Ellianna Sempek, Xiaoqing Tang

 

Michigan Technological University

 

MicroRNAs (miRNA) are a group of small non-coding RNAs that negatively regulate target gene expression in response to metabolic changes in pancreatic islets. Dysregulation of miRNAs commonly expressed in β-cells plays a valuable role in controlling the pathogenesis of type 2 diabetes. We have previously identified higher expression of miR-483 in β-cells compared to α-cells. Mice with β-cell specific deletion of miR-483 (miR483-/-) exhibited high-fat diet (HFD)-induced hyperglycemia and reduced glucose tolerance by the diminishing release of insulin. Dedifferentiation is a compensatory mechanism used by β-cells under high metabolic and mitochondrial stress to avoid apoptosis. Dedifferentiated β-cells either revert to a progenitor state or transdifferentiate into other endocrine cell types. Using immunohistochemistry and single-cell RNA sequencing (scRNAseq), we found that the endocrine cell populations within pancreatic islets of miR483-/- mice under HFD show an elevated number of dedifferentiated β-cells. Many of these dedifferentiated cells express markers of other endocrine cells, particularly glucagon. These cells also show elevated expression of dedifferentiation markers like aldehyde dehydrogenase family 1, subfamily A3 (ALDH1A3). Additionally, mitochondrial analysis using the Seahorse extracellular flux analyzer shows a decrease in aerobic capacity in miR483-/- islets. Several genes related to metabolic and mitochondrial dysfunction were found to be upregulated in our miR483-/- islets. In conclusion, miR-483 protects β-cells identity, and β-cell specific deletion of miR-483 in our mouse model under HFD induces metabolic and mitochondrial cell stress, resulting in an altered cell profile within the islet.

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Elucidating the role of GLP-1R signaling in the protective effect of Gɑ_z loss on high-fat diet-induced type 2 diabetes

 

Jojo Maier^1,2, Rodsy Modhurima^1,2, Kathryn A. Carbajal^1,2 and Michelle E. Kimple^1,2

 

¹Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, Madison, WI

²Research Service, William S. Middleton Memorial Veterans Hospital, Madison, WI

 

G Protein Coupled Receptors (GPCRs) alternatively regulate beta-cell cyclic AMP (cAMP) levels through stimulatory, Gɑ_s, or inhibitory, Gɑ_i, signaling. Agonists of the Gɑ_s-coupled GLP-1 receptor (GLP-1R) are gold-standard T2D therapeutics well-known to stimulate beta-cell function, replication, and survival. In previous studies, we used the high-fat diet (HFD)-induced mouse model of type 2 diabetes (T2D) to determine the effect of ablating cAMP-inhibitory Gɑ_z signaling on beta-cell function and mass. HFD-fed Gɑ_z-null C57Bl/6N mice were protected from T2D due to a significant increase in functional beta-cell mass, and islets from these mice had increased GLP-1-potentiated insulin secretion, with a coordinate decrease in basal glucagon secretion. We performed immunofluorescence experiments on pancreas slide sections from control-diet and HFD-fed wild-type (WT) and Gɑ_z-null mice, revealing HFD feeding reduced the percentage of GLP-1 producing alpha-cells in WT islets, with Gɑ_z loss partially ameliorating this effect. These results raise the question of whether the protective effect of Gɑ_z loss requires GLP-1R signaling. To test this hypothesis, I will surgically implant control diet-fed and HFD-fed WT and Gɑ_z-null mice with mini-osmotic pumps continuously releasing the GLP-1R antagonist, exendin 9-39, quantifying the effects of GLP-1R antagonism on beta-cell function, replication, and mass. We predict exendin 9-39-treated Gɑ_z-null HFD-fed mice will be glucose intolerant and have reduced beta-cell replication and mass as compared to vehicle-treated HFD-fed Gɑ_z-null mice. If our studies are successful, our results will provide strong support for the pursuit of the Gɑ_z signaling pathway as a novel therapeutic target for T2D.

 

This study was supported by NIH grants R01 DK137505 and R01 DK102598 (to M.E.K.) and Department of Veterans Affairs grants IK6 BX006804, I01 BX005804, and I01 BX003700 (to M.E.K.)

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Improving differentiation efficiency and functional maturation of human induced pluripotent stem cell-derived islets via epigenetic modulation

 

Savannah J. Makowski¹, Joshua A. Nord¹, Sarah L. Wynia-Smith¹, and Brian C. Smith^1,

 

¹Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI

 

Type 1 diabetes (T1D) is an autoimmune disease that leads to the destruction of insulin-producing pancreatic β-cells. Islet replacement, a T1D treatment strategy, consists of transplanting donor islets from human cadavers into diabetic patients. However, human cadaver islet quality is highly variable, as cellular stresses associated with donor cause of death and the islet isolation process disrupt β-cell function. Human induced pluripotent stem cell (hiPSC)-derived islets are an attractive alternative to human cadaver islets. However, current differentiation protocols yield less functional, fetal-like β-cells. Improving the functional maturation of hiPSC-derived islets is essential to generating islets for research and transplant use. We have shown that inhibitors of the bromodomain and extra-terminal domain (BET) protein family of bromodomains (BETi) reduce β-cell inflammation and improve β-cell function. Therefore, we hypothesize that adding BETi during differentiation will enhance hiPSC-derived islet functional maturity. We optimized a 28-day iPSC protocol that recapitulates the islet development process. We used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to verify that these cells express all appropriate differentiation markers for each differentiation stage and contain all appropriate islet cell subtypes. Our initial results show that BETi improve differentiation efficiency at several differentiation stages. We are currently identifying the optimal differentiation stage for BETi addition to improve islet maturity. Functionally mature hiPSC-derived islets will provide high-quality human islets with high scientific and translational potential for advancing T1D research and improving the efficacy of islet transplants for patients with T1D.

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Primary Cilia Regulate GLP-1 Signaling in Pancreatic Beta Cells

 

Isabella Melena^1,2, Jeong Hun Jo¹, Lifei Zhu¹, Shannon Townsend¹, Samantha Adamson¹, Jing Hughes¹

 

¹Division of Endocrinology, Metabolism & Lipid Research, Washington University School of Medicine, St. Louis, MO, USA.

²MD-PhD Program, Washington University in St. Louis, St. Louis, MO, USA.

 

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) have gained widespread attention for their pleotropic metabolic benefits and are now among the most prescribed medications for diabetes and obesity. A key therapeutic effect of GLP-1RAs is their ability to enhance postprandial insulin secretion in pancreatic beta cells. However, the signaling mechanism of GLP-1 in beta cells, including potential subcellular compartmentalization, remain incompletely understood. In this study, we investigated 1) the subcellular localization of GLP-1 receptor (GLP-1R) on primary cilia, membrane sensory organelles known to signal toward hormone secretion in beta cells, and 2) whether cilia loss disrupts GLP-1-augmented insulin secretion by altering cAMP and Ca²⁺ signaling. Using immunofluorescence imaging and immuno-electron microscopy, we detected GLP-1R protein expression on primary cilia in healthy mouse islets. Islets lacking primary cilia exhibited reduced whole-cell cAMP response to glucose and the GLP-1RA liraglutide, as measured by live FRET imaging. Similarly, whole-cell Ca²⁺ dynamics were diminished in cilia-deficient islets in response to glucose and GLP-1RA stimulation. Dynamic insulin secretion in response to co-stimulation with glucose and GLP-1 was also impaired in cilia knockout islets. Furthermore, disrupting ciliary GPCR trafficking via TULP3 knockdown reduced GLP-1-stimulated insulin secretion. These findings demonstrate that primary cilia serve as a physical platform of GLP-1R signaling and modulate beta cell responses to GLP-1. This represents a previously unrecognized mechanism of subcellular GLP-1 signaling, highlighting the potential for targeting ciliary function to enhance therapeutic outcomes.

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Understanding the Connection Between Down Syndrome and Diabetes: Insights into Insulin Secretion and Glucose Dysregulation

 

Maria del Carmen Mendoza¹, Jake Smith¹, Bayley Waters¹, Cyrus Sethna¹, Barak Blum¹

 

¹University of Wisconsin-Madison, Madison, Wisconsin, United States

 

People with Down Syndrome (DS) have an increased risk of developing diabetes mellitus (DM). Pancreatic β cells regulate glucose homeostasis through a controlled release of insulin, which is stored in dense core secretory granules. While there is an association with DS/DM and β cell dysfunction, molecular mechanisms such as insulin secretion and glucose homeostasis dysfunction in DS remain unclear. Previous experiments in our lab using Ts65Dn mice, the most comprehensive murine model of DS, revealed sex-specific differences in glucose regulation. Male Ts65Dn mice exhibit delayed glucose clearance, whereas female Ts65Dn mice maintain normal glucose clearance. In this research, we hypothesize that trisomic (ALT1) Ts65Dn β-cells display abnormal insulin vesicle secretion, which leads to dysfunction in glucose homeostasis. To test this hypothesis, we analyzed the endocrine cell type composition of Islets of Langerhans in disomic (WT) and trisomic (ALT1) Ts65Ds mice using immunohistochemistry.  We also examined organelle distribution, particularly insulin vesicles in β-cells, through transmission electron microscopy (TEM). Our findings demonstrate that ALT1 islets are smaller than WT, however, ALT1 β-cells are larger. Furthermore, differences were observed in the number of mature and immature vesicles. ALT1 β-cells had fewer immature vesicles but a greater number of mature vesicles. Collectively, these findings suggest a potential insulin release dysregulation in DS, which could lead to insulin retention inside the cells, explaining the increased size of ALT1 β-cells. There could be multiple critical factors contributing to disruptions in insulin release, leading to dysregulation. However, challenges still remain to fully understand insulin physiology in DS.

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Unraveling Mechanisms of Cholecystokinin Expression Underlying Pancreatic Beta-cell Compensation

 

Rodsy Modhurima¹, Shashank Pokhrel-Joshi¹, Molly C. Mulcahy¹, Michelle E. Kimple*^1,2, Dawn B. Davis*^1,2

 

¹University of Wisconsin - Madison, School of Medicine and Public Health, Department of Medicine

²William S. Middleton Memorial Veterans Hospital

*Co-corresponding Authors

 

Background: In the endocrine pancreas, cholecystokinin (CCK) is secreted in response to increased metabolic stress. cAMP, obesogenic stress, chronic high glucose exposure, and GLP1R signaling all upregulate islet Cck expression. We found that culturing islets in CCK confers increased beta cell survival. We aim to elucidate the mechanisms underlying islet Cck expression to discover ways to leverage the protective role of CCK in beta-cell compensation.

 

Methods: Islets from C57BL6J mice (n=6) were isolated for culture. Islets were cultured at 25 mM glucose for glucose response time-curve. Specific adenylyl cyclase inhibitors were utilized to assess the role of cAMP in glucose response. Cck expression was measured by RT-qPCR.

 

Findings: I found that islets demonstrate elevated Cck expression within two hours of exposure to high glucose media in a cAMP-independent manner. Cck is constitutively elevated in islets from leptin deficient obese Lep^ob/ob mice due to obesogenic stress, and inhibiting cAMP production by overnight culture with sulprostone does not diminish Cck expression. We identified a partial carbohydrate response element (ChoRE) site in the Cck promoter that is conserved in human, mouse, and rat genomes. The timeline of glucose response, evidence that cAMP is not necessary for Cck response to glucose, and presence of a conserved partial ChoRE site together suggest that the carbohydrate response element binding protein (ChREBP) responsible for glucose-mediated Cck expression. We plan to utilize ChREBP inhibitors and ChREBP KO mice to test whether ChREBP is a necessary transcription factor for glucose-mediated upregulation of Cck expression.           

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Evaluating the immune cell-beta cell interface in Cystic Fibrosis related Diabetes

 

Rachael Morara¹, Farooq Syed Ph.D.², Carmella Evans-Molina MD, Ph.D.¹, Amelia Linnemann Ph.D.¹

 

¹Indiana University School of Medicine

²Beckman Research Institute

 

Cystic fibrosis related diabetes (CFRD) affects 20% of adolescents and 40-50% of adults with cystic fibrosis. While the prevalence of CFRD increases, there is a poor understanding of the etiology of the disease. Although similar to other types of diabetes, CFRD is caused by insufficient insulin release from beta cells, however, the mechanism of insulin insufficiency remains unknown. To evaluate islet arrangement and composition, we initially performed immunofluorescence on a fresh-frozen, human, pancreatic donor sample with CFRD. We confirmed that the islets were present and intact, with clear insulin and glucagon staining preserved, but presented with abnormal shape. We further evaluated the presence of immune cell infiltration with CO-Detection by indEXing (CODEX) on the same sample. Using this approach, we observed infiltration of islets by CD3+, CD4+, and CD8+ T cells. Interestingly, we noticed that CD3+ and CD4+ T cells were abundant within the islets and the tissue surrounding them, but CD8+ T cells were primarily abundant within the islets. Future studies will utilize spatial transcriptomics to evaluate gene expression of single cells within the CFRD pancreatic tissue and compare this to gene expression in non-diabetic pancreatic tissue. With this information, we aim to gain insight into the interaction between the immune cell-beta cell interface in CFRD and further knowledge of the disease development.

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Evaluation of Pubertal use of GLP-1 Therapy on Cardiometabolic Health and Beta Cell Proliferation in Type 2 Diabetes

 

Molly C. Mulcahy¹, Matthew Flowers^1,2, Rodsy Modhurima¹, Sam Saghafi¹, Dawn B. Davis^1,2

 

¹University of Wisconsin - Madison, Department of Medicine

²William S. Middleton Veterans Administration Hospital

 

Pediatric type 2 diabetes (T2D) is an aggressive disease that results in rapid loss of insulin-producing pancreatic beta cells, disrupted whole-body metabolism, and lasting cardiometabolic damage to multiple organ systems. Proliferation of beta cells is rare in adulthood, but proliferative rates in beta cells increase briefly during pubertal transition in adolescence. Glucagon-like-peptide-1 (GLP-1) drugs are known to induce proliferation in animal models of diabetes and induce serotonin signaling, which contributes to beta cell proliferation. Despite the prescribing of GLP-1 drugs to adolescents with T2D, evaluations of their proliferative potency when used during the pubertal transition in murine models are absent.

 

To examine whether GLP-1 drugs employed during pubertal transition maximize treatment efficacy, we will induce type 2 diabetes in young mice by combining high fat diet feeding and low-dose streptozotocin. Using this adolescent T2D murine model, we will assess diabetes progression and serotonergic activity in vivo. I will employ immunofluorescent staining and metabolic phenotyping to understand how treatment with these drugs during adolescence impacts T2D progression and the paracrine environment surrounding proliferating cells. I will use ex vivo islet techniques to investigate the role of GLP-1 drug induced serotonergic signaling in beta cell mass and insulin secretion.

 

We will test the hypothesis that the growth-factor rich environment and serotonin-mediated increase in beta cell proliferation during the pubertal transition presents a unique opportunity for GLP-1 drugs to maximize beta cell proliferation and reduce the burden of comorbidities. Data collection is ongoing, and newest findings will be shared at MIC.

