bluuebunny/crossref_metadata_2025 huggingface dataset:
2032-P: Adipose Tissue Inflammation after Weight Loss
Authors: ESPINOSA DE YCAZA ANA E. , SØNDERGAARD ESBEN , MORGAN-BATHKE MARIA , DELIVANIS DANAE A. , CARRANZA LEON BARBARA G. , LYTLE KELLI , JENSEN MICHAEL D.
Published: June 2020

Background: Studies of changes in adipose tissue (AT) inflammation after weight loss have conflicting results. Our aim was to evaluate the changes in AT macrophages, senescence and AT insulin resistance after weight loss.

Methods: Twenty-five participants (18 women) with obesity underwent a lifestyle intervention weight loss program and abdominal-femoral AT biopsies. AT insulin resistance was estimated calculating the insulin concentration that suppresses lipolysis by 50% (IC50) during a hyperinsulinemic-euglycemic clamp. Immunohistochemistry was used to estimate total adipose tissue macrophage (ATM) content (CD68+), pro-inflammatory ATM (CD14+), anti-inflammatory ATM (CD206+). Senescent cells in AT were identified by senescence associated β-galactosidase staining. All parameters were assessed before and after weight loss.

Results: Median BMI was 34 kg/m2 (IQR, 32-35). A median percent weight loss of 10.2% (IQR, 6.5-12.1) resulted in a mean IC50 reduction of -6.2 ± 13 µIU/mL, p=0.03. There was no significant change in ATM content in the abdominal or femoral fat depots after weight loss, except for an increase in CD68 ATMs (mean change 4.5 ± 7.1 ATM/100 adipocytes, P=0.02) in the femoral depot. The proportion of senescent pre-adipocytes in abdominal and femoral depots remained unchanged after weight loss. In the abdominal depot, there was no relationship between IC 50 and CD68 or CD206 ATMs after weight loss, R2= 0.11, P=0.15 and R2= 0.08, P =0.22, for CD68 and CD206 ATMs respectively. There was a weak negative relationship between CD14 ATMs and IC 50 (R2=0.2, P =0.04). In the femoral depot, there was a weak negative relationship between IC 50 and CD68 ATMs (R2=0.23, P=0.04), and no relationship between IC 50 and CD14 ATMs (R2= 0.2, P=0.06) or CD206 ATMs after weight loss (R2= 0.06, P=0.33).

Conclusion: Moderate weight loss is not associated with reduction in ATM content or senescent pre-adipocytes, despite an improvement in AT insulin resistance. Our results suggest that recruitment of some populations of ATMs increases after weight loss.

Disclosure

A.E. Espinosa De Ycaza: None. E. Søndergaard: None. M. Morgan-Bathke: None. D.A. Delivanis: None. B.G. Carranza Leon: None. K. Lytle: None. M.D. Jensen: None.
2037-P: Islet ß-Cell Dedifferentiation Is Involved in Progression of Type 2 Diabetes
Authors: AMO-SHIINOKI KIKUKO , TANABE KATSUYA , BOUCHI RYOTARO , NISHIMURA WATARU , OGAWA YOSHIHIRO , TANIZAWA YUKIO
Published: June 2020

T2D is associated with deficits of β-cell function and number. But it remains unclear how these processes interact during disease progression. It has been described that islet cell plasticity, i.e., β-cell dedifferentiation and conversion into other islet cells, is implicated to β-cell failure in murine diabetes of insulin resistance. We aimed to examine it in human T2D and to elucidate its involvement in disease progression.

Methods: We evaluated pancreata obtained during pancreatectomy from Japanese subjects who had been over-weighed during adulthood and had no diabetic family history and analyzed patient’s clinical information. The diabetics in the early- and the advanced-stage that were defined by combining clinical information on disease duration, microvascular complications and insulin replacement were evaluated. The islet area was determined by measuring ChgA- positive area. We detected islet major 4 hormones-negative/ChgA-positive cells as dedifferentiated cells.

Results: There was a diversity in relative β-, α- and islet area to pancreatic area in the T2D subjects, suggesting metabolic heterogeneity associated with various capabilities of islet compensation in response to insulin resistance. We found that a decreased relative β-cell number and an increased relative α-cell number to entire ChgA-positive cell number in individual T2D islets. But there was no further increase in α-cells along disease progression despite progressive β-cell loss, and only little INS/GCG double positive cells. Instead, a strikingly elevated number of the dedifferentiated cells in T2D islets compared to control subjects and its significant increase with disease progression was observed. Importantly, there was a significant inverse correlation between increasing dedifferentiated cells and declining C-peptide index among the groups. These finding indicates that islet cells exert plasticity during diabetes progression and that β-cell dedifferentiation is a pathologic process leading β-cell failure.

Disclosure

K. Amo-Shiinoki: None. K. Tanabe: None. R. Bouchi: None. W. Nishimura: None. Y. Ogawa: Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker’s Bureau; Self; Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation. Y. Tanizawa: None.

Funding

Japan Society for the Promotion of Science (20390093, 25461357, 16K09752, 15K21198); Banyu International Life Science Foundation; Novo Nordisk; Japan Association for Diabetes Education and Care; Merck Sharp & Dohme Corp; Public Interest Incorporated Foundation
2039-P: Plasmin-Mediated Cleavage of Human Islet Amyloid Polypeptide (hIAPP) Protects ß-Cells from Amyloid-Induced Toxicity
Authors: ESSER NATHALIE , HOGAN MEGHAN F. , TEMPLIN ANDREW T. , CASTILLO JOSEPH J. , RALEIGH DANIEL , EDGAR JOHN S. , ZRAIKA SAKENEH , HULL REBECCA L. , KAHN STEVEN E.
Published: June 2020

