bluuebunny/crossref_metadata_2025_split huggingface dataset:
2072-P: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) in the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Function In Vitro and Strongly Impairs Glucose-Stimulated Insulin Secretion In Vivo
Authors: GEORGIADOU ELENI , CHABOSSEAU PAULINE L. , TOMAS ALEJANDRA , LECLERC ISABELLE , RUTTER GUY A. 
Published: June 2020

Background and Aims: Mitochondrial metabolism of glucose is vital for the initiation of insulin secretion in pancreatic beta cells. Whether mitochondrial ultra-structure, and the proteins controlling their fission and fusion, are essential for glucose recognition is less clear. In the present study, beta cell-selective adult mouse restricted deletion of Mfn1 and Mfn2 was used to investigate the role of mitochondrial fusion in insulin secretion and glucose homeostasis in vivo and in vitro.
Methods: C57BL6 mice bearing Mfn1 and Mfn2 alleles with FloxP sites, were crossed to animals carrying an inducible Cre recombinase at the Pdx1 locus (PdxCreERT). After induction of recombination with tamoxifen, live beta cell fluorescence imaging of mitochondrial [Ca2+] and membrane potential were performed on isolated islets. Mitochondrial ultra-structure was measured using super resolution fluorescence and transmission electron microscopy. Intraperitoneal glucose tolerance, and insulin secretion in vivo and in vitro, were measured using standard techniques.
Results: Mitochondrial length was strongly (to 77±0.9% of controls, p&lt;0.0001) and selectively diminished in beta cells from Mfn1/2 KO mice. KO animals displayed higher fasting glycaemia than control animals at 11-12 weeks (8.6 vs. 6.4 mmol/L, p&gt;0.05). An increase in circulating glucose levels was also observed (p&lt;0.05 at 30 min and p&lt;0.01 at 60 min) and was linked to a considerable (&gt;five-fold) decrease in plasma insulin (5-15 min, p&lt;0.0001) post-intraperitoneal glucose injection. Mitochondrial Ca2+ accumulation and membrane potential were significantly reduced (p&lt;0.01) in response to high glucose in KO animals.
Conclusions: Mitochondrial fusion and fission cycles are indispensable in the beta cell in order to preserve normal mitochondrial bioenergetics and glucose sensing, and may be disrupted in some forms of diabetes.

Disclosure
E. Georgiadou: None. P.L. Chabosseau: None. A. Tomas: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd.

Funding
UK Wellcome Trust (WT098424AIA, 212625/Z/18/Z); UK Medical Researh Council (MR/R022259/1, MR/J0003042/1, MR/L020149/1, MR/L02036X/1, MR/N00275X/1); Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA16/0005485)
2080-P: Lipid Profiles from Individual Cells of the Human Pancreatic Islet
Authors: RUBAKHIN STANISLAV , SWEEDLER JONATHAN V. 
Published: June 2020

Lipids play multiple roles in normal and pathological functions of cells and organs including structural, energy balance, cell signaling and maintenance of homeostasis. In the pancreatic islets, lipids have been implicated in regulating aspects of membrane fusion and hence insulin (and other hormones) release. Imbalances in lipid metabolism, accumulation, and localization have been associated with T2D progression. Wide variety of lipid classes are observed in the pancreatic islets including glycerophospholipids, glycerophospholipids, and ceramides. Complex cellular organization of these structures suggests that lipid profiles of different cell types or even functional cell subtypes, may not be the same. In this work, high throughput single cell mass spectrometry (MS) was used to analyze cellular populations of isolated human individual pancreatic islets received from the Human Pancreas Analysis Program (UPenn). Besides lipids, peptide markers for the canonical islet cell types are observed in the same cells allowing us to associate specific lipid profiles with cell types. For example, beta cells exhibited larger signals matching to PC(34:1), SM(d34:2), and SM(d38:1). Epsilon cells, among others, have enriched SM(d42:2)- and SM(d42:1)-like signals. Alpha cells demonstrate the most complex lipid profile with 10 lipid species exhibiting higher relative intensities. Current work involves matching the single cell lipid peaks to specific lipids, and understanding how the high plasticity of lipid profiles in each cell type track disease progression. These observations create a base for further investigation of roles of lipids in normal and pathological functioning of the pancreatic islets.

