bluuebunny/crossref_metadata_2025_split 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&lt;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 &lt; 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&lt;0.01), whereas that of β cells was the highest in I (p&lt;0.01). CCND2 was robustly expressed in the nucleus of β cells in F (&gt;90%) and I (&gt;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&gt;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.