Islet amyloid deposition and reduced ß-cell mass are pathologic hallmarks of type 2 diabetes. Amyloid deposits contain the 37 amino acid hIAPP, aggregation of which is toxic to ß cells. The 20-29 amino acid region of the peptide is known to confer its amyloidogenic potential. We previously reported that plasmin, a fibrinolytic protease, cleaves hIAPP thereby inhibiting fibril formation. We now sought to determine whether hIAPP fragments arising from plasmin-mediated cleavage retain the potential to aggregate and induce cytotoxicity. The ß-cell line INS-1 was treated for 24 h with full-length hIAPP (20 µM) alone or in the presence of plasmin (0.4 µM); plasmin alone or buffer were used as controls. Exposure to hIAPP alone decreased cell viability (53±1.4% of buffer, p<0.05, n=5). Addition of plasmin to hIAPP restored cell viability (90±4.3% of buffer, p=0.3 vs. buffer and p<0.05 vs. hIAPP alone, n=5), while treatment with plasmin alone had no effect (102±2.9% of buffer, p=0.7, n=5). LC/MS analysis of hIAPP cleavage products showed that plasmin induced a rapid decrease in the abundance of full-length hIAPP and appearance of the hIAPP 1-11 and 12-37 fragments. Since the hIAPP 12-37 contains the amyloidogenic region, we compared the amyloidogenic and cytotoxic properties of this fragment to full-length hIAPP. In a thioflavin T assay, hIAPP 12-37 was less amyloidogenic than full-length hIAPP. As expected, treatment of INS-1 cells for 24 h with full-length hIAPP (0-60 µM) reduced cell viability in a dose-dependent manner (67±1.3%, 30±0.4% and 16±0.7% of buffer, at 20, 40 and 60 µM respectively; p<0.05 vs. buffer for all, n=2), while hIAPP 12-37 was not toxic at any dose tested (90±3.2%, 90±3.3% and 108±3.5% of buffer, at 20, 40 and 60 µM respectively; n=2).

In summary, plasmin protects ß cells from hIAPP-induced toxicity by cleaving hIAPP between amino acids 11 and 12. Thus, increasing islet plasmin activity might be a strategy to limit ß-cell loss in type 2 diabetes.

Disclosure

N. Esser: None. M.F. Hogan: None. A.T. Templin: None. J.J. Castillo: None. D. Raleigh: None. J.S. Edgar: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. R.L. Hull: None. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck & Co., Inc., Novo Nordisk A/S, Pfizer Inc.

Funding

U.S. Department of Veterans Affairs (I01BX001060)
2048-P: DNA Methylation Changes Reveal Distal Regulatory Regions in Islets of Intrauterine Growth Restricted Rats
Authors: LIEN YU-CHIN , LU XUEQING M. , SIMMONS REBECCA A.
Published: June 2020

Intrauterine growth retardation (IUGR) is associated with development of type 2 diabetes in adulthood. Using a rat model of IUGR induced by bilateral uterine artery ligation, we performed RNA-Seq and HELP-TagSeq, a restriction enzyme-based genome-wide approach, to assess the association of DNA methylation changes in islets at 2 weeks of age, prior to the onset of diabetes. We identified over 2,000 transcripts differentially expressed in IUGR (q<0.05 vs. control) and 512 differentially methylated regions (DMRs->3 contiguous CpGs within 500 bp) mapped to 481 genes (q <0.05). 23 and 489 loci showed significantly increased and decreased DNA methylation in IUGR islets, respectively (q <0.05). Integrating methylome and transcriptome datasets showed alterations in multiple genes and canonical pathways that are critical for normal islet function. DMRs were enriched with transcription factor binding motifs, such as Elk1, Etv1, Foxa1, Foxa2, Pax7, Stat3, Hnf1, and AR. These transcription factors were also identified as top upstream regulators by Ingenuity Pathway Analysis of islet transcriptomes. To determine if identified DMRs represented regulatory regions for non-proximal genes, we performed in silico analysis of 3D chromosomal interactions between conserved DMRs and interacting regions of the genome using published human pancreas and islet Hi-C datasets. Hi-C analysis identified interactions between 14 highly conserved DMRs and 35 genes (key islet genes) with expression changes (q < 0.05). Changes in gene expression for many of these persisted until adulthood. In adulthood, there were far more HiC interaction genes associated with altered expression and most of these were key islet genes. Our findings have identified novel DMRs with potential distal regulatory functions, and suggest alterations of DNA methylation in key transcription factor binding motifs and genes in early life that may contribute to gene dysregulation and an abnormal islet phenotype in IUGR rats.

Disclosure

Y. Lien: None. X.M. Lu: None. R.A. Simmons: None.

Funding

National Institutes of Health (DK114054)
2053-P: Integrated Blood Flow between Endocrine and Exocrine Pancreas Is Conserved among Multiple Species
Authors: BUTTERFIELD JOHN K. , HARA MANAMI
Published: June 2020

Pancreatic islets compose the endocrine pancreas and are considered distinct from the exocrine pancreas in terms of both function and regulation of blood flow. Indeed, enzymatically isolated islets directly respond to glucose and other secretagogues in vitro. The islet has been regarded as an enclosed micro-organ, in which blood flows uni-directionally from an afferent arteriole to an efferent venule. This blood circulation pattern has been termed as “the insulo-acinar portal system.” Historically, the endocrine and exocrine pancreas have been studied separately by different fields of investigators. Using intravital analysis of pancreas blood flow in mice, we have recently reported that islet capillaries are continuously integrated with those in the exocrine pancreas, which makes the islet circulation open, not self-contained. Furthermore, by tracking fluorescently-labeled red blood cells revealed bi-directional blood flow between the endocrine and exocrine pancreas. In the present study, in order to verify whether this integrated vascular network is conserved among different species, we carried out three-dimensional morphological analysis of pancreatic capillary networks using thick pancreatic tissue slices (600-800 µm) in ferrets, rabbits, pigs, dogs and monkeys, besides mice and humans. We have confirmed that the integrated blood flow between endocrine and exocrine pancreas is conserved among multiple species that we studied. Here we propose that the open circulation model physically links both endocrine and exocrine parts of the pancreas as a single organ through the integrated vascular network. This new paradigm may provide revived or contrasting insights into the findings of past studies, as well as a novel approach for prospective research projects.

