bluuebunny/crossref_metadata_2025_split huggingface dataset:
2168-P: A Novel Method for Efficient and Homogeneous Viral Transduction of Pancreatic Islets
Authors: YESILDAG BURCAK , MIR-COLL JOAN , NEELAKANDHAN APARNA , FORSCHLER FELIX , BIERNATH ADELINN , LEIBIGER INGO B. , LEIBIGER BARBARA , BERGGREN PER-OLOF , MOEDE TILO , AMMALA CARINA 
Published: June 2019

Modification of gene expression in pancreatic islets can be a powerful strategy for understanding the pathology of diabetes and developing novel therapeutic strategies against it. However, amenability of the isolated islets to genetic manipulation has been limited to only a subset of cells at the periphery due to poor penetration of transduction particles. To address this issue, we developed a standardized islet model, produced by optimized dissociation and controlled scaffold-free reaggregation of primary human islet cells. This process allowed for an ideal experimental window for accessing and manipulating the pancreatic endocrine cells at their single cell state, while enabling production of uniform islet microtissues displaying long-term (&gt;28 days) and robust function. We used an adenovirus that allows tracking of transduced total cells, endocrine cells and beta cells by labeling them with three specific fluorescent reporters expressed from a single back-bone. To define the optimal transduction conditions, we introduced the virus at various titers during three different production stages; after islet dispersion, during and post reaggregation. We quantified transduction efficiency and viral penetration via 3D confocal microscopy followed by assessment of insulin secretory function, insulin content, and cell viability of transduced islet microtissues. Highly efficient (&gt;75%) and uniform transduction was achieved when the virus was added after cell dispersion and during reaggregation. Approximately 80-95% of transduced cells were endocrine cells, of which 50-63% corresponded to β-cells. Although highly transduced islet microtissues displayed decreased chronic (35-50%), basal (55-62%) and stimulated (65-75%) insulin secretion, a significant fold induction of insulin secretion and unaltered insulin/ATP content was observed. Here we present efficient genetic manipulation of functional reaggregated islets by viral transduction as a novel tool for diabetes research.

Disclosure
B. Yesildag: None. J. Mir-Coll: Employee; Self; InSphero. Employee; Spouse/Partner; Roche Pharma. A. Neelakandhan: None. F. Forschler: Employee; Self; InSphero. A. Biernath: None. I.B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. P. Berggren: None. T. Moede: None. C. Ammala: None.
217-OR: Islet Transplantation vs. Standard of Care for Type 1 Diabetes Complicated by Severe Hypoglycemia from the Collaborative Islet Transplant Registry (CITR) and T1D Exchange (T1DX) Registry
Authors: RICKELS MICHAEL R. , FOSTER NICOLE C. , BALLOU CASSANDRA M. , HERING BERNHARD J. , ALEJANDRO RODOLFO , BECK ROY , MILLER KELLEE , BARTON FRANCA B. 
Published: June 2019

Islet transplantation is an emerging treatment for T1D, in particular for those with long standing disease experiencing severe hypoglycemia events (SHE). A phase 3 trial of islet transplantation in T1D complicated by SHE demonstrated &gt; 80% of islet recipients achieved HbA1c &lt; 7.0% and absence of SHE at 1 year. To better understand the long-term benefit for glycemic control and risk of immunosuppression to kidney function associated with islet transplantation vs. standard of care, we selected patients from CITR with at least one SHE in the year prior to transplant, and compared them to patients from T1DX with at least one SHE in the prior year and followed over 5 years. The cohorts from CITR (n = 58) and T1DX (n = 213) were aged 42 ± 9 and 43 ± 17 with disease duration 29 ± 10 and 26 ± 15 years (P &lt; 0.01) and experienced 5 ± 11 and 2 ± 1 SHE (P &lt; 0.01) in the year prior to baseline with HbA1c 7.3 ± 1.0 vs. 7.8 ± 1.5 (P &lt; 0.001). Mean HbA1c decreased to 6.3% vs. ranging 7.8-7.6% over 5 years (P &lt; 0.001), and the % experiencing recurrent SHE was less in CITR than T1DX (P &lt; 0.01), such that 78-77% vs. 23-28% met the primary endpoint of HbA1c &lt; 7.0% and absence of SHE (P &lt; 0.001). Insulin requirements were slightly less at baseline in CITR than T1DX (0.52 ± 0.14 vs. 0.64 ± 0.31 U/kg/d; P &lt; 0.001), and decreased to 0.06-0.17 vs. remaining 0.66-0.56 U/kg/d over 5 years (P &lt;0.001) with 80-48% insulin independent in CITR. GFR was 92 ± 17 and 90 ± 29 ml/min/1.73 m2 at baseline, and decreased to 83-78 vs. 87-81 ml/min/1.73 m2 over 5 years (P &lt; 0.001). These data demonstrate long-term durability of islet transplantation to achieve near-normal glycemic control in the absence of SHE more often than that observed with standard of care delivered by specialized diabetes practice. Initial reduction in GFR observed during the first-year post-transplant may be attributable to the initiation of immunosuppression, but was not associated with further decline during 5 year follow-up.