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Interferon Signaling in Type 1 Diabetes

 

Charanya Muralidharan¹, Emily C. Elliott², Soumyadeep Sarkar², Xiaoyan Yu³, Jacob R. Enriquez¹, Decio L. Eizirik³, Ernesto S. Nakayasu², Sarah A. Tersey¹, Raghavendra G. Mirmira¹

 

¹Department of Medicine and the Kovler Diabetes Center, The University of Chicago, Chicago, IL, USA

² Pacific Northwest National Labs

³ ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium.

 

In type 1 diabetes (T1D), pancreatic β cell destruction is driven by immune cells and exacerbated by β cell stress. Cytokines, particularly interferons, contribute to autoimmunity by activating interferon signaling and the integrated stress response (ISR), which regulates mRNA translation to support cellular adaptation. However, chronic activation can be detrimental. Upon interferon receptor activation, STAT proteins are phosphorylated and regulate interferon-responsive genes. STAT proteins also undergo S-palmitoylation, a lipid modification mediated by palmitoyltransferases and depalmitoylases, but its role in β and immune cells in T1D remains unclear.

 

We hypothesized that interferon signaling and protein palmitoylation are key regulators of β cell dysfunction and T1D progression. To investigate, we analyzed published RNA sequencing (RNAseq) and single-cell RNAseq datasets, including human islets exposed to cytokines (IFN-γ + IL-1β), palmitate, and prediabetic NOD mouse islets treated with a PERK (ISR kinase) inhibitor. RNAseq revealed robust interferon responses in cytokine-exposed human islets. Cytokine exposure also increased endothelial lipase and palmitate levels. Lipidomics on plasma samples from TEDDY study showed elevated palmitate at the time of AAb seroconversion, suggesting a role for S-palmitoylation early in the disease. Palmitate exposure enriched protein trafficking pathways in human islets and enhanced interferon response in cytokine treated MIN6 mouse β cells. Inhibition of palmitoylation in EndoC-βH1 human β cells reduced STAT1 levels, suggesting S-palmitoylation’s role in interferon-mediated STAT signaling. Sc-RNAseq of PERK-inhibited islets showed reduced enrichment of genes involved in palmitoylation and depalmitoylation in β and immune cells.

 

Our findings suggest a link between palmitoylation, ISR, and interferon signaling in β cell dysfunction in T1D. Future research will define their specific roles in disease progression.

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Decoding CHD5: Unveiling Its Role in Pancreatic Beta Cell Health and Function

 

Snehasish Nag^1,2,3, Avinil Das Sharma^1,2,3, Abigail Taylor¹, Jason M. Spaeth^1,2,3

 

¹Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN

²Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN

³Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN

 

Pancreatic and duodenal homeobox 1 (PDX1) is a critical transcription factor essential for pancreatic beta cell development, function, and maintenance, with its dysregulation leading to beta cell dysfunction and diabetes. Chromodomain Helicase DNA-binding Protein 5 (CHD5), a member of the nucleosome remodeling and deacetylase (NuRD) complex, has been implicated in transcriptional regulation, but its role in beta cells remains unclear. Previously, we demonstrated CHD4, another NuRD complex member, interacts with PDX1 in beta cells and CHD4 knockout leads to CHD5 upregulation, suggesting a possible compensatory mechanism. Co-immunoprecipitation  and Proximity Ligation Assays demonstrated a direct interaction between CHD5 and PDX1, similar to CHD4. To investigate the function of CHD5, we generated a beta cell-specific CHD5 knockout mouse model (Chd5^Δbeta) using mice with loxP sites flanking Chd5 exon 2 and tamoxifen-inducible MIPCre-ER. Immunofluorescence confirmed successful knockout of CHD5 in beta cells, and glucose tolerance tests conducted four weeks post-tamoxifen treatment showed glucose intolerance in both male and female Chd5^Δbeta mice. Protein analysis of Chd5^Δbeta pancreata revealed significant downregulation of insulin, MAFA, Urocortin 3, Chromogranin A and B, while PDX1 levels remained unchanged. These findings highlight the crucial role of CHD5 in beta cell function, where its absence leads to beta cell dysfunction and glucose intolerance. Future investigations into the role of CHD5 in controlling gene expression, chromatin accessibility and subsequent insulin secretion in Chd5^Δbeta mice, as well as its relevance to human beta cell biology and type 2 diabetes, will provide deeper insights into its role in maintaining beta cell health.

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Mitochondrial Nrf2 Activation and Its Role in Mitigating Oxidative Damage in β-Cells

 

Arun Nandwani^1,2, Alissa Muncy^1,2 and Amelia K. Linnemann^1,2,3

 

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN

²Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN

³Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN

 

Pancreatic β-cells have relatively low levels of antioxidant response, making them more vulnerable to oxidative damage induced cell death. During diabetes pathogenesis, pro-inflammatory cytokines mediate the generation of reactive oxygen species (ROS) that when unmitigated, can induce β-cell death by increasing DNA damage and mitochondrial damage. Nuclear factor erythroid 2–related factor 2 (NRF2) plays a critical role in cellular defense against ROS-induced oxidative damage in part by regulating the expression of genes responsible for detoxifying and eliminating ROS and electrophilic agents. Despite its importance, the exact molecular mechanisms involved in activating NRF2 remain incompletely understood. We have previously shown that the cytokine interleukin 6 (IL-6) initiates the antioxidant response by stimulating NRF2 translocation to mitochondria, which was associated with the induction of mitophagy. We hypothesized that post-transcriptional modulation of NRF2 leading to changes in interaction with potential mitochondrial proteins might facilitate this translocation. To this end, here we used in silico protein-protein

 

interaction tools and databases to identify potential interactors of NRF2. We identified a series of putative proteins that may be involved in NRF2 mitochondrial translocation, including both known mitochondrial proteins and stress response proteins. We then used co-immunoprecipitation (co-IP) and western blot to validate potential interactors in a β-cell line under conditions of cytokine stress leading to NRF2 mitochondrial translocation. This revealed a set of interactors that co-translocate to the mitochondria with NRF2. Experiments are now ongoing to determine the necessity of these interactors for translocation and the functional consequences of their impairment.

 

Funding: R01DK124380

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The Rnf20 epigenetic modifier is necessary for pancreatic organogenesis and postnatal islet function

 

Catherine Nelms, Tanya Pierre, Samuel O. Poole, Chad S. Hunter

 

Department of Internal Medicine - Division of Endocrinology, Diabetes and Clinical Pharmacology, University of Kansas Medical Center, 1000 Hixon, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA

 

Introduction. Pancreatic islets are essential for insulin secretion and maintaining glucose homeostasis, with diabetes mellitus arising from autoimmune destruction (T1D) or dysregulation (T2D) of pancreatic β-cells. While T1D patients require exogenous insulin, future therapies using stem cell-derived islet-like cells offer a promising cure but require a deeper understanding of pancreas and islet development. Pancreas organogenesis occurs during two phases – the primary transition (embryonic day (E) 9.5-12.5) initiates multipotent progenitor cells (MPCs), while the secondary transition (E12.5-16.5) drives differentiation, followed by postnatal islet maturation. Pancreas development is controlled by transcription factors and less understood co-regulators such as Rnf20, an E3 ubiquitin ligase that monoubiquitinates histone H2B. We hypothesize Rnf20 is required for pancreas development and gene regulation.

 

Methods. We used a pancreas-specific (Pdx1) Cre-Lox recombination approach to delete Rnf20 in embryonic mice. To evaluate developmental Rnf20 loss, we utilized immunofluorescence to examine transcription factor and hormone expression. Fasting/fed blood glucose and plasma insulin were measured in neonates. 

 

Results. We observed pancreatic agenesis (females) and dysglycemia (males/females) in Rnf20 neonatal knockouts. However, an overtly normal primary transition was suggested by unaffected Pdx1 expression (an MPC marker). Histology suggested impaired pancreas development in Rnf20-deficient females during the secondary transition with insulin/glucagon expression loss and reduced pancreas size.

 

Conclusions. Rnf20 is required for pancreas organogenesis, as its loss leads to agenesis and/or islet dysfunction. Overall, these data support a novel role for Rnf20 at least during the secondary transition of pancreas development and subsequent neonatal islet function. 

 

Conflicts of Interest: None

 

Support: R01-DK128132

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Effects of Maternal obesity on metabolic impairment in the second-generation offspring

 

Marzieh Nemati¹, Kelli DeVanna¹, Kok Lim Kua¹

 

¹Wells Center for Pediatric Research, School of Medicine

 

Background:  Preclinical models have shown that first-generation (F1) offspring of obese mothers exhibit glucose intolerance and increased adiposity in a sex-dependent manner; raising critical questions about whether these metabolic impairments can be transmitted to second-generation offspring (F2) and how they respond to an obesogenic diet. We aim to investigate impacts of maternal obesity (MatOb) on the likelihood of metabolic dysfunction in next generations.

 

Method:  F1 male (MM) and female (MF) obese mice were mated to control male (CM) or female (CF) mice, generating four F2 groups: CMCF (control), MMCF, CMMF, and MMMF. All F1 mice remained on standard chow during pregnancy and lactation. F2 offspring were weaned to chow until 8 weeks, then switched to either chow or a western diet (WD, TD88137) for 4 weeks (n=3-5/sex/group). Glucose tolerance tests (GTT) and body composition were assessed.

 

Results:  At postnatal day 21 glucose levels during GTT and adipose mass were higher in both sexes of F2 offspring born to obese mice compared to same-sex controls. After 4 weeks on high-fat diet, both males and females in the MMCF group showed exacerbated glucose intolerance. Adipose mass was higher in males than females, with a significant increase in males in MMCF group on high-fat diet compared to normal diet.

 

Conclusion:   MatOb transmits metabolic risks to F2 offspring, particularly in response to an obesogenic diet. These findings highlight the importance of maternal health and diet on future generations' metabolic outcomes, underscoring the need for further research and interventions to mitigate MatOb-associated risks.

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Sex-dependent effects of circadian rhythm disruption on glucose homeostasis, insulin secretion, and islet circadian gene expression

 

Thanh Nguyen, Zhang Luhui, Kuntol Rakshit, Satish Sen, Ananya Bharath, Kazuno Omori, and Aleksey Matveyenko

 

Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science, Rochester, MN, USA.

 

Disruption of circadian rhythms (CD) promotes development of T2DM through deleterious effects on the β-cell. Studies have shown the sexual dimorphism in glucose homeostasis and islet function in health, CD, and T2DM, implying potential sex-specific differences in the physiological and molecular responses to CD. Therefore, we utilized mouse models of environmental (e.g., shift work and light at night) CD to determine sex-dependent effects of CD on glucose homeostasis, insulin secretion, and circadian islet gene expression. We demonstrate that in response to 10-week shift work-like conditions both male and female C57BL6 mice show 1) disrupted activity and feeding circadian behaviors; 2) disrupted circadian regulation of glucose tolerance and glucose-stimulated insulin secretion; and 3) a reduction in the number of circadian-regulated genes in isolated islets (p<0.05 for all parameters vs. Control). However, the extent of CD-induced glucose intolerance and β-cell dysfunction was significantly greater in male than female mice (p<0.05 for male vs. female). In addition, bioinformatics analysis of islet’s circadian transcriptional profiles revealed that CD led to a comparable decrease in circadian-regulated islet genes in male (~75%) and female (~90%) mice. However, whereas exposure to CD in males predominantly impacted islet genes enriched for metabolic pathways, exposure to CD in female mice preferentially disrupted circadian expression of genes regulating islet protein processing and ER function. Our studies highlight unique sex-dependent effects of CD on glucose homeostasis and suggest the need for additional studies in identifying mechanisms underlying the protection of female mice from CD-induced glucose intolerance and β-cell dysfunction. 

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BET bromodomain inhibitors selectively attenuate IL-1β-induced transcription of NF-κB targets in β-cells

 

Joshua A. Nord¹, Paul F.W. Sidlowski¹, Savannah J. Makowski¹, John A. Corbett¹, and Brian C. Smith¹

 

¹Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI

 

Cytokine-stimulated transcription of inflammatory NF-κB gene targets is linked to the development of autoimmune diabetes. Inhibitors of bromodomain and extraterminal domain (BET) epigenetic reader proteins attenuate inflammatory gene transcription and diabetic onset in non-obese diabetic (NOD) mice. We have shown that BET bromodomain inhibitors (BETi) disrupt the interaction between BET family member BRD4 and NF-κB transcription factor p65, thus attenuating transcription of inflammatory NF-κB target genes and ameliorating cytokine-mediated functional deficits in β-cells. However, the role of NF-κB in developing autoimmune diabetes is controversial, as other studies have shown that NF-κB inhibition in β-cells increases diabetic onset in NOD mice. Therefore, some cytokine-induced NF-κB targets likely have physiological or protective roles in β-cells. Here, we performed RNA-sequencing to investigate whether BETi selectively attenuate NF-κB targets with roles in disease progression while sparing those important for β-cell homeostasis. We discovered that NF-κB targets exhibit variable dependence on BET proteins for full transcriptional activation (classified as BET-dependent or -independent). We found that BET-dependent targets participate in intercellular processes and promote inflammation. In contrast, BET-independent targets are primarily involved in intracellular signaling cascades and function to maintain the integrity of intracellular processes and defenses against injury. These studies define a novel and selective role for BET bromodomain-containing proteins in regulating inflammatory gene activation and suggest applications of BETi in the transcriptional regulation of inflammation in β-cells and autoimmune diabetes.

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Investigating effects of modulating glucokinase activity on human islet and mitochondrial function under diabetogenic conditions

 

Kazuno Omori^1,2, Thanh Nguyen¹, Luhui Zhang¹, Satish K Sen¹, Kuntol Rakshit¹ and Aleksey Matveyenko¹

 

¹Department of Physiology and Biomedical Engineering, Mayo Clinic School of Medicine, Mayo Clinic, Rochester, MN, USA

²Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan

 

Glucokinase (Gck) is critical for glucose-stimulated insulin secretion (GSIS). Recent clinical trials demonstrated the transient efficacy of Gck activator (GKA) in treating type 2 diabetes (T2DM). Interestingly, studies in rodent T2DM models (db/db mice) report that Gck haploinsufficiency paradoxically improves glucose tolerance and preserves functional beta cell mass. These data suggest that prolonged hyperactivation of Gck is detrimental, whereas partial inhibition is protective against beta cell failure. To test this hypothesis in human islets, we obtained cadaveric islets from 8 (Age=47±4.1; HbA1c=5.4±0.4%) non-diabetic and 3 T2DM donors (Age=49±;3.2; HbA1c=6.6±0.6%). We assessed GSIS and gene expression (RNAseq) in human islets under 1) control conditions (CON: 48h incubation at 4mM glucose), 2) glucotoxicity (GLT: 48h incubation at 16mM glucose) and either 3) GLT + GKA (RO-28-0450) and/or 4) GLT + Gck inhibitor (GKI: Mannoheptulose). GLT and GLT+GKA conditions were associated with a reduction in GSIS (~50%, ~80% decrease vs. CON, p<0.01). Administration of GKI fully reversed the deleterious effects of GLT on GSIS (p=ns for CON vs. GLT+GKI). GLT and GLT + GKA conditions were also associated with a reduction in the network area of Tomm20 (marker of mitochondrial integrity) in beta cells (~55% vs. CON, p<0.05), whereas GLT+GKI fully reversed this effect. Interestingly, RNAseq analysis revealed reduced expression of transcripts involved in mitochondrial function in GLT+GKI vs. GLT. Our studies support the hypothesis that partial inhibition of Gck attenuates deleterious effects of hyperglycemia on beta-cell function and demonstrates potential benefits of GKI on protecting mitochondrial integrity in human beta cells.