Disclosure

J.K. Butterfield: None. M. Hara: None.

Funding

National Institutes of Health (DK117192, DK020595)
2055-P: S100A8 Suppresses ß-Cell Proliferation in Diet-Induced Obese Mice
Authors: MIYASHITA DAISUKE , SHIRAKAWA JUN , TOGASHI YU , OKUYAMA TOMOKO , KYOHARA MAYU , TERAUCHI YASUO
Published: June 2020

S100 calcium-binding protein A8 (S100A8), a member of damage-associated molecular pattern molecules (DAMPs), reportedly contribute to the islet inflammation by mediating interaction between β-cells and macrophages (J Biol Chem., 2018). However, the role of S100A8 in the regulation of β-cell function and mass remain unclear. To clarify the role of S100A8 in β-cell functionality, we generated β-cell-specific S100A8 knockout mice (KO) by crossing S100A8-floxed mice (control) and Ins1-cre mice. In the presence of high glucose (200 mg/dL) and palmitic acid, the expression of S100A8 in KO islets significantly reduced (0.025-fold, p<0.05) compared to control islets. Islets and peritoneal macrophage (Mφ) were isolated from control and KO, and cultured under the conditions of glucose concentration, presence or absence of palmitic acid, single culture or co-culture. When control Mφ co-cultured with KO islets in the presence of high glucose and palmitic acid, the expression of TNFα, IL-6, or CCL2 in Mφ was significantly reduced (0.17-fold, p<0.05; 0.69-fold, p<0.05; 0.22-fold, p<0.05) compared to that when co-cultured with control islets. KO fed normal chow showed comparable body weight, insulin sensitivity, and glucose tolerance with control. In contrast, high-fat diet-fed KO demonstrated improved glucose tolerance, increased β-cell mass (2.65-fold, p<0.05), and enhanced β-cell proliferation (1.91-fold, p<0.05) compared to control. Thus, β-cell-derived S100A8 mediates the islet inflammation induced by glucolipotoxicity through the interaction with infiltrated Mφ. Furthermore, S100A8 seemed to negatively regulate β cell proliferation in diet-induced obese mice, and could be a novel therapeutic target to reverse β-cell loss in subjects with diabetes.

Disclosure

D. Miyashita: None. J. Shirakawa: None. Y. Togashi: None. T. Okuyama: None. M. Kyohara: None. Y. Terauchi: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Novo Nordisk A/S, Sanofi. Consultant; Self; Astellas Pharma Inc. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Merck Sharp & Dohme Corp., Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Kowa Company, Ltd., Medscape, Medtronic, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation.
2059-P: Gene Repression during Maturation of Pancreatic ß-Cells Mirrors Those Upregulated in Islets of Diabetic db/db Mice
Authors: NISHIMURA WATARU , TUMURKHUU MUNKHTUYA , NODA YASUKO , YASUDA KAZUKI
Published: June 2020

Pancreatic β-cells are specialized to properly regulate glucose metabolism. Loss of individual transcription factors that are critical for endocrine differentiation embryonically causes a deep loss of cell identity and upregulation of genes normally repressed in mature β-cells. We hypothesized that the factors important for maintenance of mature β-cell homeostasis are those that are lost in the identity loss or dedifferentiation. To identify genes critical for acquisition and loss of β-cell identity, we examined islets and β-cells by comprehensive gene expression analyses. Islets isolated from 8 wk (mature) and 4 wk (immature) old C57BL/6J mice were analyzed by microarray, and FACS-sorted mature β-cells (Mafa+7AAD-) and other islet cells including Mafa negative β-cells (Mafa-7AAD-) purified from Mafa reporter mice by RNAseq. We found that the maturation process involved genome-wide downregulated gene expression. Mature islets and Mafa+ β-cells had suppression of many genes including “β-cell disallowed genes” such as Igfbp4 and Nfib, but little to noupregulation of genes critical for β-cell function such as Ins1, Slc2a2, Ucn3 and Sytl4 from 4 wk to 8 wk. In contrast, most of the suppressed genes were significantly upregulated (by RNAseq) in islets from 21 wk old diabetic BKS-db/db mice compared with BKS-db/+ (misty). Comprehensive methylation screening (MeDIP-seq) of DNA from islets of 4 and 8 wk old C57BL/6J mice specifically identified highly methylated regulatory regions in some “repressed” genes in 8 wk old islets.

In conclusion, the gain of identity during maturation phase of β-cells mirrors that of the dedifferentiation during diabetes.

Disclosure

W. Nishimura: None. M. Tumurkhuu: None. Y. Noda: None. K. Yasuda: None.

Funding

Japan Society for the Promotion of Science (JP16H01647, JP26461351, JP17K09843); International University of Health and Welfare; Japan Diabetes Foundation; Novartis Pharmaceuticals
206-LB: Survival after Islet Transplantation in Subjects with Type 1 Diabetes: 20-Year Follow-Up
Authors: RICORDI CAMILLO , LEMOS JOANA , BAIDAL DAVID , ALVAREZ ANA M. , ALEJANDRO RODOLFO
Published: June 2020

