bluuebunny/crossref_metadata_2025 huggingface dataset:
2034-P: ß Cell miRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and Risk
Authors: SYED FAROOQ , KRISHNAN PREETHI , RESTREPO IVAN , MIRMIRA RAGHAVENDRA , EVANS-MOLINA CARMELLA
Published: June 2020

Biomarkers capable of monitoring β cell stress during the evolution of type 1 diabetes (T1D) are currently lacking. MicroRNAs (miRNAs) are a class of small non-coding RNAs ∼22 nucleotides in length that modulate gene expression by binding to the 3’untranslated region of target mRNAs. Given their stability in biological fluids and enrichment in cell-derived EVs, we hypothesized that miRNAs from human islet and islet-derived EVs could identify β-cell stress/death and be leveraged in T1D biomarker strategies. To test this, human islets were obtained from 10 cadaveric donors (5 male/5 female) and treated with or without cytokines (IL-1β and IFN-γ) for 24 hrs, as an ex vivo model of T1D. Small RNA sequencing was performed and identified 1110 and 890 miRNAs in total and 20 and 14 differentially expressed (DE) miRNAs (fold change≥1.5 and p<0.05) from islets and EVs, respectively. These findings were validated in an independent set of cytokine-treated islets and islet-derived EVs (7M and 5F donors). Interestingly, miRNA expression pattern was strikingly different between male and female donors at baseline and under cytokine stress with < 10% overlap among the DE miRNAs. miR-155-5p and miR-146a-5p were the only two miRNAs that were upregulated in cytokine-treated islets and EVs in both the sexes. Functional enrichment analysis of DE miRNAs identified pathways such as insulin signaling, ER stress and apoptosis. Taken together, these data suggest that miRNA expression patterns change dynamically in both human islets and islet-derived EVs in response to pro-inflammatory cytokine stress. EV miRNAs were largely distinct from those in the islet fraction, suggesting that miRNAs are selectively packaged into EVs in response to extrinsic cues. Finally, these data highlight the importance of considering sex as a biological variable when defining T1D biomarkers.

Disclosure

F. Syed: None. P. Krishnan: None. I. Restrepo: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, UC4-DK104166 to C.E-M., R.M.); U.S. Department of Veterans Affairs (I01BX001733 to C.E-M.); JDRF (3-SRA-2014-41-Q-R); Sigma Beta Sorority; Ball Brothers Foundation; George and Frances Ball Foundation (to C.E-M.); Indiana Diabetes Research Center (P30DK097512)
2036-P: I-BET 762 Inhibits Inflammation-Induced Pancreatic Beta-Cell Apoptosis by Controlling Inflammatory Pathways
Authors: NEGI VINNY , LEE JEONGKYUNG K. , LIU RUYA , JAGANNATHAN RAJAGANAPATI , LI FENG , YANG PING , PEREZ-GARCIA MELISSA , MOULIK MOUSUMI , YECHOOR VIJAY
Published: June 2020

Diabetes is one of the most prevalent disease in the United States, affecting 9% of the population, which is expected to increase by 54% by 2030. It occurs due to the body’s inability to make or respond to insulin, leading to hyperglycemia and significant risk of chronic complications. Pre-clinical investigations have reported the beneficial effect of an anti-cancer drug, I-BET (inhibitor of bromodomain and extra-terminal protein) on β-cell function but the underlying mechanisms remain unknown. As apoptosis of β-cells is a characteristic event in both T1D and T2D, we were interested in determining the effect of I-BET762 on inflammation induced β-cell apoptosis. In the present study, we found that I-BET was able to reduce the inflammation induced apoptosis in insulinoma cell line (Ins1); as determined by Annexin V-PI staining using flow cytometry (Figure 1) and cleaved caspase-3 quantitation using western blot. In addition, we also found that I-BET was able to reduce hyperglycemia in vivo in the low dose STZ (streptozotocin) model of T1D. This anti-apoptotic effect of I-BET is mediated via a reduction in the cytokine-induced activation of NF-kB and STAT1 signaling.

