bluuebunny/crossref_metadata_2025_split huggingface dataset:
2077-P: Can Small ß-Cell Subpopulations Control Islet Dynamics? A Theoretical Study
Authors: DWULET JAEANN M. , BENNINGER RICHARD K. 
Published: June 2020

Electrical activity of a pancreatic islet is globally activated under high glucose conditions and globally suppressed under low glucose conditions. This homogeneous behavior occurs due to the highly-coupled nature of the islet, despite heterogeneity of individual β-cells. Using hyperpolarization to silence β-cell activity, studies have shown that small subpopulations of highly functional β-cells can disproportionately control whole islet dynamics. Using a computational model of islet electrophysiology, we studied the theoretical basis of how small highly functional subpopulations of β-cells may regulate the global activity of islets. We modeled metabolic activity of β-cells as a continuous distribution and as a bimodal distribution. Then, in each case, we silenced the highest metabolically active cells and quantified the response of the islet. These highly metabolic cells (GKHigh) were silenced by hyperpolarization, similarly to experimental data. Only under a bimodal distribution, did a small (10%) population of GKHigh cells dramatically reduce islet activity, as experimentally demonstrated. Next, a simulation was run without GKHigh cells present in the islet. When GKHigh cells were absent, the islet remained globally active and had near normal duty cycle, suggesting that these cells are unnecessary for maintaining islet dynamics. Similarly, we investigated another functional subpopulation, β-cells with higher Ca2+ oscillation frequencies which could potentially act as pacemaker cells. When simulating the absence of 10% of these higher frequency cells, the global frequency of the islet was not significantly altered. These results indicate that small highly functional subpopulations are unable to drive islet function under normal conditions, but when targeted under hyperpolarizing conditions may cause dysfunction.

Disclosure
J.M. Dwulet: None. R.K. Benninger: None.

Funding
National Institutes of Health (R01DK106412, R01DK102950)
2085-P: Effects of Intermittent Caloric Restriction on Body Composition and Glucose Tolerance after Fasting and Refeeding
Authors: HARER CLEMENS , KRSTIC JELENA , KOTZBECK PETRA , KARACAY CEREN , PROKESCH ANDREAS , PIEBER THOMAS 
Published: June 2020

Intermittent caloric restriction has the potential for improving metabolic and cardiovascular markers in rodents and humans, as well as longevity (in rodents). Furthermore, an intermittent caloric restriction regime that mimics prolonged fasting (fasting mimicking diet, FMD) has been linked to proliferation of β-cells in diabetic mice. Aim of the study was to assess the effect of FMD in female C57BL/6J mice on glucose tolerance, body and organ weight, measured at the end of the fasting and at the end of the refeeding period. We administered three weekly cycles of FMD (1st day 50%-, 2nd-3rd day 10% of daily calorie intake) followed by 4 days of refeeding ad libitum after every cycle to 20 mice at 12 weeks of age. In the second FMD cycle an intraperitoneal glucose tolerance test (IGTT) was performed either after fasting or after the 3rd refeeding day. Control animals (n=10) were fed a standard chow ad libitum. Animals were sacrificed and tissues collected after the 3rd FMD cycle, precisely 7 days after the IGTT. Compared to controls, FMD animals weighed less after the 1st, 2nd and 3rd cycle (mean differences ±SE (%): 18.9±1.8, 17.2±1.8, 15.1±1.8; p&lt;0.001). After refeeding, bodyweight returned to levels of age matched control animals. IGTT revealed impaired glucose tolerance after fasting compared to refeeding (mean difference ±SE (mg/dL): min30: 76.4±21.8, min60: 68.7±14.3; p&lt;0.05). Liver weight was reduced by 21.6±4.1% (p&lt;0.001) after fasting and increased by 9.7±2.5% (p&lt;0.001) after refeeding compared to age matched controls. Our study reveals impaired glucose tolerance at the end of the fasting period compared to the end of the refeeding period in healthy mice exposed to three cycles of FMD. Our data support previous findings that prolonged fasting rather reduces insulin secretory capacity of the ß-cell than changing insulin sensitivity, indicating to a fast adaptive cellular mechanism in ß-cells during prolonged fasting.