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Using congenital hyperinsulinism models to identify novel therapeutics that suppress insulin secretion

 

Andrea Ordóñez¹, Salem Kifle¹, Rachel Kuntz¹, Nida Ajmal², Gitanjali Roy¹, Diva De León³, Michael A. Kalwat^1,4

 

¹Indiana Biosciences Research Institute, Indianapolis, IN

²Ohio University, Athens, OH

³Children’s Hospital of Philadelphia, Philadelphia, PA

⁴Center for Diabetes and Metabolic Disease, Indiana University, Indianapolis, IN

 

Congenital hyperinsulinism (HI) is a disease that causes inappropriately high insulin secretion. HI is caused by mutations in the KATP channel genes or in metabolic genes like glutamate dehydrogenase 1 (GLUD1). Insulin hypersecretion can lead to β-cell dysfunction. New HI treatments are direly needed. To address this, we established human and mouse genetic HI models in β-cells and identified new therapeutic molecules. To create genetic models, we used lentivirus in human EndoC-βH1 and mouse MIN6 β-cells to either transgenically express activating mutants of GLUD1 or knockout KATP channel genes. We are using these models to test novel and repurposed small molecules for their ability to prevent hypersecretion. We also use a chemical model that employs a hypersecretion-inducing compound that phenocopies HI. To identify new therapeutic molecules, we used a combination of human HI islet gene expression data and phenotypic screening in our model systems. Our candidate insulin secretion suppressors include inhibitors of Ca²⁺ channels (nifedipine), serine biosynthesis, histamine receptors, GLP1R, and some novel compounds (SW269324 and SW297577). We tested these candidates for their ability to counteract the effects of HI. We found that exposure to some of these inhibitors resulted in dose-dependent suppression of insulin secretion. SW269324 and SW297577 were also confirmed to suppress insulin secretion in human islets and WT EndoC-βH1 cells, potentially via affecting Ca²⁺ influx. Future directions include characterizing ABCC8 (SUR1) knockout human EndoC-βH1 cells to test novel treatments in vitro to enable downstream in vivo testing in HI mouse models.

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Prediabetic stage β-cell Xbp1 deficiency confers protection against T1D in NOD mice

 

Qiaodan Ou¹, Vibha Sathesh Kumar¹, Mridula Srivathsan¹, Feyza Engin^1,2,3

 

¹Department of Biomolecular Chemistry, University of Wisconsin-Madison, School of Medicine and Public Health. Madison, WI

²Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health. Madison, WI

³Department of Cell and Regenerative Biology, University of Wisconsin-Madison, School of Medicine and Public Health. Madison, WI

 

Accumulating preclinical and clinical findings suggest that β-cell endoplasmic reticulum (ER) stress and the aberrant unfolded protein response (UPR) contributes to pathogenesis of type 1 diabetes (T1D). ER stress, instigated by various insults including chronic inflammation, toxins, and misfolded proteins, triggers the UPR. The UPR is mediated by three sensors, ATF6, IRE1α, and PERK. IRE1α excises 26nt intron of X-box binding protein 1 (Xbp1), leading to translation of spliced XBP1 (sXbp1), a critical transcription factor for adaptive stress responses. We have previously shown that β-cell-specific deletion of Ire1α  or Xbp1 prior to insulitis protects NOD mice against T1D owing to a transient β-cell dedifferentiation and immune evasion. However the role of Xbp1 in β-cells of NOD mice at postinsulitis/prediabetic stage has remained unknown. To address this, we induced β-cell-specific deletion of Xbp1in NOD mice at the prediabetic stage. Remarkably, similar to early stage Xbp1 deficiency model, later stage loss of function of beta cell Xbp1 also provided protection from T1D, despite the presence of established insulitis. Histological analyses revealed transient β-cell dedifferentiation following Xbp1 deletion and significantly reduced insulitis compared to control mice. Unexpectedly, unlike the previous model, these mice exhibited unique islet morphological changes, including a thickened peri-islet basement membrane and increased extracellular matrix deposition. Overall, our study suggests the β-cell-specific deficiency of sXbp1 both at preinsulitis and prediabetes stages protects NOD mice against T1D through overlapping, but not identical mechanisms. Further mechanistic studies will allow better understanding of the stage specific roles of Xbp1 during T1D progression.

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eIF5A-mediated mRNA translation regulates the balance of differentiated cells in the developing pancreas

 

Danielle L. Overton¹, Catharina BP Villaca¹, Caleb D. Rutan¹, Dorian J. Dale¹, Morgan A. Robertson¹, Craig T. Connors¹, Emily K. Anderson-Baucum¹, Teresa L. Mastracci^1,2

 

¹Department of Biology, Indiana University Indianapolis IN, USA;

²Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA.

 

The pancreas contains two compartments: the endocrine, responsible for hormone production and the exocrine, responsible for the secretion of digestive enzymes. Although functionally distinct, the cells within these compartments originate from a common pancreatic progenitor cell. These progenitor cells proliferate and organize into a ductal epithelium that contains endocrine progenitors within the ductal trunks and exocrine progenitors at the ductal tips, which subsequently differentiate during the secondary transition (embryonic day (E) 12.5 – E16.5) into the islets and acinar cells, respectively. Our lab is investigating how mRNA translation regulates this differentiation and has implicated the translation factor eukaryotic initiation factor 5A (eIF5A) in the process. To decipher the role of eIF5A, we generated a mouse model with a genetic deletion of Eif5a in the Ptf1a-expressing pancreatic progenitor cells. Preliminary data showed a significant reduction in exocrine with a concomitant significant increase in beta cell mass at the completion of pancreas development. Therefore, we hypothesized that eIF5A functions to regulate the balance of differentiated acinar and beta cells. To test this, we evaluated ductal branching, ductal tip formation, and Ngn3-expressing endocrine progenitor cell number during the secondary transition. Our data showed reduced ductal branching and tip cells, with a significant increase in Ngn3-expressing cells. Interestingly, Notch signaling influences Ngn3 expression as well as the balance of acinar and beta cell differentiation. Preliminary data revealed a reduction in cells expressing the Notch effector Hes1 at E14.5, suggesting that eIF5A-mediated translation may regulate Notch signaling and thus the differentiation of exocrine and endocrine cells.

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Pancreatic β cell PMCA1-mediated reductions of glucose-stimulated Ca²⁺ influx and oscillations results in glucose intolerance

 

Spencer J. Peacheé¹, Prasanna K. Dadi¹, Jordyn R. Dobson¹, Shannon E. Gibson¹, & David A. Jacobson¹

 

¹Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee

 

While a great deal of islet research has focused on glucose-mediated control of Ca²⁺ influx, there remains a gap in our understanding of the mechanisms that regulate islet-cell Ca²⁺ extrusion and how they contribute to hormone secretion. Plasma membrane Ca²⁺ ATPase isoform 1 (PMCA1) is the highest expressed Ca²⁺ extrusion protein in β cells, but its role in islet cell function has yet to be investigated. PMCA1 maintains low concentrations of cytoplasmic Ca²⁺ and has also been shown to modulate Ca²⁺ oscillations in oocytes. Thus, we utilized β cell selective ablation or KD of PMCA1 (βPMCA1KO/KD) to determine its role in Ca²⁺ handling, insulin secretion, and glucose homeostasis. Mouse βPMCA1KO islets showed an increase in glucose-stimulated Ca²⁺ oscillation frequency and decreased slope from peak to trough of each oscillation. Moreover, the glucose-stimulated (20 mM) Ca²⁺ response amplitude was greater in βPMCA1KO islets compared to controls. As insulin secretion is dependent on Ca²⁺, this likely contributes to the significant improvement in glucose tolerance observed in the βPMCA1KO mice. Interestingly, human pseudoislets with β cells transduced with an shRNA targeting PMCA1 showed elevated basal insulin secretion but depletion of insulin content resulting in diminished secretagogue-stimulated insulin secretion. Therefore, β cell PMCA1 limits secretagogue-stimulated Ca²⁺ influx leading to reduced insulin secretion and diminished glucose tolerance.

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Tomosyn-2 Regulates β-Cell Proliferation and Functional Maturity in Neonatal Islets

 

Katherine C. Perez¹, Justin Alexander¹, Md Mostafizur Rahman¹, Haifa A. Alsharif¹, Yanping Liu¹, Jeong-A Kim¹, Chad S. Hunter^{1, 2}, Thanh Nguyen³, and Sushant Bhatnagar^1*

 

¹Heersink School of Medicine, Division of Endocrinology, Diabetes, & Metabolism, Comprehensive Diabetes Center, University of Alabama, Birmingham, AL, 35294, USA

²Current Address: Department of Internal Medicine, Division of Endocrinology, Diabetes and Clinical Pharmacology, University of Kansas Medical Center, Kansas City, KS 66160, USA

³Department of Biomedical Engineering, University of Alabama, Birmingham, AL, 35233, USA

 

Proliferating neonatal β-cells are functionally immature, whereas mature β-cells exhibit enhanced glucose-stimulated insulin secretion (GSIS) with reduced proliferation. The transition from neonatal to adult β-cells requires a balance between proliferation and insulin secretion, yet the molecular mechanisms governing this process remain unclear. Here, we identify tomosyn-2 as a key regulator of β-cell proliferation and maturation. Tomosyn-2 expression declines in islets from 2 to 14 weeks of age, coinciding with increased biphasic GSIS and reduced β-cell proliferation. Tomosyn-2-deficient mice display improved glucose clearance and elevated plasma insulin levels without altering insulin sensitivity. Loss of tomosyn-2 enhances biphasic GSIS in ex vivo islets while maintaining basal insulin secretion. Mechanistically, tomosyn-2 inhibits insulin granule exocytosis by interacting with syntaxin-1A, preventing fusion complex formation. Transcriptomic analysis reveals that tomosyn-2 loss strengthens connectivity between insulin secretion pathways and proliferative processes. Further, tomosyn-2 loss upregulates genes promoting β-cell identity, maturation, and insulin secretion while downregulating proliferation and immaturity-related genes. This is accompanied by reduced AKT signaling, decreased CyclinD1 (cell-cycle activator), and increased Cdkn1b (cell-cycle inhibitor), correlating with fewer Ki67+ β-cells and reduced β-cell mass. Additionally, tomosyn-2 deficiency enhances β-cell identity, marked by elevated insulin and UCN3, reduced immaturity markers, and altered islet architecture, with α-cells appearing within the islet core. These findings position tomosyn-2 as a key regulator of β-cell proliferation and insulin secretion during postnatal maturation, essential for establishing functional β-cell populations. This study provides novel insights into postnatal β-cell remodeling, with implications for diabetes therapies targeting β-cell dysfunction.

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Mechanisms by which RA signaling and GATA6 synergize during pancreas development

 

Daria Podgorski^1,2, Kristen Wells-Wrasman³, Dylan Sarbaugh³, Andrea Laurin^1,2, Andrea

Alvarez^2,4, Paul Gadue⁵, Lori Sussel³, David Lorberbaum^1,2,4

 

¹Department of Pharmacology, University of Michigan, Ann Arbor

²Caswell Diabetes Institute, University of Michigan, Ann Arbor

³Barbara Davis Center for Diabetes, University of Colorado, Denver, Anschutz Medical Campus, ⁴Cellular and Molecular Biology, University of Michigan, Ann Arbor

⁵Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia

 

The pancreas is a multifunctional organ that regulates blood glucose homeostasis and facilitates

digestion. Disruptions to either function can lead to life threatening complications. In order to better understand these complications and improve treatments for these diseases, this project examines genetic mutations impacting the pancreas to define the molecular relationships

underpinning pancreas development and function. Mutations found within the coding and noncoding regions of the transcription factor GATA6 are associated with cardiac and pancreatic defects. Of 78 characterized heterozygous GATA6 mutations, 87% of patients displayed cardiac

defects, ~60% of these were impacted by pancreatic agenesis and/or diabetes, while ~40% did not overtly affect the pancreas. The heterogeneity of outcomes associated with these mutations suggests GATA6 likely interacts with other genetic or environmental signals for proper pancreatic specification and/or regulation. The Lorberbaum lab has begun probing this potential synergy between the transcriptional effector of RA signaling, RARa, and GATA6. The main objective of this project is to determine how GATA6 synergizes with RA signaling during pancreas development in health and disease. To address this question, we have established a physical interaction between GATA6 and RARa through co-immunoprecipitation followed by Western blot analysis. Furthermore, we used molecular cloning to mutate the GATA6 zinc finger domains and observed a partial disruption of its protein-protein interaction with RARa. By defining these mechanisms by which RA signaling and GATA factors interact, we can better understand pancreatic development and promote improvements for treatment of pancreatic disease.

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β-cell dysfunction in O-GlcNAcylation deficiency is partially rescued by blocking ULK1-mediated autophagy

 

V Pszczolkowski¹, Seokwon Jo¹, Grace Chung¹, Emilyn Alejandro¹

 

¹Department of Integrative Biology and Physiology, University of Minnesota Twin Cities, Minneapolis MN

 

O-GlcNAc transferase (OGT) is a key regulator of β-cell function, with reduced OGT activity resulting in β-cell failure (PMID: 26673325). This failure may be partially driven by excessive autophagy, as long-term absence of OGT in β-cells is associated with heightened autophagy (PMID: 39388284). However, O-GlcNAcylation of the autophagy kinase ULK1 contributes to autophagosome formation in multiple tissues (PMIDs: 28903979, 30517873), and whether the β-cell dysfunction under OGT deficiency is driven by elevated autophagy remains untested. To interrogate the OGT-autophagy relationship, we crossed mice expressing rat insulin 2 promoter (RIP)-Cre with mice expressing floxed OGT and ULK1, thereby generating mice with β-cell-specific deletions of OGT (βOGTKO), ULK1 (βULK1KO), or both (βOGT/ULK1KO). Floxed Cre-negative littermates were pooled as control (Ctrl). In vivo glucose tolerance and insulinemia were partially rescued from βOGTKO by βOGT/ULK1KO, with both metrics for βOGT/ULK1KO falling midway between Ctrl and βOGTKO. In vitro GSIS was decreased from Ctrl under βOGTKO, with βOGT/ULK1KO restored to Ctrl level. Contrastingly, β-cell mass and islet insulin content were decreased in both βOGTKO and βOGT/ULK1KO compared to Ctrl. Likewise, the mitochondrial dysfunction observed in βOGTKO was also present in βOGT/ULK1KO, as preliminary OCR analysis revealed similarly blunted responsiveness compared to Ctrl. Currently underway studies delineating other factors such as ER and oxidative stress may offer insight into the mechanisms behind increased the autophagy observed in OGT deficiency.

