bluuebunny/crossref_metadata_2025 huggingface dataset:
2062-P: Pancreatic Inflammation Implicates Increased Cellular Plasticity in the Pancreatic Duct Glands in Type 1 Diabetes
Authors: KULKARNI SHWETA S. , BUTLER ALEXANDRA E. , ATKINSON MARK A.
Published: June 2020

Type 1 diabetes (T1D) is an autoimmune disease characterized by endocrine/exocrine pancreas inflammation, fibrosis and, ultimately, decreased organ mass. Accumulating evidence suggests that this chronic inflammation may play a pivotal role in the initiation and progression of endogenous pancreatic cell plasticity. Pancreatic duct glands (PDGs), a potential stem/progenitor cell niche, represent a distinct anatomical compartment of the pancreas, forming blind-ending outpouchings from the main pancreatic duct and its branches. PDGs become activated in response to inflammation in T1D, evidenced by a high frequency of epithelial cell replication and an increase in progenitor-like cells. We addressed the hypothesis that in T1D, perturbation of hormone and non-hormone expressing cells in the interlobular ducts (ILD) and PDGs is a consequence of inflammation. Pancreatic sections from 21 T1D (disease duration 3 - 84 y) and 22 age-matched nondiabetic (ND) donors were evaluated for replication and expression of known pancreatic endocrine hormones in ducts and PDGs. We confirmed increased duct and PDG replication in T1D (T1D vs. ND— ILD: 2.8 ± 0.9 vs. 0.7 ± 0.1%, p<0.05; PDG: 3.4 ± 1.0 vs. 1.1 ± 0.4%, p<0.05) and reduced frequency of insulin expressing cells (T1D vs. ND— ILD: 0.0 ± 0.0 vs. 0.4 ± 0.1%, p<0.001; PDGs: 0.01 ± 0.01 vs. 0.8 ± 0.3%, p<0.05). The frequency of PDG cell replication was highest in T1D donors with acute pancreatitis. No differences were found in the frequency of other endocrine cell types regardless of the presence of pancreatic inflammation. These data suggest that increased pancreatic ductal cell replication in the PDG compartment of subjects with T1D is associated with sustained autoimmunity-related inflammation. Efforts are underway to determine specificity of pancreatic ductal cell changes to islet or acinar inflammation and the identity of replicating ILD and PDG cells, to better understand the mechanisms underlying pancreatic ductal cell plasticity.

Disclosure

S.S. Kulkarni: None. A.E. Butler: None. M.A. Atkinson: None.
2074-P: Histone Deacetylase Inhibitors Improve Store-Operated Calcium Entry and Glucose-Stimulated Insulin Secretion in Cytokine-Stressed Pancreatic ß-Cells
Authors: LEE CHIH-CHUN , KONO TATSUYOSHI , WEAVER STACI A. , SOHN PAUL , EVANS-MOLINA CARMELLA
Published: June 2020

Store-operated calcium entry (SOCE) is a mechanism that functions to refill endoplasmic reticulum (ER) Ca2+ stores in response to ER Ca2+ depletion, through gating of plasmallemal Ora channels by the ER Ca2+ sensor STIM1. Dysfunctional β cell SOCE has been observed in response to cytokine-mediated stress and in models of diabetes. To identify compounds capable of modulating SOCE, a screening was performed in INS-1 cells treated with interleukin-1β (IL-1β). Two HDAC inhibitors (HDACi), sodium butyrate (NaB) and MS275, rescued SOCE in a dose-dependent manner in IL-1β-treated INS-1 cells. While dysregulation of HDACs has been linked with metabolic disease, the relationship between HDACs and SOCE has not been tested in the pancreatic β cell. To this end, we explored the effect of HDACi on Ca2+ signaling, insulin secretion, and gene expression in cytokine-treated islets and β cells. In mouse islets treated with IL-1 β and interferon-𝛾;, NaB and MS275 increased first phase Ca2+ responses in response to glucose, while NaB restored glucose-induced Ca2+ oscillations. Consistent with this, NaB, but not MS275, restored GSIS in IL-1β-treated INS-1 cells and in human islets from donors with type 2 diabetes. Chronic (24 hrs) but not acute (8 hrs) HDACi treatment restored cytokine-mediated reductions in SOCE, raising the possibility that changes in gene expression may underlie these effects. To test this, qRT-PCR for SOCE molecular components was performed. Both NaB and MS275 restored STIM1 expression but had no effect on Orai1, Orai2 and Orai3 levels. Consistent with this, HDACi were unable to restore SOCE in IL-1β-treated STIM1 knockout INS-1 cells.

Taken together, these data suggest that HDAC inhibition restores insulin secretion under pro-inflammatory conditions through improved SOCE and upregulated STIM1 expression. Thus, our findings identify a novel pathway through which HDAC inhibition exerts protective effects in the diabetic β cell.

Disclosure

C. Lee: None. T. Kono: None. S.A. Weaver: None. P. Sohn: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (2R01DK093954)
2075-P: Testing the First Selective Talk-1 Inhibitors for Their Ability to Improve ß-Cell Function in Diabetes
Authors: NAKHE ARYA Y. , DADI PRASANNA , WESTOVER DAVID , DAYS EMILY , DENTON JEROD , JACOBSON DAVID
Published: June 2020

Insulin levels are inadequate to maintain euglycemia in patients with type-2 diabetes (T2D), which is due in part to perturbations in pancreatic ꞵ-cell Ca2+ homeostasis. TALK-1 K+ channels are key regulators of ꞵ-cell electrical excitability, Ca2+ handling and glucose-stimulated insulin secretion (GSIS). KCNK16, which encodes TALK-1 is the most abundant ꞵ-cell K+ channel transcript and also the most islet-restricted ion channel transcript. Furthermore, a nonsynonymous coding sequence polymorphism in KCNK16 causes a predisposition for developing T2D. However, the ability to utilize TALK-1 as a therapeutic target to normalize ꞵ-cell Ca2+ homeostasis in T2D has not been determined. Here, we optimized a high throughput fluorescence-based thallium (TI+) flux assay (z’ =0.64) to screen for TALK-1 inhibitors, which resulted in identification of 286 TALK-1 inhibitors with potent drug-like properties (from a 50,000 compound screen). Of these hits, 78 exhibit TALK-1 selectivity and do not influence the activity of other K+ channels including TALK-2, TREK-2, TASK-1, TASK-3, KATP or hERG channels. We then performed dose response curve analyses and identified 9 potent inhibitors with IC50s < 2.5μM that inhibit TALK-1 activity (>65%). To test the efficacy of the inhibitors for their ability to augment islet function, we assessed their impact on mouse islet Ca2+ handling. TALK-1 inhibitors significantly increased islet Ca2+ oscillation frequency (e.g., VU0233408, 2.47 peaks/min vs. DMSO, 1.82 peaks/min, p-value= 0.011), similar to what was previously observed in TALK-1 KO islets. Moreover, the inhibitors did not increase glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. Thus, pharmacological inhibition of TALK-1 is predicted to increase GSIS.

