bluuebunny/crossref_metadata_2025 huggingface dataset:
1666-P: ScRNAseq–Based Analysis of the Stromal Immune Cell Composition in Murine Preweaning and Adult White Adipose Tissues
Authors: YAN XIAOCHENG , MIOK VIKTORIAN , KARLINA RUTH , BÖTTCHER ANIKA , LUTTER DOMINIK , LICKERT HEIKO , GARCIA CACERES CRISTINA , USSAR SIEGFRIED
Published: June 2023

Early postnatal adipose growth is essential to establish a functional metabolism. Conversely, proportional adipose expansion in adults results in insulin resistance. Despite adipocytes, immune cells play a pivotal role in orchestrating adipose tissue function and expansion. To this end, we performed an in-depth analysis of scRNAseq data of immune cell populations of 2 weeks (pre-weaning) and 8 weeks old (adult) murine subcutaneous and perigonadal adipose depots. Our analysis revealed 15 distinct immune cell clusters. Most immune cell types were found in all depots and ages. Among the detected T-cell populations, we only observed differences in the abundance of naïve T cells, which were predominantly found in in subcutaneous white adipose tissue, irrespective of age. Similar to human, we could define several murine macrophage subtypes. Among them, perivascular macrophages (PVM) were predominantly found in subcutaneous white adipose tissue of 2 weeks old mice. RNA-velocity analysis and pseudotime trajectory construction suggested a developmental trajectory from lipid-associated macrophages (LAM) to PVM, which was further corroborated by analysis of gene expression dynamics resolved along latent time of white adipose tissue macrophages from 2 weeks old mice. Further analysis revealed that the top-ranked 100 genes of LAM were associated with the regulation of endothelial cell migration and proliferation (Nrp1, Grn, Glul, Sash1, Ccl24), blood vessel morphogenesis (Nrp1, Tgfbr2) and the regulation of epithelial cell proliferation.

In summary, we provide a comprehensive overview of the immune cell composition of pre-weaning and adults murine adipose tissues. Moreover, we provide in-depth analysis of T-cell and macrophage populations and their relationship and dependency based on mRNA expression analysis.

Disclosure

X. Yan: None. V. Miok: None. R. Karlina: None. A. Böttcher: None. D. Lutter: None. H. Lickert: Research Support; Eli Lilly and Company. C. Garcia Caceres: None. S. Ussar: Research Support; Novo Nordisk.

Funding

China Scholarship Council (201908370218)
168-LB: A Randomized Crossover Trial Comparing Glucose Control between Different Exercise Types in Adolescents with Type 1 Diabetes Using a Hybrid Closed-Loop System
Authors: SECKOLD ROWEN , MORRISON DALE , RAFFERTY JORDAN , VALKENBORGHS SARAH R. , PALDUS BARBORA , ZAHARIEVA DESSI P. , RIDDELL MICHAEL C. , SMART CARMEL E. , O'NEAL DAVID N. , KING BRUCE R.
Published: June 2023

Background: Hybrid closed loop (HCL) systems may mitigate the glycemic impact of different exercise types in youth with type 1 Diabetes.

Aims: Glucose was compared during and following moderate intensity exercise (MIE), high intensity interval exercise (HIE) and resistance exercise (RE) in adolescents using HCL insulin delivery.

Methods: Adolescents using HCL (Medtronic Minimed 670G) undertook 40 min of MIE, HIE and RE in random order. A temporary glucose target was set 2 hours prior and during exercise with 15g carbohydrate given pre-exercise if glucose level was <126mg/dL. A standardized dinner was eaten 4 hours after exercise. The primary outcome was sensor glucose percentage time in range 70-180mg/dL (TIR) for 12 hours from exercise onset.