Type 1 diabetes (T1D) is associated with premature mortality due to T1D-related acute and chronic complications, including severe hypoglycemic events (SHE), cardiovascular (CVD) and renal disease. Islet transplantation (ITx) has been shown to provide near-normal glycemic control and protection from SHEs in a select group of patients with T1D. We retrospectively evaluated the survival of ITx recipients (n= 49) followed up to 20 years. Subjects were transplanted and followed from 2000-2020 at our institution. Study cohort included 29 females (59.2%). Median age at T1D diagnosis was 13.2 (6.7 - 20.0) years while mean age and median duration of T1D at ITx was 42.8 ± 8.3 years and 29.5 (17.2 - 38.2) years, respectively. Two subjects (4.08%) died during follow-up. Duration of graft function was 4.4 (1.3 - 12.2) years. Cumulative survival post-ITx was 100%, 96% and 85%, at 10, 15 and 20 years follow-up, respectively. Results suggest that despite chronic immunosuppression, near-normal metabolic control achieved following ITx can reduce the increased mortality risk associated with T1D. Possible factors accounting for these results include lower cumulative hyperglycemic exposure and prevention of SHE. Subjects selected for ITx represent a highly vulnerable group at risk of recurrent SHE and death. ITx may prevent mortality by preventing SHE and reducing CVD risk factors in selected patients with T1D.

Disclosure

C. Ricordi: None. J. Lemos: None. D. Baidal: None. A.M. Alvarez: None. R. Alejandro: None.

Funding

Diabetes Research Institute Foundation; State of Florida; JDRF; The Leona M. and Harry B. Helmsley Charitable Trust; University of Miami Clinical and Translational Science Institute
2063-P: Quantitative Analysis of Human and Mouse Islet Tissue Structure
Authors: CREASEY OLIVIA A. , SNEDDON JULIE B. , GARTNER ZEV J.
Published: June 2020

The pancreatic islet is a complex tissue consisting of multiple endocrine cell types, capillaries, and a peri-islet basement membrane. The 3D organization of cells, blood vessels, and basement membrane (BM), enhances islet function by (1) supporting beta cell electrochemical coupling and synchronization through gap junctions between adjacent cells and (2) providing access to nutrients and signals for beta cell survival and function through contact with capillaries and BM. Though a quantitative understanding of islet structure is lacking, there is strong evidence linking islet structural changes to diabetes progression. Studies of these changes in islet structure will enhance understanding of diabetes-related losses in islet function and lead to the development of new therapeutics for diabetic and prediabetic patients. Here, we have quantified healthy adult islet tissue structure through a single-cell analysis of the cell-cell and cell-BM interactions at individual endocrine cell plasma membranes. We have used this single-cell data to identify endocrine cell subtypes, as defined by cell morphology and structural niche. We have also identified spatially-defined clusters of beta cells through a graph analysis of cell-cell connectivity. We report a baseline quantification of structure for healthy adult human and mouse islets. These data both provide an answer to the ongoing debate regarding the differences in structure between human and mouse islets and set the stage for future quantification of islet structure in diabetes.

Disclosure

O.A. Creasey: None. J.B. Sneddon: Advisory Panel; Self; Encellin. Advisory Panel; Spouse/Partner; Encellin. Z.J. Gartner: None.

Funding

National Science Foundation (1650113, DBI-1548297); National Institutes of Health (P30DK063720); Chan-Zuckerberg Biohub; Bakar Foundation
2064-P: Pharmacological Modulation of Prostaglandin E2 Receptor Activity Enhances ß-Cell Mass and Maintains ß-Cell Identity in a Mouse Model of Type 2 Diabetes
Authors: CHRISTENSEN ASHLEY A. , ANDREI SPENCER R. , DUNN JENNIFER C. , RICCIARDI VALERIE F. , PACE WILLIAM A. , BREYER RICHARD M. , GANNON MAUREEN
Published: June 2020

Type 2 diabetes (T2D) is a chronic condition characterized by hyperglycemia and inflammation. This condition arises in response to a failure to increase β-cell mass in the face of insulin resistance. Prostaglandin E2 (PGE2), which signals through four receptors (EP1-4), is a mediator of inflammation that has been shown to be upregulated in diabetes. Previous data from our lab has shown that EP3 receptor blockade promotes β-cell proliferation and survival in isolated mouse and human islets ex vivo. In the current study, six week-old db/db mice were treated with the EP3 antagonist DG-041 (20 mg/kg) daily for two weeks. Pancreata were excised and analyzed using immunohistochemistry. We found that treatment with DG-041 leads to improved β-cell mass (2.8 ± 1.5 mg) compared to mice treated with vehicle alone (1.0 ± 0.7 mg). Additionally, we also observed increased β-cell proliferation in DG-041-treated mice (2.2 ± 0.6% Ki67+/Ins+ β-cells) compared to vehicle-treated mice (1.6 ± 0.3%). Further analysis demonstrated that the β- to α-cell ratio was altered in mice treated with DG-041 (2.13:1) compared to vehicle-treated mice (1.78:1). We also immunolabeled for MafA and Nkx6.1, as loss of these transcription factors has been correlated with β-cell dysfunction and diabetes progression in db/db mice and humans. DG-041-treated mice had a higher percentage of MafA positive β-cells (50.3 ± 3.8%) compared to vehicle-treated mice (40.7 ± 3.8%) while there were similar numbers of Nkx6.1 positive β-cells in DG-041-treated and vehicle-treated groups (81.3 ± 4.7% and 81 ± 5.1%, respectively). The current results provide evidence that EP3 receptor blockade promotes β-cell mass expansion and proliferation while preserving homeostatic endocrine islet composition.

Disclosure

A.A. Christensen: None. S.R. Andrei: None. J.C. Dunn: None. V.F. Ricciardi: None. W.A. Pace: None. R.M. Breyer: None. M. Gannon: None.