 

This work was supported by the National Institutes of Health funding grants: T32 Postdoctoral Fellowship (T32DK083250) to VLP; F31 Predoctoral Fellowship (F31DK131860) to SJ; and R56 (R56DK136293) to EA, as well as the University of Minnesota Department of Integrative Biology and Physiology Accelerator Program and the McKnight Land-Grant Professorship and Presidential Fellowship to EA.

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Dysregulated regulatory B cell function may drive type 1 diabetes in non-obese diabetic mice

 

Rachel N. Ramos¹, Jessica K. Bernard¹, Jamie L. Felton¹

 

¹Indiana University Dept of Pediatrics, Center for Diabetes and Metabolic Diseases, Wells Center for Pediatric Research

 

Background:  Interleukin-10 (IL-10) is an anti-inflammatory cytokine secreted by Bregs (regulatory B cells) associated with the regulation of immune responses. Previous studies have shown reduced Breg frequencies in peripheral blood of individuals with  (type 1 diabetes) T1D compared to healthy controls. We compared Breg frequencies and IL-10 secretion using the non-obese diabetic (NOD) mouse as a model for T1D and C57Bl6/J (B6) mouse as a non-autoimmune control. We hypothesized autoimmune Bregs would have decreased IL-10 frequency and secretion compared to non-autoimmune controls.

 

Methods:  Bregs were defined as CD19+B220+CD5+CD1d^hiIL-10+ via flow cytometry. Splenocytes and isolated splenic B cells from female NOD or B6 control mice (10-17 weeks) were assessed. For Breg frequency, cells were stimulated with LPS (10ug/mL), PMA (50 ng/mL), and ionomycin (500 ng/mL), monensin (2uM) for four hours at 37C and 5% CO₂. For IL-10 secretion, cells were cultured with LPS (10ug/mL) for 48 hours and supernatant IL-10 was measured by ELISA.

 

Results:  Compared to non-autoimmune controls, the frequency of NOD Bregs was significantly higher.  However, compared to non-autoimmune controls, secretion of IL-10 from NOD splenocytes was not significantly different.

 

Conclusions:  Despite the significantly increased frequency of Bregs in NOD compared to B6 mice, IL-10 secretion was not significantly different. This may suggest that while NOD Bregs are increased in response to inflammation, secretion of IL-10 in NOD mice is dysregulated.

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Extracellular Vesicle Heterogeneity from Beta Cells: A New Perspective on Type 1 diabetes Dynamics

 

Chaitra Rao¹, Saptarshi Roy², Carmella Evans-Molina^1,4, Jon D. Piganelli², Decio L. Eizirik³, Raghavendra G. Mirmira⁴, Emily K. Sims¹

 

¹Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA

²Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

³ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles (ULB), Brussels, Belgium.

⁴Department of Medicine and the Kovler Diabetes Center, The University of Chicago, Chicago, IL, USA

 

Intercellular communication between pancreatic beta cells and immune cells is critical in Type 1 Diabetes (T1D) progression. Beta cells release extracellular vesicles (EVs), lipid-bound nanoparticles that carry molecular cargo reflecting the inflammatory microenvironment. We previously demonstrated that the checkpoint protein PD-L1 is present on beta cell EV surfaces, is induced by interferon treatment, and suppresses CD8⁺T cell activity. However, the broader functional roles of EV PD-L1 and the effects of interferon treatment on other EV-associated proteins remain unclear.

 

We hypothesized that EV PD-L1 could inhibit immune cell types beyond CD8⁺T cells, and that interferon treatment might regulate EV-associated HLA-ABC class I proteins, which are implicated in beta cell death during T1D. Functional co-culture assays with EVs and immune cells revealed that PD-L1 on beta cell EVs suppresses both the proliferation and activation of CD4⁺T cells. Furthermore, interferon-treated human islets showed increased EV HLA-ABC cargo without altering EV numbers. In contrast to other EV proteins, PD-L1 and HLA-ABC were rarely colocalized on the same EVs (<0.5%).

 

To investigate local EV secretion in humans, we tested whole pancreas slice perifusates from T1D and nondiabetic donors. Elevated levels of both EV PD-L1 and HLA-ABC were observed in T1D donors, with heterogeneity among individuals. Consistent with a role in beta cell protection, higher EV PD-L1 correlated with increased insulin secretion, while higher EV HLA-ABC levels showed an inverse relationship.

 

In conclusion, our findings highlight the immunomodulatory effects of beta cell EV PD-L1 and reveal heterogeneity in pancreas EV cargo linked to T1D clinical phenotypes.

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Sarco/Endoplasmic Reticulum Ca²⁺ ATPase (SERCA2) Dysfunction Contributes to β Cell Stress and the Development of Chemically Induced Type 1 Diabetes

 

Robert N. Bone^1,5,6, Cameron R. Rostron^4,5,6, Solaema Taleb², Xin Tong⁸, Tatsuyoshi Kono^1,5,6, and Carmella Evans-Molina^{1,2,3,4,5,6,7}

 

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202

²Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

³Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202

⁴Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

⁵The Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN 46202

⁶Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

⁷Richard L. Roudebush VA Medical Center, Indianapolis, IN 46202

⁸Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37240.

 

Increasing evidence suggests that stress pathways intrinsic to the β cell contribute to their destruction during type 1 diabetes (T1D) development. Our group has previously shown that loss of sarco-endoplasmic reticulum calcium (Ca²⁺) pump 2 (SERCA2) exacerbates high-fat diet-induced diabetes in murine models. Here, we assessed whether SERCA2 haploinsufficiency exacerbates loss of β cell function in a murine model of T1D. Multiple low-dose streptozotocin (STZ) injections were used to induce diabetes in mice haploinsufficient for SERCA2 (S2^+/-) and in wild type (WT) C57BL/6J littermate controls. Next, to determine whether SERCA2 modulation could improve glycemia, STZ-WT mice were treated for 5 days with an allosteric SERCA activator or vehicle. Intraperitoneal glucose tolerance tests were performed, and proinsulin and insulin were measured by ELISA.  STZ treatment reduced endoplasmic reticulum Ca²⁺ levels in islets from both WT and S2^+/- mice, with S2^+/- islets showing a greater reduction. STZ-S2^+/- mice also had worsened glucose tolerance as compared to STZ-WT mice. The non-fasting proinsulin to insulin ratio was increased in STZ-S2^+/- mice, but not in STZ-WT mice, compared to untreated controls, indicating a potential defect in β cell protein processing.  Importantly, SERCA activator-treatment in STZ-WT mice improved glucose tolerance compared to vehicle-treated mice. Taken together, these data indicate that SERCA2 dysregulation may be an important component of β cell stress during T1D development and that SERCA2 activation may improve glucose tolerance and insulin secretion.

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Utilizing redox modulation with Novel Catalytic Antioxidants to protect human β-cells from Tacrolimus-induced toxicity in New-Onset Diabetes mellitus After Transplantation (NODAT)

 

Saptarshi Roy¹, Quosena Mir¹, Megan Proffer¹, Virginie Lazar¹, Jon D. Piganelli¹*

 

¹Department of Endocrinology, Indiana University School of Medicine, Indianapolis, IN

 

New-onset diabetes mellitus (NODAT) is a serious and common complication following kidney and liver transplantation, with an incidence of 30–35% among transplant recipients. Among various risk factors, immunosuppressive drugs, particularly tacrolimus, significantly contribute to the development of NODAT, accounting for approximately 74% of cases. Tacrolimus is associated with a higher incidence of NODAT (16.6–33.6%) compared to cyclosporine (9.8–26%). This risk is particularly concerning in pediatric transplant recipients, as lifelong exposure to tacrolimus can lead to progressive β-cell dysfunction and insulin deficiency. The mechanism underlying tacrolimus-induced diabetes involves increased mitochondrial-derived reactive oxygen species (ROS) production, impaired insulin secretion, and β-cell apoptosis. Furthermore, tacrolimus disrupts Nuclear Factor of Activated T cells (Cn/NFAT) signaling, which is critical for pancreatic β-cell development and function. It also impairs mitochondrial calcium uptake, disrupts autophagy, and promotes oxidative stress, ultimately leading to β-cell dysfunction and death.

 

To mitigate these deleterious effects, metalloporphyrin’s (MnPs), such as BuOE, have been explored as potential therapeutic agents due to their ability to scavenge ROS and block NF-κB dependent proinflammatory cytokines. This study investigates the protective effects of MnPs on pancreatic islets in the presence of tacrolimus, with a focus on insulin secretion, β-cell survival, and immunosuppression maintenance. Experimental findings demonstrate that MnPs effectively reduce ROS-induced β-cell damage, preserve glucose-stimulated insulin secretion (GSIS), and prevent apoptosis. Experimental findings further reveal that MnP enhances Tacrolimus-mediated immunosuppression even under Concanavalin A stimulation, a general activator of all T cells, demonstrating a similar effect as observed with BDC 6.9 Peptide. Additionally, MnPs maintain T-cell suppression when co-administered with tacrolimus, ensuring continued immunosuppressive efficacy. These findings suggest that MnPs can serve as a novel adjunct therapy to mitigate tacrolimus-induced β-cell toxicity, potentially preventing the onset of NODAT in transplant recipients while preserving immunosuppression.

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Nutritional regulation of stress responses controlling proinsulin levels for insulin biosynthesis in pancreatic beta cells

 

Yuting Ruan, Rubing Shao, and Peter Arvan

 

Division of Metabolism, Endocrinology & Diabetes; University of Michigan, Ann Arbor

 

Humans respond to circadian-based feeding/fasting cycles.  Even beta cells in culture experience nutrient abundance and depletion, but the effects of such nutrient fluctuations have rarely been studied.  Using INS1E rat beta cells, we established a fasting-refeeding protocol to assess nutrient-dependent responses.  Cells were fed 48h before the experiment and fed again 4h before the experiment with a limited volume of fresh medium to control nutrient abundance.  At time zero, phospho-eIF2α was low and proinsulin high.  The medium was then either retained ("old medium") or replaced with fresh components.  After five hours, old medium led to eIF2α re-phosphorylation and proinsulin decline.  Similarly, replacing the medium with glucose-free RPMI, or 11mM glucose lacking amino acids, both induced eIF2α re-phosphorylation with a decline of proinsulin.  In media with 100% of the amino acids contained in RPMI at physiological (5.5 mM) glucose, beta cells maintained low eIF2α phosphorylation and high proinsulin levels.  We then compared this media (100% of RPMI amino acids) to those containing only 50% or 25% of the amino acids, with glucose clamped at 5.5 mM.  As amino acid availability decreased, we observed an increase of phospho-eIF2α and a decline of proinsulin levels.  Regardless of the RPMI dilution, extracellular glucose levels changed negligibly over the 5h time course.  Evidently, amino acid availability influences these responses.  Pharmacological GCN2 inhibition reversed eIF2α phosphorylation and restored proinsulin levels.  These experiments are designed ultimately to define the amino acid requirement for maintenance of proinsulin and insulin levels in pancreatic beta cells under euglycemic conditions.

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Loss of SPCA1 Impairs Insulin Secretion in the Pancreatic Islet

 

 

Robert N. Bone^1,5,6, David Sanchez Rodriguez^2,5,6, Prudhvi Raj Terli^2,5, Xin Tong⁸, Staci A. Weaver⁴, Charanya Muralidharan⁴, Preethi Krishnan^1,5,6, Lata M Udari^1,4,5,6, Chih-Chun Lee^1,5,6, Marjan Slak Rupnik^9,10, Tatsuyoshi Kono^1,5,6, Carmella Evans-Molina^{1,2,3,4,5,6,7}

 

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202

² Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

³ Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202

⁴ Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

⁵Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN 46202

⁶Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

⁷Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, IN 46202

⁸Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37240

⁹Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

¹⁰Institute of Physiology, Faculty of Medicine, University of Maribor, Slovenia

Objective: The β-cell Golgi apparatus serves as a critical intracellular Ca²⁺ reservoir and a key site for proinsulin maturation. However, its role in diabetes pathophysiology remains unclear. The Secretory Pathway Ca²⁺ ATPase (SPCA1) is the primary regulator of Golgi Ca²⁺ homeostasis, and its dysfunction has been linked to impaired Golgi function in other cell types. This study aims to determine whether SPCA1 and Golgi Ca²⁺ are essential for β-cell Ca²⁺ homeostasis and insulin secretion.

Methods: We investigated the role of SPCA1 and Golgi Ca²⁺ in β-cell function using INS-1 β cells lacking SPCA1 (SPCA1KO) and mice haploinsufficient for SPCA1 (SPCA1⁺/⁻).

 

Results: SPCA1 expression was decreased in islets from diabetic mice and human organ donors with type 2 diabetes. SPCA1KO INS-1 cells exhibited reduced intraluminal Golgi Ca²⁺ levels, impaired glucose-stimulated insulin secretion (GSIS), and increased insulin content. Similarly, islets from SPCA1⁺/⁻ mice displayed reduced GSIS, altered glucose-induced Ca²⁺ oscillations, and disrupted insulin granule maturation.

 

Conclusions: These findings indicate that SPCA1 is essential for maintaining β-cell Golgi Ca²⁺ homeostasis and that reduced Golgi Ca²⁺ levels impair insulin granule maturation and secretion, potentially contributing to diabetes pathogenesis.

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Small molecule allosteric activators of SERCA improve β-cell viability and calcium homeostasis

 

David Sanchez Rodriguez¹, Tatsuyoshi Kono^2,6,7, Renato C. S. Branco^2,5,7, Russel Dahl⁸, and Carmella Evans-Molina^{1,2,3,4,5,6,7}

 

¹Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202

²Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202

³Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

⁴Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

⁵Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN 46202

⁶Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

⁷Roudebush VA Medical Center, Indianapolis, IN 46202

⁸Neurodon, Crown Point, IN 46307

 

Pancreatic β-cells regulate insulin production and secretion; however, pathological stressors encountered during diabetes development, including endoplasmic reticulum (ER) stress, contribute to decreased β-cell mass, function, and identity. The ER serves as the central Ca²⁺ storage organelle for the β-cell, and Ca2+ is actively transported from the cytosol into the ER by the Sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA). We have shown that pancreatic islet expression of SERCA2 is decreased in mouse models of obesity and in organ donors with T2DM. Furthermore, loss of SERCA2 activity impairs insulin secretion and β-cell survival. Here, we determined whether ER stress-induced β-cell death is ameliorated through SERCA activation. INS-1 cells and human pancreatic islets were co-treated with tunicamycin (300 nM) and a series of small-molecule allosteric SERCA activators for 24 hours to determine their effect on ER Ca²⁺ dynamics and β-cell viability. To determine ER Ca²⁺ concentrations, cells were transfected with pCMV G-CEPIA1er and imaged using an upright confocal laser scanning microscope. Cell viability was determined by Sytox green and quantified with a Sartorius IncuCyte S3/SX1 live cell imaging and analysis system. As expected, tunicamycin treatment of INS-1 cells and islets increased cell death and lowered ER Ca²⁺ levels; however, when cells were co-incubated with tunicamycin and small-molecule SERCA allosteric activators, cell survival was increased, and ER Ca²⁺ levels were maintained compared to cells and islets treated with tunicamycin alone. Our findings suggest that SERCA activation with small molecules holds promise as a therapeutic strategy to treat ER stress-mediated β-cell dysfunction during T2DM.

