bluuebunny/crossref_metadata_2025_split huggingface dataset:
1722-P: RIPK1 Promotes Thapsigargin-Induced ß-Cell Death Independent of Caspase 3/7 Activity In Vitro
Authors: MUKHERJEE NOYONIKA , CONTRERAS CHRISTOPHER J. , LIN LI , CAI ERICA P. , TEMPLIN ANDREW T. 
Published: June 2023

Endoplasmic reticulum (ER) stress promotes β-cell dysfunction and death in diabetes. Although studies of ER stress-induced β-cell death have focused on mechanisms of apoptosis, we recently showed that β cells are also susceptible to a form of programmed cell death called necroptosis. Necroptosis occurs downstream of receptor interacting protein kinase 1 (RIPK1), does not require caspase activation, and results in lytic cell death. Studies in non-islet cell types have linked ER stress-induced cell death to necroptosis. Thus, we hypothesized that RIPK1 promotes ER stress-induced β-cell death in vitro. To test this hypothesis, we quantified cell death and caspase 3/7 activity in NIT-1 β cells, human iPSC-derived β-like cells and intact mouse islets following treatment with thapsigargin (a SERCA2b inhibitor and ER stress-inducing agent, 50 nM or 500 nM) and zVAD-FMK (a pan-caspase inhibitor, 40 μM). After quantifying cell death hourly for 48 hrs, we found that thapsigargin significantly increased cell death in NIT-1 cells, β-like human iPSCs and intact mouse islets, and that this occurred in conjunction with increased caspase 3/7 activity. We next asked whether co-treatment with zVAD was sufficient to prevent thapsigargin-induced cell death. In each cell type evaluated, we found that zVAD inhibited caspase 3/7 activity but failed to protect from thapsigargin-induced cell death. To establish the role of RIPK1 in this process, we next evaluated cell death in gene-edited NIT-1 control (non-targeting gRNA) and NIT-1 Ripk1Δ (gRNA targeting Ripk1 exons 2-3) β cells. After 24 hrs, we found that NIT-1 Ripk1Δ cells were strongly protected from thapsigargin-induced cell death both when caspases were active (n=6, p&lt;0.001) and when they were inhibited (n=6, p&lt;0.001). These data indicate that RIPK1 regulates thapsigargin-induced β-cell death independent of caspase 3/7 activation. Future studies will examine the mechanisms by which RIPK1 regulates ER stress-induced β-cell death in vitro and in vivo.

Disclosure
N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. A.T.Templin: None.

Funding
U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)
1723-P: Dual-Label Kinetic Assay to Monitor ß-Cell Death and Proliferation
Authors: KATZ LIORA S. , WANG PENG , KANISKAN HUSNU U. , GELLES JESSE D. , EDWARD CHIPUK JERRY , STEWART ANDREW F. , SCOTT DONALD 
Published: June 2023

A deficit of β-cell mass is common to all types of diabetes. Thus, many research efforts are dedicated to developing therapies to preserve and expand β-cell mass. Current methods assessing β-cell proliferation and cell death are limited due to reliance on a single time point, and usually cannot measure proliferation and death simultaneously. Here we have developed and optimized a novel method, based on the single-cell and population-level analyses using real-time kinetic labeling (SPARKL), to study kinetics of cell proliferation and death in β cells. SPARKL uses two fluorescent dyes - SYTO21 labels all cells in green, and YOYO3, a red cell-impermeable viability dye that stains dead cells. We continuously imaged cells every few hours over numerous days and have optimized growth conditions and dye concentrations to monitor INS1 or primary rodent or human islet cells on single cell and population levels. As a first step, we compared growth under 6, 11 and 20 mM glucose and glucolipotoxicity (20 mM glucose and 1 mM palmitate). We found a doubling time of 52±2, 48±4, 44±3 and &gt;120 h, respectively, in INS1 cells. At 48 h, fewer than 16% of the cells are dead in varying glucose concentrations compared to 69±5% dead cells in the glucolipotoxic condition (N=8). Harmine, a known mitogen of β-cells, enhanced proliferation in all glucose concentrations by 5-15% and protected from glucolipotoxic cell death (N=3). In primary islets, as expected, the proliferation rates were extremely low and often masked by cell death in varying glucose concentrations. Glucolipotoxicity led to &gt;80% cell death in mouse and human islets (N=3). Finally, we screened a library of 73 epigenetic modifiers to screen for compounds that rescued INS1 cells from glucolipotoxic-like stress (overexpression of ChREBPβ). Our screen yielded four epigenetic modifiers that reduced cell death to less than 2% at 48 h. In conclusion, SPARKL is a novel and useful method that is accurate, unbiased, and requires minimal handling to simultaneously measure β-cell proliferation and death.