Results: Twenty-eight participants (50% female); age [mean (SD)] 16.3(2.1) years; diabetes duration 9.4(4) years; HbA1c 7.3(0.6)% [56(7)mmol/mol], completed the study. In the 12 hrs following exercise onset the TIR for MIE was [median (IQR)] 76(60-92)%, HIE 87(78-96)% and RE 85(69-94)% (MIE v HIE; (p=0.015). Time >180mg/dL (TAR) was greater for MIE v HIE (18[3-25]% v 7[0-15]%; p=0.041). Time <70mg/dL (TBR) did not differ between MIE, HIE and RE [0 (0-7)%; 2(0-9)%; and 2(0-10)% respectively]. After the post-exercise meal (280-715mins), TIR for MIE was lower than HIE [86(76-96) v 67(49-87)%; p=0.003] and TAR was higher for MIE than HIE [5(0-19) v 28(0-41)%; p=0.012]. There was no significant difference between exercise types in TIR, TAR and TBR during exercise (0-40min) and before dinner (40-280mins). There was a total of 8 episodes of hypoglycemia (15mins <54mg/dL) each for MIE and RE and 5 for HIE.

Conclusion: Glucose control in the post meal period following exercise differed between HIE and MIE but not RE. Hypoglycemia occurred across all exercise types.

Disclosure

R. Seckold: None. D. Morrison: None. J. Rafferty: None. S. R. Valkenborghs: None. B. Paldus: None. D. P. Zaharieva: Speaker's Bureau; American Diabetes Association. Research Support; Hemsley Charitable Trust, International Society for Pediatric and Adolescent Diabetes, Insulet Corporation. Advisory Panel; Dexcom, Inc. Speaker's Bureau; Ascensia Diabetes Care, Medtronic. M. C. Riddell: Stock/Shareholder; Supersapiens. Advisory Panel; Zealand Pharma A/S. Speaker's Bureau; Dexcom, Inc. Consultant; Lilly Diabetes. Speaker's Bureau; Novo Nordisk, Sanofi. Stock/Shareholder; Zucara Therapeutics. Advisory Panel; Zucara Therapeutics, Indigo Diabetes. Consultant; Eli Lilly and Company, Jaeb Center for Health Research. C. E. Smart: None. D. N. O'Neal: None. B. R. King: None.

Funding

JDRF (3-SRA-2018-532-M-B)
168-OR: Marked Variability of Starting Insulin Algorithms for Noncritical Illness—A Survey of 32 Academic Hospitals in the United States
Authors: CHIANG HOU-HSIEN , KAHN STEVEN E. , HIRSCH IRL B.
Published: June 2023

Glycemic control immediately after hospitalization is difficult, with blood glucose within 24 hours after admission often above recommended targets. Further, it takes time to adjust and determine individual insulin requirements. Endocrine Society guidelines suggest starting scheduled insulin therapy at 0.2–0.5 units/kg body weight as the total daily dose, but there has been no rigorous study to support this guideline and other institutional algorithms. To understand the variability of current practice, we conducted a survey of starting insulin algorithms for non-critically ill patients among top-ranking hospitals and endocrinology programs in the US.

Among the 32 hospitals surveyed, replies were received from 25. Of these, 19 provided their algorithms, 5 stated they did not have such, and one used a proprietary (commercial) algorithm. The figure details the number of algorithms specifying basal/nutritional insulin regimens versus only correction insulin, approaches to calculating the basal/nutritional dose, and guides to choosing correction algorithms.

In summary, amongst US academic hospitals there is great variability in methods for determining starting insulin dosing on admission for non-critically ill patients. This inconsistency suggests that a clinical trial is required to determine the best approach(es) to estimating initial insulin requirements.

Disclosure

H.Chiang: None. S.E.Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck & Co., Inc., Other Relationship; Novo Nordisk. I.B.Hirsch: Consultant; Abbott Diabetes, Lifecare, Inc., Hagar, Research Support; Beta Bionics, Inc., Insulet Corporation, Dexcom, Inc.

Funding

National Institutes of Health (5T32DK007247-45)
169-LB: Neuropsychological Consultation and Glycemic Control in Pediatric Patients
Authors: GONZALEZ JUANA E. , STEIN CHERYL R. , ILKOWITZ JENIECE , POWELL SARAH , PAT GALLAGHER MARY
Published: June 2023

Executive function underlies the ability to plan, reason, and problem solve - skills essential to diabetes management. Children with T1D are at elevated risk of executive dysfunction. Providing families with supports to acknowledge and address these deficits may improve diabetes outcomes. In a randomized controlled trial, we evaluated whether neuropsychological consultation improved glycemic control.