In conclusion, our results indicate that inhibition of bromodomain proteins could be a therapeutic strategy in all forms of diabetes by protecting β-cells from cytokine-induced apoptosis.

Disclosure

V. Negi: None. J.K. Lee: None. R. Liu: None. R. Jagannathan: None. F. Li: None. P. Yang: None. M. Perez-Garcia: None. M. Moulik,: None. V. Yechoor: None.

Funding

National Institutes of Health (R01DK097160)
204-OR: Inhibition of Hepatic ACC on a High-Fat Diet Results in Hyperglycemia and Hepatomegaly Due to Excess Energy Generation
Authors: DEJA STANISLAW , FLETCHER JUSTIN A. , KUCEJOVA BLANKA , FU XIAORONG , KIM CHAI-WAN , BROWNING JEFFREY , HORTON JAY D. , BURGESS SHAWN C.
Published: June 2020

Liver triglyceride accumulation associated with insulin resistance is partially mediated by elevated de novo lipogenesis (DNL); thus, limiting this pathway is a promising therapeutic strategy for NAFLD. Targeted suppression of acetyl-CoA carboxylase (ACC) activity depletes malonyl-CoA resulting in lower DNL and a simultaneous increase in fat oxidation due to activation of CPT-1. Recently developed ACC1/2 liver specific double KO mice (LDKO) are protected from hepatic steatosis and closely recapitulate pharmacological inhibition of ACC1/2 in humans with NAFLD. However, hepatic ACC inhibition causes surprising elevations in circulating triglyceride and glucose concentrations. LDKO mice had worsened glucose tolerance, which was most consequential during a HFD and especially prominent during fasting. Glucose labeling from 2H2O and [U-13C3] lactate was higher in LDKO hepatocytes, an effect that was dependent on the availability of long chain fatty acids, suggesting that loss of malonyl-CoA and activation of CPT-1 promote gluconeogenesis (GNG). Ketogenesis was increased in LDKO hepatocytes and showed limited response to insulin. Acetyl-CoA was elevated in the fed state, as previously reported, but was normal during fasting when hyperglycemia was most prominent. LDKO mice exhibited massive accumulation of liver citrate and a high fasted hepatic energy charge, which are both known to activate GNG. Hence, despite improved hepatic lipid profile and insulin sensitivity, LDKO liver activates GNG. The resulting hyperinsulinemia partially ameliorates the glycemia phenotype, but may also induce hepatomegaly through canonical signaling pathways and effects on energy status. Thus, activation of fat oxidation in liver lowers lipid accumulation, but has downstream metabolic effects on energetically dependent pathways, such as GNG, that may promote elevated glucose levels in contexts of reduced insulin action, such as fasting and diet induced obesity.

Disclosure

S. Deja: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. C. Kim: None. J. Browning: None. J.D. Horton: Board Member; Self; Pfizer Inc. Consultant; Self; Gilead Sciences, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc. Stock/Shareholder; Self; Catabasis Pharmaceuticals. Other Relationship; Self; Regeneron Pharmaceuticals, Sanofi. S.C. Burgess: None.

Funding

National Institutes of Health (R01DK078184, P41EB015908); Robert A. Welch Foundation (I-1804)
2041-P: Engineered Glycopolymers Preserve Pancreatic Beta-Cell Function and Survival by Inhibiting Heparanase
Authors: ZHANG KEZHONG , NGUYEN HIEN M.
Published: June 2020

Human pancreatic β cells contain high levels of heparan sulfate (HS) polysaccharide that is lost in type 1 diabetes (T1D) patients. Heparanase is the only human enzyme that degrades HS. During T1D and diabetic nephropathy, the immune system produces heparanase that destroys HS within β cells and causes their death. We synthesized two carbohydrate molecules by incorporating multiple sugar units or highly tunable sulfation patterns, Glycopolymer GPM and Trisaccharide 1α. GPM and Trisaccharide 1α efficiently mimic the properties of naturally existing HS, and thus act as specific inhibitors of heparanase. We treated mouse pancreatic β cell line MIN-6 and human pancreas islets with GPM or Trisaccharide 1 in the presence of heparanase, and then evaluated β cell or islets morphology, function, and survival. The results showed that GPM of nenomole (nM) doses or Trisaccharide 1α of micromole (µM) doses can effectively preserve cellular HS contents, prevent mitochondrial ROS damage, and help survival of human or mouse pancreas β cells under heparanase challenge. Protein affinity and toxicity analyses suggested that GPM and Trisaccharide 1α possess highly specific binding activities to heparanse but low affinity to many HS-binding proteins responsible for adverse effects.