Disclosure
L.S.Katz: None. P.Wang: None. H.U.Kaniskan: None. J.D.Gelles: None. J.Chipuk: None. A.F.Stewart: None. D.Scott: None.

Funding
National Institutes of Health (R01DK130300)
1725-P: The Role of miR-146a-5p on Cytokine-Induced ß-Cell Mitochondrial Dysfunction and Death
Authors: KRISHNAN PREETHI , BRANCO RENATO C.S. , WEAVER STACI A. , LEE CHIH-CHUN , SYED FAROOQ , EVANS-MOLINA CARMELLA 
Published: June 2023

MicroRNAs are small non-coding RNAs with ~22 nucleotides in size involved in the modulation of gene expression. Previously, we observed that miR-146a-5p is upregulated during inflammatory stress in pancreatic islets and pancreatic tissue sections from T1D donors. Although others have linked overexpression of miR-146a-5p to β cell death and impaired insulin secretion, the molecular mechanisms mediating these effects remain unclear. To further understand miR-146a-5p role in β cell health and disease, we have generated stable cell lines that are either overexpressed (OE) or inhibited (ZIP) for miR-146a-5p and assessed for β cell function under inflammatory (IL-1β, IFN-γ, and TNF-α) conditions to model T1D in vitro. Treatment of OE cells with or without pro-inflammatory cytokines increased expression of cleaved caspase-3 and Annexin-V-positive cell death and reduced insulin secretion in both untreated and cytokine treatment. However, ZIP cells are protected from inflammatory stress showing decreased apoptosis and improved insulin secretion. Furthermore, ZIP cells were found to have increased mitochondrial DNA copy numbers, mitochondrial membrane potential, and basal and maximal respiration rate. Notably, cytokines-treated OE cells showed reduced basal respiration rate and ATP production compared to cytokines-treated ZIP cells. In addition, mitochondria isolated from MIN6 cells showed enriched miR-146a-5p under cytokine treatment, suggesting inflammation-mediated enrichment of miR-146a-5p. Finally, RNA sequencing analysis of OE and ZIP cells identified the RNA transcripts regulated by miR-146a-5p, including MAPT, MYRIP, NRG1, and GPX1; pathway enrichment analysis showed pathways related to insulin secretion, apoptosis, and mitochondrial function. Taken together, findings from our study show for the first time that miR-146a-5p upregulation promotes β cell dysfunction and death by suppressing mitochondrial function during T1D pathogenesis.

Disclosure
P.Krishnan: None. R.C.S.Branco: None. S.A.Weaver: Employee; Eli Lilly and Company. C.Lee: None. F.Syed: None. C.Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc.

Funding
JDRF (2-SRA-2019-834-S-B, 2-SRA-2018-493-A-B); National Institutes of Health (DK127308, U01DK127786, UC4DK104166)
1728-P: Transcriptional Activation of Myc by Glucose Requires Recruitment of ChREBP to the Pvt1 Promoter Regulating Proliferation in ß-Cells
Authors: KATZ LIORA S. , WANG PENG , BRILL GABRIEL , ZHANG PILI , HALDEMAN JONATHAN , NEWGARD CHRISTOPHER B. , STEWART ANDREW F. , GARCIA-OCANA ADOLFO , SCOTT DONALD 
Published: June 2023

Myc and Carbohydrate Response Element (ChoRE) Binding Protein (ChREBP) are glucose-responsive transcriptional regulators of β-cell proliferation. Myc requires ChREBP to drive glucose-mediated proliferation in β-cells, yet no ChoREs are present in Myc’s promoter. In primates and rodents, the Myc gene and the long non-coding RNA, PVT1 gene are syntenic, separated by approximately 60 kbp. Mutations in this loci are associated with malignancies and complications of diabetes but are unexplored in β-cells. In INS-1 cells, Myc and PVT1 mRNAs were induced in response to glucose by 3.5±0.02and 2.9±0.1 fold, respectively, and Myc protein levels increased by 4-fold ±0.17. Silencing of ChREBPβ significantly blocked the glucose response of Myc and Pvt1. ChREBP ChIPseq revealed a single ChoRE located approximately 100 bp upstream of the Pvt1 transcription start site and is highly conserved in primates and rodents. By Chromosome Conformation Capture (3C), we demonstrated that high glucose increased interaction between the Pvt1 and Myc promoters by a factor of 3.5±0.4. Using adenoviral delivery of CRISPR-xCas9-sgRNA targeting the Pvt1 ChoRE, we specifically attenuated ChREBP recruitment by 2.8±0.8 fold, while other known ChoREs were unaffected. We demonstrated by 3C that the chromatin looping in response to glucose was abolished by the Pvt1 ChoRE disruptor. Both in primary rat islets and in INS-1 cells, disruption of Pvt1’s ChoRE resulted in complete and significant blockage of Pvt1 and Myc mRNA upregulation in response to glucose, with no effect on other ChoRE-containing genes. Finally, disruption of the ChoRE on the Pvt1 promoter eliminated glucose-mediated proliferation of primary rat β-cells (a decrease of 9.8±1.9 fold on Ki67+ Ins+ staining). In conclusion, we identify a novel mechanism driving the ChREBP-dependent activation of the Myc and Pvt1 genes and proliferation of β-cells.