From July 2019-September 2020 we enrolled patients aged 10-17 years with T1D ≥6 months. Participants were randomized at enrollment to neuropsychological consultation (n=26) or usual care (n=24). Both groups completed executive function and psychosocial assessments at baseline and follow-up plus received diabetes management education. The intervention group also underwent neuropsychological consultation followed by focused feedback. Follow-up for both groups extended for 12 months. We assessed whether neuropsychological consultation was associated with change in HbA1C from baseline to follow-up using age and sex-adjusted linear regression models in intention-to-treat and per protocol analyses.

At baseline, the mean (SD) age of participants was 13.8 (2.4) years with 51% in grades 5-8 and 49% in grades 9-12. Mean (SD) HbA1c was 7.7% (1.7); most participants used CGMs (94%) and insulin pumps (87%). Thirty-five percent of the intervention group met criteria for executive dysfunction. HbA1C was lower at follow-up in the treatment group according to both the intention-to-treat (-1.3%; 95% CI -2.5, -0.1) and per protocol (-1.3%; 95% CI -2.6, 0.1) analyses.

This small randomized controlled trial performed primarily during the COVID-19 pandemic demonstrates that neuropsychological consultation may contribute to improvement in glycemic control among pediatric patients with T1D. Next steps include examining changes in psychosocial outcomes, identifying the most impactful aspects of the consultation, and studying this intervention in a larger patient population.

Disclosure

J. E. Gonzalez: None. C. R. Stein: None. J. Ilkowitz: None. S. Powell: None. M. Gallagher: None.
169-OR: SGLT2 Inhibitor Prescribing Discrepancies in Patients with Diabetes Mellitus Type 2 and Heart Failure at an Academic Medical Center
Authors: HANSEN JESSICA , KULASA KRISTEN , SANTOS CAVAIOLA TRICIA
Published: June 2023

With new indications for SGLT-2 inhibitors (SGLT2i) and efforts to minimize cardiovascular risk, there are concerns about equitable and timely distribution of SGLT2i. Inpatient initiation of SGLT2i is an important opportunity to optimize medical therapy. To better understand current prescribing practices, we compared patients started on a SGLT2i at time of discharge across two hospital sites in the 15 months after inclusion in the inpatient medication formulary. A retrospective chart review from February 2021 to May 2022 was performed of patients admitted to UC San Diego and discharged with a SGLT2i. Diagnosis was determined by ICD-10 code, and/or A1c during admission 6.5%. This cohort was compared to all patients admitted with an ICD-10 diagnosis of heart failure and/or DMT2. We examined patient demographics including age, gender, race/ethnicity, and differences between hospital sites (H1, H2). An SGLT2i was prescribed for 178 patients out of 1160 total admissions for CHF and 52,291 total admissions of individuals with DMT2. Of those who received an SGLT2i, 105 (58%) had diabetes, 151 (85%) had CHF and 82 (46%) had both. Between hospitals, 22% of patients with CHF at H1 v. 5% at H2 received an SGLT2i, and 0.22% of patients with diabetes at H1 and 0.17% at H2. The average age of those prescribed compared to the eligible population was 59 v. 64 for CHF and 60 v. 47 for DMT2. Women were less likely to be prescribed than men in both the heart failure (7% v. 16%) and DMT2 groups (0.1% v. 0.32%). The percentage of recipients (CHF, DMT2) by ethnicity was Asian (6, 0.08%), American Indian 0%, Black (7, 0.23%), Hispanic (19, 0.23%), Pacific Islander (26%, 0.81%), White 10, 0.17%). This analysis of SGLT2i initiation at hospital discharge indicates there are likely key differences in prescribing frequency between gender, ethnicities, and hospital sites. Continued collaboration amongst specialties and attention to implicit bias in prescribing practices is warranted.