Disclosure
C. Harer: None. J. Krstic: None. P. Kotzbeck: None. C. Karacay: None. A. Prokesch: None. T. Pieber: Advisory Panel; Self; ADOCIA, Arecor, AstraZeneca, Eli Lilly and Company, Novo Nordisk A/S, Sanofi. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker’s Bureau; Self; Novo Nordisk A/S, Roche Diagnostics K.K.
209-LB: Truncated SOCS3 Overcomes Insulin Resistance Associated With Type 2 Diabetes
Authors: JEON JISOOK , LEE KUYSOOK , KIM SU JEONG , KANG HYEONTAE , KWAK JEONGHUN , CHOI YOUNGSIL , JO DAEWOONG 
Published: June 2020

Aim: Insulin resistance, the reduced ability of tissues to respond to insulin, is a defining feature of T2DM. Leptin, hormone signals though the JAK/STAT pathway and enhances insulin sensitivity. Leptin signaling is negatively regulated by suppressor of cytokine signaling 3 (SOCS3) that binds to the leptin receptor and promotes leptin resistance. Prolonged leptin stimulation is further associated with hyperinsulinemia and insulin resistance. To investigate the interplay between leptin and insulin signaling with the goal of suppressing leptin-induced insulin resistance in pathological settings we developed a cell-permeable dominant-interfering SOCS3 [(∆) SOCS3].
Method: Cell-permeable, dominant-interfering ΔSOCS3 protein consists of the SOCS3 SH2 domain, a sequence optimized-advanced macromolecule transduction domain (aMTD) to deliver the protein into cells and tissues to facilitate soluble purification, biologically active proteins. Intracellular delivery of ΔSOCS3 was tested in diet-induced obese (DIO) mice to evaluate the increment of insulin sensitivity and glucose control.
Result: CP-ΔSOCS3 was delivered into cells and tissues including brain, liver, spleen and lung. Insulin pretreatment attenuates insulin-mediated Akt signaling in HepG2 cells; whereas, insulin sensitivity was restored by CP-ΔSOCS3. DIO mice treated with CP-ΔSOCS3 showed 12.1% body weight loss in 14 days. In addition, groups of mice were examined with the glucose tolerance test. Blood glucose at 2 hrs decreased by 66.7% and area under curve decreased by 40.3% in treatment group as compared to the diluent group. CP-ΔSOCS3 also reduced blood cholesterol by 31% and fatty liver by 92.6% and was accompanied by significant increases in Akt phosphorylation in the hypothalamus, fat and muscle consistent with enhanced insulin sensitivity.
Conclusion: CP-ΔSOCS3 has therapeutic effect on obesity and blood glucose control. These results suggest CP-ΔSOCS3 may provide a mechanism-specific therapy for T2DM.

Disclosure
J. Jeon: None. K. Lee: None. S. Kim: None. H. Kang: None. J. Kwak: None. Y. Choi: None. D. Jo: None.
2098-P: Transcriptomic Changes Associated with Oleate-Induced ß-Cell Proliferation in Rat Islets
Authors: VIVOLI ALEXIS , CASTELL ANNE-LAURE , PACIS ALAIN , SLADEK ROBERT M. , GHISLAIN JULIEN , POITOUT VINCENT 
Published: June 2020

Background: In nondiabetic obese individuals, β-cell mass accretion maintains glucose homeostasis by balancing levels of circulating insulin and insulin resistance. Type 2 diabetes appears when these compensatory mechanisms fail. Deciphering the pathways controlling β-cell proliferation has thus become a major research goal in the hope to identify therapeutic targets to expand β-cell mass and prevent or delay the onset of diabetes. Previously we demonstrated that fatty acids potentiate glucose induced β-cell proliferation in response to nutrient surfeit in rats. In recent studies we identified the mono-unsaturated fatty-acid oleate as a major fatty acid driving β-cell proliferation in rat islets ex vivo.
Aim: To identify the transcriptional mechanisms underlying oleate-induced β-cell proliferation in rats.
Method: Rat islets were exposed to 16.7mM glucose +/- oleate (0.5mM) for 48h. Proliferation was assessed by flow cytometry using c-peptide antibodies and EdU. Single cell cDNA libraries were generated using 10X Genomics technology and sequenced on the Illumina platform. Bioinformatic analyses were performed using the Cell Ranger pipeline, Seurat and Monocle.
Results: Oleate induced a 3.1+/-0.4 fold increase (p&lt;0.01; n=4) in β-cell proliferation. Following single-cell RNA sequencing, t-SNE plots revealed several β-cell sub-populations, including proliferating β-cells. Comparison between proliferative and non-proliferative β-cells identified more than 300 differentially expressed genes. Gene ontology suggested an increase in mitochondrial activity and reactive oxygen species (ROS) in proliferating β-cells. Pseudotime ordering of β cells revealed that ER-stress related genes are up-regulated prior to proliferation in response to oleate.
Conclusion: Our study suggests an involvement of ROS and ER-stress in oleate-induced β-cells proliferation. Further functional analyses will substantiate the importance of these stress responses.