Disclosure
L.S.Katz: None. P.Wang: None. G.Brill: None. P.Zhang: None. J.Haldeman: Employee; Janssen Research & Development, LLC. C.B.Newgard: Advisory Panel; Eli Lilly and Company, Novo Nordisk, AstraZeneca. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D.Scott: None.

Funding
National Institutes of Health (R01DK130300, P30DK020541)
1729-P: Role of ß-Cell Senescence in Islet Transplantation
Authors: IWASAKI KANAKO , CARAPETO PRISCILA , AGUAYO-MAZZUCATO CRISTINA 
Published: June 2023

Islet transplantation (Tx) is an effective intervention to prevent severe hypoglycemia and provide insulin independence in type 1 diabetes (T1D). Previous studies have shown that islets from older donors are less effective in reversing blood glucose (BG) than those of younger ones. Cellular senescence leads to age-related β-cell dysfunction and drives alloimmune responses to organs from older donors, but can be targeted with senolysis. However, the mechanism of pancreatic β-cell senescence in transplant grafts and its pathological significance are unknown. Therefore, we performed two types of islet Tx to test the hypothesis that cellular senescence may play a role in loss of graft functionality/viability and that the use of senolysis may ameliorate it: 1) overnight cultured 600 IEQ islets from normoglycemic rats were transplanted into kidney capsule of streptozotocin-induced diabetic immunodeficient mice (Stz-Ms). After 2 weeks, the grafts were removed and compared with the cultured islets (control). In Stz-Ms, the nuclear exclusion rate of Hmgb1 and p21-positive cells tended to increase, and the mRNA levels of p16, Il-6, and β-cells related genes were significantly higher than in control islets indicating β-cell senescence after transplantation. To test if the elimination of p16 positive senescent β-cells from transplanted islets improved BG levels, 2) 440 IEQ islets from INK-ATTAC mice (p16Ink4a mediated apoptosis through targeted activation of caspase) were transplanted into Stz-Ms and treated intraperitoneally with BB Homodimerizer (BB) or vehicle for 2 terms of 3 consecutive days, of which 2 weeks of rest. With senolysis, there was a decrease in fed glucose levels and p16 mRNA expression while expression of hallmark β-cell genes increased by 74%, which correlates with improved response to high glucose. In conclusion, islet transplantation increases β-cell senescence in grafts and senolysis improves β-cell function. This has important implications in increasing graft function and survival in the treatment of T1D.

Disclosure
K.Iwasaki: None. P.Carapeto: None. C.Aguayo-mazzucato: None.

Funding
Manpei Suzuki Diabetes Foundation
1731-P: Determining the Mechanistic Relationship between Maternal Diet and Postnatal Beta-Cell Function
Authors: NEVILLS SIMONE , GU GUOQIANG 
Published: June 2023

Previous studies have shown that intrauterine nutrient levels impact the risk of diabetes in offspring postnatally, but the molecular mechanisms underpinning this phenomenon remain poorly characterized. We hypothesize that maternal malnutrition during gestation may disrupt the fetus’ epigenetic program during development, leading to insufficient functional beta-cell mass postnatally. To this end, we show that: 1) endocrine progenitor cells with different DNA methylation signatures give rise to beta-cell subsets with different functional and proliferative capacity; 2) de novo DNA methyltransferases Dnmt3a and Dnmt3b are downregulated in embryonic endocrine progenitors exposed to a low-protein or high-fat diet in utero, while DNA demethyltransferases Tet1 and Tet2 are upregulated; 3) beta cells in offspring of dams fed a low-protein or high-fat diet during gestation demonstrate reduced expression of genes associated with beta cell maturation and function both during development and postnatally (e.g. Pdx1, MafA); 4) islets from adult mice exposed to maternal high-fat diet in utero have compromised insulin secretion when treated with sustained high glucose or free fatty acids in vitro. These findings are consistent with a model in which abnormal DNA methylation in islet progenitors is a key mechanism by which maternal nutrients mediate postnatal functional beta-cell mass and diabetes risk.