 

Key Words: SERCA, Tunicamycin, ER Ca^2+, ER stress.

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TM4SF4 promotes glucagon secretion and α-cell identity

 

Madushika Wimalarathne¹, Soham Saraf^{1, 2}, Nitin Shankar¹, Anna Marie R. Schornack³, Katelyn Sellick¹, Paola Bisignano³, Derek Claxton³, E. Danielle Dean^1,3

 

¹ Vanderbilt University Medical Center, Department of Medicine, Division of Diabetes, Endocrinology, & Metabolism, Nashville, TN. 

² The SyBBURE Searle Undergraduate Research Program, Vanderbilt University, Nashville, TN.

³ Vanderbilt University School of Medicine, Department of Molecular Physiology and Biophysics, Nashville, TN. 

 

The transmembrane 4 cell surface protein superfamily member TM4SF4 is highly expressed in pancreatic α-cells and considered an “α-cell identity gene” based on single cell RNASeq studies. Known for its role in cancer, TM4SF4 inhibitors slow the growth of hepatocellular carcinoma. TM4SF4 gene expression is significantly decreased in α-cells from donors with type 1 diabetes with impaired glucagon secretion. This leads us to hypothesize that TM4SF4 plays a critical role in α-cell function, including proliferation, glucagon secretion, and differentiation. We find that Tm4sf4 shRNA knockdown in αTC1-6 cells reduces glucagon secretion.Live confocal imaging reveals TM4SF4 protein translocates from the cell membrane to lysosomes when briefly starved of arginine and glutamine and then restimulated with both, suggesting a role in amino acid sensing. To further investigate, we developed a mouse model to knockout TM4SF4 expression in pancreatic α-cells, αTm4sf4 (GcgCre^ERT2; Tm4sf4^fl/fl) mice. We observe 85.4-95.5% reduction in TM4SF4 expression in α-cells when compared to Tm4sf4^fl/fl mice from female and male mice, respectively. αTm4sf4 mice display decreased α-cell proliferation (4.24 fold) and significantly reduced glucagon levels when fasted and stimulated with an arginine bolus, indicating TM4SF4 regulates both glucagon secretion and cell growth. Moreover, our findings reveal a ten-fold increase in the number of glucagon⁺somatostatin⁺ bihormonal cells in αTm4sf4 mice suggesting that TM4SF4 is partially responsible for maintaining a differentiated α-cell phenotype. In summary, these findings highlight the key role of TM4SF4 in pancreatic α-cell function and differentiation, suggesting potential applications for diabetes treatments and therapies that target TM4SF4.

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Late-life caloric restriction reduces insulin secretory demand and alters islet immune composition by limiting antigen presentation and T-lymphocyte infiltration

 

Michael Schleh¹, Amanda Cambria¹, Melanie Cutler¹, Christopher Acree¹, Gabriel Ferguson¹, Rafael Arrojo e Drigo¹

 

Molecular Physiology and Biophysics, Vanderbilt University School of Medicine; Nashville, TN

 

Caloric restriction (CR) can extend organismal lifespan and/or healthspan, and some of these effects rely on improved peripheral insulin sensitivity. We showed young mice exposed to CR for 2 months reduces the demand for beta cell insulin release and prolongs beta cell longevity. However, whether CR triggers similar responses in beta cells from aging mice remains unknown. To address this gap, we exposed 70-week-old C57BL/6J male mice to 20% CR for two months and assessed in vivo glucose homeostasis and islet transcriptional regulation using single-nucleus RNA-seq. We found aged mice exposed to CR improved glucose tolerance and insulin sensitivity, which correlated with a twofold reduction in glucose-stimulated insulin secretion and lower beta cell mass. At the molecular level, late-life CR increased islet autophagy, and altered the transcriptional profile of both islet alpha and beta cells. This was marked by a significant downregulation of ER-dependent protein processing in beta cells, and antigen presentation pathways in both alpha and beta cells. As a result, CR reduced the population of adaptive immune cells in islets, including antigen-presenting dendritic cells, B lymphocytes, and a threefold decrease in islet T cells marked by a near elimination of cytotoxic CD8⁺ T cells. In addition, intercellular communication network analysis revealed a loss of cell-to-cell communication between antigen-presenting cells, adaptive immune (B and T lymphocytes), and alpha cells. These findings highlight how late-life CR reshapes islet endocrine cell function and the immune landscape to mitigate age-dependent immune activation and islet inflammation to enhance metabolic healthspan.

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The Role of Glutamine Metabolism in Pancreatic Islet α-Cells

 

Anna Marie R. Schornack¹, Mathew Shou², Joshua Debo², Walter Siv², Varsha Chigurupathi², Katelyn Sellick², Jade E. Stanley¹, Austin Reuter², E. Danielle Dean^1,2

 

¹Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN

²Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, TN

 

Pancreatic islets integrate glucose, amino acid, and fatty acid signals to coordinate hormone secretion for metabolic homeostasis via target tissues such as the liver. Dysregulated glucagon secretion from α-cells contributes to metabolic disorders such as diabetes, exacerbating hyperglycemia. Interrupting glucagon receptor (GCGR) signaling can improve glycemia but also results in hyperaminoacidemia and hyperglucagonemia. We find that high glutamine levels stimulate α-cell proliferation. We observe that glutaminase (Gls), including both alternatively spliced variants KGA and GAC, expresses at much higher levels in α-cells than other pancreatic cells in humans and mice. To test the role of glutamine metabolism in α-cell proliferation, we have used both pharmacological targeting in whole islets and genetic targeting of GLS activity specifically in α-cells using GcgCre^ERT2; Gls^fl/fl mice (αGls^KO) with reduced GLS expression after tamoxifen treatment (αGls^WT: 95% GLS⁺, αGls^KO: 5% GLS⁺). While αGls^WT mice treated with GCGR mAb for 10 days have robust α-cell proliferation, αGls^KO mice treated with GCGR mAb showed a 4.5-fold decrease in α-cell proliferation and were similar to mice treated with the control IgG (αGls^WT  Ab: 17.0±4.6% α-cell proliferation, αGls^WT IgG: 1.0±0.4%***, αGls^KO IgG: 0.2±0.2***, αGls^KO Ab: 3.8±1.6%**; n=4-6, **p<0.01, ***p<0.001). Similarly, αGls^WT mice treated with GCGR mAb show robust activation of mTOR in α-cells, but this is lost in αGls^KO mice treated with GCGR mAb. Interestingly, the glutamine transporter, Slc38a5, expression was also greatly decreased in αGls^KO mice treated with GCGR mAb. Together, these data suggest a critical role for glutamine metabolism via glutaminase in α-cell proliferation.

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Sex-Specific Metabolic and Endocrine Dysregulation in Down Syndrome: Insights from the Ts65Dn Mouse Model and iPSC-Derived Islet Cells

 

Cyrus R. Sethna¹, Bayley J. Waters¹, Jacob M. Smith¹, Maria de Carmen Mendoza Niemes¹, Sutichot D. Nimkulrat¹, Anita Bhattacharyya^1,2, Valentina Lo Sardo¹, Barak Blum¹

 

¹Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA.

²Waisman Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison WI 53705, USA

 

Individuals with Down Syndrome (DS), caused by trisomy of human chromosome 21, exhibit an increased susceptibility to all forms of diabetes. This heightened risk is thought to arise from aberrant β-cell development, function, and metabolism. Post-mortem analyses of pancreatic islets from individuals with DS have revealed mitochondrial fragmentation, impaired insulin secretion with increased proinsulin secretion, and elevated islet amyloid polypeptide (IAPP) plaques, all of which suggest significant β-cell dysfunction. Here, using the Ts65Dn mouse model, we identify striking sex-specific defects in glucose homeostasis, alongside alterations in islet cell-type composition, increased ER stress, and potential disruptions in insulin processing and secretion. To further dissect islet-intrinsic dysfunction independent of systemic DS physiology, we have established an islet-specific disease model by differentiating isogenic, patient-derived iPSCs into stem cell-derived islets (SC-Islets). This dual-model approach enables us to interrogate the molecular mechanisms driving β-cell dysfunction in DS, offering new insights into the pathogenesis of diabetes in this population.

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C-peptide mutants that impair the folding of proinsulin

 

Rubing Shao and Peter Arvan

 

Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor

 

The mammalian INS gene coding sequence is highly conserved, and over 90 mutations have been identified as contributors to diabetes.  Some (but not all) INS gene variants lead to monogenic diabetes.  Of these, Mutant INS-gene-induced diabetes of youth (MIDY) is an autosomal dominant form of diabetes caused by mutations altering the coding sequence of proinsulin so as to cause its misfolding, with activation of endoplasmic reticulum (ER) stress response, and insulin deficiency.  Most MIDY mutants affect the primary structure of the insulin B-chain or A-chain.  In contrast, the exact biological function of C-peptide remains unknown, and only one C-peptide missense mutation other than those creating an extra unpaired cysteine has been reported in MIDY patients.   Additionally, with few exceptions, residues of the connecting C-peptide are less highly conserved than those of the B- or A-chains.  However, a recent report has identified a diabetic patient carrying a non-Cys missense mutation:  Q65R, with multiple additional diabetic first-degree relatives.  Here we have sought to test the role that C-peptide plays in the folding and trafficking of proinsulin.  Specifically, missense mutations have been introduced into selected conserved C-peptide residues.  Our preliminary findings already indicate several mutants that (A) induce proinsulin misfolding and may impair proinsulin protein stability; (B) impair the proper trafficking of proinsulin, and (C) appear to interact with co-expressed wild-type proinsulin.  These are among the first studies to examine the role of the C-peptide in insulin biosynthesis, and its possible role in the development of pancreatic beta cell dysfunction in diabetes.

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Dietary Protein Tunes the Liver-Alpha Cell Axis

 

Taverlyn Shepard¹, Katelyn Sellick², Jade E. Stanley¹, Anna Marie R. Schornack¹, Madushika Wimalarathne², E. Danielle Dean^1,2

 

¹Vanderbilt University School of Medicine, Department of Molecular Physiology and Biophysics, Nashville, TN

²Vanderbilt University Medical Center, Department of Medicine, Division of Diabetes, Endocrinology, & Metabolism, Nashville, TN 

 

The liver-alpha cell axis is a classical endocrine feedback loop where glucagon signaling in the liver regulates hepatic glucose production via catabolism of glycogen and amino acids. Amino acids feedback to the pancreatic alpha cells to stimulate glucagon secretion. Impaired glucagon signaling in the liver, such as small molecule targeting or genetic deletion of glucagon receptors, leads to hyperaminoacidemia, hyperglucagonemia, and alpha cell proliferation. We hypothesize alteration of dietary protein impacts alpha cell mass and glucagon secretion via circulating amino acid levels. To test this, we weaned 3-week-old C57Bl/6J male mice onto a diet with 13% fat content and either 6, 20, or 40% protein (PD) for 14 weeks. As predicted, fasting and arginine-stimulated glucagon levels in serum are significantly lower in 6% PD mice. While random-fed levels were not different, serum amino acid levels are significantly increased in fasting 6% PD mice above both 20% and 40% PD. These data contradict our understanding of the liver alpha cell axis, so we measured changes in pancreas morphology in these mice. Pancreas mass is significantly smaller in both 6% PD and 20% PD versus 40% PD mice. Again, surprisingly, we observe increased alpha cell area in both 6% and 40% PD mice versus 20% PD mice. While these data suggest that alpha cell area correlates with circulating amino acid levels, we interpret these data to suggest the liver alpha cell axis is metabolically flexible to dietary inputs remodeling to sense amino acids when dietary protein is limiting. 

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Synchrotron X-ray fluorescence in single pancreatic beta-cells reveals novel stress-associated iron-regulating structures

 

Kira Slepchenko¹, Si Chen², Robert Colvin¹, Craig Nunemaker¹

 

¹Ohio University, Athens, OH; ²Advanced Photon Source, Lemont, IL.

 

The pancreatic beta-cell is a unique cellular model to study iron dynamics because these cells are highly susceptible to oxidative stress associated with ferroptosis, which is a novel form of iron-induced cell death. Our group has used synchrotron X-ray fluorescence (SXRF) to describe for the first time distinct rounded cellular structures inside beta-cells that are 0.5-1.5µm in diameter and contain high density of iron (10 times more than cytosol), we termed these structures iron puncta. About 25% of cellular iron is in these iron-dense structures. On average, there are 40 iron puncta per cell, with different iron densities that are heterogeneously dispersed throughout the cytosol but not present in the nucleus. Similar structures have been reported in neurons and hepatocytes. The unique aspect of using SXRF is that this technique unambiguously describes metal distribution and accurately quantifies metal amounts in single cells with 100nm resolution. Exposing beta-cells to low grade inflammation (cytokines IL-6 and IL-1beta), as well as iron-loading (50µM iron), both produce significant increases in iron density inside iron puncta, suggesting that iron puncta are physiologically relevant and respond to cellular stress by accumulating iron; the number of iron puncta in cells is not altered by cellular stress. Interestingly, we showed that iron puncta may be involved in protecting beta-cells from erastin-induced ferroptosis, further suggesting physiologically relevant function of these newly discovered iron-regulating structures. Iron puncta thus represent a novel structure in cells that may play a role in cellular stress responses by regulating intracellular availability of iron. 

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Loss of the mitochondrial inorganic phosphate transporter impairs β cell glucose-stimulated insulin secretion despite a maintenance of ATP levels

 

Ava M. Stendahl¹, Emily M. Walker¹, Mabelle Pasmooij¹, Benjamin Thompson², Jeremias Corradi², Leslie S. Satin², and Scott A. Soleimanpour¹

 

¹University of Michigan; Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes

²University of Michigan; Department of Pharmacology

 

The classical model for β cell glucose-stimulated insulin secretion (GSIS) depends on increases in the ATP/ADP ratio, thus leading to closure of the ATP sensitive potassium channel. Recent studies have challenged the importance of mitochondrial ATP in the regulation of GSIS. Slc25a3 encodes the mitochondria-specific inorganic phosphate transporter, PiC, which is vital for phosphate transport, and ultimately ATP generation. Mutations in SLC25A3 lead to cardiomyopathy and premature mortality in humans; however, the role of PiC in pancreatic β cells and diabetes is unclear. We aimed to determine the importance of PiC on ATP generation and GSIS in β cells.

 

To characterize the role of Slc25a3 in β cell function in vivo, we generated β cell specific knockout mice which developed elevated random blood glucose levels at 2 weeks of age and glucose intolerance beginning at 3 weeks. Despite increases in mitochondrial mass, β-Slc25a3^KO islets had significantly impaired glucose-stimulated oxygen consumption. Further, we detected a 2-fold decrease in β cell mass at 6 weeks of age that was associated with impaired β cell replication. Contrary to our expectations, ATP concentrations were not reduced in β-Slc25a3^KO islets. In addition, ATP/ADP ratio was elevated at 2 mM glucose and was not significantly induced following glucose stimulation.

 

Together, our data will determine how β cells compensate for the severe bioenergetic changes following Slc25a3 loss. Additionally, insights garnered from this model may further clarify mechanisms underlying β cell GSIS, particularly in the contribution of mitochondrial ATP production to the triggering pathway.