In summary, we engineered and characterized glycopolymers as a new class of non-toxic, specific heparanase inhibitors to preserve pancreatic β cell function and survival, thus positioning these molecules as potential therapeutics for diabetes and its complications.

Disclosure

K. Zhang: None. H.M. Nguyen: None.

Funding

National Institutes of Health (GM098285, DK090313)
2046-P: Retinoic Acid Signaling in Pancreatic Endocrine Cells Regulates Islet Cell Specification
Authors: LORBERBAUM DAVID , SUSSEL LORI
Published: June 2020

Diabetes is characterized by β cell death and/or dysfunction. Currently, one promising treatment option for patients with diabetes involves replenishing the body with β cells to restore glucose homeostasis. Despite great progress using in vitro differentiation protocols to generate human β cells from stem cell populations, there are still challenges associated with generating fully functional β cells. To improve this therapeutic strategy, my studies are focusing on defining the role of cell signaling pathways in pancreatic islet cell specification. Genetic studies in fish, mice, and humans have demonstrated that retinoic acid (RA) is broadly required for pancreas development; however, the role of RA signaling in pancreatic endocrine cell specification remains unclear. By examining the role of RA using in vivo endocrine-specific inhibition of RA signaling at critical time points during pancreas development, we demonstrate that inhibition of RA signaling in the endocrine progenitor population results in the generation fewer β and increased Δ cell genes by embryonic day (e)16.5. These defects persist after birth and are accompanied by overt hyperglycemia in the adult. Whole transcriptome analysis further indicates that cross-talk between RA signaling and the Wnt pathway is important for islet cell specification. In addition, we show that RA signaling is also important for human β cell specification. Together, these data demonstrate the importance of RA signaling in the endocrine progenitor population and provide a framework to more clearly define the mechanisms by which RA signaling directs endocrine cell fate in mice and humans. Future studies on the role of RA and its interacting pathways during pancreas development will better inform strategies to treat diabetes using cell replacement therapies.

Disclosure

D. Lorberbaum: None. L. Sussel: None.

Funding

American Diabetes Association (1-18-PDF-107 to D.L.)
2047-P: Possible Implication of Cyclin D2 in Beta-Cell Proliferation of Human Perinatal Islet
Authors: OSONOI SHO , ICHINOHE HIROSHI , KUDO KAZUHIRO , YAGIHASHI SOROKU , MIZUKAMI HIROKI
Published: June 2020

Cyclin D2 (CCND2) is an essential cell cycle regulator of rodent pancreatic β cells, while its expression in human islets is still controversial in the past studies. It may be because few proliferative β cell is generally present in human adult islets. In contrast, expansion of β cell population mainly depends on a cell proliferation in human perinatal islets. Therefore, CCND2 is expected to be implicated in β cell proliferation in human perinatal islets. The aim of this study is to explore the implication of CCND2 in proliferation of human perinatal β cells. We evaluated pancreata of 39 autopsy cases from prenatal 24 weeks old to postnatal 19 years old. The subjects were divided into fetus (F: prenatal 24-31 weeks), infant (I: 0-1 years), child (C: 2-12 years) and adolescence (A: 13-19 years). Quintuple-immunohistochemistry (IHC) for glucagon, insulin, somatostatin, pp and Ki-67 was performed for the measurement of islet cell volume density (V) and cell replication status. Double-IHC for insulin with CCND2, 5-hydroxy tryptophan (5-HT) and p16 were performed. Double positive cells for each staining were counted. Morphometric analysis revealed that Vα and Vβ were increased with maturation. Ki-67 positivity for α and Δ cells was the highest in F (p<0.01), whereas that of β cells was the highest in I (p<0.01). CCND2 was robustly expressed in the nucleus of β cells in F (>90%) and I (>90%). Thereafter, its expression was dramatically declined in C and A (2.25±1.4%). 5-HT was highly expressed in perinatal subjects but not in C and A. Conversely, p16 expression was minimum before adolescence and its expression was evident in A. Our results clearly suggested that CCND2 could be implicated in the development of human β cell population in perinatal stage. The expression of CCND2 might be independently regulated by 5-HT and p16, while other factors may be implicated in the expression mechanism. To elucidate the regulation mechanism of CCND2 in human islets may lead to develop a novel strategy for diabetes treatments.