Disclosure
S.Nevills: None. G.Gu: None.

Funding
National Institutes of Health (5R01DK125696-03, 5T32HD007502-25)
1747-P: Islet Ca2+ Dynamics, Heterogeneity, and Consistency in Three Dimensions with Activators of Pyruvate Kinase
Authors: BRIGGS JENNIFER , JIN ERLI , MERRINS MATTHEW J. , BENNINGER RICHARD K. 
Published: June 2023

Pancreatic β-cell heterogeneity is important for Ca2+ triggering and insulin secretion and is affected in diabetes. Organization and consistency of β-cell subpopulations are poorly understood despite being key in understanding how individual cells contribute to islet function and diabetes. Existing studies analyze single 2D planes of the islet. However, subpopulation organization cannot be fully understood without examining β cell dynamics throughout the entire islet in 3D. Here, we imaged islets from β cell specific GCaMP6s Ca2+ reporter mice using high-speed light-sheet microscopy to analyze the 3D organization and consistency in high glucose and with pharmacologic activators of pyruvate kinase (PK), a glycolytic enzyme that controls βcell excitability. Phase analysis revealed that cells which initiated islet depolarization (leaders) are not highly consistent across oscillations, but the 3D region where the calcium wave originates is consistent, according to a metric based upon the KL-divergence (P&lt;0.0001 over 11 oscillations). Using functional network analysis, β-cell hubs which drive islet synchrony are more consistent over an islet region than across individual cells (P&lt;0.0001). The wave origin was more regionally dependent than the β-cell hubs (P&lt;0.0001). We also found that network analysis is similar (P=0.04) and phase analysis is different between 2D and 3D (P&lt;0.0001). Following quantification of heterogeneity in the 3D islet, certain sub-populations of β cells are similar throughout the islet, while others are not. Acute PK activation decreased the consistency of wave initiators (P&lt;0.0001), but not the consistency of hub cells in the functional network (P=0.15), shedding new light onto the mechanism behind these subpopulations. Islet regions, rather than individual cells, are more consistent and thus likely responsible for initiating Ca2+ triggering and insulin secretion. These mechanisms help render the islet robust to single β cell failures.

Disclosure
J.Briggs: None. E.Jin: None. M.J.Merrins: None. R.K.Benninger: None.

Funding
William S. Middleton Memorial Veterans Hospital; U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01B005113); National Institutes of Health (R01DK113103, R01DK127637, R01DK102950, R01DK106412); National Science
1751-P: SERCA2 Regulates ER Lipid Composition and Proinsulin Processing
Authors: HARTLEY MARYALICE , PEARSON MACKENZIE J. , SYED FAROOQ , BUI HAI , KONO TATSUYOSHI , ROTH KENNETH D. , EVANS-MOLINA CARMELLA 
Published: June 2023

Endoplasmic reticulum calcium (ER Ca2+) levels are primarily maintained through the sarco-ER Ca2+ ATPase (SERCA) pump. We have demonstrated reduced SERCA2 expression in islets from human cadaveric donors with Type 2 Diabetes (T2D) compared to normoglycemic controls and linked β-cell specific deletion of SERCA2 with impaired proinsulin processing. To test mechanisms of how reduced ER Ca2+ and SERCA2 deficiency regulate proinsulin processing, a SNAP-tagged human proinsulin adenovirus (SNAP-proinsulin) was overexpressed in rat insulinoma β-cells where SERCA2 had been deleted (S2KO INS-1) using CRISPR/Cas9 and in islets from C57BL/6 mice with SERCA2 haploinsufficiency fed a high fat diet or treated ex vivo with glucolipotoxicity (GLT). Immunoblotting SNAP-tagged cleavage products showed reduced proinsulin processing in SERCA2 deficient models compared to wildtype (WT), these defects were exacerbated by GLT treatment. To determine whether reduced processing could be related to impaired trafficking of proinsulin within the β-cell secretory pathway, SNAP-proinsulin was fluorescently labelled in C57BL/6 mouse islets treated with GLT. The colocalization of SNAP-proinsulin with Golgi immuno-marker, Giantin, suggested an accumulation in this compartment. The ER is the main site of lipid synthesis and lipid remodeling is known to regulate β-cell function. Preliminary mass spectrometry data revealed that loss of SERCA2 increases ER levels of phosphatidylcholine and decreases ceramide and sphingomyelin in S2KO INS-1 cells compared to WT cells. Taken together, these results demonstrate that ER Ca2+ deficiency changes ER lipid composition, which may affect proinsulin processing and trafficking. Characterizing this novel mechanism could provide valuable insight into β-cell dysfunction in T2D.