Disclosure

J.Hansen: None. K.Kulasa: None. T.Santos cavaiola: Consultant; 9am Health.
1718-P: Association between Sleep Habits and Overweight among Adolescents in Japan
Authors: SHIOZAKI HARUKA , FUJIHARA KAZUYA , TAKEDA YASUNAGA , IKEDA IZUMI , MORIKAWA SAKIKO Y. , TSURUOKA KAHORI , KODERA REMI , MON KHIN LAY , WU SIJIA , FERREIRA EFREM D. , ISHIGURO HAJIME , OGAWA YOHEI , SONE HIROHITO
Published: June 2023

Insufficient sleep and late bedtime are associated with deteriorating glucose tolerance status, diabetes and overweight. Although lifestyle in adulthood is influenced by childhood and adolescent experiences, little is known about the association between sleep habits and glucose status in children. Thus, we investigated the association between sleep habits and overweight in 1050 Japanese adolescents (497 boys) considering the effects of diet, physical activity and screen time. Overweight was defined by the International Obesity Task Force. Sleep duration and wake/sleep time were based on questionnaires, and their association with overweight was analyzed by logistic regression. On weekends, girls with overweight had short sleep duration. In a univariate analysis of participants divided into 4 groups, the relationship between sleep habits and overweight did not present a U- or J-shaped curve. We conducted a multivariate logistic regression analysis based on the median bedtime of 23:00 and sleep duration of 9 h. In boys, there was no association between sleep habits and overweight. The odds ratios for overweight in girls with long sleep duration were high regardless of bedtime (early bedtime: odds ratio 3.42 [95% CI 1.15-10.17]; late bedtime 3.08 [1.14-8.33]). Gender differences exist in the association between sleep habits and overweight. In Japanese adolescent girls, sufficient sleep duration on weekends may be necessary.

Disclosure

H.Shiozaki: None. E.D.Ferreira: None. H.Ishiguro: None. Y.Ogawa: None. H.Sone: Research Support; Novo Nordisk, Astellas Pharma Inc., Kyowa Kirin Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Ono Pharmaceutical Co., Ltd., Eisai Co., Ltd., Takeda Pharmaceutical Co., Ltd. K.Fujihara: None. Y.Takeda: None. I.Ikeda: None. S.Y.Morikawa: None. K.Tsuruoka: None. R.Kodera: None. L.Khin: None. S.Wu: None.
172-LB: Traditional Risk Factors for Elevated A1C Persist among Youth with T1D Using CGM—Results from the 4T Study 1
Authors: KIM JAEHYUN , ZAHARIEVA DESSI P. , BISHOP FRANZISKA K. , SCHEINKER DAVID , JOHARI RAMESH , DESAI MANISHA , HOOD KOREY K. , MAAHS DAVID M. , ADDALA ANANTA
Published: June 2023

Continuous glucose monitoring (CGM) use soon after T1D diagnosis in the 4T Study was associated with improved glycemic outcomes. We evaluated participant factors associated with elevated versus in target A1c for youth in the 4T Study.

All youth from the 4T Study 1 (n=133) were evaluated. In this analysis, the 110 youth who had a final A1c between 6-13 months were included in a complete case analysis. These 110 youth were comparable to the 133 4T Study 1 youth by race/ethnicity, insurance, preferred language, and age. Group differences by non-ordered A1c categories were evaluated for categorical (race/ethnicity, insurance, gender, and language) and continuous (age and time from CGM start) variables via chi-square and ANOVA, respectively.

A majority of youth in the 4T Study 1 met glycemic targets (65% with A1c ≤7% between 6-13 months post-diagnosis). Age, race/ethnicity, and insurance status were significantly associated with A1c categories (p=0.02 for all; Table). Higher A1c categories were more likely to include Hispanic youth and youth with public insurance.

In the 4T Study 1, Hispanic youth and youth with public insurance had higher A1c categories despite similar CGM access and training. These findings suggest the need to address additional drivers of disparities in addition to CGM access. Approaches focused on health equity are required to improve glycemic outcomes in all youth newly diagnosed with T1D.