Disclosure

S. Osonoi: None. H. Ichinohe: None. K. Kudo: None. S. Yagihashi: None. H. Mizukami: None.
2057-P: Atypical Trefoil Factor Family 2 Expressing ß-Cells of Diabetic Mutant CRY1 Transgenic Mice with Intraislet Ducts
Authors: OKANO SATOSHI , YASUI AKIRA , KANNO SHIN-ICHRO , SATOH KENNICHI , IGARASHI MASAHIKO , NAKAJIMA OSAMU
Published: June 2020

We have demonstrated that cysteine414 (zinc-binding site of mCRY1)-alanine mutant mCRY1 transgenic mice (Tg mice) show diabetes characterized by the reduction of β-cell proliferation and by β-cell dysfunction due to SASP (senescence-associated secretory phenotype)-like characters of β-cells. In addition, our previous studies showed that mucinous pancreatic duct glands (PDGs) occur in exocrine areas of aged Tg mice. Further, we also showed that mucin-producing intra-islet ducts develope age-dependently in Tg mice (Okano S. et al., 2019). To explore the molecular mechanisms for developing the intra-islet ducts, we conducted DNA microarray analysis using RNA from islets of aged Tg mice. The islet samples were obtained by paraffin-embedded pancreas sections by laser-capture microdissection. The result showed that the mRNA expression of Trefoil factor family 2 (TFF2), which is reportedly expressed in PDGs, was up-regulated in the islet of aged Tg mice. Immunostaining experiments with TFF2 antibodies revealed that, inside of islet of aged Tg mice, TFF2 was expressed by considerable rate in β-cells. TFF2 was located in speckle-like granules in the cytoplasm in the β-cells. In wild type controls, no TFF2 was observed in β-cells. These results suggest that the atypical β-cells have both endocrine and ductal characteristics. In α-cells, essentially no TFF2 protein expression was observed in both wild type controls and Tg mice. Taken together, our results strongly suggest that β-cells are major sources for intra-islet ductal cells in Tg mice. Our results also suggest that direct trans-differentiation of β-cells to ductal cells is involved in the generation of the intra-islet ductal cells.

Disclosure

S. Okano: None. A. Yasui: None. S. Kanno: None. K. Satoh: None. M. Igarashi: None. O. Nakajima: None.

Funding

Japan Society for the Promotion of Science (19K07498); IDAC; Tohoku University
206-OR: Generation of Induced Pluripotent Stem Cells Derived from a Patient with PIK3R1 Mutation and Analysis of Defects in Insulin Action in Hepatocytes Differentiated from These Cells
Authors: HAMAGUCHI TETSUSHI , HIROTA YUSHI , TAKEUCHI TAKEHITO , KOYANAGI-AOI MICHIYO , AOI TAKASHI , OGAWA WATARU
Published: June 2020

Background: A protocol of differentiation form induced pluripotent stem cells (iPSCs) to hepatocytes has been previously reported, and such iPSCs-derived hepatocytes possessed morphological and biochemical characteristics similar to authentic hepatocytes. It is however unknown whether iPSCs-derived hepatocytes normally respond to metabolic regulatory hormones including insulin. Mutations in the gene for p85α subunit of PI3K (PIK3R1), an essential element of insulin action, give rise to severe insulin resistance in human. Such cases are very rare, and only ∼30 families with PIK3R1 mutations have been reported as of today.