Disclosure
M.R. Rickels: None. N.C. Foster: None. C.M. Ballou: None. B.J. Hering: Other Relationship; Self; Diabetes-Free, Inc. R. Alejandro: None. R. Beck: Other Relationship; Self; Abbott Laboratories, Ascensia Diabetes Care, Bigfoot Biomedical, Dexcom, Inc., Insulet Corporation, Lilly Diabetes, Roche Diabetes Care, Tandem Diabetes Care. K. Miller: None. F.B. Barton: None.

Funding
National Institutes of Health; Leona M. and Harry B. Helmsley Charitable Trust
2171-P: Excessive Drinking Impairs FGF-21/Beta-Klotho-Mediated Protective Effects against Islet Dysfunction and Apoptosis in Mouse Pancreatic Islets
Authors: YANG BAOCHEN , LEUNG POSING 
Published: June 2019

Excessive alcohol consumption has negative effects on β-cell insulin secretion and survival, thus increasing the risks of type 2 diabetes mellitus (T2DM). Meanwhile, fibroblast growth factor 21 (FGF-21) is a potent metabolic regulator for lipid and glucose homeostasis. Recently, FGF-21 and its obligate co-receptor β-klotho (KLB) have been shown to inhibit alcohol ingestion and metabolism in mice and human, probably via the mediation of gastric emptying rate and initial alcohol metabolism. Despite this evidence, whether heavy alcohol intake modulates the expression and function of FGF-21 in mouse islets is still ambiguous. Therefore, this study aimed to investigate this issue using in vitro/ex vivo and in vivo cell/animal models. In ex vivo studies, islets isolated from of C57BL/6J mice were cultured and treated with 80 mM alcohol for 24 h, mimicking the excessive alcohol condition. In vivo studies, the mice were gavaged with 3 g/kg ethanol/respective saline up to 3 weeks. Ex vivo results showed that the expression of FGF-21 and KLB was upregulated, whereas its receptor FGFR1, FGFR2, FGFR3 and FGFR4 were downregulated. In addition, islet apoptosis was increased as demonstrated by ELISA kit. Moreover, the expression of bax and SIRT1 were also downregulated. In corroboration, in vivo studies showed that glucose homeostasis and islet function were impaired by excessive alcohol drinking as demonstrated by OGTT and GSIS results. Consistently, the expression profile of FGF-21/its receptors was similar to those observed in ex vivo condition. Meanwhile, the expression of islet function-related genes (insulin1/2, IRS, PDX1, GLUT2, etc.) was also downregulated in in vivo experiments. Our preliminary data indicate that excessive drinking may induce β-cell dysfunction and apoptosis through the inhibition of FGF-21/KLB expression, and that islet FGF-21 signaling is associated with alcohol homeostasis and T2DM.

Disclosure
B. Yang: None. P. Leung: None.

Funding
Research Grants Council of Hong Kong (CUHK14107415)
2179-P: Overexpression Screening Identifies Mitochondrial Carbonic Anhydrase 5b as an Important Regulator of Glucose-Stimulated Insulin Secretion in Beta Cells
Authors: KOSUDA MINAMI , YAMAGUCHI SUGURU , NAGASAWA AKIKO , YAMANA MIDORI , KOIKE MASAO , NISHIOKA HIDENORI , ISHIHARA HISAMITSU 
Published: June 2019