Disclosure
A. Vivoli: None. A. Castell: None. A. Pacis: None. R.M. Sladek: None. J. Ghislain: None. V. Poitout: None.
210-OR: Activation of GFRAL Neurons Decreases Food Intake via Aversive Pathways
Authors: SABATINI PAUL V. , SEELEY RANDY J. 
Published: June 2020

Growth and differentiation factor 15 (GDF15), an anorexigenic peptide that represents a promising candidate for anti-obesity treatment, acts via GDNF Family Receptor Alpha Like (GFRAL), which is expressed almost exclusively on a subset of neurons in the area postrema (AP). To determine the function and mechanisms of action for GFRAL neurons, we generated Gfralcre and conditional GfralCreERT mice. Although their chemogenetic (DREADD-mediated) activation promoted FOS in a variety of brainstem, hypothalamic, and limbic nuclei, GFRAL neurons projected only to the nucleus of the solitary tract (NTS) and the parabrachial nucleus (PBN), where they innervated and activated aversive/anorexigenic GCRP-expressing cells. Tetanus-toxin-mediated silencing of PBN CGRP neurons abrogated the aversive and anorexic effects of GDF15. Furthermore, while non-gastrointestinal (GI) stimuli (e.g., GDF15 and LPS, but not feeding or gut peptide mimetics) activated GFRAL neurons, chemogenetically activating these cells decreased gastric emptying, suppressed feeding, and promoted a conditioned taste aversion. These findings suggest that GFRAL neurons link non-GI anorexigenic signals to the control of gut physiology and to the aversive suppression of food intake. Additionally, because the chemogenetic activation of GFRAL neurons suppressed food intake more strongly than GDF15 in lean mice, additional modes of activating GFRAL neurons may augment the anorectic potential of GDF15.

Disclosure
P.V. Sabatini: None. R.J. Seeley: Consultant; Self; Ionis Pharmaceuticals, Inc., Kintai, Kintai, Novo Nordisk A/S, Sanofi, Scohia Pharma Inc. Research Support; Self; AstraZeneca, Novo Nordisk A/S, Pfizer Inc. Stock/Shareholder; Self; Redesign Health.

Funding
American Diabetes Association (1-19-PDF-099 to P.V.S.)
2104-P: Proinflammatory Cytokines Impair Human Islet and Beta-Cell Glucose Oxidation
Authors: MELO JOANA , JENSEN VERENA HIRSCHBERG , STØRLING JOACHIM 
Published: June 2020

Pro-inflammatory cytokines contribute to beta cell dysfunction in type 1 diabetes (T1D), but the exact underlying mechanisms remain unclear. It was previously shown that cytokines inhibit mitochondrial glucose oxidation leading to impaired glucose-stimulated insulin secretion in rat INS-1E cells, however, the effect of cytokines on mitochondrial respiration in human islets has not been explored. In this study, we investigated mitochondrial dysfunction in human islets and rat beta cells following cytokine treatment. We exposed isolated human pancreatic islets and rat INS-1E cells for up to 24 h to IL-1β and IFN-γ to mimic early beta cell destruction in T1D. Mitochondrial bioenergetics were analyzed by Seahorse XFe24 technology. Human islets from three healthy organ donors exposed to cytokines showed a reduction in glucose-induced oxygen consumption and an increase in ATP-uncoupled respiration. Higher basal mitochondrial oxygen consumption was also observed, which was not associated with an increase in ATP production. In INS-1E cells, cytokines exerted a strong inhibitory effect on mitochondrial glucose oxidation in a dose-independent manner. In acute exposure experiments, no effect on oxygen consumption was observed up to eight hours after cytokine exposure. This finding proposes that cytokine-induced inhibition of mitochondrial respiration is dependent on secondary signaling mechanism and/or changes in gene transcription. To conclude, glucose-induced oxygen consumption is reduced by cytokines in both human islets and INS-1E cells indicative of impaired mitochondrial respiration. Moreover, cytokines augment mitochondrial uncoupled respiration in human islets, indicating mitochondrial dysfunction that may contribute to islet failure in T1D. Ongoing work is investigating gene expression of genes involved in mitochondrial respiration processes. Further studies are needed to clarify the possible pathogenic role of mitochondrial dysfunction in T1D and the underlying mechanisms.