Methods and Results: We established a protocol of differentiation from iPSCs to hepatocytes that respond to insulin. In hepatocytes differentiated from iPSCs derived from healthy subject with this protocol, phosphorylation of AKT and the expression of the lipogenic genes FASN as well as SREBF1c were induced by insulin. Furthermore, the expression of the gluconeogenic gene G6PC was upregulated by a combination of cAMP and dexamethasone, which was markedly impaired by the addition of insulin. We then generated iPSCs from a severe insulin resistant diabetic patient with the PIK3R1 c.1945C>T mutation, a hot-spot mutation in this gene. Insulin-induced gene expression along with phosphorylation of AKT was impaired in hepatocytes differentiated from the patient-derived iPSCs. We also repaired the mutation of PIK3R1 in the patient-derived iPSCs by the CRISPR/Cas9 genome editing. Insulin responsiveness was recovered in hepatocytes differentiated from the resultant iPSCs in which the mutation was repaired.

Conclusion: We established a differentiation protocol of hepatocytes in which insulin’s metabolic actions can be evaluated. The usefulness of this protocol was validated with the use of iPSCs derived from a severe insulin-resistant patient with PIK3R1 mutation.

Disclosure

T. Hamaguchi: None. Y. Hirota: Other Relationship; Self; Sanofi. T. Takeuchi: None. M. Koyanagi-Aoi: None. T. Aoi: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.
2061-P: Islet Single Cell Transcriptomes Reveal Cellular and Gene Regulatory Alterations in Type 2 Diabetes Pathogenesis
Authors: LAWLOR NATHAN , KURSAWE ROMY , STITZEL MICHAEL L.
Published: June 2020

Pancreatic islet (dys)function and failure is central to both type 2 diabetes (T2D) genetic risk and pathogenesis. Islet cellular heterogeneity has impeded precise understanding of the specific gene(s) and pathway(s) in each cell type that are altered by and/or may contribute to pathophysiology in the prediabetic (PD) and type 2 diabetic (T2D) states. To identify cellular and molecular features of islet dysfunction in PD and T2D states, we completed single cell transcriptome profiling of 137k human islet cells from 11 ND, 6 PD, and 11 T2D donors (⋃5k/individual).

Single cell transcriptomes aggregated into distinct clusters of endocrine (alpha, beta, delta, PP/gamma), exocrine (acinar, stellate, ductal), endothelial, and immune cell types. Targeted analysis of each endocrine cell type identified a proliferative alpha cell sub-population comprising ∼0.15-0.3% of endocrine cells and variable insulin production and endoplasmic reticulum stress beta cell states. We compared the proportion of islet endocrine cell type (sub)populations sampled between T2D, PD, and ND donors and found that T2D samples contained significantly fewer beta cells and more alpha cells than ND and, to a lesser degree, PD samples. Delta and PP/gamma cell proportions were similar between disease states. Importantly, no (sub)populations were ND, PD, or T2D-specific.

We compared aggregate T2D, PD, and ND cell type-specific transcriptomes to identify differentially expressed genes in each cell type. Genes related to exocytic granule assembly/organization, including those induced by T2D risk variants (DGKB) or T2D state (GAP43), were among 99 genes induced in T2D beta cells. 103 repressed genes included those associated with glucose transport and implicated as T2D GWAS effector genes such as SLC2A2, SLC5A1, and STARD10, a gene whose deletion in mice impairs islet insulin secretion. Studies are ongoing to assess the functional role(s) of these and additional genes in islet (dys)function.