Funding

U.S. Department of Veterans Affairs (1I01BX003744-01); National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK120626-01A1)
2070-P: KCNJ11 (Kir6.2) Gain-of-Function Mutation in Zebrafish Leads to Transient Neonatal Diabetes
Authors: IKLE JENNIFER , SINGAREDDY SOMA S. , TRYON ROBERT C. , YORK NATHANIEL , YIN LINLIN , CHEN WENBIAO , NICHOLS COLIN
Published: June 2020

The majority of neonatal diabetes (NDM), defined by early onset of hyperglycemia due to impaired β-cell function, is caused by gain-of-function (GOF) mutations in the ATP-sensitive potassium (KATP) channel genes (ABCC8 encoding SUR1 and KCNJ11 encoding Kir6.2). These GOF mutations lead to increased KATP channel activity, decreased β-cell excitability, and impaired insulin secretion. A zebrafish model of NDM can provide a high throughput platform for understanding mechanisms of NDM and testing potential therapeutic approaches. To create such a model, we used CRISPR mutagenesis to introduce the most common human Kir6.2 GOF mutation (R201H) into the equivalent zebrafish locus. Patch-clamp analysis demonstrates decreased ATP sensitivity in β-cells isolated from mutant fish (K1/2 for ATP inhibition = 17.21 ±1.66 mM in WT, 40.38 ± 7.6 mM in heterozygous R201H). Increasing [glucose] causes a robust increase of [Ca]i in WT islets expressing a GCaMP6s fluorescent reporter, but there is no glucose-dependent increase in R201H islets. R201H fish have smaller overall body size, indicative of failure to thrive, and β-cell density is reduced ∼50% in R201H compared to WT. Whole body glucose was measured from larval zebrafish using a fluorometric assay. Starting at 4 weeks of age, blood glucose was directly measured by glucometer from the transected cardiac cavity. While R201H animals are markedly hyperglycemic (BG > 250 mg/dl) at 4 weeks, blood sugar normalizes (BG <120 mg/dl) by ∼12 weeks of age.

In conclusion, R201H mutants demonstrate the expected KATP GOF, loss of glucose-dependence of [Ca]i, and marked hyperglycemia early in life, proving a viable model of the molecular and cellular consequences of NDM. Intriguingly, early hyperglycemia is followed by normalization of blood sugar in older fish. This resolution of hyperglycemia is an unexpected finding, and may provide further insight into physiologic differences between transient and permanent NDM.

Disclosure

J. Ikle: None. S.S. Singareddy: None. R.C. Tryon: None. N. York: None. L. Yin: None. W. Chen: None. C. Nichols: None.

Funding

National Institutes of Health (DK109407); Pediatric Endocrine Society; Endocrine Fellows Foundation
2079-P: Racial Differences in Gut Microbiota, ß-Cell Function, Body Fat Distribution, and Their Relation with Incretins in Subjects with Normal Glucose Tolerance
Authors: NIZIGIYIMANA PAUL , LIU LERONG , XU BOYA , LIU TINGTING , GAO LIN , LIU ZEHAO , LI CHANGJUN , LUO XIANGHAN , LEI MINXIANG
Published: June 2020

Racial differences in the gut microbiota and its influence in development of diabetes have been previously reported. However, whether differences exist between Chinese and Africans, currently no study. We investigated whether differences in body fat may account for microbial communities to expound racial disparities in insulin secretion, sensitivity or resistance, and that GLP-1 may play a role. We measured fasting and stimulated GLP-1, insulin and glucose levels during a 3h OGTT in 27 Han-Chinese university students(HC) and 29 International students from East Africa(EA) healthy subjects with normal glucose tolerance. We also measured the whole body fat and the regions of interest of local body composition, and gut microbiota composition. HC group showed significantly higher triglyceride levels, total bile acids, glucose response at 30min, incremental glucose level at 30min, HOMA-IR at 30 min, body fat distribution and abundance of microbial phyla, but lower hip circumference and β-cell function than EA. Interestingly, HC had lower bacterial taxa and majority of measures of microbial diversity than EA. GLP-1 correlated with metabolic risk factors and microbial communities. The results suggest that body fats are associated with gut microbiota and that GPL-1 enhances their association, which in turn may result in Chinese developing T2DM.

Disclosure

P. Nizigiyimana: None. L. Liu: None. B. Xu: None. T. Liu: None. L. Gao: None. Z. Liu: None. C. Li: None. X. Luo: None. M. Lei: None.
2089-P: Islet GLUT2 Plasticity Improves Glycemic Control in Insulin-Resistant KK Mice but Not T2DM KKAy Mice
Authors: LEPARD KATHY J. , SONDGEROTH KORIE L.
Published: June 2020

Beta cells secrete insulin in response to glucose entry by GLUT transporters. GLUT are reduced in beta cells from T2DM rodents. We hypothesized that glucose entry into islets would be reduced in T2DM mice. Islets were isolated from euglycemic mice (C57/Bl6, ICR), insulin-resistant but euglycemic mice (4 and 8 wk KK), prediabetic mice (4 wk KKAy), and T2DM mice (8 wk KKAy). Islets were exposed to the fluorescent glucose analogue 2-NBDG in presence or absence of high glucose or the GLUT inhibitor phloretin. Islet fluorescence was visualized using confocal microscopy and quantified from 3-D images using FIJI Image J. GLUT2 protein was determined in isolated islets by ELISA. Islet GLUT2 location was determined using IF sections with blinded observers assigning a score from 0 (cytosol) to 10 (cell membrane). *p<0.05. Using islets from euglycemic mice, 2-NBDG entry was competitively reduced 59% by high glucose and 30% by phloretin. Despite a 44% decrease in islet GLUT2 protein in prediabetic mice (KK, 4.0±0.5; KKAy, 2.3±0.3*), 2-NBDG entry into islets was similar (KK: 7.32±0.86; KKAy: 7.25±0.86). More GLUT2 transporters were located in the membrane of islets from KKAy (5.88±2.14*) as compared to KK (4.44±3.06) mice. Phloretin significantly decreased 2-NBDG entry in both KK (6.31±0.61*) and KKAy (6.52±0.69*) mice. By 8 wks of age, islet GLUT2 protein had decreased by 51% in KK mice (1.97±0.50*) with no change in KKAy mice (2.58±0.22), yet 2-NBDG entry was not altered (KK: 7.32±0.63; KKAy: 7.35±0.62). More GLUT2 was located in the membrane of 8 wk KK (7.08±1.68*) as compared to 4 wk KK mice. Membrane GLUT2 was unchanged in KKAy mice. Phloretin increased 2-NBDG entry into islets from both KK (8.05±0.48*) and KKAy (7.95±0.79*) mice. With age, HbA1c did not change in KK mice (4.53-4.97%) but increased in KKAy mice (5.27-6.57%). Plasticity in islet GLUT2 content, location and transport was evident in islets from KK mice but not KKAy mice. Plasticity may contribute to better glycemic control in aging KK mice.