Disclosure
J. Melo: None. V. Hirschberg Jensen: None. J. Størling: None.
2106-P: Glucagon-Like Peptide-1 Receptor Agonist Exenatide Promotes ß-Cell Function via Activation of PPARd/UCP-2 Pathway
Authors: SU YANNA , XU WEN , LIN BEISI , LIU ZIYU , CHEN YALAN , YE ZHIWEI , GAN LU , YAO BIN 
Published: June 2020

Background: Accumulating evidence indicates that glucagon-like peptide-1 receptor agonists (GLP-1 RAs) lower glucose mainly by enhancing glucose-dependent stimulation of insulin secretion. However, the underlying mechanisms remain to be further investigated.
Methods: C57BL6/J mice fed with 12-week high-fat diet (HFD) were treated with exenatide (a GLP-1 RA, 24nmol/kg/d), GW501516 (a PPARΔ agonist, 2mg/kg/d) or saline injection as control for 8 weeks. Phenotypic evaluations were performed during and after the intervention. Tolerance tests and insulin measurement were performed after 8-week treatment. The mouse pancreatic β-cell line MIN6 was used as an in vitro model. The MIN6 cells were treated with GW501516 (10uM), exendin-4 (a GLP-1 RA, 20nM) combined with GSK0660 (a PPARΔ antagonist, 10uM) or not. Furthermore, mediums containing various concentrations of exendin-4 (1, 20 and 100 nM) or GW501516 (0.1, 1, 10 and 100uM) were applied. And the cells were incubated for 24 hours. PPARΔ and uncoupling protein-2 (UCP-2) protein expression were detected.
Results: Compared with the control group, exenatide and GW501516 treatment led to decreased body weight and improved insulin sensitivity in HFD mice. The mice in control group had higher serum insulin levels during the tolerant tests, but their early-phase insulin secretion was lacking, indicating the impaired insulin sensitivity and β-cell function. Both exenatide and GW501516 decreased the serum insulin levels in the HFD mice. Importantly, the lacking early-phase insulin secretion were recovered by both exenatide and GW501516. The protein expression of PPAR Δ was promoted while that of UCP-2 was decreased by exendin-4 in a dose-dependent manner. Similar results were found in cells treated with GW501516. However, these salient effects were partly reversed by GSK0660.
Conclusions: The effect of the GLP-1 RA exenatide on promoting β-cell function might be related to the activation of PPARΔ/UCP-2 pathway.

Disclosure
Y. Su: None. W. Xu: None. B. Lin: None. Z. Liu: None. Y. Chen: None. Z. Ye: None. L. Gan: None. B. Yao: None.

Funding
National Natural Science Foundation of China (81770821 to W.X.)
2107-P: A Human Pancreatic Spheroid Platform for Toxicology and Diabetes Research
Authors: MORA-CASTILLA SERGIO , GAERTNER BJOERN , BARBOSA-FARIAS INGRID , SI WONJONG , ZANELLA FABIAN 
Published: June 2020

Diabetes mellitus (DM) is a prevalent and chronic metabolic disorder that affects more than 300 million patients in the world. One of the major obstacles in DM is the lack of in vitro models that can capture the physiological features of the in vivo environment. Therefore, to identify new treatments for DM there is an increasing demand for functional in vitro pancreatic islet models in which the cellular microenvironment, consistency and long-term culture is more fully preserved. More recently, functional beta-cells derived from human induced pluripotent stem cells (hiPSCs) have gained prominence as an enticing system for the study of DM as a human, tissue-specific model to model the disease and identify potential therapeutic intervention. Here we describe and characterize a novel micro-engineered 384 well plate platform in which each well contains a single size-controlled hiPSC-pancreatic islet equivalent spheroid that emulates key characteristics of adult human pancreas. Gene expression and immunofluorescence analysis indicated that these islet-equivalents express key markers of pancreas cellular identity such as the master pancreatic transcription factor PDX-1, glucose-sensing and insulin-secreting genes such as GLUT2, ABCC8 and SYT4, and importantly, secreted proteins produced in mature humans islets, like Insulin, glucagon and somatostatin. RNAseq data indicated larger transcriptional consistency from different batches of microPancreas production than traditional cadaveric islet samples. In addition, cells show glucose dependent stimulated insulin secretion (GSIS), highlighting the functionality of the platform. Altogether, we describe a novel human pancreatic platform applicable to gene-expression, imaging and insulin-secretion studies as a reliable research source for diabetes drug discovery, disease modeling and toxicology applications.