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Uncovering how β-cell hypersecretory stress affects protein translation and loss of insulin secretory responses

 

Kaitlyn Stickel¹, Jagannath Misra², Ron Wek², Michael A. Kalwat^1,3

 

¹Indiana Biosciences Research Institute, Indianapolis, IN

²Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

³Center for Diabetes and Metabolic Disease, Indiana University School of Medicine, Indianapolis, IN.

 

Insulin hypersecretion causes β-cell dysfunction during T2D and obesity pathogenesis, yet the underlying mechanisms remain unclear. We identified a compound, SW016789, that induces insulin hypersecretion via enhancing Ca²⁺ influx while allowing adaptation via loss of secretory responses without cell death. The mechanisms of how the cessation of insulin secretion responses occurs during this process is an open question. We hypothesize that β-cells undergoing insulin hypersecretion lose responsiveness due to negative feedback from insulin exocytosis and downstream alterations in the protein translation. To identify differently translated mRNAs in hypersecretion models, we performed polysome profiling in MIN6 cells treated with SW016789 to measure mRNA translation. We have found that hypersecretion causes a transient drop in the polysome: monosome ratio in β-cells. Second, we treated β-cells with alkyne-labeled puromycin (OP-Puro), which incorporates into peptide chains. We have optimized OP-Puro labeling to be used with SW016789 or thapsigargin treatment for click-chemistry and proteomics to determine the immediate mechanisms responsible for how the β-cell recognizes hypersecretion, shuts down exocytosis, and avoids cell death. We confirmed that 4 h treatment with SW016789 or cycloheximide is sufficient to repress insulin secretion in MIN6 cells. In parallel, to investigate the source of negative feedback, we used tetanus toxin light chain (to abolish β-cell insulin exocytosis by cleaving the SNARE protein VAMP2 without affecting glucose-stimulated Ca²⁺ influx. SW016789 treatment under these conditions causes enhanced Ca²⁺ influx but not enhanced exocytosis and differential gene expression will elucidate the feedback mechanism. This understanding is crucial for developing β-cell therapeutics.

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CHD3 helicase modulates pancreatic β-cell function and identity in absence of CHD4

 

Sukrati Kanojia^1,2, Avinil Das Sharma^1,2, Abigail Taylor^2,#, Rajani M. George², Rebecca Davidson^1,2, Kayla Huter², Meredith Osmulski², Kassandra Sandoval², Jason Spaeth^1,2*

 

¹ Department of Biochemistry and Molecular Biology, Indiana University School of Medicine

² Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine

 

^# Presenting Author

* Corresponding Author

 

PDX1, an essential transcription factor during β-cell development, controls expression of genes required to maintain β-cell function through selective recruitment of transcriptional coregulators. We found CHD4, a helicase within the Nucleosome Remodeling and Deacetylase (NuRD) complex, modulates a subset of PDX1 transcriptional activity in β-cells, partly by controlling chromatin accessibility. This investigation revealed removing CHD4 from mature β-cells (Chd4^Δβ) led to increased expression of CHD3, an alternate NuRD complex helicase subunit. We uncovered PDX1 and CHD3 also interact in mature β-cells; their interactions are increased in Chd4^Δβ β-cells, posing whether CHD3 alone influences β-cell function and/or compensates in the absence of CHD4. We generated tamoxifen-inducible, β-cell-specific deletions of Chd3 (Chd3^Δβ) and Chd3/Chd4 (Chd3/4^Δβ). Four weeks-post CHD3 removal, Chd3^Δβ mice showed no changes in glucose homeostasis, whereas Chd3/4^Δβ mutants exhibited severe glucose intolerance. Chd3/4^Δβ mutants displayed elevated ad libitum fed blood glucose levels, reduced β-cell mass, increased apoptosis, and compromised glucose induced insulin secretion (GSIS), significantly more severe phenotypes than Chd4^Δβ mutants. RNA-Seq and ATAC-Seq of flow-sorted β-cells identified differentially expressed genes and altered chromatin accessibility regions, including reduced expression of Mafa, Ucn3, Chga, Chgb, and increased expression of non-β-cell gene markers, Gcg, Sst, Arx, Hhex. Immunofluorescence analysis revealed increased GCG expression in lineage-labeled CHD3/4-deficient β-cells. Human pseudoislets generated following shRNA lentiviral infection against CHD3 and/or CHD4 reveal CHD3 knockdown has normal insulin secretion, whereas CHD4 and CHD3/4 knockdown present with impaired secretion. Collectively, our data reveals CHD3/4 are essential for modulating genes required to maintain β-cell function and identity.

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The RNA binding protein hnRNPK regulates b-cell insulin secretion and endocrine cell mass in pancreatic islets

 

Matthew Varney¹, Bareket Daniel¹, Austin Good¹, Jeff Ishibashi¹, Xander Utecht¹, Matthew Haemmerle¹, and Doris Stoffers¹

 

¹Institute for Diabetes, Obesity, and Metabolism, Division of Endocrinology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

 

Dysfunction and death of the insulin secreting b-cells are the central cause of hyperglycemia and diabetes mellitus. It is therefore essential we understand mechanisms of b-cell demise to develop new treatment strategies to preserve b-cell function. b-cells harness posttranscriptional controls to ensure sufficient expression of proteins critical for function and survival. Our group discovered the RNA binding protein (RBP), hnRNPK, was activated by glucolipotoxic stress in b-cells and that it binds mRNA transcripts critical for insulin secretion and cell survival. However, significant gaps remain in our understanding of how hnRNPK regulates b-cell dynamics in vivo. To understand hnRNPK activity in b-cells, we created a novel hnRNPK conditional allele mouse to generate an animal model with hnRNPK deletion specifically in b-cells. We then performed metabolic physiology tests and morphometric analyses to determine the impact hnRNPK b-cell depletion has on glucose homeostasis. Mice with hnRNPK deficient b-cells had diminished glucose tolerance in both males and females that was accompanied by a reduction in insulin secretion but normal insulin sensitivity. The islets from mice devoid of b-cell hnRNPK had decreased b-cell but unchanged a-cell mass. However, the ratio of b- to a-cells in islets from hnRNPK b-cell deficient mice had a marked increase in the number of a-cells. Furthermore, these islets displayed an abnormal distribution of endocrine cells where glucagon expressing a-cells were at the core of the islet. These findings support a critical role for hnRNPK in regulating insulin secretion via its requirement for functional b cell mass and normal islet architecture.

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Modulation of the Biomechanical Environment alters β-cell Function and Maturity

 

Dr. Kelly J. Vazquez^1,2, Carsten Schumm¹, Julianna Norman, Andrea L. Laurin², and Dr. Richard K. Benninger^1,2

 

¹ Department of Physics and Engineering, Wheaton College, Wheaton, IL

²Barbara Davis Center for Childhood Diabetes

³Department of Bioengineering, University of Colorado-Anschutz Medical Campus, Aurora, CO.

 

It is well-established that cells respond to mechanical cues via cytoskeletal remodeling. However, it is not known whether the mechanical environment influences β-cell maturity and function. The coordination between β-cells is necessary for proper oscillatory insulin release in the presence of elevated glucose. Here we examine how mechanical stimuli impact coordinated β-cell Ca²⁺ oscillatory dynamics and expression of β-cell maturity markers.

To examine how mechanical cues influence the β-cell, we used biocompatible substrates with tunable mechanical properties synthesized with elastic moduli ranging from ~3kPa–33 kPa. The surface was coated with extracellular matrix and MIN6 β-like cells were seeded. To independently examine a link between β-cell function and maturity, we examined islet cells from mice in which β-cells express CaMPARI, a photoconvertible fluorescent protein that in the presence of high Ca²⁺ activity changes from green to red. The CaMPARI islets were gently dissociated, and individual islet cells were seeded atop substrates with varied stiffness. For all conditions in MIN6 cells and CAMPARI-expressing dissociated islets, we imaged Ca²⁺ dynamics via Fluo4 at low (2mM) and high glucose (11, 20mM). qPCR was conducted on MIN6 cells (6, 22 kPa) to examine expression of genes linked to β-cell function and maturity. With increased substrate stiffness, MIN6 cell Ca²⁺ oscillations were more robust, including a significant increase in duty cycle (p < 0.01) and were more coordinated, consistent with increased excitability. With increasing stiffness, there was significantly decreased expression of Sur1, Kir6.2, Ins2, Pdx1, Fltp, and Ecad (p < 0.01). Thus, the dynamics of Ca²⁺ activity is mechanoresponsive and may result from altered β-cell maturity. In CaMPARI islets, Ca²⁺ oscillations were more robust and coordinated for photo-converted red cells which marks cells with higher Ca²⁺, compared to green cells. With increased substrate stiffness, the fraction of these red cells that exhibit higher Ca²⁺ was increased, again showing that stiffness influences β-cell excitability. In conclusion, we show evidence in both MIN6 cells and isolated primary β-cells that with increased stiffness cells are more excitable and may have altered maturity. Further, our results suggest there may be an inverse relationship between β-cell maturity and function.

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Beta cell translation capacity drives beta cell function

 

Catharina BP Villaca¹, Teresa L. Mastracci^1,2

 

¹Department of Biology, Indiana University Indianapolis IN, USA;

²Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA.

 

Beta cells can modulate mRNA translation to generate increased quantities of specific proteins, notably insulin, in response to metabolic need. Our lab discovered that the specialized mRNA translation factor, eukaryotic initiation factor 5A (eIF5A), regulates this process and specifically the synthesis of critical beta cell proteins including insulin. For eIF5A to perform its mRNA translation function, it must be post-translationally modified by the enzyme deoxyhypusine synthase (DHPS) to generate the active form, eIF5A^HYP. Our published data shows that beta cell-specific Dhps deletion in mice (Dhps^LoxP;Ins1-cre; denoted DHPS^ΔBETA) results in diabetes onset at 5 weeks-of-age. Moreover, in the mutant islets we found beta cells with and without insulin expression. Published studies have categorized beta cells into subtypes based on gene/protein features i.e. β_HIGH and β_LOW cells; β_HIGH cells had lower Insulin1 transcript but higher insulin protein abundance, suggesting a higher translational rate. Moreover, an altered β_HIGH/β_LOW ratio in islets was shown to correlate with diabetes. Considering published and preliminary data, we hypothesized that eIF5A^HYP facilitates the higher translation rate of β_HIGH cells. To test this hypothesis, we analyzed pancreata from 4-week-old DHPS^ΔBETA mice, before diabetes onset, to determine if changes in subtype abundance contribute to disease development. Compared with controls, we observed decreased β_HIGH cells in mutant islets, revealing a loss of highly translationally active beta cells before diabetes onset. Ongoing studies are investigating protein folding, UPR activation, and coordinated secretion. These studies will reveal how eIF5A^HYP mediated on-demand protein synthesis maintains beta cell translational capacity and facilitates islet function.

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β-Cell Heterogeneity in the Acute IFN-α Response

 

Leslie E. Wagner¹, Olha Melnyk², Charanya Muralidharan¹, Matthew C. Arvin², Michelle M. Martinez-Irizarry³, Bryce E. Duffett², Justin J. Crowder², Elisabetta Manduchi³, Klaus H. Kaestner³, Joseph T. Brozinick⁴, and Amelia K. Linnemann^1,2,5

 

Departments of ¹Biochemistry and Molecular Biology and ²Pediatrics, Indiana University School of Medicine (IUSM), ³Department of Genetics, University of Pennsylvania, ⁴Eli Lilly and Company, and ⁴Indiana University Center for Diabetes and Metabolic Diseases

 

Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors, including viral infection. We investigated the impact of interferon alpha (IFN-α), a cytokine produced during immune responses, on human β-cell physiology, specifically evaluating reactive oxygen species (ROS) production. ROS serve as crucial signaling molecules in β-cells, regulating proliferation and glucose-stimulated insulin secretion; however, excessive ROS can cause cellular dysfunction. Intravital microscopy on transplanted human islets using a β-cell-selective ROS biosensor (RIP1-GRX1-roGFP2) revealed a subset of β-cells acutely producing ROS in response to IFN-α. Comparison to Integrated Islet Distribution Program (IIDP) data showed healthier donors had more ROS-accumulating cells. In vitro IFN-α treatment of human islets similarly increased superoxide production, and this response was ablated in islets from type 2 diabetes donors. Using EndoC-βH1 cells, we identified mitochondria as the source of IFN-α-stimulated ROS. To determine the molecular signature predisposing cells to IFN-α-stimulated ROS production, we flow sorted IFN-α-treated human islets. RNA sequencing identified inflammatory and immune response genes in ROS-producing cells. Comparison with single-cell RNA-Seq datasets from the Human Pancreas Analysis Program (HPAP) showed these genes were enriched in control β-cells rather than T1D β-cells. Together, these data suggest IFN-α stimulates mitochondrial ROS production in healthy β-cells, potentially predicting a more efficient antiviral response. This IFN-α–mediated mechanism may serve as a marker of β-cell resilience, offering insight into β-cell susceptibility in T1D.

 

Funding: R01, R03DK127766, and a Human Islet Research Network (HIRN, RRID:SCR_014393; https://hirnetwork.org) New Investigator Award (AKL), F31DK137567 (LEW).

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Investigating expression of Robo1/2 and Slit2/3 in Obesity-Induced Islet Expansion

 

Matthew R. Wagner¹, Bjorn M. Schoff¹, Alessandra L. Murray¹, Barak Blum¹

 

¹Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI

 

Obesity is the leading risk factor for developing Type 2 diabetes mellitus (T2DM). T2DM is a progressive metabolic condition defined by peripheral insulin resistance and hyperglycemia due to pancreatic islet dysfunction. Pancreatic islets are clusters of endocrine cells that regulate glucose homeostasis through the secretion of hormones such as insulin, glucagon, and somatostatin. While islet structure is remarkably stable in homeostatic conditions, islets dramatically expand during obesity and prediabetes to compensate for increasing metabolic demands. In mice, islet architecture is organized as a core of insulin-secreting beta cells surrounded by a mantle of glucagon-secreting alpha cells and somatostatin-secreting delta cells. Evidence suggests that this cell-type arrangement is necessary for optimal islet function. Formation of islet architecture requires expression of Roundabout receptors 1 and 2 (Robo1/2) in endocrine cells and of Slits 2 and 3 (Slit2/3) in the mesenchyme. Robo2 expression is known to decrease in islets of diabetic mouse models, however, we do not know the cell types in which Robo expression decreases or if the reduction is uniform throughout the islet. To address these gaps, we performed RNAscope. Here, we show Robo1/2 expression in islet cells and Slit2/3 expression in the surrounding stromal cells. Furthermore, we observed a uniform reduction of Robo2 throughout the islet in obese mice. Understanding how Slit and Robo work to regulate islet architecture and expansion during obesity may inform the development of new preventative or therapeutic strategies for treating T2DM.