Disclosure
S. Rubakhin: None. J.V. Sweedler: None.

Funding
American Diabetes Association/Pathway to Stop Diabetes (1-18-VSN-19 to J.V.S.)
2086-P: Peptidyl Hormone Detection in Formaldehyde-Fixed Pancreas Using Mass Spectrometry Imaging
Authors: LEE DONGKYU , RUBAKHIN STANISLAV , SWEEDLER JONATHAN V. 
Published: June 2020

The availability of well-documented high-quality clinical pancreas specimens in tissue depositories such as the nPOD allows multiplatform comparative investigations of the biochemical correlates of T1D and T2D disease progression. Mass spectrometry imaging (MSI) is an emerging technique that allows us to determine, in an untargeted manner, the proteins and peptides and monitor their localization within the pancreas. However, its application to specimens preserved using standard formaldehyde-induced crosslinking is limited to molecules that do not have primary amines, and thus excludes endogenous peptides and proteins. Here we are creating a MSI-compatible chemical decrosslinking to enable us to use the tissue banked materials. We found that hydroxylamine hydrochloride in acidic tris(hydroxymethyl)aminomethane reverses the crosslinking between model peptides in vitro. Among amino acid moieties affected by formaldehyde-fixation, arginine, lysine and cysteine demonstrate best performance leading to the restoration of peptide detection in the treated samples. This treatment is compatible with formaldehyde-fixed pancreas and successfully enables the detection of glucagon, insulin and pancreatic polypeptides localized in the α-, β- and γ-cells. Our approach opens important opportunities for multimodal investigation of large sets of well preserved and documented clinical specimens.

Disclosure
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.)
209-OR: Evidence that Agrp Neuron Activation Drives Cold-Induced Hyperphagia
Authors: DEEM JENNIFER D. , FABER CHELSEA L. , PEDERSEN CHRISTIAN , PHAN BAO ANH N. , OGIMOTO KAYOKO , LARSEN SARAH A. , TRAN MEGAN A. , DAMIAN VINCENT , KAIYALA KARL , SCARLETT JARRAD , BRUCHAS MICHAEL R. , SCHWARTZ MICHAEL W. , MORTON GREGORY J. 
Published: June 2020

Although hyperphagic feeding during cold exposure is widely viewed as a response to the negative energy state that results from increased thermogenesis, the underlying mechanisms are unknown. We investigated an alternative hypothesis, that during cold exposure, hyperphagia is mounted in anticipation of homeostatic need, rather than as a compensatory response. In support of this hypothesis, we report that in chow-fed mice acutely housed at 14°C, increases of food intake and thermogenesis occur both rapidly and simultaneously, implying hyperphagia is not mounted as a compensatory response. We next asked whether this cold-induced feeding response involves activation of agouti-related peptide (Agrp) neurons, which are implicated in other forms of hyperphagic feeding. Consistent with this hypothesis, we report that Agrp neurons are activated within 1-2 min of cold exposure and prior to the onset of hyperphagia, as judged by in vivo fiber photometry and confirmed by cFos induction (Agrp+/cFos+, 22°C: 5.3±4.5% vs. 14°C: 20.2±6.1%; p&lt;0.01). To test whether Agrp neuron activation is required for cold-induced hyperphagia, we employed a chemogenetic strategy to silence Agrp neurons. As predicted, their silencing prevented hyperphagic feeding (2h food intake after housing at 14°C: 0.26±0.06 g vs. 0.10±0.04 g; p&lt;0.05), but not the associated thermogenic response (0.63 vs. 0.62 kcal/hr; p=ns) during cold exposure. Interestingly, cold-induced hyperphagia is absent in mice with diet-induced obesity (DIO). Consequently, unlike chow-fed mice, these animals lose weight during cold exposure. These observations suggest that 1) Agrp neurons lie downstream of thermoregulatory neurocircuits, 2) during cold exposure, increased Agrp neuronal firing occurs rapidly as part of a homeostatic response that preserves body temperature and energy stores, and 3) this response appears to be impaired in mice with DIO. Future studies will map the neurocircuitry underlying these responses and clarify how DIO impairs their activation.