In conclusion, we have identified the first potent and selective TALK-1 inhibitors that can be utilized to assess the role of TALK-1 during human ꞵ-cell GSIS under physiological and diabetic conditions.

Disclosure

A.Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (R01DK115620VDC); Vanderbilt University Diabetes and Research Training Center (P60DK20593)
2078-P: APPL2 Deficiency Diminishes Glucose-Stimulated Insulin Secretion in Pancreatic Beta Cells
Authors: LIN HUIGE
Published: June 2020

In response to elevated postprandial blood glucose, remodeling of F-actin enables exocytosis of insulin granules from pancreatic β cells. Dysregulation of F-actin remodeling contributes to defective glucose-stimulated insulin secretion (GSIS) in β cells and subsequent type 2 diabetes. The adaptor protein APPL1 potentiates GSIS and prevents β cell loss in diabetes, but the role of its close homolog APPL2 in β cell function remains unknown. Here we demonstrated that APPL2 controls GSIS by Rac1-mediated F-actin depolymerization via interaction with Rac GTPase activating protein 1 (RacGAP1). β-cell specific deletion of APPL2 induced defective GSIS and glucose intolerance in mice. Ex vivo study revealed a dramatic reduction in both first- and second-phase GSIS in islets, accompanied by a dysregulation of F-actin remodeling. Consistently, knockdown of APPL2 caused impaired glucose-induced F-actin remodeling and insulin secretion in INS-1E β cells. The defects were mainly due to the inactivation of Rac1- the key player controlling insulin granule secretion via F-actin remodeling. Ectopic expression of constitutively active Rac1 mutant rescued the defective GSIS and F-actin remodeling in APPL2 deficient β cells. Furthermore, we identified RacGAP1 as an interacting partner of APPL2. RacGAP1 interacted with and regulate activity of Rac1, and overexpression of RacGAP1 significantly suppressed glucose-induced F-actin depolymerization, whereas co-expression with APPL2 antagonized this suppressive effect. In addition, the defects in APPL2 knockdown β cells were largely reversed by simultaneous downregulation of RacGAP1 or co-expression of constitutively active Rac1 mutant.

In summary, our study suggest that APPL2 potentiates GSIS by antagonizing the inhibitory effect of RacGAP1 on Rac1 activation and F-actin remodeling and insulin secretion in β cells, highlighting the importance of APPL2 in the regulation of GSIS in pancreatic β cells and glucose homeostasis.

Disclosure

H. Lin: None.

Funding

National Natural Science Foundation of China (81970675)
208-LB: Stimulation of the ß3 Adrenergic Receptors Prime Human White and Brown Adipocytes for Increased Lipolysis and Thermogenesis
Authors: LEA HANNAH J. , ZHU KENNETH Y. , CERO CHERYL , CYPESS AARON M.
Published: June 2020

Recent clinical trials show that chronic activation of the β3-adrenergic receptor (AR) by mirabegron leads to numerous beneficial physiological responses, including increased energy expenditure, insulin sensitivity, and improved lipoprotein profile. Preliminary evidence suggests that they are mediated in part through adaptations by adipose tissue, but little is known about how mirabegron affects the cellular metabolism of human white or brown adipocytes (hWA and BA, respectively). We therefore treated paired immortalized hWA and hBA for 6 hours with a range of mirabegron doses (1 nM - 10 µM), as well as the β1- and β2-AR selective agonists, dobutamine and terbutaline, at 10 µM. We found that in untreated cells, ADRB1 and ADRB2 were higher in hWA than hBA, but ADRB3 was higher in hBA. However, mirabegron at all concentrations increased the expression of ADRB3 in hWA, and the highest dose of mirabegron boosted expression above that in hBA. While an increase in ADRB3 was detected in hWA with β1- and β2-agonist treatment, no changes occurred to the other two β-ARs when exposed to their selective agonists. Downstream, the β1- and β2-agonists increased thermogenic UCP1 expression in hWA, but mirabegron raised UCP1 expression only in hBA. Among the genes associated with lipolysis, glucose metabolism, and insulin signaling, mirabegron 10 µM promoted the expression of ATGL and HSL encoding genes, as well as GLUT4 in hBA only. In contrast, no changes occurred in hWA.

In summary, chronic exposure to a β3-AR agonist primes human white adipocytes to respond more potently to adrenergic-mediated lipolysis and enables human brown adipocytes to increase their thermogenic capacity.

Disclosure

H.J. Lea: None. K.Y. Zhu: None. C. Cero: None. A.M. Cypess: None.
2083-P: Dynamics of D-Amino Acids in Mammalian Pancreatic Islets
Authors: LEE CINDY J. , PATEL AMIT , LEE DONGKYU , RUBAKHIN STANISLAV , SWEEDLER JONATHAN V.
Published: June 2020