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Retrograde mitochondrial signaling governs the identity and maturity of metabolic tissues

 

Emily M. Walker; Gemma L. Pearson; Nathan Lawlor; Ava M. Stendahl; Anne Lietzke; Vaibhav Sidarala; Jie Zhu; Tracy Stromer; Emma C. Reck; Jin Li; Elena Levi-D’Ancona; Mabelle B. Pasmooij; Dre L. Hubers; Aaron Renberg; Kawthar Mohamed; Vishal S. Parekh; Irina X. Zhang; Benjamin Thompson; Deqiang Zhang; Sarah A. Ware; Leena Haataja; Nathan Qi; Stephen C.J. Parker; Peter Arvan; Lei Yin; Brett A. Kaufman; Leslie S. Satin; Lori Sussel; Michael L. Stitzel; and Scott A. Soleimanpour

 

Mitochondrial damage is a hallmark of metabolic diseases, including type 2 diabetes (T2D) and metabolic dysfunction-associated steatotic liver disease (MASLD). Defects in mitochondrial structure, gene expression, and energetics previously observed in T2D could arise due to impairments in mitochondrial quality control. We hypothesized that models of impaired mitophagy, mitochondrial genome integrity, or mitochondrial fusion in isolation would allow us to parse the contribution of each mitochondrial quality control defect to β-cell failure in T2D. We utilized transcriptomic profiling, lineage tracing, and assessments of chromatin accessibility, to determined how deficiency anywhere in the mitochondrial quality control pathway (e.g., genome integrity, dynamics, or turnover) would affect function and maturity of β cells, hepatocytes, and brown adipocytes. In pancreatic β-cells of donors with T2D we observed impairments in mitochondrial genome integrity, mitochondrial RNA expression, and mitophagy. Furthermore, comparing the top 500 up- and down-regulated genes across our models of impaired mitochondrial quality control revealed a common activation of the mitochondrial integrated stress response (mtISR), a retrograde (mitonuclear) signaling program. Single cell sequencing studies also showed robust changes in chromatin accessibility and gene expression consistent with mtISR engagement. Loss of cell identity and maturity was due to dedifferentiation, confirmed by genetic lineage tracing, rather than apoptosis. Finally, the mtISR was ultimately induced following defects in the electron transport chain/oxidative phosphorylation (ETC/OXPHOS) system in mouse and human β-cells. Importantly, pharmacologic blockade of mitochondrial retrograde signaling in vivo restored β-cell mass and identity following mitochondrial damage. Targeting mitochondrial retrograde signaling in metabolic tissues may be promising in the treatment or prevention of diabetes and other metabolic disorders.

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Exploring the Role of Transcriptional Enhancers in the Regulation of Islet-Specific G6PC2 Expression

 

Tenzin Wangmo¹, Alec S. Rodriguez¹, Mark P. Keller², and Richard M. O’Brien¹§

 

¹Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232

²Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706

 

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that is predominantly expressed in pancreatic islet beta cells where it opposes the action of glucokinase, thereby modulating fasting blood glucose (FBG) by regulating the sensitivity of insulin secretion to glucose. The proximal G6pc2 promoter binds multiple islet-enriched transcription factors, and this region is sufficient to drive islet-specific transgene expression in newborn mice. However, that expression is lost in adult mice. Using HiC, ATAC and fusion gene assays we and others have identified ten putative enhancers, designated A-J, in the vicinity of the G6pc2 gene that are presumably required for sustained G6pc2 expression in adult mice. Two of these enhancers (I and J) are located in introns of the 3’ Abcb11 and 5’ Nostrin genes, respectively. Deletion of G6pc2 enhancer I reduces G6pc2 expression but also expression of the neighboring Spc25 gene. In on-going experiments, we are exploring the effects of deleting G6pc2 enhancer J, specifically whether it affects G6pc2 expression in adult but not newborn mice. We are also examining the effects of multiple common human single nucleotide polymorphisms (SNPs) on G6PC2 enhancer activity. The impact of causative SNPs on human health will then be investigated using BioVU to determine whether altered enhancer activity affects just FBG or also other aspects of human health. The existence of multiple enhancers that regulate G6PC2 expression and the ~80,000 bp distance between enhancers I and J highlights the complexity of G6PC2 transcriptional regulation and emphasizes the difficulty in studying enhancer versus non-synonymous SNPs.

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Human islets in a hyperglycemic microenvironment disrupt identity and function after transplantation into kidney capsule

 

Rashaun Williams¹, Esmeralda Castelblanco¹, Sumit Patel¹, Maria Remedi¹

 

¹Department of Medicine, Division of Endocrinology, Metabolism and Lipid Research, Washington University In St. Louis

 

Pancreatic islet transplantation is a promising approach for β-cell replacement in insulin-deficient diabetes, yet maintaining islet viability post-transplantation remains a challenge. A supportive microenvironment, including effective vascularization, paracrine-autocrine signaling, and normoxia, is essential for islet survival. We hypothesize that human islets from non-diabetic donors, when transplanted into diabetic mice, lose β-cell identity and function due to glucotoxicity.

 

Human islets (250, 400, and 600) were transplanted into STZ-induced diabetic immunocompromised NSG mice. Prior to transplantation, islets exhibited normal levels of C-peptide, insulin, and β-cell identity marker NKX6.1. Transplantation of 400 or 600 islets resulted in occasional hyperglycemia reduction, with 600-islet transplants demonstrating better kidney engraftment and stronger graft staining. Immunostaining of kidney sections from euglycemic control mice confirmed insulin and NKX6.1 presence, indicating preserved islet identity. STZ-diabetic mice that achieved euglycemia post-transplant exhibited improved β-cell marker expression. However, diabetic mice remaining hyperglycemic showed no islet function improvement, likely due to glucotoxicity.

 

These findings highlight the impact of a hyperglycemic microenvironment on islet survival and function post transplantation, offering insights to enhance islet transplantation outcomes. Ensuring a euglycemic microenvironment may prevent further dysfunction and loss of islets post transplantation.

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Role of Soluble Adenylate Cyclase in the regulation of cAMP levels and glucose-stimulated insulin secretion in ß-cells of wild-type and ob/ob Mice

 

Gisela F Wilson, Liam D Hurley, and Michelle E Kimple

 

Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, Madison, WI, USA; Research Service, William S. Middleton Memorial Veterans Hospital, Madison, WI, USA

 

G-protein-coupled receptor (GPCR) regulation of transmembrane adenylate cyclases (tmACs) plays a key role in the second phase of glucose-stimulated insulin secretion (GSIS). We investigate whether soluble adenylate cyclase (sAC, Adcy10) also plays a role using wild type (WT) C57BL/6J islets and islets from normoglycemic (NGOB) or hyperglycemic (HGOB) mice homozygous for the leptin ob mutation. Early studies used inhibitors that have since been found to have off-target effects that complicate interpretation. Here we employ a new class of inhibitors that allosterically inhibit sAC by interacting with its bicarbonate binding site. In ELISA assays of islets, we found that the sAC inhibitors either increased cellular cAMP levels and glucose-stimulated insulin secretion or had no effect, whereas 2’,5’-dideoxyadenosine (ddA), a tmAC inhibitor, reliably lowered cAMP and GSIS. Next, we shifted to interrogating ß-cells directly with a ß-cell-specific cAMP FRET biosensor in real-time live cell imaging, which also allowed us to simultaneously monitor changes in Ca²⁺_i with fura red. We were able to identify subpopulations of WT and NGOB islets for which the sAC inhibitor, TDI-11861, decreased ß-cell cAMP levels, while only increases were observed in islets from HGOB mice suggesting sAC contributions to ß-cell cAMP homeostasis fail in HGOB mice. Finally, we explored whether increases in cAMP observed in response to sAC inhibitors were a result of paracrine signaling through islet ɑ- or δ-cells using the hyperpolarization K_ATP channel opener diazoxide and G protein coupled receptor (GPCR) antagonists for the glucagon-like peptide-1 receptor (GLP1R), somatostatin receptor (SST2R), and glucagon receptor (GCGR).

 

This study was supported by NIH grants R01 DK137505 (to M.E.K.) and Department of Veterans Affairs grants IK6 BX006804 and I01 BX005804 (to M.E.K.)

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A transcriptomic comparison of potential type 1 diabetes therapeutic compounds: identifying key shared pathways using RNA sequencing

 

Akua Pomaah Wiredu^1,2, Nida Ajmal^3,4, Palwasha Khan^3,4, Kathryn L. Cobrin³, Craig Nunemaker^2,3,4

 

¹Biological Sciences department, College of Arts and Sciences, Ohio University, Athens, OH, 45701

²Molecular and Cellular Biology (MCB) Graduate Program, Graduate College, Ohio University

³Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, 45701

⁴Translational Biomedical Sciences (TBS) Graduate Program, Graduate College, Ohio University

 

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic β-cells leading to insulin deficiency. Cytokines are key modulators of inflammation during an immune response. We are developing compounds that protect islets against pro-inflammatory cytokine damage and enhance insulin secretion. Transcriptomic profiling of pancreatic islets using bulk RNA sequencing provides information on expressed genes and highlights potential therapeutic targets for drug development. We compared gene expression patterns on mRNA collected from mouse islets following one-hour exposure to several potential therapeutic compounds for T1D using RNA sequencing.

 

We identified 12 differentially expressed genes that were upregulated and 5 differentially expressed genes that were downregulated across all compounds using IDEP from a total of 56,886 genes. From the results, dual specificity phosphatase 1 (Dusp1) was amongst the topmost upregulated genes with a high protein fold change, and a high p-value across all compounds. Thioredoxin interacting protein (Txnip), a key regulator of pancreatic β-cell dysfunction and apoptosis, was downregulated across all compounds. Dusp1 is a promising therapeutic target because of its ability to inhibit Mitogen-activated protein kinases (MAPKs). MAPKs regulate cellular processes including proliferation, inflammation, survival, differentiation and apoptosis. The negative correlation of Dusp1 on the MAPK cascade results in the negative regulation of its cellular processes. The upregulation of Dusp1 and the down regulation of Txnip upon treatment with our multiple T1D compounds show that these compounds may contribute to protecting against cytokine-mediated destruction and restoring pancreatic β-cell function, indicating a promising therapeutic effect for T1D.

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Investigating the roles of the Pdx1 C-terminal protein interaction domains during pancreas development

 

Anthony Wokasch^1,2, Jennifer Fuhr^1,3, Scott Soleimanpour⁴, Doris Stoffers⁵, Maureen Gannon^1,2,3,6,7

 

¹ Program in Developmental Biology, Vanderbilt University School of Medicine

²Cell and Developmental Biology, Vanderbilt University

³Division of Medicine, Vanderbilt University School of Medicine

⁴Department of Internal Medicine, University of Michigan

⁵Department of Medicine, Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania

⁶Department of Molecular Biology and Biophysics, Vanderbilt University

⁷Department of Veteran Affairs Tennessee Valley

 

Baseline b-cell mass is established early in life through two key events: embryonic b-cell differentiation and peri-natal b-cell proliferation. A key member of the transcriptional network guiding these processes is the homeobox transcription factor pancreatic and duodenal homeobox 1 (Pdx1). Global loss of Pdx1 results in pancreatic agenesis while b-cell specific Pdx1 inactivation during embryogenesis results in decreased b-cell proliferation and thus decreased b-cell mass at birth. Many transcription factors contain domains that mediate physical interactions with other transcriptional regulators and post-translational modifiers, that directly affect target gene selection, protein stability, or function. Pdx1 cooperates via its C-terminus with the onecut transcription factor Oc1 in multipotent pancreatic progenitor cells to promote endocrine specification and differentiation by activating expression of the master endocrine transcription factor neurogenin 3. Furthermore, the E3 ubiquitin ligase substrate adapter protein, speckle-type POZ protein (SPOP) also interacts with the Pdx1 C-terminus to promote proteasomal degradation of Pdx1. Importantly, we previously showed that a decrease in Pdx1 is required for b-cell proliferation. Preliminary data indicate that SPOP and Oc1 interactions with the Pdx1 C-terminus are partially overlapping and potentially competitive, based on domain mapping studies and our observation that Oc1overexpression protects Pdx1 from SPOP-mediated degradation. Using unique in vivo Pdx1 C-terminal mutant mouse models, I am investigating the role of these Pdx1 C-terminal interaction domains at key stages of pancreas development. My preliminary data suggests that deletion of the Pdx1-Oc1 C-terminal interaction domain alone leads to severe pancreas hypoplasia and a significant decrease in endocrine differentiation and proliferation.

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Single-Cell Profiling Reveals Altered Natural Killer Cell Subsets and Their Cytotoxic Role in Early-Onset Type 1 Diabetes

 

Wenting Wu^1,2,*, Tingbo Guo², Cameron R.Rostron^1,3,6,  Raghavendra, Mirmira⁴, Jia Shen^2,5, Chi Zhang², Carmella Evans-Molina^1,3,6,7*

 

¹Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, Indiana

²Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA

³Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA

⁴Kovler Diabetes Center and Department of Medicine, The University of Chicago, Chicago, IL, USA

⁵Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN

⁶Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA

⁷Richard L. Roudebush VA Medical Center, Indiana University School of Informatics and Computing, Indianapolis, IN, USA

 

* Corresponding author                                                                 

 

Single-cell RNA sequencing (scRNA-seq) was conducted to profile 108,795 peripheral blood mononuclear cells (PBMCs) obtained from 5 youth within 48 hrs of Stage 3 Type 1 diabetes (T1D) onset and 5 age- and sex-matched healthy controls (HC) and identified 31 distinct immune cell clusters. We apply our procedure for another CITE-seq datasets of 335,381 human pancreatic lymph node (pLN) cells obtained from 19 human donors with panels extending to 163 antibodies to contract a multimodal atlas of the circulating immune system. Within PBMCs, cell-specific differential expression analysis showed that Natural Killer (NK) cells had the largest number of differentially expressed genes (n=363). Response to virus and response to cytokines were two notable pathways that showed strong, specific enrichment in NK cells from T1D donors. Strikingly, multi-modal cell-specific gene expression revealed the most robust similarities between NK cells in the circulation and the pLN (r = 0.97, P = 1.38×10^-5 by fold change (FC) ≥1.19 and FDR≤0.05 threshold), followed by CD4⁺ TCM cells. Two major NK cell types, CD56^brightCD16^lo and CD56^dimCD16^hi, key players in immune surveillance and cytotoxicity, exhibit altered subset compositions and functional shifts in individuals with recently diagnosed T1D. Protein disulfide isomerase family A member 3 (PDIA3) gene has been found upregulated (T1D vs HC) in NK cell across two tissues, and statistically significantly upregulated (average FC= 1.53) by multiple NK cytokines, especially type I interferons. By SCENIC prediction into upstream regulator, PDIA3 is significantly enriched of IRF1. To validate these findings, a CD56^brightCD16^lo cell line (NK-92) was treated with type 1 interferon. RT-qPCR analysis showed increased expression of IRF1 (FC= 3.48, adjusted P =0.008) and markers of activation and maturity. Single-cell profiling reveals an increasing trend in NK subsets with heightened cytotoxic activity, which may contribute to pancreatic islet destruction, highlighting a potential link between NK cell dynamics and disease progression. Understanding these alterations at the single-cell level could offer novel perspectives for early diagnosis and targeted immunotherapies in T1D.