Despite extensive research efforts, a precise picture of how glucose itself and its metabolites initiates insulin secretion in a concentration-dependent manner has unveiled. To identify candidate genes important for glucose-stimulated insulin secretion (GSIS), we have conducted screening of differentially expressed genes identified by microarray analyses between highly glucose-responsive and less responsive MIN6 subclones. For this purpose, a platform containing zeocin resistant gene flanked with a wild type and mutated flippase recognition target sites was integrated in the genome of MIN6 cells expressing Tet3G. The modified MIN6 cells have been used to generate stable transfectants expressing mRNA or shRNA of genes of interest by means of the recombinease-mediated cassette exchange method. Using this system, more than ten stable MIN6 cell transformants can be gnenerated at a time. By creating approximately 100 overexpressing transfectants, we have identified mitochondrial carbonic anhydrase 5b (Car5b) as an important regulator of GSIS. Car5b is a one of 15 members of carbonic anhydrases, which catalyze the interconversion of carbon dioxide and water to bicarbonate and protons, and modulate various intracellular processes, such as urea formation, HCO3- supply for carboxylation and regulation of intracellular acid-base balance. Car5b overexpression in MIN6 cells increased 14CO2 formation from H14CO3- (37 ± 14% increases, p &lt; 0.05) in isolated mitochondria and resulted in 32 ± 12% increases (P &lt; 0.05) in GSIS at 20 mM, while shCar5b expression reduced Car5b protein levels to 31.4 ± 4.4% (p &lt; 0.05), 14CO2 formation to 68.7 ± 8.1% (p &lt; 0.05), and GSIS at 20 mM to 77 ± 5% (p &lt; 0.05). In addition, we found that Car5b expressions in ob/ob mice were 3.7 ± 0.4-fold higher (p &lt; 0.01) and tended to be higher (p = 0.073) in HFD-mice. These data indicated that mitochondrial carbonic anhydrase Car5b plays a role in GSIS in pancreatic β-cells.

Disclosure
M. Kosuda: None. S. Yamaguchi: None. A. Nagasawa: None. M. Yamana: None. M. Koike: None. H. Nishioka: None. H. Ishihara: Research Support; Self; Daiichi Sankyo Company, Limited, Lilly Diabetes. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.
2180-P: HERG2 Channel Plays a Key Role in Insulin Secretion
Authors: ZHAO MIAOMIAO , YANG JINKUI 
Published: June 2019

Background: Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is mainly mediated by KATP channels. A problem commonly associated with drugs like the sulfonylureas, KATP channel inhibitor, is that KATP current inhibition is independent of the basal glucose levels and hypoglycemia is frequently observed. Nevertheless, the role of non-KATP K+ channels in insulin secretion is largely unknown. Here, we show that a voltage-gated K+ (Kv) channel, HERG2, plays a key role in insulin secretion.
Aims: To investigate the effects of HERG2 channel in β-cells.
Methods: Using TALEN-based method, we created Herg2 gene knock out (KO) mice. Patch-clamp experiments were performed to record Kv currents and action potential in primary cultured β-cells from wild type (WT) and KO mice. INS-1 cells, rat β-cell line, were transfected with shRNA of Herg2 gene or scramble shRNA to generated Herg2 transiently knock down (KD) or WT cells. Glucose-stimulated calcium levels were measured and GSIS experiments were performed in INS-1WT and INS-1KD cells.
Results: The Kv currents of β-cells from the KO mice decreased approximately 37.6% compared with that of the WT mice, indicating HERG2 channel constitutes 37.6% of all Kv currents. The action potential duration was significantly prolonged in KO β-cells, whereas the action potential magnitude and resting potential remained unchanged compared with WT β-cells. INS-1KD showed enhanced glucose-induced elevation of the intracellular Ca2+ concentrations, the most important ion that triggers insulin secretion in β-cell. Consistently, Herg2 knockdown significantly enhanced insulin secretion at a high glucose concentration, but did not potentiate insulin secretion at basal glucose concentration, suggesting the glucose-dependent insulin secretion of HERG2 channel.
Conclusions HERG2, as a non-KATP K+ channel, regulates the action potential duration of β-cells and plays a key role in insulin secretion. Inhibition of HERG2 currents leads to enhanced insulin secretion only in high glucose levels.

Disclosure
M. Zhao: None. J. Yang: None.
2181-P: Glucose-Induced Hyperphosphorylation of Microtubule-Associated Protein Tau Potentiates Islet Beta-Cell Insulin Secretion by Destabilizing the Microtubule Network
Authors: HO KUNG-HSIEN , GU GUOQIANG , KAVERINA IRINA 
Published: June 2019

The microtubule (MT) cytoskeleton of pancreatic β cells is important for glucose-stimulated insulin secretion (GSIS). We have recently shown that the MT-mediated transportation of insulin vesicles away from the plasma membrane limits insulin secretion, and that the disassembly of MTs is necessary for robust GSIS. Here, we report that high glucose induces rapid MT disassembly at the cell periphery measured by real-time imaging of photoconverted MTs in islet β cells. The MT-associated protein Tau regulates this process. Tau is known to stabilize MTs by direct association, and Tau hyperphosphorylation leads to MT disassembly. Along this line, we found that high glucose induced Tau hyperphosphorylation and dissociation from MTs in mouse islet β cells. ShRNA-based Tau knockdown in islets directly destabilized MTs, eliminating the glucose responsiveness of the MT dynamics, increasing basal insulin secretion, and impairing GSIS. Moreover, we showed that the glucose-induced Tau hyperphosphorylation relied on kinase GSK3, which is likely regulated by the PI3K-Akt, but not insulin signaling. We propose that Tau stabilizes MTs in low glucose to restrict basal secretion. In high glucose, Tau is hyperphosphorylated by GSK3 to promote MT disassembly at cell periphery to facilitate the release of the secretable insulin pool.