Evidence has shown the presence of D-amino acids (D-AAs), unusual cell-to-cell signaling molecules, in the pancreatic islets of rodents and humans. In particular, D-alanine (D-Ala) and serine racemase, the enzyme that converts L- to D-serine (D-Ser), colocalize within insulin-producing beta cells. Both D-Ala and D-Ser function as co-agonists of the N-methyl-D-aspartate (NMDA) receptor, which is also present in islets and modulates insulin release, suggesting potential autocrine or paracrine signaling role of these compounds. Here, we investigated the relationship between the endogenous levels of D-AAs and islet regulation of glucose homeostasis. The glucose-stimulated D-AA profiles in the rat islets revealed that the L-AA forms were released from the pancreatic islets in a dose-dependent manner. Interestingly, we observed between a 20% and 45% reduction of D-Ala and D-Ser levels, respectively, in islets after the stimulation which appears to be independent of the extracellular glucose levels. In addition to islets, significant D-AAs were detected in the surrounding acinar tissue; only D-Ala was abundant at high percentage of 11.6 ± 1.5% to total Ala in the tissue, while other D-AAs were not measured at high levels. As beta cells appear not to be able to synthesize their own D-Ala, this suggests that the D-Ala detected in these cells can be supplied by nearby acinar tissue. We also have measured the D-Ser percentages in healthy and type 2 diabetes (T2D)-affected islets received from the Human Pancreas Analysis Program (UPenn). Preliminary results revealed the lower D-Ser level in the T2D-affected islets compared to healthy islets. We are continuing to accumulate more data to determine the potential correlation of D-AAs, including D-Ala and D-Ser, to diabetes progression. The continued characterization of endogenous D-AAs levels will shed light on their roles in islet normal and pathological functions.

Disclosure

C.J. Lee: None. A. Patel: None. D. Lee: None. S. Rubakhin: None. J.V. Sweedler: None.

Funding

American Diabetes Association/Pathway to Stop Diabetes (1-18-VSN-19 to J.V.S.)
2087-P: Functional Genomics of Human Islet Gluco/Lipotoxicity and Type 2 Diabetes
Authors: YI XIAOYAN , MARSELLI LORELLA , PIRON ANTHONY , SULEIMAN MARA , SZYMCZAK FLORIAN , EIZIRIK DECIO L. , MARCHETTI PIERO , CNOP MIRIAM
Published: June 2020

Gluco- and lipotoxicity impair β cell function and survival, potentially contributing to type 2 diabetes onset and progression. Here we profiled transcriptomes of human islets in gluco/lipotoxic and type 2 diabetes conditions. Human islets from 26 nondiabetic donors were exposed to 0.5 mM palmitate (P), 22.2 mM glucose (G), P+G, 1 mM palmitate+oleate, (1:2 ratio, P+O) and P+O+G for 2 days (n=2-5/condition), followed by 4 days of washout. Glucose-stimulated insulin secretion and RNA-seq transcriptomes were assessed. Data were compared to RNA-seq of islets from 34 type 2 diabetic and 62 nondiabetic donors by rank-rank hypergeometric overlap (RRHO), and to the Connectivity Map to identify therapeutic targets. PO did not impair insulin secretion, P or G induced transient dysfunction (resolved after washout), while PG or POG induced persistent β cell dysfunction. Among common upregulated pathways in lipo- and gluco/lipotoxicity were PPAR signaling, protein processing in the endoplasmic reticulum and aminoacyl tRNA biosynthesis (gene set enrichment analysis). The greatest overlap by RRHO between upregulated genes in gluco/lipotoxicity and type 2 diabetes was seen for washout of PG and POG (conditions of persistent β cell dysfunction), with gene set enrichment for inflammatory responses and extracellular matrix organization. Comparison of gluco/lipotoxic and type 2 diabetes gene signatures with the Connectivity Map identified endoplasmic reticulum stress signaling and knockdown of the cholesterol sensor SCAP as strongly positively correlated pathways. A negative correlation was found for JNK and DNA protein kinase inhibitors, pointing to compounds that could revert gluco/lipotoxic and type 2 diabetes gene expression signatures.

In conclusion, human islet transcriptome signatures of transient or persistent gluco/lipotoxicity identify similarities to type 2 diabetes islet transcriptomes and point to novel therapeutic targets.

Disclosure

X. Yi: None. L. Marselli: None. A. Piron: None. M. Suleiman: None. D.L. Eizirik: None. P. Marchetti: Consultant; Self; Menarini Group. Research Support; Self; Janssen Pharmaceuticals, Inc. M. Cnop: None.
2094-P: Effects of Irisin on Human Pancreatic Islets from Type 2 Diabetic Subjects
Authors: MARRANO NICOLA , NATALICCHIO ANNALISA , BIONDI GIUSEPPINA , CIGNARELLI ANGELO , VINCENTI LEONARDO , LUPO LUIGI G. , PERRINI SEBASTIO , LAVIOLA LUIGI , GIORGINO FRANCESCO
Published: June 2020

Irisin is a hormone secreted by skeletal muscle following physical activity and excess of saturated fatty acids, able to improve metabolic homeostasis and promote energy expenditure. In a previous study, we have demonstrated that, in beta-cells, irisin promotes proliferation and glucose-stimulated insulin secretion (GSIS), increases insulin content levels, and reduces lipotoxicity-induced apoptosis. The aim of this study was to evaluate the effects of irisin on the function and survival of pancreatic islets isolated from patients with type 2 diabetes mellitus (T2DM), characterized by reduced ability to secrete insulin and high levels of apoptosis, and to investigate the molecular mechanisms underlying this action. Pancreatic islets isolated from T2DM patients (n=7) and nondiabetic subjects (ND) (n=3), as well as INS-1E cells, were exposed to 100 nM irisin for different times. GSIS, insulin content, apoptosis and cytoplasmic calcium levels were measured by ELISA assays; the activation of the main mediators of beta-cell intracellular signalling (AKT, CREB and PKA) was evaluated by immunoblotting. Irisin increased GSIS approximately 2-fold (p<0.05) in both ND and T2DM islets. Notably, in T2DM islets, characterized by reduced GSIS, this was restored to the levels of ND islets. In addition, irisin increased insulin content (1.6-fold in ND, 1.9-fold in T2DM, p<0.05) and tendentially decreased apoptosis levels only in T2DM islets. Moreover, irisin was able to activate AKT and CREB under conditions of high glucose in the ND islets, without changing cytoplasmic calcium levels. In contrast, in the T2DM islets, irisin did not induce AKT or CREB activation.

In conclusion, the myokine irisin is able to increase the secretory function and survival of human pancreatic islets and to improve the functional defects typical of the T2DM islets. The mechanisms underlying the secretagogue effects of irisin in the T2DM islets do not apparently involve the known signaling machinery of this novel hormone.