Disclosure
M.Hartley: Employee; Eli Lilly and Company. M.J.Pearson: None. F.Syed: None. H.Bui: None. T.Kono: None. K.D.Roth: Employee; Eli Lilly and Company. C.Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc.

Funding
National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, R01DK127308, R01DK127236)
1766-P: Pick1 Regulates Glucose Homeostasis and Insulin Secretion through the MAPK Pathway
Authors: LU JING , ZHAO RUXUAN , XIONG FENGRAN 
Published: June 2023

Adult patients with a dysfunctional Protein interacting with C-kinase 1 (PICK1) present with hyperinsulinemia and hyperglycemia. PICK1 was reported to be a peripheral membrane protein that controled insulin granule formation, trafficking, and maturation in INS-1E cells. However, the mechanism of Pick1 in glucose metabolism disorders has not been clearly defined. Here, we demonstrate that Pick1 β-cell-specific knockout (βKO) had impaired glucose tolerance and decreased insulin serection. By contrast, overexpression of Pick1 in primary pancreatic β-cells led to apoptosis. Similarly, induction of Pick1 by tail vein injection into βKO mice improved glucose tolerance by reducing apoptosis. Furthermore, we show that Pick1 alleviated apoptosis and inflammation via the mitogen activated protein kinase (MAPK) pathway both in vitro and in vivo. In summary, our study provides new insights into the causes of primary pancreatic β-cells apoptosis.

Disclosure
J.Lu: None. R.Zhao: None. F.Xiong: None.

Funding
National Natural Science Foundation of China (82070890)
1770-P: Islet ß-Cells Shed DOC2B-Laden EVs via Sorting Signals Present in DOC2B Protein
Authors: ESPARZA DIANA , OH EUNJIN , GHAELI IMA , JOVANOVIC-TALISMAN TIJANA , THURMOND DEBBIE C. 
Published: June 2023

Loss of β-cell function occurs early in type 1 diabetes (T1D). However, C-peptide is insufficient to detect early pre-type 1 diabetes-related β-cell dysfunction. Thus, the discovery of clinically accessible β-cell health biomarkers may enable early therapeutic interventions, mitigate T1D, and prevent complete loss of β-cell mass. The exocytosis regulatory protein double C2-containing protein β (DOC2B) abundance is closely related to β-cell health and function. Our recent publication demonstrated the correlation between the decline in DOC2B abundance in blood-derived platelets and the loss of β-cell function in early-onset T1D and pre-diabetic nonobese diabetic (NOD) mice. Indeed, preliminary data show that DOC2B reduction in plasma precedes C-peptide reduction in prediabetic mice, suggesting that reduced DOC2B levels in plasma may serve as an early biomarker of β-cell dysfunction. However, the mechanisms by which β-cells export DOC2B into the circulation remain unknown. We hypothesize that DOC2B is uniquely sorted into β-cell extracellular vesicles (EVs). Preliminary data show DOC2B is detected in EVs isolated from human plasma, and that DOC2B-laden EVs can stem from β-cells. Because DOC2B is a broadly expressed protein, we questioned whether DOC2B-laden EVs are predominantly released by β-cells, relative to that of other cell types known to express DOC2B protein. Biochemical analyses reveal that DOC2B largely exists as luminal cargo in β-cell-derived EVs and is largely absent from EVs released by liver, brain, and skeletal muscle cells. Mechanistically, the research has delineated that the sufficient structural domain enabling DOC2B sorting into β-cell-derived EVs is that which encompasses the tandem C2 domains at the C-terminus of DOC2B. Taken together, these data indicate that DOC2B in circulation could originate from DOC2B-laden EVs released from β-cells, and that the mechanism by which DOC2B is sorted into those EVs involves one or both C2 domains of the DOC2B protein.

Disclosure
D.Esparza: None. E.Oh: None. I.Ghaeli: None. T.Jovanovic-talisman: None. D.C.Thurmond: Consultant; Immutics, Inc.

Funding
National Institute of Diabetes and Digestive and Kidney Diseases (DK067912, DK112917, DK102233); Wanek Family Project for Type 1 Diabetes (to D.C.T); Ford Foundation (to D.E.)