Disclosure
K. Ho: Research Support; Self; Eli Lilly and Company. G. Gu: None. I. Kaverina: None.
2183-P: miR-125b Is Regulated by Glucose via AMPK and Impairs ß-Cell Function
Authors: CHEUNG REBECCA , PIZZA GRAZIA , ROLANDO DELPHINE M. , CHABOSSEAU PAULINE L. , NGUYEN-TU MARIE-SOPHIE , LECLERC ISABELLE , RUTTER GUY A. , MARTINEZ-SANCHEZ AIDA 
Published: June 2019

miRNAs are tiny RNAs that repress gene expression post-transcriptionally, necessary for normal endocrine function. AMPK is essential for glucose homeostasis and a suggested target of metformin. β-cell-specific inactivation of AMPK in mice (AMPKdKO) impairs glucose homeostasis and β-cell function. AMPKdKO mice present dysregulation of several miRNAs that may contribute to these defects, including miR-125b (∼2.5 fold upregulated). High levels of miR-125b are associated with hyperglycaemia (HbA1c) in prediabetic, T2D and T1D subjects, suggesting miR-125b as a biomarker or contributor to the development of diabetes. The role of miR-125b in β-cells remains elusive although in other cell types regulates apoptosis, growth and differentiation. We aim to determine miR-125b function in β-cells and the role of glucose and AMPK in the regulation of its expression. miR-125b level in human islets strongly correlates with BMI and is induced by glucose in both mouse and human islets, while feeding mice a ketogenic (low sugar) diet reduced islet miR-125b. These effects disappeared in islets with β-cell specific deletion of AMPK whereas treatment of human islets with highly specific AMPK activators significantly decreased miR-125b, indicating that glucose regulates miR-125b via AMPK in both mice and humans. miR-125b overexpression in MIN6 cells limited cytokine-induced apoptosis whilst strongly reducing insulin content. RNA-Seq on these cells suggests a role for miR-125b in metabolism, respiration and cytokine receptor interactions and RNA-IP of the miRNA-induced silencing complex revealed novel miR-125b targets such as mannose-6-phosphate receptor (MPR), important for enzyme sorting within secretory granules. Preliminary data show that β-cell-specific inhibition of miR-125b using a molecular “sponge” improves glucose tolerance in mice fed a high-fat diet.
In summary, we reveal miR-125b as an important regulator of β-cell function that might contribute to the deleterious effects of hyperglycaemia on β-cells.

Disclosure
R. Cheung: None. G. Pizza: None. D.M. Rolando: None. P.L. Chabosseau: None. M. Nguyen-Tu: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma. A. Martinez-Sanchez: None.

Funding
Medical Research Council UK
2184-P: Foxo1-CoF Repressor (FCoR) Regulates Pancreatic Alpha- and Beta-Cell Identity by both DNA and Histone Methylation
Authors: KODANI NORIKO , KOBAYASHI MASAKI , KIKUCHI OSAMU , KITAMURA TADAHIRO , ITOH HIROSHI , NAKAE JUN 
Published: June 2019

We have previously identified a Foxo1 binding protein, FCoR, which suppresses Foxo1 activity by increasing acetylation. FCoR is expressed in pancreatic α- and β-cells. FCoR expression is decreased in high-fat fed mice and in Leprdb/db mice, and FcorKO mice exhibited decreased insulin secretion, increased α-cell mass and β to α-cell conversion, suggesting FCoR involvement in the pathophysiology of diabetes. In FcorKO islets, nucleus localization of Foxo1 was increased, and Foxo1 acetylation was significantly decreased compared to WT. These data suggest that FCoR regulates acetylation and activity of Foxo1 in islets. The qPCR in isolated islets from FcorKO showed increased Arx, Mafb and Gcg expression compared to WT, supporting the phenotype of increased α-cell mass. Interestingly, expression level of Mafa and Neurod1, which are the β-cell markers and target genes of Foxo1, were also significantly increased, confirming activation of Foxo1 in the absence of FCoR. FCoR suppresses the expression of Aristaless related homeobox (Arx), which is essential for α-cell differentiation, via increased methylation of the CG rich promoter region of Arx. One of the epigenetic modifications of histone, the tri-methylation at H3K4 (H3K4me3), is an important marker of Arx expression in α-cells. Chromatin immunoprecipitation assay revealed that H3K4me3 was significantly decreased in αTC cells transduced with Flag-FCoR compared to LacZ-transduced-cells, suggesting that FCoR suppresses Arx expression through epigenetic regulation involving both DNA and histone methylation. Our data show that FCoR regulates Foxo1 activity in islets, and that FCoR regulates pancreatic α- and β-cell identity by epigenetic regulation of DNA and histone methylation.