Disclosure

N. Marrano: None. A. Natalicchio: Consultant; Self; AstraZeneca, Novo Nordisk Inc., Sanofi-Aventis. G. Biondi: None. A. Cignarelli: None. L. Vincenti: None. L.G. Lupo: None. S. Perrini: None. L. Laviola: Advisory Panel; Self; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis. Speaker’s Bureau; Self; Abbott, Medtronic. F. Giorgino: Advisory Panel; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi. Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., LifeScan, Inc., MedImmune, Merck Sharp & Dohme Corp., Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Development Centre Europe Ltd.

Funding

European Union (AIM1810057)
2099-P: Combined Proteomics and Lipidomics Studies Uncovered New Molecular Alterations Underlying Beta-Cell Glucotoxicity
Authors: WOODS JAMES , PAN BO , CHEN XUEQUN
Published: June 2020

In T2D, chronic hyperglycemia causes beta cell dysfunction and failure. To systematically characterize the molecular alterations underlying this detrimental process, quantitative proteomic studies were carried out to compare global protein changes in INS-1 832/13 cells in normal (11mM) or high glucose (HG, 25mM) for 48 hours. In parallel, untargeted lipidomic studies were carried out to compare global lipid changes. In four experiments, a total of 4088 proteins were quantified, among which 184 proteins were significantly altered upon HG treatment. While we have confirmed some known hallmark changes (e.g., PDX1 and insulin) in beta cell glucotoxicity, most of these protein changes have not been reported previously. These proteins involve in key biological processes such as cell cycle regulation, calcium homeostasis, insulin biogenesis, protein folding, and ER export. Consistent with the significantly increased triglycerides in the lipidomic study, fatty acid synthase and acetyl-CoA carboxylase, two key enzymes in fatty acid synthesis, were significantly upregulated. In addition, for the first time in beta cells, MIG12 was found highly upregulated (4.50±1.50, n=4) in HG treated cells. This change was confirmed by Western blotting in mouse and human islets. MIG12 was previously reported in the liver to induce ACC1 polymerization and enhance its enzymatic activity. However, its role in beta cell is unknown. Our preliminary results showed that MIG12 knockdown inhibited HG-induced lipid droplet formation in beta cells. Among other novel findings, ether lipids and its regulating enzymes were significantly altered. This lipid change was confirmed by thin layer chromatography, and the enzyme change confirmed by Western blotting in beta cells and immunofluorescence in db islets. Functional consequences of several key protein changes are being investigated. Taken together, our study provides new targets for mechanistic studies of beta cell glucotoxicity.

Disclosure

J. Woods: None. B. Pan: None. X. Chen: None.

Funding

National Institutes of Health (R01DK110314)
21-LB: Derived Time-in-Range Is Associated with MACE in T2D: Data from the DEVOTE Trial
Authors: BERGENSTAL RICHARD M. , HACHMANN-NIELSEN ELISE , KVIST KAJSA , BUSE JOHN B.
Published: June 2020

Background: There is a need to validate time-in-range (TIR; percentage of time with plasma glucose between 70 and 180 mg/dL (3.9-10.0 mmol/L) as a surrogate endpoint for long-term clinical outcomes.

Methods: We used data from patients with 8-point glucose profiles (8pp) from the double-blind cardiovascular outcomes trial, DEVOTE (NCT01959529). In total, 7637 patients with T2D and either established CVD or at high risk for CVD were included in the trial. The primary endpoint in DEVOTE was time to first MACE. The 8pp were collected at 1 year, 2 years and end-of-trial. Median length of follow-up was 2 years. For 5644 patients, 8pps with at least 7 points existed. Among the 681 major adverse cardiovascular events (MACEs) in DEVOTE, 360 were among patients with 8pps. Individual TIR was derived as the proportion of the 8pp within range. A Cox model was used to estimate the association between derived TIR and time to first MACE. Hazard ratios (HR) were estimated for patients with TIR>70% vs. TIR≤70%, and for TIR>70% and TIR 50 −70% vs. TIR≤50%.

Results: Derived TIR was >70% for 65% of the patients. Estimated rate of first MACE was lower for TIR >70% and TIR 50-70% vs. TIR≤50% (Figure) and for TIR>70% vs. TIR≤70% (HR: 0.74 [0.60;0.91]95% CI; p<0.01). The associations were maintained when analyses were adjusted for baseline characteristics.

Conclusions: Derived TIR was associated with rate of first MACE for T2D patients in DEVOTE.

Disclosure

R.M. Bergenstal: Consultant; Self; Ascensia Diabetes Care, Johnson & Johnson. Other Relationship; Self; Abbott, Dexcom, Inc., Hygieia, Lilly Diabetes, Medtronic, Novo Nordisk A/S, Onduo, Roche Diabetes Care, Sanofi, UnitedHealth Group. E. Hachmann-Nielsen: Employee; Self; Novo Nordisk A/S. K. Kvist: Employee; Self; Novo Nordisk A/S. J.B. Buse: Consultant; Self; Cirius Therapeutics, CSL Behring, Neurimmune. Research Support; Self; American Diabetes Association, National Institutes of Health, Novo Nordisk A/S, Patient-Centered Outcomes Research Institute, Sanofi, Tolerion, Inc., vTv Therapeutics. Stock/Shareholder; Self; Mellitus Health, Pendulum Therapeutics, PhaseBio Pharmaceuticals, Inc., Stability Health. Other Relationship; Self; ADOCIA, AstraZeneca, Dance Biopharm Holdings, Inc., Eli Lilly and Company, MannKind Corporation, NovaTarg Therapeutics, Novo Nordisk A/S, Senseonics, Inc, vTv Therapeutics, Zafgen, Inc.