Disclosure
S. Mora-Castilla: Employee; Self; StemoniX. B. Gaertner: Employee; Self; StemoniX. I. Barbosa-Farias: Employee; Self; StemoniX. W. Si: Employee; Self; StemoniX. F. Zanella: Employee; Self; StemoniX.
2109-P: Type 2 Diabetic Human Beta Cells Are Characterized by Marked Expression of the Na+-nHCO3- Cotransporter NBCe1 and Intracellular Alkalization
Authors: BROWN MATTHEW , STILLER ALISON , ROMERO MICHAEL F. , MATVEYENKO ALEKSEY 
Published: June 2020

Metabolic stressors common to type 2 diabetes (T2D) lead to perturbations of β-cell’s transcriptional identity which provide β-cells an adaptive survival advantage at the expense of compromised function (i.e., dedifferentiation). Increase in the intracellular pH (pHi alkalization) is a characteristic feature of cancer cell dedifferentiation and survival. Interestingly, a recent single-cell transcriptomic study of human pancreas identified SLC4A4 (encoding Na+-coupled HCO3- cotransporter, NBCe1) as one of the few genes enriched in dedifferentiated T2D β-cells. Indeed, NBCe1 is a key regulator pHi alkalization; however, its role in human β-cell dysfunction in T2D is unknown. To address this we first defined whether aberrant SLC4A4/NBCe1 expression marks failing human β-cells in T2D. First, utilizing immunofluorescence analysis of human autopsy pancreas we observed near complete absence of NBCe1 expression in β-cells of lean nondiabetic individuals (ND) and robust expression of NBCe1 in obese T2D β-cells (∼10 fold increase in T2D vs. ND, p&lt;.01). Moreover, NBCe1 expression occurred in conjunction with the nuclear to cytoplasmic redistribution of NKX6.1 and with an increase in ALDH1a3-positive β-cells, both key markers of β-cell dedifferentiation in T2D. Moreover, we independently procured ND and T2D isolated human islets (12 independent shipments) and observed ∼ 3-fold increase in SLC4A4 mRNA in T2D islets (p&lt;.05 vs. ND). Since the primary function of NBCe1 is HCO3- import, T2D islet cells also exhibited significantly higher pHi (p&lt;.001 vs. ND). Finally, NBCe1 activity inhibition (with selective inhibitor, S0859) reversed β-cell dysfunction in isolated T2D islets by significantly enhancing β-cell GSIS (p&lt;.01 vs. UT). Taken together, our study suggests that induction of β-cell SLC4A4/NBCe1 expression and consequent increase in pHi may play a previously unappreciated role in β-cell dysfunction and dedifferentiation in T2D.

Disclosure
M. Brown: None. A. Stiller: None. M.F. Romero: None. A. Matveyenko: None.
2115-P: Beta-Cell Autophagy Is Reduced in Type 1 Diabetes
Authors: MURALIDHARAN CHARANYA , MARASCO MICHELLE , CROWDER JUSTIN , CONTEH ABASS M. , LINNEMANN AMELIA K. 
Published: June 2020

Autophagy is a dynamic recycling mechanism that maintains beta cell homeostasis, whereas the related process of crinophagy also plays a specific role in insulin granule regulation. Impairment of autophagy and crinophagy have been implicated in beta cell death and dysfunction in the context of type 2 diabetes; however, there is currently no literature describing beta cell autophagy in type 1 diabetes pathogenesis. Therefore, we set out to determine if beta cell autophagy and crinophagy are impaired in the context of type 1 diabetes. We utilized immunofluorescent staining of LC3 (autophagosome marker), Lamp1 (lysosome marker), and proinsulin to analyze pancreatic islet autophagy and crinophagy in the NOD (non-obese diabetic) mouse model of type 1 diabetes as well as in human pancreas tissue obtained from the network for Pancreatic Organ donors with Diabetes (nPOD). We observed evidence of reduced macroautophagy in the residual proinsulin-positive islet cells of human donors with type 1 diabetescompared to controls, as measured by significantly reduced autophagosome:lysosome colocalization (p=0.034). Similarly, there was a trend towards reduced autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to nondiabetic NOR mice (p=0.08). We also observed significantly reduced colocalization of proinsulin with lysosomes in the proinsulin positive cells from human donors with type 1 diabetes in comparison to both controls without diabetes (p=0.0014) and autoantibody positive donors (p= 0.0278), as well as in diabetic NOD vs. nondiabetic NOR mice (p=0.0121), suggesting that crinophagy is also reduced as a function of type 1 diabetes. Collectively, we provide evidence that macroautophagy and crinophagy are reduced in the context of type 1 diabetes. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development.

Disclosure
C. Muralidharan: None. M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. J. Crowder: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. A.K. Linnemann: None.