Disclosure
N. Kodani: Research Support; Self; Lilly Diabetes, Novartis Pharmaceuticals Corporation. M. Kobayashi: None. O. Kikuchi: None. T. Kitamura: None. H. Itoh: None. J. Nakae: None.
2188-P: Altered Transcription Factor Binding and Gene Bivalency May Contribute to Long-Term Gene Dysregulation in Intrauterine Growth-Retarded Rat Islets
Authors: LIEN YU-CHIN , LU XUEQING MAGGIE , WANG ZHIPING , SIMMONS REBECCA A. 
Published: June 2019

Intrauterine growth retardation (IUGR), which induces epigenetic modifications and permanent changes in gene expression, has been associated with the development of type 2 diabetes. Using a rat model of IUGR induced by bilateral uterine artery ligation, we performed ChIP-Seq and RNA-Seq to assess the association of genome-wide histone modifications and gene dysregulation in islets from 2 and 10 week rats. IUGR induced significant changes in enrichment of H3K4me3 and H3K27me3 marks in both 2-week and 10-week islets, which were correlated with expression changes of multiple genes critical for normal islet function in IUGR islets. HOMER analysis showed that IUGR-induced histone mark changes were enriched at transcription factor binding motifs, such as Nkx2-1, Nkx2-5, FoxL2, Pax7, Sox4, Mitf, and Oct4-Sox2-Nanog. These transcription factors were also identified by Ingenuity Pathway Analysis of islet transcriptomes as top upstream regulators that regulate the changes in gene expression and biological activities. In addition, our ChIP-seq data revealed more than 1,000 potential bivalent genes as identified by enrichment of both H3K4me3 and H3K27me3. No expression or very low expression of these genes was consistent with a poised state of transcription. The poised state of many potential bivalent genes was altered by IUGR. Interestingly, 3 genes important for islet function, Acod1, FGF-21, and Serpina11, which were poised in 2-week control islets, lost their poised state in 10-week control islets, but gained bivalency in 10-week IUGR islets. In contrast, another 3 genes, Cdh16, Lrrc27, and Lrrc66, which were not poised in control 2-week islets, gained de novo bivalency in 10-week control islets, but lost their poised state in 10-week IUGR islets. Collectively, our findings suggest alterations of histone modification in key transcription factors and genes which may contribute to long-term gene dysregulation and an abnormal islet phenotype in IUGR rats.

Disclosure
Y. Lien: None. X.M. Lu: None. Z. Wang: None. R.A. Simmons: None.

Funding
National Institutes of Health
2193-P: SREBP1c-PAX4 Axis Mediates Pancreatic Beta-Cell Compensatory Responses upon Metabolic Stress
Authors: KIM JAE BUM 
Published: June 2019

Sterol responsive element-binding protein 1c (SREBP1c) is a key transcription factor for de novo lipogenesis. Although SREBP1c is expressed in pancreatic islets, its physiological roles in pancreatic beta cells are largely unknown. In this study, we demonstrate that SREBP1c regulates beta cell compensation under metabolic stress. SREBP1c expression level was augmented in pancreatic islets from obese and diabetic animals. In pancreatic beta cells, SREBP1c activation promoted the expression of cell cycle genes and stimulated beta cell proliferation through its novel target gene, PAX4. Compared to SREBP1c+/+ mice, SREBP1c-/- mice showed glucose intolerance with low insulin levels. Moreover, beta cells from SREBP1c-/- mice exhibited reduced capacity to proliferate and secrete insulin. Conversely, transplantation of SREBP1c overexpressing islets restored insulin levels and relieved hyperglycemia in streptozotocin-induced diabetic animals. Collectively, these data suggest that pancreatic SREBP1c is a key player in mediating beta cell compensatory responses in obesity.

Disclosure
J. Kim: None.

Funding
Korean National Research Foundation (2011-0018312)