Funding

Novo Nordisk A/S
2101-P: Roles of Glycolysis Distinct from Mitochondrial Metabolism for Signal Generation in Insulin-Secreting Cells
Authors: ISHIHARA HISAMITSU , NISHIOKA HIDENORI , YAMANA MIDORI , NAGASAWA AKIKO , KOSUDA MINAMI , KOIKE MASAO , KOHNO GENTA , SAITO HITOKI
Published: June 2020

Glucose produces, through glycolysis, pyruvate, a substrate for mitochondrial metabolism. Mitochondrial metabolism generates signals for insulin exocytosis, which include ATP and other as yet unidentified molecules. Although important roles of mitochondrial metabolism of glucose for insulin secretion is established, roles of glycolysis are not completely understood. Does glycolysis merely produce pyruvate for mitochondrial metabolism or generate its own signal? To study this question, we have created mitochondrial pyruvate carrier 2 (Mpc2) knockout MIN6 cells by the Crispr technology. We first generated MIN6 cells expressing MPC2-Flag under the control of Tet3G transcriptional activator, to assure mitochondrial metabolism of glucose in a condition of Mpc2 alleles being knocked out. Then, stop codons followed by the zeocin or puromycin-resistant gene units were introduced in the second exons of the gene. We have confirmed a complete loss of endogenous MPC2 expression by Western blot analyses and these cells were maintained in the presence of doxycycline, expressing MPC2-Flag. Upon withdrawal of doxycycline, glycolytic flux estimated by 5-[3H]glucose utilization was unaltered at 5 mM glucose but reduced by 37.2% ± 1.2% (Mean ± SE, n = 3, p < 0.05) at 12.5 mM and 55.3% ± 9.6% (p < 0.05) at 20 mM. In MPC2-deficient MIN6 cells, glucose-stimulated insulin secretion was completely abolished at 12.5 and 20 mM glucose, while glutamine (5 mM) plus leucine (5 mM) induced insulin secretion was comparable to that by doxycycline-treated MPC2-Flag expressing cells. Interestingly, when MPC2-deficient MIN6 cells were activated with glutamine plus leucine, 5-20 mM glucose was able to cause further dose-dependent increases in insulin secretion with 1.9 ± 0.3-fold (n = 4, p < 0.05) augmentation at 20 mM. These data suggested that glycolysis generates signals for insulin secretion distinct from those produced by the mitochondria in glucose-stimulated insulin secreting cells.

Disclosure

H. Ishihara: Advisory Panel; Self; Astellas Pharma Inc. Research Support; Self; Daiichi Sankyo, Eli Lilly Japan K.K., Kowa Company, Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi. H. Nishioka: None. M. Yamana: None. A. Nagasawa: None. M. Kosuda: None. M. Koike: None. G. Kohno: None. H. Saito: None.
2105-P: Beta Cell PCSK1 Deficiency Increases Diabetes Susceptibility in Male Mice
Authors: TAYLOR AUSTIN , CHEN YI-CHUN , VERCHERE BRUCE
Published: June 2020

Proinsulin (PI) is processed to mature insulin by prohormone convertases 1/3 (Pcsk1) and 2 (Pcsk2). In individuals with T1D and T2D, there is an increase in the circulating PI:insulin ratio. We hypothesized that beta-cell Pcsk1 deletion would result in beta-cell dysfunction and hyperglycemia.

Male and female Pcsk1flox/flox Ins1cre/+ (Pcsk1beta-KO) and Pcsk1+/+ Ins1cre/+ (Pcsk1beta-WT) mice were fed either a chow or a high fat diet (HFD; 45% fat) and monitored until 30 weeks of age. Despite an approximately 150-fold increase in fasting plasma PI (p < 0.001) and only immature insulin granules detectable by electron microscopy, chow-fed Pcsk1beta-KO male mice had normal glucose tolerance early in life and only developed glucose intolerance by 26 weeks of age (AUC: 934±294 vs. 597±223; p = 0.008). Histological analysis showed that male Pcsk1beta-KO mice displayed a trend toward increased beta-cell area (1.3±0.6 vs. 0.8±0.4%; p = 0.08). The unexpectedly mild hyperglycemia in Pcsk1beta-KO mice is likely due to incomplete disruption of proinsulin processing, as mass spectrometry and western blot analysis of Pcsk1beta-KO islets confirmed markedly elevated proinsulin, but detectable mature insulin. In HFD-fed cohorts, 3/10 Pcsk1beta-KO male mice developed sustained fasting hyperglycemia (>16 mM) after 19 weeks of diet. Chow and HFD-fed female Pcsk1beta-KO mice displayed no differences in glycemia or body weight from Pcsk1beta-WT controls. Deletion of both Pcsk1 and Pcsk2 in beta cells (Pcsk1flox/flox Pcsk2flox/flox Ins1cre/+) resulted in undetectable mature insulin in islets (western blot), and impaired glucose tolerance at 10 weeks of age in both chow-fed male and female mice.

Our results suggest that beta-cell Pcsk1 deficiency is not sufficient to drive diabetes development, but increases diabetes susceptibility in conditions of increased insulin secretory demand. Our data in mice support the idea that beta-cell prohormone processing errors likely contribute to pathogenesis of diabetes but not obesity.

Disclosure

A. Taylor: None. Y. Chen: None. B. Verchere: Advisory Panel; Self; Integrated Nanotherapeutics Inc., Sirona Biochem. Stock/Shareholder; Self; Integrated Nanotherapeutics Inc.

Funding

Canadian Institutes of Health Research (PJT153156)
211-LB: Branched-Chain Amino Acid Levels Negatively Associate with Postprandial ß-Cell Function in Type 1 Diabetes
Authors: KARUSHEVA YANISLAVA , STRASSBURGER KLAUS , MARKGRAF DANIEL F. , ZAHARIA OANA P. , BODIS KALMAN , TURA ANDREA , PACINI GIOVANNI , BURKART VOLKER , SZENDROEDI JULIA , RODEN MICHAEL
Published: June 2020