Disclosure

K.J. LePard: None. K.L. Sondgeroth: None.

Funding

Midwestern University (to K.J.L.)
2092-P: Invalidation of GSK3B in Pancreatic Beta Cell as a Therapeutic Target for T2D
Authors: MURRAY SUE , ZHANG JIANGWEI , FERNG ANNIE , PRAKASH THAZHA , LEE RICHARD , ANDERSSON EVA-MARIE , JANZÉN DAVID , KNERR LAURENT , AMMALA CARINA , GUO SHULING
Published: June 2020

Insufficient pancreatic β-cell mass or function is one of the main causes underlying diabetes mellitus. Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed serine/threonine kinase. GSK3 is encoded by two known genes, GSK3 alpha (GSK3A) and GSK3 beta (GSK3B), and small molecule inhibitors targeting both are known to induce micronucleation. Isoform specific small molecule inhibitors targeting GSK3B have demonstrated increased cell proliferation and expansion of islets mass in human and rodents. However, many of the inhibitors target dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) in addition to GSK3B making the data difficult to interpret (Shen et al, Nature comms 2015). Mice with β-cell overexpression of GSK3B have reduced β-cell mass and proliferation through modulation of PDX-1 stability. Recently, Ämmälä et al. demonstrated conjugation of ASOs to glucagon-like-peptide-1 receptor (GLP-1R) ligand can productively deliver ASO cargo to the insulin secreting β-cells. The doses needed for GLP-1-conjugated ASOs to silence target genes in the β-cells are below what is needed to affect target gene expression elsewhere allowing for evaluation of specific inhibition in the β-cells. In lean C57B6 mice a single administration of GLP-1-conjugated GSK3B ASO achieved an ED50=0.016µmol/kg with no change to DYRK1A RNA expression. Here we demonstrate robust, specific GSK3B reduction in the β-cells after GLP-1-conjugated GSK3B ASO treatment in db/db mice. In contrast to the SMI data, specific inhibition of GSK3B in β-cells worsened the health based on reductions in β-cell specific genes, PDX1 and MAFA, and decreased the β-cell mass (2.9 ± 1.3mg GLP-1-Con ASO vs. 1.4 ± 0.6mg GLP-1-GSK3B ASO). In addition, no effect on glucose or insulin was observed suggesting specific inhibition of GSK3B in the β-cell is not enough to preserve the function and increase β-cell survival.

In summary, we have invalidated GSK3B as a therapeutic target for protecting the β-cell against glucotoxicity and T2D.

Disclosure

S. Murray: Employee; Self; Ionis Pharmaceuticals. J. Zhang: None. A. Ferng: None. T. Prakash: None. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E. Andersson: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. D. Janzén: None. L. Knerr: None. C. Ammala: None. S. Guo: Employee; Self; Ionis Pharmaceuticals, Inc.
2095-P: ß-Cell Glucose-Dependent Insulinotropic Polypeptide Receptors Are Necessary for Improved Islet Function following Sleeve Gastrectomy
Authors: DOUROS JONATHAN D. , CAMPBELL JONATHAN , TONG JENNY , D’ALESSIO DAVID
Published: June 2020

Bariatric surgeries improve glucose control in part by enhancing the glucose sensitivity of insulin secretion. In a mouse model of sleeve gastrectomy (SG), prandial secretion of the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are increased and positively correlated with ex vivo insulin secretion. However, several previous studies suggest that GLP-1 action is dispensable for the effects of SG on glucose homeostasis. Therefore, we hypothesized that improved glucose control and islet function after SG require β-cell GIP receptors (GIPR). We tested this hypothesis using an inducible, β-cell specific GIPR knockout mouse line (GIPRβcell-/-). Within two weeks of surgery control mice with SG have reductions of bodyweight and glucose excursion during either a mixed-meal (MMTT) or intraperitoneal glucose tolerance test (IPGTT) compared to sham-operated animals. Islets isolated from control SG mice have enhanced glucose stimulated insulin secretion (GSIS) compared to islets from sham-operated controls in perifusion experiments, similar to our previous reports. While GIPRβcell-/- mice given SG evince improvements in bodyweight and MMTT glucose disposal compared to sham operated GIPRβcell-/- animals, they do not display the postoperative improvement of IP glucose tolerance seen in control mice given SG. Moreover, islets from GIPRβcell-/- SG mice do not have the improved GSIS typical of this surgery. These data indicate that changes in bodyweight and prandial glucose homeostasis after SG are not dependent on GIPR signaling. However, β-cell GIPR are necessary for enhancing intrinsic islet function after SG. These results support enteroendocrine signaling as a mediator for one of the key components of metabolic improvement after bariatric surgery.

Disclosure

J.D. Douros: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. J. Tong: None. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.