Disclosure

J. Kim: None. D. P. Zaharieva: Advisory Panel; Dexcom, Inc., Research Support; Hemsley Charitable Trust, International Society for Pediatric and Adolescent Diabetes, Insulet Corporation, Speaker's Bureau; American Diabetes Association, Ascensia Diabetes Care, Medtronic. F. K. Bishop: None. D. Scheinker: None. R. Johari: None. M. Desai: None. K. K. Hood: Consultant; Cecelia Health. D. M. Maahs: Advisory Panel; Medtronic, LifeScan Diabetes Institute, MannKind Corporation, Consultant; Abbott, Research Support; Dexcom, Inc. A. Addala: None.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (K23DK13134201, R18DK122422)
1720-P: Metabolic Response to Bariatric Surgery of Novel High-Risk Prediabetes Subphenotypes
Authors: SZCZERBINSKI LUKASZ , GOSCIK JOANNA , KWEDLO WOJCIECH , WOJCIECHOWSKA GLADYS E.P. , CITKO ANNA , KONOPKA PAULINA , PASZKO ADAM , TAYLOR MARK , HADY HADY R. , KRETOWSKI ADAM J.
Published: June 2023

A novel pathophysiology-based method to subphenotype individuals at elevated risk for type 2 diabetes has recently identified 6 subgroups, 2 of which constitute obese, high-risk subpopulations with different glycemic, renal, cardiovascular and all-cause mortality risk profiles. The aim of our study was to evaluate differences in response to weight-loss surgery between these clusters, termed clusters 5 & 6 by Wagner et al. We performed this clustering on the Bialystok Bariatric Surgery Study Cohort consisting of nondiabetic patients with at least one year of follow-up data who underwent sleeve gastrectomy surgery. We used generalized linear models to test for differences between clusters in baseline parameters and changes after one year. At baseline, 48 (47%) patients were classified as cluster 5 and 54 (53%) as cluster 6. Before the surgery, cluster 5 subjects had significantly (q-value<0.05) higher levels of fasting and 2-hour glucose and insulin, HbA1c, triglycerides and visceral fat mass, and lower levels of HDL-cholesterol and Matsuda Index. There were no significant differences in age, weight, BMI or body composition. Further, we found that cluster 5 presented a greater reduction in fasting and 2-h glucose, but lesser in hip and waist circumferences, compared to cluster 6. However, after adjusting for baseline values of these parameters, only change in hip and waist circumferences remained significant. We did not find any differences in body composition or lipid parameter responses between clusters. Our study confirms that cluster 5 shows more severe glycemic and lipid deterioration compared to cluster 6 despite their shared obese phenotypes. However, bariatric surgery seems to be equally beneficial in both clusters, except for small differences in hip and waist circumference response.

Disclosure

L. Szczerbinski: None. J. Goscik: None. W. Kwedlo: None. G.E.P. Wojciechowska: None. A. Citko: None. P. Konopka: None. A. Paszko: None. M. Taylor: None. H.R. Hady: None. A.J. Kretowski: None.
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<0.001) and when they were inhibited (n=6, p<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 >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 >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<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<0.0001). The wave origin was more regionally dependent than the β-cell hubs (P<0.0001). We also found that network analysis is similar (P=0.04) and phase analysis is different between 2D and 3D (P<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<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.)
1771-P: STIM1 Maintains Beta-Cell Identity and Function
Authors: KONO TATSUYOSHI , SOHN PAUL , MCLAUGHLIN MADELINE , WU WENTING , POSTIC SANDRA , KRISHNAN PREETHI , LEE CHIH-CHUN , SLAK RUPNIK MARJAN , EVANS-MOLINA CARMELLA , SLAK RUPNIK MARJAN , KRISHNAN PREETHI
Published: June 2023