Understanding the role of insulin resistance (IR) in type 1 diabetes (T1D) may help to improve its treatment. Postprandial insulin secretion stimulates amino acid (AA) uptake and protein synthesis. Consequently, impaired insulin-mediated AA clearance could raise postprandial branched-chain AA (BCAA: valine (VAL), leucine (LEU), isoleucine (ILE)) levels and in turn contribute to BCAA-induced IR. We hypothesized that BCAA levels associate negatively with postprandial ß-cell function already in recent-onset T1D. In a cross-sectional study, volunteers of the German Diabetes-Study with T1D (n=10, diabetes duration <1 year) and excellent glycemic control (HbA1C 6.6±0.9%/48±9 mmol/mol), and age-, sex- and BMI- matched glucose tolerant humans (control, CON, n=10, HbA1C 5.1±0.3%/32±3 mmol/mol) underwent a mixed-meal tolerance test (MMT) to assess ß-cell function through disposition index (DI, the product of insulin sensitivity (IS) and acute insulin response to glucose) and postprandial IS (PREDIM index, predicted M-value). Adipose tissue (AT) IR index was calculated as fasting plasma levels of FFA (mmol*l-1) x insulin (pmol*l-1). Postprandial total BCAA, VAL and LEU levels were 25% higher in patients with T1D compared with CON (AUC 11167±5136 vs. 8410±3298, 14363±959 vs. 11754±3890, 11980±4317 vs. 8476±4257 µmol*l-1*3 h-1, p<0.05, respectively). PREDIM was lower in T1D (3.3±1.1 vs. CON 7.0±1.4 ml*min-1*m-2, p<0.01), while fasting and postprandial AT IR were not different. Persons with T1D showed an inverse relationship between BCAA concentrations and C-peptide levels (total BCAA: r=-0.76, p<0.01; VAL: r=-0.80, p<0.01; LEU: r=-0.71, p<0.05; ILE: r=-0.67, p<0.05) and DI (total BCAA: r=-0.71, p<0.05; VAL: r=-0.78, p<0.01; LEU: r=-0.75, p<0.01; ILE: r=-0.69, p<0.05).

In conclusion, persons with even well-controlled recent-onset T1D exhibit elevated postprandial BCAA concentrations, likely due to impaired ß-cell function, which contribute to postprandial IR.

Disclosure

Y. Karusheva: None. K. Strassburger: None. D.F. Markgraf: None. O.P. Zaharia: None. K. Bodis: None. A. Tura: None. G. Pacini: None. V. Burkart: None. J. Szendroedi: None. M. Roden: Advisory Panel; Self; Servier. Board Member; Self; Poxel SA. Consultant; Self; Eli Lilly and Company, Gilead Sciences, Inc., ProSciento, TARGET PharmaSolutions. Research Support; Self; Boehringer Ingelheim International GmbH, Novartis Pharma K.K., Sanofi US. Speaker’s Bureau; Self; Novo Nordisk A/S.
2112-P: Sarco/Endoplasmic Reticulum ATPase (SERCA2) Deficiency in the Nonobese Diabetic (NOD) Mouse Accelerates Type 1 Diabetes Development
Authors: BONE ROBERT N. , REISSAUS CHRISTOPHER A. , KONO TATSUYOSHI , EVANS-MOLINA CARMELLA
Published: June 2020

Recent evidence has implicated pathways intrinsic to the β cell, such as endoplasmic reticulum (ER) stress, as potential triggers of T1D. ER stress is increased by the loss of ER Ca2+, leading to decreased β cell function and increased β cell apoptosis. Maintenance of intraluminal ER Ca2+ stores is primarily regulated by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) pump, and we have shown that β cell SERCA2 expression is reduced in islets from mouse models of T1D prior to, and after, disease onset. Thus, we hypothesized that SERCA2-mediated ER Ca2+ dyshomeostasis could be a major contributor to T1D development. To test this, we generated a mouse model haploinsufficient for SERCA2 on the NOD background (NOD-S2+/-). Compared to wild type littermates (NOD-WT), NOD-S2+/- mice had a higher incidence of diabetes (p<0.0001) and incidence was accelerated (14.5 wks vs. 19 wks, p<0.0001). Prior to diabetes onset, NOD-S2+/- islets also had increased MHC-I expression, which has been reported to follow increased ER stress and lead to increased β cell immunogenicity. To test whether SERCA2 activation may have beneficial therapeutic effects, we treated NOD-WT and NOD-S2+/- mice with CDN1163, an allosteric activator of SERCA2, from 6-8 wks of age and observed a two week delay in NOD-S2+/- average onset (p<0.05). To characterize patterns of islet immune infiltration in response to SERCA2 loss, we are utilizing the CODEX multiplexed tissue imaging platform. Our pilot study used a ten antibody panel to label and image spleen, pancreatic lymph nodes, and islets from NOD-WT. Using Indiana University-developed software, Volumetric Tissue Exploration Analysis, our preliminary analysis yields a high fidelity of nuclear segmentation and low (∼10%) false positive identification of markers. Taken together, our data suggest that loss of SERCA2 exacerbates T1D development and indicates SERCA2 may be a novel T1D therapeutic target.

Disclosure

R.N. Bone: None. C.A. Reissaus: None. T. Kono: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb.
212-LB: Gestational Exposure to Low-Dose Benzene Predisposes to Diabetes in Adulthood
Authors: KOSHKO LISA , DEBARBA LUCAS K. , DIDYUK OLESYA , FAKHOURY PATRICK I. , SADAGURSKI MARIANNA , KLUEH ULRIKE
Published: June 2020

Benzene is a well-known human carcinogen that is one of the major components of air pollution. Sources of benzene in ambient air include cigarette smoke, e-cigarettes, vaping, and evaporation of benzene-containing petrol processes. Epidemiological studies demonstrated the association with type 2 diabetes (T2D) and the development of insulin resistance (IR). Because maternal environmental stressors can greatly affect offspring health, we hypothesized that exposure to low concentrations of benzene during critical windows of development may contribute to the escalating prevalence of metabolic imbalance and diabetes in offspring. Specifically, we tested the hypothesis that prenatal exposure to benzene at concentrations relevant to cigarette smoking or vaping, would prime hypothalamic gliosis, resulting in exacerbated metabolic outcomes following exposure to a high-fat diet in adulthood. Pregnant C57BL/6JB dams were exposed to benzene at 50ppm or filtered air throughout gestation, and offspring metabolic phenotype was evaluated before and after high-fat-diet (HFD) feeding for 6 months. The male offspring of benzene-exposed dams exhibited exaggerated hyperglycemia on a chow diet compared to the male offspring of control dams, whereas female offspring did not differ according to prenatal treatment. No changes in body weight, body composition, or early postnatal hypothalamic glial activation were observed between groups. Regardless, adult male offspring of benzene-exposed dams exhibited severe impairment in the parameters of energy homeostasis (Vo2, VCO2, and heat production), suggesting the effect of metabolic reprogramming in whole-body metabolic regulation in adult offspring.