Funding

1F32DK115031-01
210-LB: Combined Use of Oral Standard Cookie Meal (OSC) and the Newly Revised Insulin Suppression Test for the Evaluation of Whole Body (WB), Muscle, and Hepatic Glucose Clearance (GC) Under Endogenous or Exogenous Insulin and Its Clinical Usefulness
Authors: HARANO YUTAKA
Published: June 2020

Adequate insulin action (sensitivity: IS) in muscle, liver and WB is the most important metabolic factor in diabetes, especially in S-GLUT-2 user. Oral intake of hypoglycemic agents followed by the standard 75g starch including 15% maltose (OSC, Saraya Co.), which is digested and absorbed into blood glucose at a constant rate for 2 hours, minus urinary glucose loss, divided by the steady state PG at 2h equals the GC in WB under the endogenous insulin secretion. In SGLUT-2 user(HbA1c, 6.8%, BMI, 25.3), this was significantly low(4.2±0.5,SE ml/kg/min, n=6 vs. normal, 8.7,n=10). Average of 1 and 2h insulin was low,10.5 vs. normal,30μU/ml. They are recommended to keep BMI below 23, upper normal range of IS, and use GLP-1 analog or insulin to compensate insufficient IS. This approximation is further evaluated using the revised half dose of glucose and insulin infusion, 3mg, 0.39mU/kg/min, with octreotide 50μg SC for muscle, 100μg for whole body, 30min before the test. Under this exogenous insulin(19.1μU/ml, at 2-4h), 2h GC is confirmed significantly low (2.3±0.1,n=6 vs. control, 3.7±1.0, n=10) reflecting impaired muscle GC in SGLUT-2 user. Additional intake of OSC under continued subcutaneous insulin(1.5 rate over iv), gives the 2h decreased GC (5.84, vs. normal, 9.5,) of WB, and the difference corresponding to hepatic GC, which was significantly low. Better GC was obtained for non-SGLUT groups on oral agents with or without insulin secretagogues. Conclusion Combined use of OSC and simplified IS test enables to evaluate muscle, hepatic and WB glucose utilization under endogenous or exogenous insulin. The method is useful to detect and clarify the various mechanisms of insulin resistance in diabetes, metabolic syndrome, heart failure, Alzheimer disease or fatty liver, etc.

Disclosure

Y. Harano: None.
2102-P: Regulation of Alpha-Cell Function by Beta-Cell Extracellular Vesicles
Authors: KALIVARATHAN JAGAN , SARAVANAN PRATHAB BALAJI , LEVY MARLON F. , KANAK MAZHAR A.
Published: June 2020

People with severe type 1 diabetes (T1D) are known to experience life-threatening hypoglycemic events as a result of alpha cell dysfunction. The phenomenon that causes α cell dysfunction is still unclear. It has been reported that β-cells regulate α cells via paracrine signaling but the underlying mechanism still remains elusive. Extracellular vesicles (EVs) are a class of small vesicles (30-2000nm) that contain a variety of biomolecules. α and β-cells in the islets have been shown to release EVs. It is hypothesized that β-cells transfer signals via EVs in order to regulate α-cell function. Loss of β-cells results in a lack of exosomal communication that causes α-cell dysfunction. Here, we investigated the role of β-cell EVs in modulating α cell function. Mouse TC1-α and βTC6 cell lines were stimulated with low and high glucose media for 24h after exosome depletion. EVs were isolated from conditioned media using differential centrifugation methods. EVs were characterized using TEM, NTA analysis, confocal microscopy, and blotting for surface markers. Alpha cells were challenged with low and high glucose conditions in the presence or absence of EVs from β- cells in order to evaluate the role of EVs in glucagon secretion. ELISA was used to measure glucagon levels in the media, q-PCR was performed on glucagon and other regulatory genes such as MafB and c-Maf. The size of EVs based on NTA analysis was found to be 50 - 200nm. Confocal images reveal that the uptake of BODIPY labeled β-cell EVs after 3h was found localized in the nucleus and cytoplasm of α-cells. Under low glucose stimulation, mRNA and protein expression of glucagon in α-cells was significantly increased in the presence of β-cell EVs (6h: P = 0.0134). Expression of mafB which is a regulator of glucagon transcription correlated with glucagon expression. These results suggest a putative role of β-cell EVs in regulating α-cell function. Our findings will aid in developing new approaches for preventing severe hypoglycemic episodes by reversing α-cell dysfunction in T1D.

Disclosure

J. Kalivarathan: None. P. Saravanan: None. M.F. Levy: None. M.A. Kanak: None.
2110-P: Correlation between Ex Vivo Islet Proteomic Analysis and In Vivo Secretory Function in Humans
Authors: ASSUNTA CEFALO CHIARA MARIA , MEZZA TERESA , CINTI FRANCESCA , MOFFA SIMONA , CAPECE UMBERTO , KULKARNI ROHIT , GIACCARI ANDREA
Published: June 2020

Insulin resistance and β-cell dysfunction are the main actors in the pathogenesis of type 2 diabetes mellitus, causing both quantitative and qualitative loss of insulin secretion and therefore hyperglycemia. The molecular mechanisms underlying progressive β-cell dysfunction, however, are still unknown. In order to correlate changes in the insular proteome to different metabolic conditions in vivo, a mass spectrometry analysis was performed on human islets, extracted by Laser Capture Microdissection, from surgical samples of 14 subjects who underwent duodeno-cephalopancreasectomy, and previously studied through OGTT and mixed meal test. The subjects were classified based on glucose tolerance into normal (n: 7 NGT), intolerant (n: 5 IGT) and diabetic (n: 2 DM2). A first quantitative analysis showed about 150 proteins differently expressed in the 3 groups. Among these, a protein, SEL1L, which regulates the differentiation of pancreatic precursors in the endocrine line was significantly increased in IGT rather than DM2 and NGT subjects and was inversely correlated with β-cell glucose sensitivity (r = -0.81, p = 0.01). The TCPD protein, involved in the folding and transport of neosynthesized proteins, which progressively decreases from NGT to DM2, was inversely correlated with the AUC of glycemia during OGTT (r = 0.5, p = 0.04). Finally, the expression of IQAG1 which was significantly reduced in DM2 compared to the other groups, and implicated in GLP-1 induced insulin production was inversely correlated with insulin resistance evaluated using the HOMA-IR index (r = -0.70, p = 0.004). The results suggest that both insulin resistance and/or β-cell dysfunction in vivo are closely linked to the intrainsular proteome in diabetic subjects.