 

We acknowledge grant support from National Institute of Diabetes, Digestive and Kidney Diseases via the NIDDK Information Network's (dkNET) New Investigator Pilot Program in Bioinformatics U24DK097771, NIH Center for Diabetes and Metabolic Diseases Pilot and Feasibility program P30DK097512.

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Proinflammatory stress activates neutral sphingomyelinase 2 based generation of a ceramide-enriched β β-cell EV subpopulation

 

Jerry Xu^1,2, Irene Amalaraj^1,2, Andre De Oliveira^1,2, Arianna Harris-Kawano^1,2, Jacob R. Enriquez⁴, Raghavendra G. Mirmira⁴, Josie Eder⁵, Meagan Burnet⁵, Ivo Diaz Ludovico⁵, Javier Flores⁵, Ernesto Nakayasu⁵, Emily K. Sims^1,3

 

¹Indiana University School of Medicine, Department of Pediatrics, Herman B Wells Center for Pediatric Research

²Center for Diabetes and Metabolic Diseases

³Pediatric Endocrinology and Diabetology, Indianapolis, IN

⁴Kovler Diabetes Center and Department of Medicine, The University of Chicago, Chicago, IL 60637, USA

⁵Pacific Northwest National Laboratory

 

 

β-cell extracellular vesicles (EVs) play a crucial role as paracrine effectors in maintaining islet health by facilitating intercellular communication. These vesicles carry bioactive molecules, such as proteins, lipids, and RNAs, that can influence the function of neighboring cells, including other β-cells and insulin-sensitive tissues. However, the mechanisms linking β-cell stress to alterations in EV cargo and the potential impacts on diabetes remain poorly defined. We hypothesized that β-cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched small EVs that could impact surrounding β-cells. Consistent with this, proinflammatory cytokine treatment of INS-1 β-cells and human islets concurrently increased β-cell nSMase2 and ceramide expression, as well as small EV ceramide species. Direct chemical activation or genetic knockdown of nSMase2, treatment with ZVAD to inhibit apoptosis, or treatment with a GLP-1 receptor agonist also modulated cellular and EV ceramide. RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to β-cell function and identity. EV treatment from cytokine-exposed parent cells inhibited peak GSIS in wild-type recipient cells; this effect was abrogated when using EVs from nSMase2 knockdown parent cells. Finally, plasma EVs in children with recent-onset T1D showed increases in multiple ceramide species. These findings highlight nSMase2 as a regulator of β-cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β-cell inflammatory stress and death.

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Atf4 protects islet β-cell identity and function under acute glucose-induced stress but promotes b-cell failure in the presence of free fatty acid

 

Mahircan Yagan^1,2, Sadia Najam^1,2, Ruiying Hu^1,2, Yu Wang³, Mathew Dickerson⁴, Prasanna Dadi⁴, Yanwen Xu^1,2,5, Alan J. Simmons^1,2,5, Roland Stein⁴, Christopher M. Adams⁶, David A. Jacobson⁴, Ken Lau^1,2,5, Qi Liu³, Guoqiang Gu^1,2,*

 

¹Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

²Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

³Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt Medical Center, Nashville, TN37232, USA

⁴Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

⁵Epithelial Biology Center, Vanderbilt Medical Center, Nashville, TN 37232, USA

⁶Division of Endocrinology, Diabetes, Metabolism and Nutrition, Mayo Clinic, Rochester, MN 55902, USA

 

Glucolipotoxicity, caused by combined hyperglycemia and hyperlipidemia, results in β-cell failure and type 2 diabetes via cellular stress-related mechanisms. Activating transcription factor 4 (Atf4) is an essential effector of stress response. We show here that Atf4 expression in β-cells is minimally required for glucose homeostasis in juvenile and adolescent mice but it is needed for β-cell function during aging and under obesity-related metabolic stress. Henceforth, Atf4-deficient β-cells older than 2 months after birth display compromised secretory function under acute hyperglycemia. In contrast, they are resistant to acute free fatty acid-induced dysfunction and reduced production of several factors essential for β-cell identity. Atf4-deficient β-cells down-regulate genes involved in protein translation. They also upregulate several lipid metabolism or signaling genes, likely contributing to their resistance to free fatty acid-induced dysfunction. These results suggest that Atf4 activation is required for β-cell identity and function under high glucose. But Atf4 activation paradoxically induces β-cell failure in high levels of free fatty acids. Different transcriptional targets of Atf4 could be manipulated to protect β-cells from metabolic stress-induced failure.

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Glucocorticoid receptor deficiency impairs gestational β-cell compensation and contributes to gestational diabetes

 

Hsuan Yeh, Taofeek Usman, Chenglin Pan, Wen Quan Zheng, Goma Chhetri, and Henry Dong

 

Division of Endocrinology, Department of Pediatrics, UPMC Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA 

 

Gestational β-cell compensation is characterized by enhanced insulin secretion to counteract pregnancy-induced insulin resistance in the mother. Defective β-cell compensation leads to gestational diabetes mellitus (GDM). Glucocorticoid (GC) is a hormone whose production surges in late pregnancy, coinciding with the induction of β-cell compensation. To understand the mechanism of gestational β-cell compensation, we investigated the role of glucocorticoid receptor (GR) in regulating β-cell mass and function in female mice in virgin vs. pregnant status. We found that β-cell GR activity is markedly upregulated, correlating with the gestational surge of GC in pregnant mice. Pregnant β-cell GR-knockout (βGR-KO) mice developed GDM characterized by glucose intolerance, reduced glucose-stimulated insulin secretion at gestational day 15.5, and higher newborn weight (macrosomia) compared to WT dams. Histomorphometry analysis showed that WT dams had 2-fold β-cell mass expansion in late pregnancy. This effect was abrogated in βGR-KO dams, alongside an increased α/β-cell ratio and the dispersal of α-cells in the center of islets. βGR-KO vs. WT maternal islet RNA-seq assay unveiled 231 upregulated genes, whereas 10 genes were downregulated, suggesting that GR mainly functions as a trans-repressor in β-cells during pregnancy. GR deficiency results in the induction of genes involved in extracellular matrix deposition, coinciding with distorted islet architecture in maternal islets of βGR-KO dams. Furthermore, GR-deficient islets had a marked upregulation of disallowed genes, including Aldob, whose overproduction in β-cells is associated with β-cell dysfunction. We conclude that GR signaling is essential for gestational β-cell compensation and its deficiency leads to GDM. 

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MCU overexpression and NCLX ablation in β-cells: How increasing mitochondrial calcium affects glucose homeostasis

 

Yu-Jin Youn^1,2, Seokwon Jo¹ and Emilyn U Alejandro^1,2

 

¹Department of Integrative Biology & Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA

²Minnesota Postbaccalaureate Readiness in Metabolism, Endocrinology, and Diabetes (PRIMED), University of Minnesota, Minneapolis, Minnesota, USA

 

Calcium is a key regulator of aerobic metabolism; yet, regulation of mitochondrial calcium handling is understudied in pancreatic β-cells. The mitochondrial calcium uniporter (MCU) and mitochondrial sodium-calcium exchanger (NCLX) were identified as the major routes for calcium influx and efflux across the inner mitochondrial membrane, respectively. Previous studies have shown that reduced mitochondrial calcium in β-cells by MCU ablation does not cause glucose intolerance, but few studies focus on mitochondrial calcium overload. Here, we assessed whether calcium overload by MCU overexpression or NCLX deletion in β-cells is sufficient to perturb glucose homeostasis in vivo. Given that mitochondrial metabolic activity is calcium-dependent, we hypothesized that MCU overexpression or NCLX ablation would lead to excess mitochondrial calcium, thus causing perturbations in glucose homeostasis by β-cell failure. We found that MCU overexpression led to significant glucose intolerance that progressively worsened with age in both male and female mice in vivo (p<0.05), but the mechanisms by which intolerance developed were sexually dimorphic; intolerance was driven by reduced β-cell mass in males and reduced β-cell function in females. In contrast, βNCLX-KO mice presented comparable glucose tolerance to littermate controls but exhibited significantly reduced glucose-stimulated insulin secretion (p<0.05) and OCR (p<0.05) at the islet level in vitro. Collectively, these data suggest that NCLX ablation is insufficient to induce glucose intolerance in vivo, whereas regulation of calcium entry into the mitochondria via the MCU is critical in glucose homeostasis in a sexually dimorphic manner.

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Identify proinsulin regulators with both CRISPR screen and an in vivo mouse genetic QTL mapping

 

Sisi Lai^1,2,*, Mark P. Keller^3,*, Zhou Fang^1,*, Jinglin Zhang^1,*, Ying Xie¹, Chen Weng^1,2 Saixian Zhang¹, Shanshan Zhang^1,2, Peidong Gao¹, Yuntong Wang¹, Kelly A. Mitok³, Lauren Clark³, Kathryn L. Schueler³, Yuanyuan Chen⁴, Anath Shalev⁵, Fulai Jin^1,6,7,$, Alan D. Attie^3,$, Yan Li^1,$

 

¹Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio 44106, USA

²The Biomedical Sciences Training Program (BSTP), School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA

³Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA

⁴Department of Ophthalmology and Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

⁵Comprehensive Diabetes Center, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35249, USA

⁶Department of Computer and Data Sciences and Department of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH 44106, USA

7 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA

^*These authors contribute equally.

^$Correspondence authors: Fulai Jin (fxj45@case.edu); Alan D. Attie (attie@biochem.wisc.edu); Yan Li (yxl1379@case.edu).

 

Altered proinsulin levels in β-cells and the bloodstream are hallmarks of diabetes and other metabolic disorders. However, the regulatory mechanism of proinsulin remains poorly understood. In this study, we conducted a genome-wide CRISPR screen to identify 84 candidate genes that modulate the intracellular proinsulin-to-insulin ratio in a mouse β-cell line. Notably, these proinsulin regulators are distinct from insulin regulators identified in a previous orthogonal CRISPR screen. Functional annotation of the proinsulin regulators highlights the Golgi apparatus as the primary organelle for proinsulin storage and regulation. Increased trafficking toward the Golgi elevates the intracellular proinsulin-to-insulin ratio, whereas trafficking away from the Golgi, including exocytosis and Golgi-to-ER retrograde transport, reduces intracellular proinsulin levels. To bridge genetic insights with biological mechanism, we mapped mouse quantitative trait loci (QTLs) associated with plasma proinsulin levels and leveraged our CRISPR screen results to pinpoint causal genes within these loci. Interestingly, Pdia6, a protein disulfide isomerase, emerged as the strongest hit from both the CRISPR screen and QTL mapping. Knockdown of Pdia6 significantly decreased proinsulin accumulation in the Golgi and secretory granules. Intriguingly, depletion or inhibition of PDIA6 in both human and mouse β-cell lines did not disrupt proinsulin folding but resulted in a severe reduction in proinsulin production through a UPR-independent mechanism. Taken together, our findings provide a comprehensive genetic profile of proinsulin regulation, conveying novel mechanistic insights into proinsulin-to-insulin homeostasis and potential therapeutic targets for diabetes. 

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Time-restricted feeding prevents deleterious effects of diet-induced obesity on circadian regulation of β-cell function and transcription

  

Luhui Zhang^1,2, Satish K. Sen¹, Kuntol Rakshit¹, Thanh T. Nguyen¹, Kazuno Omori¹, Ananya Bharath¹, Aleksey V. Matveyenko^1,3

 

¹Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA

²Biochemistry and Molecular Biology track, Mayo Clinic College of Medicine and Science, Rochester, MN, USA

³Division of Endocrinology, Diabetes & Metabolism, Mayo Clinic, Rochester, MN, USA

 

Diet-induced obesity (DIO), modeled by a high-fat diet, adversely affects circadian glucose homeostasis partly by dysregulating fasting/feeding cycles. However, its impact on circadian β-cell function and gene expression remains unclear. To investigate this, we exposed male and female C57BL/6 mice to either chow or DIO for eight weeks and assessed behavioral (feeding/activity), physiological (glucose tolerance/insulin secretion), transcriptomic (RNA-seq), and epigenomic (ATAC-seq) circadian rhythms. Additionally, we tested whether normalizing fasting/feeding cycles via time-restricted feeding (tRF) mitigates DIO's effects. DIO disrupted circadian fasting/feeding rhythms, with greater disruption in males than females (p<0.05). In males, DIO abolished circadian regulation of glucose tolerance and in vivo glucose-stimulated insulin secretion (GSIS) (p<0.05 vs. chow), whereas females maintained circadian glucose homeostasis. RNA-seq analysis of male islets revealed a ~50% reduction in circadian-regulated transcripts under DIO (p<0.05 vs. chow), with weakened rhythms in key pathways such as protein processing in the ER and cAMP signaling. Besides, ATAC-Seq analysis indicated reduced chromatin accessibilities of genes/pathways related to insulin secretion and glucose hemostasis under DIO (p<0.05 vs. chow) during both day and night. Notably, restoring fasting/feeding cycles in DIO males via tRF rescued circadian glucose tolerance, GSIS, and islet gene expression (p<0.05 vs. chow). Motif analysis identified transcription factors DBP and NRF1 as potential mediators of tRF’s beneficial effects in DIO islets. Our study demonstrates that DIO disrupts circadian β-cell function and gene expression by dysregulating fasting/feeding cycles and reveals significant sex differences in how DIO affects circadian glucose homeostasis and β-cell function.

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IRE1α Signaling is Required for Islet Alpha Cell Function

 

Wenzhen Zhu, Nina Frias, Annissa Sisson, Brent Pederson, and Rachel B. Reinert

 

Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan

 

Islet endocrine cells rely on endoplasmic reticulum (ER)-resident quality control systems, including ER-associated degradation (ERAD) and the unfolded protein response (UPR), to manage misfolded proteins and optimize hormone synthesis. We previously found that ERAD dysfunction alters α cell identity and limits glucagon (Gcg) production. This was accompanied by increased activity of the UPR sensor IRE1α; however, whether IRE1α-mediated signaling affects α cell function is unknown. Thus, we deleted IRE1α (Ern1) in Gcg+ cells, with or without co-inactivation of the ERAD component Sel1L, and evaluated α cell identity and function. Sel1L^ΔGcg mice had impaired glucagon secretion following insulin-induced hypoglycemia, along with reduced pancreatic glucagon content and decreased α cell mass. Sel1L^ΔGcg α cells showed loss of classic identity markers MafB and TTR, dilation of ER cisternae, and upregulation of IRE1α expression and splicing of its downstream effector, Xbp1. Sel1L;Ern1^ΔGcg mice showed a similar reduction in both stimulated glucagon secretion and pancreatic glucagon content as Sel1L^ΔGcg mice, without recovery of α cell marker expression. In contrast, inactivation of IRE1α alone in Ern1^ΔGcg mice did not appear to cause overt ER stress in α cells, as assessed by expression of the ER chaperone BiP. However, Ern1^ΔGcg mice had impaired glucagon secretion in response to arginine stimulation but not following insulin-induced hypoglycemia, suggesting that impaired IRE1α signaling may alter α cell nutrient sensing. Together, these data demonstrate that IRE1ɑ activity is required for normal islet α cell function, but that IRE1ɑ-mediated signaling does not alter the profound ɑ cell defects caused by ERAD deficiency.