In summary, prenatal benzene exposure at low dose programs offspring for increased susceptibility to diet induced metabolic changes in adulthood in a sexually dimorphic manner.

Disclosure

L. Koshko: None. L.K. Debarba: None. O. Didyuk: None. P.I. Fakhoury: None. M. Sadagurski: None. U. Klueh: Research Support; Self; Abbott. Other Relationship; Self; Cell and Molecular Tissue Engineering, LLC.
2121-P: Proinflammatory Cytokines Alter Beta-Cell Exosome Cargo and Function to Enhance Activation of the CXCL10-CXCR3 Axis in Diabetes
Authors: JAVEED NAUREEN , HER TRACY K. , BROWN MATTHEW , VANDERBOOM PATRICK M. , EGAN AOIFE M. , VELLA ADRIAN , LANZA IAN R. , PATEL TUSHAR , MATVEYENKO ALEKSEY
Published: June 2020

Coordinated activity among islet cells is necessary for glucose homeostasis. Chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturbs islet cell crosstalk and β-cell function in diabetes. Secretory nanovesicles - exosomes (EXO) derived from cytokine exposed β-cells modulate physiological and pathological responses to β-cell stress. However, the mechanisms governing this process remain unknown. We tested the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory EXO to promote inflammation and inhibit β-cell function. Assessment of EXO concentrations from Min6 β-cells exposed to diabetogenic cytokines (IL-1β, TNF-α, and IFNγ, cytoEXO) revealed a 2-fold increase in EXO secretion (p<.05, vs. control (ctl) EXO). While acute inhibition of EXO formation with the compound GW4869 (neutral sphingomyelinase inhibitor) showed significant restoration in β-cell function (GSIS) in cytokine-exposed mouse and human islets (∼7 and 2 fold vs. cytokines alone, p<.05). Moreover, functional assessment of mouse islets exposed to cytoEXO (48h) resulted in suppression of GSIS (∼55%, vs. untreated, p<.05). Identification of cytoEXO content through proteomic analysis revealed a significant upregulation of the chemokine, CXCL10 (∼40 fold vs. ctlEXO) and RNA-Seq analysis of cytoEXO treated mouse islets depicted a marked upregulation of transcripts associated with CXCL10-CXCR3 signaling (p<.001) and subsequent downstream pathways (e.g., NFκB; p=.016 and JAK/STAT; p=.021). Furthermore, inhibition of cytoEXO (GW4869) with cytokines markedly decreased CXCL10 (∼30%) and CXCR3 (∼65%) expression in Min6 cells. These data suggests that cytokines enhance CXCL10 expression in β-cell EXO to enhance inflammation-induced diabetes through EXO-autocrine release of CXCL10 consequently activating CXCR3 signaling and downstream pathways to impair β-cell function.

Disclosure

N. Javeed: None. T.K. Her: None. M. Brown: None. P.M. Vanderboom: None. A.M. Egan: None. A. Vella: Advisory Panel; Self; vTv Therapeutics, Zealand Pharma A/S. Research Support; Self; Novo Nordisk A/S, Xeris Pharmaceuticals, Inc. I.R. Lanza: None. T. Patel: None. A. Matveyenko: None.
2136-PUB: GLP-1 Agonist Exendin-4 Protects Endothelial Cells against Hyperhomocysteinemia by Alleviating ER Stress and Enhancing Protein Folding Machinery
Authors: CHENG CHAK KWONG , LUO JIANG-YUN , LAU CHI WAI , TIAN XIAO YU , HUANG YU
Published: June 2020

Hyperhomocysteinemia (HHcy) is a risk factor for higher mortality in type 2 diabetic patients. HHcy induces endothelial dysfunction, which precedes overt hyperglycemia in type 2 diabetic patients. In addition, endothelial dysfunction predisposes diabetic patients to cardiovascular disorders, such as atherosclerotic cardiovascular diseases and retinopathy. HHcy induces oxidative stress and endoplasmic reticulum (ER) stress to impair endothelial cells. GLP-1 agonist exendin-4 was shown to attenuate ER stress in endothelial cells although detailed mechanism remains elusive. The current study intends to reveal the beneficial effects of exendin-4 on HHcy-impaired endothelial cells.

HHcy was induced by feeding C57BL/6 mice a high methionine low folate diet for 12 weeks. To assess endothelial function, mouse aortae and mesenteric arteries were mounted on wire myograph to determine acetylcholine-induced endothelium-dependent relaxations (EDRs). OGTT and ITT were conducted to study in vivo glucose metabolism. Dihydroethidium staining and lucigenin-enhanced chemiluminescence were used to measure ROS production. The ER stress markers expression was measured by biochemical assays. The role of ER chaperone was studied by adenoviral overexpression and siRNA-mediated knockdown.

A 4-week subcutaneous injection of exendin-4 significantly improved glucose tolerance, insulin sensitivity and EDRs in arteries of diet-induced HHcy mice. Exendin-4 inhibited ROS production and ER stress markers expression in mouse aortae and human endothelial cells. Such inhibitions were ablated by compound C, an AMPK inhibitor. By AMPK activation, exendin-4 up-regulated endothelial expression of ER oxidoreductase (ERO1α), an ER chaperone. In short, through AMPK-dependent upregulation of ERO1α in endothelial cells, GLP-1 agonist exendin-4 lowers HHcy-induced ER stress and oxidative stress to restore endothelial function.

Disclosure

C. Cheng: None. J. Luo: None. C. Lau: None. X. Tian: None. Y. Huang: None.

Funding

Health and Medical Research Fund (05162906); Early Career Scheme (24122318); Hong Kong Research Grants Council (14112919); Collaborative Research Fund (C4024-16W)
2137-PUB: Exercise and Stevia R. Extracts Attenuate Diabetic Cardiomyopathy in Type 2 Diabetic Rats via Upregulation of Nrf2 and Suppression of Oxidative Stress, Fibrosis, Apoptosis, and Sympathetic Overactivity
Authors: HUSSEIN ABDELAZIZ , EID ELSAYED A. , TAHA MEDHAT , LASHIN LASHIN
Published: June 2020

Objectives: In the current work, we studied the effects of exercise and stevia extracts on diabetic cardiomyopathy in type 2 diabetic rats and their possible underlying mechanisms.