Disclosure

C. Cefalo: None. T. Mezza: None. F. Cinti: None. S. Moffa: None. U. Capece: None. R. Kulkarni: None. A. Giaccari: Speaker’s Bureau; Self; Amgen, AstraZeneca, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi-Aventis.
2111-P: Role of G-Protein Coupled Receptor 31 (GPR31) in ß-Cell Health and Disease
Authors: HERNANDEZ-PEREZ MARIMAR , HAIDER ISRA , ANDERSON RYAN M. , TERSEY SARAH A. , MIRMIRA RAGHAVENDRA
Published: June 2020

Islet β-cell inflammation, dysfunction, and death are characteristic of diabetes. 12/15-Lipoxygenase (12/15-LOX) produces 12-HETE and is found activated under conditions of insulin resistance and systemic inflammation. 12-HETE acts as an inflammatory mediator that promotes β-cell dysfunction and death through oxidative stress. Since 12-HETE has been shown to bind the G-protein coupled receptor 31 (GPR31), we hypothesized that this binding activates pathways leading to β-cell dysfunction. To test our hypothesis, we screened primary islets and β-cell lines for the presence of GPR31 and studied islets and glucose homeostasis in Gpr31b-/- mice. We observed that GPR31 is present in human and mouse islets, and the mouse β cell-derived cell line (βTC3). Consistent with a role in inflammation, GPR31 expression was increased upon stimulation with a cocktail of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β). We also created a germline knockout of the gene encoding GPR31 (Gpr31b) in mice. Similar to mice with deletion of the gene for 12/15-LOX (Alox15-/-), Gpr31b-/- mice were viable and healthy, with body weight and glucose tolerance indistinguishable from control littermates. To mimic conditions of insulin resistance in vitro, we treated isolated islets Gpr31-/- and control littermates with 10 µM S961, an insulin receptor inhibitor. Treated control islets showed an expected decrease in β-cell identity genes (Ins1/2, Pdx1), genes encoding antioxidant enzymes (Gpx1), and an increase in genes associated with ER stress (Chop, sXbp1). In contrast, Gpr31b-/- islets expression of these genes was resistant to S961. Compared to control littermates, Gpr31-/- mice were resistant to glucose intolerance induced by the β-cell inflammatory toxin streptozotocin. Together these data support the hypothesis that the major product of 12/15-LOX, 12-HETE, acts via GPR31 in promoting β-cell dysfunction in the setting of insulin resistance and inflammation.

Disclosure

M. Hernandez-Perez: None. I. Haider: None. R.M. Anderson: None. S.A. Tersey: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company.

Funding

JDRF (3-PDF-2019-750-A-N); National Institutes of Health (T32DK064466, R01DK105588, (P30DK097512)
2113-P: Discrepancy between Single-Cell RNA Sequencing and Protein Expression Assessments of GLP-1Rr Heterogeneity in Beta Cells
Authors: GRAY SARAH M. , CHAZOTTE BRYANNA M. , ROSS ELIZABETH C. , SVENDSEN BERIT , RAVN PETER , SLOOP KYLE , CAMPBELL JONATHAN , D’ALESSIO DAVID
Published: June 2020

The glucagon-like peptide 1 receptor (GLP-1R) is insulinotropic in β-cells and the target of multiple classes of diabetes drugs. People with T2D secrete normal amounts of GLP-1, but are less responsive to its actions; the mechanisms for this are unknown. Studies have shown that metabolic stress decreases GLP-1R in islets, however, it is unknown if this occurs in all, or just a subset, of β-cells. We hypothesized that GLP-1R expression is heterogeneous in β-cells, such that not all β-cells express the GLP-1R, and that metabolic stress would decrease the number of GLP-1R+ β-cells. To test this, we first performed single-cell RNA sequencing (scRNAseq) in mouse and human islet cells and found that the majority of β-cells were GLP-1R negative, supporting significant heterogeneity. To validate the scRNAseq results, we generated a GLP-1R reporter mouse by crossing GLP-1R-Cre with mTmG mice. Using FACS to separate and qPCR to characterize positive (GFP+) from negative (tdTomato+) dispersed islet cells, we found GLP-1R+ cells were enriched for Ins2 and Sst, while GLP-1R- cells were enriched for Gcg, suggesting GLP-1R promoter activity is localized to β- and d-cells. In addition, β-cell markers were not present in the tdTomato+ population, suggesting very few GLP-1R negative β-cells. Next, we stained dispersed islets from the reporter mice with a validated GLP-1R antibody and found >90% congruence with GFP+ cells and no staining in tdTomato+ cells. In dispersed islets from wild type mice, ∼90% of β-cells were GLP-1R+; very few of the α- or d-cells were GLP-1R+. Metabolic stress induced through chronic high-fat feeding or using multiparous models did not alter the number of GLP-1R+ β-cells.

In conclusion, assessment of GLP-1R expression and GLP-1R protein revealed the majority of β-cells are GLP-1R+, a finding that conflicts with the interpretation of scRNAseq data. These findings suggest caution in interpreting the results of scRNAseq for low abundant transcripts such as the GLP-1R.

Disclosure

S.M. Gray: None. B.M. Chazotte: None. E.C. Ross: None. B. Svendsen: None. P. Ravn: Employee; Self; AstraZeneca. K. Sloop: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.