Impairments in endoplasmic reticulum (ER) Ca2+ homeostasis have been linked to β cell dysfunction and diabetes development; however, the pathways mediating these effects are not fully understood. Store-operated Ca2+ entry (SOCE) replenishes ER Ca2+ levels through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). We found that female, but not male, mice with β cell-specific STIM1 deletion (STIM1Δβ) had reduced β cell mass, increased α cell mass, and reduced expression of markers of β cell identity. To determine the mechanisms underlying this sexually dimorphic phenotype, RNA sequencing, immunofluorescent staining, and Ca2+ imaging experiments were performed using islets from control and STIM1Δβ mice and WT and STIM1 KO INS-1 cells. RNA sequencing of islets from STIM1Δβ mice identified 55 pathways that were significantly modulated (P<0.05), including “estrogen receptor signaling”, “mitochondrial dysfunction”, and “endocytosis”, and “estrogen receptor signaling”. Consistent with a role for SOCE in mitochondrial function, STIM1 KO cells decreased mitochondrial Ca2+ levels. Furthermore, we used the novel fluorescent time construct, syncollin-dsRedE5TIMER, to demonstrate an abnormal accumulation of aged insulin granules in STIM1 KO cells. Lastly, to test the relationship between STIM1 expression and glucose regulation in humans, we queried a publicly available NCBI GEO dataset derived from organ donors with and without type 2 diabetes (T2D). In this dataset, islet STIM1 expression was negatively correlated with HbA1c levels in female organ donors (n=35, r=-0.38, p=0.035), while there was no correlation in male donors. Together, these data highlight a novel role for STIM1 in the maintenance of mitochondrial function and insulin granule turnover. These findings confirm the importance of Ca2+ regulation within the β cell and highlight the significant role of sex as a biological variable in diabetes pathogenesis.

Disclosure

T.Kono: None. M.Slak rupnik: None. P.Krishnan: None. P.Sohn: None. M.Mclaughlin: None. W.Wu: None. S.Postic: None. P.Krishnan: None. C.Lee: None. M.Slak rupnik: 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

National Institutes of Health (R01DK093954, R01DK127236, U01DK127786, R01DK127308, UC4DK104166); U.S. Department of Veterans Affairs (I01BX001733)
1775-P: Liraglutide Corrects Impaired Insulin Secretion in 14-3-3-ζ Overexpressing Mice
Authors: SHISHANI RAHAF , TUCKER MADELYNN , MORENO ROSALINDA , BUSTAMANTE JESSICA M. , LIM GARETH E. , CUMMINGS BETHANY
Published: June 2023

Crosstalk between α-cells and β-cells is an important regulator of healthy islet function. α-cells display heterogeneity in their hormone profile. α-cells typically produce glucagon but, under certain conditions, can produce glucagon-like peptide-1 (GLP-1). However, the mechanisms regulating α-cell GLP-1 production are poorly understood. We found that increased β-cell GLP-1R signaling via the GLP-1 analog, liraglutide, activates α-cell GLP-1 production through a paracrine signaling pathway. We recently identified the protein 14-3-3-ζ as a key downstream mediator of this pathway. Specifically, liraglutide downregulates β-cell 14-3-3-ζ expression, which activates α-cell GLP-1 production. Consistent with this, we found that β-cell 14-3-3-ζ ablation enhances glucose-stimulated insulin secretion (GSIS). Furthermore, mice with 14-3-3-ζ overexpression (OE) have impaired GSIS. Therefore, we tested the hypothesis that liraglutide can correct the effect of 14-3-3-ζ OE to impair GSIS. Male and female wild-type (WT) or 14-3-3-ζ OE mice maintained on high-fat diet underwent an oral glucose tolerance test (OGTT) after 2 weeks of liraglutide (200 μg/kg sc twice daily) or saline treatment. Liraglutide improved glucose tolerance in WT mice and tended to improve glucose tolerance in OE mice. Saline-treated 14-3-3-ζ OE mice exhibited lower GSIS compared with WT (P<0.05). Liraglutide treatment of 14-3-3-ζ OE mice normalized GSIS to match that of liraglutide-treated WT mice (Insulin secretion index [Insulin AUC/Glucose AUC] *10-9: Saline WT = 38 ± 4, Lira WT = 37 ± 5, Saline OE = 25 ± 4, Lira OE = 39 ± 7; n=7-8 per group, P<0.05 Saline WT vs. Saline OE, P=0.06 Saline OE vs. Lira OE). Overall, these data suggest that liraglutide can rescue deficits in GSIS induced by 14-3-3-ζ OE.

Disclosure

R.Shishani: None. M.Tucker: None. R.Moreno: None. J.M.Bustamante: None. G.E.Lim: None. B.Cummings: None.

Funding

Hartwell Foundation; National Institute of Diabetes and Digestive and Kidney Diseases (R56DK124853)