Methods: Thirty-two male Sprague Dawley rats were randomly allocated into 4 equal groups; a) normal control group, b) DM group, type 2 diabetic rats received 2ml oral saline daily for 4 weeks, c) DM+ Exercise, type 2 diabetic rats were treated with exercise for 4 weeks and d) DM+ stevia extracts: type 2 diabetic rats received methanolic stevia extracts. By the end of experiment, serum blood glucose, HOMA-IR, insulin and cardiac enzymes (LDH, CK-MB), cardiac histopathology, oxidative stress markers (MDA, GSH and CAT), myocardial fibrosis by Masson trichrome, the expression of p53, α-SMA and tyrosine hydroxylase (TH) by immunostaining in myocardial tissues were measured. Also, the expression of nuclear factor erythroid-like factor 2 (nrf2) was measured in myocardial tissues.

Results: We found that T2DM caused significant increase in blood glucose, HOMA-IR index, serum CK-MB and LDH, myocardial damage and fibrosis, myocardial MDA, myocardial α-SMA, p53, nrf2 and TH density with significant decrease in serum insulin and myocardial GSH and CAT. On the other hand, treatment with either exercise or stevia extracts caused in significant improvement in studied parameters.

Conclusions: exercise and methanolic stevia extracts have cardioprotective effects against DCM, which might be due to attenuation of fibrosis, oxidative stress, p53, TH and upregulation of nrf2. Moreover, stevia extracts offered more protective effect than exercise.

Disclosure

A. Hussein: None. E.A. Eid: None. M. Taha: None. L. Lashin: None.
2141-PUB: Advanced Age, Higher FPG, and Conservative Treatment Increases in Hospital Mortality Risk in Female Patients with Acute Myocardial Infarction: A Retrospective Study
Authors: LUO SHUNKUI , LUO GUOJING , WANG LINGLING , ZHAN YANLI , LI TENGLING , PANG LICHUN , LU HONGYUN
Published: June 2020

Background: previous researches demonstrated that gender affected the in-hospital prognosis of acute myocardial infarction (AMI). Recent studies showed conflicting results. This study aimed to investigate the influence of gender, age, glycemia, treatment, and some risk factors on the in-hospital mortality after a first AMI.

Method: Consecutive patients admitted with AMI from 2002 to 2018 were included in this study. Analysis were conducted with the SPSS 25.0 software.

Results:1607 cases were enrolled in this study. Female patients had worse outcome, higher rate of cardiac shock, heart failure, Unadjusted in-hospital mortality was higher in women. Beides, female was older than male , had higher rate of diabetes, and preferred to take conservative treatment. Even though different gender had significant different outcome after AMI attack, logistic analysis revealed that gender itself was not the risk factor of AMI mortality. It showed that FPG≥7.0mmol/l,conservative treatment and age≥75 years-old were the risk factor of AMI mortality. Previous studies considered that gender is the important factor that affects the mortality rate of AMI, our study found it was not gender itself made that difference, it related to age, treatment and glycemia.

Disclosure

S. Luo: None. G. Luo: None. L. Wang: None. Y. Zhan: None. T. Li: None. L. Pang: None. H. Lu: None.

Funding

Five & Five Youth Talent Scientist Programme
2144-PUB: Elevated Carotid Intima-Media Thickness Is an Important Predictor of Major Cardiovascular Events and All-Cause Death in Elderly Women with Diabetes
Authors: DABROWSKI MARIUSZ , DEREZINSKI TADEUSZ , URUSKA ALEKSANDRA , ZOZULINSKA-ZIOLKIEWICZ DOROTA
Published: June 2020

Background and Aims: The presence of diabetes mellitus (DM) is associated with unfavorable long-term outcomes. The aim of this study was to assess the risk factors of clinically significant end points associated with the presence of diabetes in the 6.5-year follow-up of a group of elderly women.

Material and Methods: A group of 364 women aged 65-74 (98 with DM) were included in the study in 2012. After 6.5 years of follow-up we evaluated risk factors (anthropometric, metabolic and clinical, including carotid intima/media thickness - CIMT) associated with the occurrence of composite major cardiovascular (CV) event (MACE) (non-fatal myocardial infarction [MI], non-fatal stroke or CV death), and all-cause death depending on the baseline presence of DM.

Results: During follow-up MACE occurred in 26 women (12 with DM) and 40 females died (15 with DM). Survival data were available for all 364 women, while MACE data for 290. Baseline DM was associated with significantly higher relative risk (RR) of MACE, RR 1.69 (95% CI 1.07-2.68), P=0.043, and CV death, RR 2.53 (1.65-3.89), P=0.004. In a group with DM older age, higher CIMT and history of CV event were associated with both MACE and all-cause death. We established the best cut-off point for CIMT at 1.2 mm. Women with CIMT ≥1,2 mm had significantly higher risk of MACE, RR 5,13 (1.85-14.27), P=0.003, and of all-cause death, RR 6.24 (2.53-15.39), P<0.001, compared to those with lower CIMT. In the multiple logistic regression analysis significant predictors of MACE were both CIMT ≥1,2 mm, RR 5.09 (1.15-22,57), P=0.032 and history of MI or stroke, RR 5.47 (1.10-27.07), P=0.037, while CIMT ≥1,2 mm was the only independent predictor of all-cause death, RR 7,29 (1,99-26,67), P=0.003.

Conclusions: CIMT ≥1.2 mm seem to be an important predictor of MACE and the best predictor of all-cause death in elderly women with diabetes. Thus, CIMT assessment can be useful in identifying persons at very high risk of these outcomes.

Disclosure

M. Dabrowski: Other Relationship; Self; Ascensia Diabetes Care, AstraZeneca, Berlin-Chemie AG, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Mundipharma International, Novo Nordisk Inc., Sanofi-Aventis, Servier. T. Derezinski: None. A. Uruska: None. D. Zozulinska-Ziolkiewicz: None.