Disclosure

N. Lawlor: None. R. Kursawe: None. M.L. Stitzel: None.

Funding

American Diabetes Association/Pathway to Stop Diabetes (1-18-ACE-15 to M.L.S.)
2069-P: Distribution of Vimentin in the Human Pancreatic Islets in Type 2 Diabetes
Authors: KRIVOVA YULIYA , PROSHCHINA ALEXANDRA , OTLYGA DMITRY , SAVELIEV SERGEY
Published: June 2020

As it was recently shown, vimentin is expressed in some epithelial and endocrine cells of the pancreas during its development, regeneration and in type 2 diabetes (DM2). It is suggested, that activation of vimentin expression may occurs during the neogenesis of β-cells from epithelial progenitors or during dedifferentiation of β-cells and their reprogramming into α-cells. Our aim was to study the distribution of vimentin in the endocrine pancreas in DM2. The study was performed on pancreatic autopsies from 13 persons with DM2 (DM 2 group) and 9 persons without carbohydrate metabolism disorders (control group) using double immunohistochemistry and immunofluoresence with antibodies to vimentin and either insulin or glucagon, morphometric methods and statistical analysis. Vimentin+ cells, some of which simultaneously contain insulin or glucagon, were observed in the pancreatic islets both in DM2 and control groups. In the DM2 group, all the parameters reflecting the amount of vimentin+ cells were significantly higher than in control group (Table 1). In the DM2 group, we also found characteristic changes in the endocrine pancreas - an increase in α-cell density and a decrease in the β- to α-cell ratio (Table 1). We assume that an increase in the amount of vimentin+ cells in the pancreatic islets may reflect alterations in the endocrine cell mass, and vimentin may be used as a marker of endocrine pancreas disorders.

Disclosure

Y. Krivova: None. A. Proshchina: None. D. Otlyga: None. S. Saveliev: None.
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<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<0.05). Liver weight was reduced by 21.6±4.1% (p<0.001) after fasting and increased by 9.7±2.5% (p<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<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<.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<.05 vs. ND). Since the primary function of NBCe1 is HCO3- import, T2D islet cells also exhibited significantly higher pHi (p<.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<.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.
212-OR: Testosterone Deficiency Increases Hypothalamic Gliosis and Metabolic Risk
Authors: DORFMAN MAURICIO D. , FREY JEREMY , NESS KELLY M. , NIRAULA ANZELA , KANTER JENNY E. , BORNFELDT KARIN , RUBINOW KATYA , SCHUR ELLEN , THALER JOSHUA
Published: June 2020

Obesity is associated with metabolic syndrome and gliosis, the activation of microglia (brain macrophages) and astrocytes, in the mediobasal hypothalamus (MBH) of humans and rodents. Similarly, hypogonadism in males is associated with increased susceptibility to weight gain, metabolic syndrome and MBH gliosis. Interventions that reduce glial activation in rodents result in protection from diet-induced obesity. Thus, we hypothesize that the combination of low testosterone and western diet causes gliosis, which in turn increases obesity susceptibility. To test this hypothesis in a rodent model, we combined gonadectomy (GDX) or sham surgery with high-fat diet (HFD) feeding in adult male mice. GDX and HFD feeding synergistically induced a striking activation of MBH astrocytes along with increased numbers of MBH microglia. This gliosis response was reversed to sham levels by testosterone replacement. After only 4 weeks of HFD feeding, differences in adiposity between GDX and sham groups were minimal with only a mild worsening of glucose tolerance. Thus, testosterone deficiency combined with HFD exposure elicits gliosis prior to substantial weight gain, suggesting a direct impact on glial cell function. To characterize the transcriptional response to testosterone deprivation and HFD exposure, we have developed mouse strains with GFP-tagged ribosomes (L10A) in astrocytes and microglia to allow purification of glial cell-specific mRNA from whole hypothalamic tissue samples (tagged ribosome affinity purification (TRAP)). In preliminary analyses, we have confirmed >10-fold enrichment of cell-specific mRNAs with de-enrichment of neuronal transcripts. These studies help reveal novel mechanisms by which hypogonadism increases the predisposition to metabolic disease.

Disclosure

M.D. Dorfman: None. J. Frey: None. K.M. Ness: None. A. Niraula: None. J.E. Kanter: None. K. Bornfeldt: None. K. Rubinow: None. E. Schur: None. J. Thaler: None.

Funding

American Heart Association (16SDG27010018); Diabetes Research Center (P30DK017047); Novo Nordisk (A116079); National Institutes of Health (R01DK119754)