Disclosure
J.D. Deem: None. C.L. Faber: None. C. Pedersen: None. B.N. Phan: None. K. Ogimoto: None. S.A. Larsen: None. M.A. Tran: None. V. Damian: None. K. Kaiyala: None. J. Scarlett: None. M.R. Bruchas: None. M.W. Schwartz: Research Support; Self; Novo Nordisk A/S. G.J. Morton: Research Support; Self; Novo Nordisk A/S.

Funding
American Diabetes Association (1-19-PDF-103 to J.D.D.); Dick and Julia McAbee Foundation; National Institutes of Health (T32-HL007028-39, R01DK089056, R01DK101997, R01DK124238, R01DK089056, P30DK017047, P30DK035816)
2091-P: Effects of Caveolin-1 on the Synthesis and Decomposition of Islet ß-Cell IAPP and Its Mechanism
Authors: LIU KUNYING , ZENG WEN , LIN SHUO , LIN CHUWEN , XU FEN , CAI NAN , ZENG LONGYI 
Published: June 2020

Objective: Islet amyloid polypeptide(IAPP) is a major component of islet amyloid deposition in type 2 diabetic patients. Our preliminary study showed that membrane protein Caveolin-1 (Cav1) silencing inhibited β-cell apoptosis and promote insulin secretion. The purpose of this study was to investigate the regulation of Cav1 on islet β-cell IAPP and its mechanism.
Materials and Methods: Firstly, IAPP secretion of each group of primary islets was detected by ELISA, then Western blot and real-time quantitative PCR (qPCR) were used to detect the expression levels of IAPP, PAM(Peptidyl-glycine alpha-amidating monooxygenase), PC1(Prohormone Convertase-1), BACE2(Beta-secretase 2), IDE(Insulin-Degrading Enzyme), TXNIP(Thioredoxin interacting protein), and finally, it confirmed that Cav1 interacted with the protein of TXNIP by immunofluorescence confocal and co-immunoprecipitation (Co-IP).
Results: The secretion of IAPP was decreased in primary pancreatic islets and Cav1 deficiency significantly down-regulated the expression of IAPP in NIT-1 cells, while over-expression of Cav1 in βTC-6 cells significantly up-regulated the expression of IAPP. This effect correlated with Cav1 silencing leading to down-regulation of PAM expression associated with IAPP synthesis and over-expression of Cav1 leading to up-regulation of PC1 associated with IAPP synthesis. On the other hand,Cav1 deficiency resulted in the up-regulation of the expression of BACE2 and IDE, the enzymes associated with IAPP decomposition. While over-expression of Cav1 leaded to down-regulation of BACE2 and IDE. Further studies indicated that Cav1 co-localized with TXNIP, and it interacted with and regulated the expression of TXNIP, which regulates IAPP gene transcription.
Conclusion: Cav-1 can regulate the synthesis and decomposition of IAPP in islet β cell by interacting with TXNIP. This study suggests that Cav-1 may protect diabetic islet beta cells by reducing islet amyloid deposition.

Disclosure
K. Liu: None. W. Zeng: None. S. Lin: None. C. Lin: None. F. Xu: None. N. Cai: None. L. Zeng: None.

Funding
National Natural Science Funding of China (51873071); Natural Science Foundation of Guangdong Province (2018B030311012)
2103-P: Glucose-Dependent Insulinotropic Polypeptide (GIP) Protects against Cytokine-Induced Cell Death and Exerts Both Insulinotropic and Glucagonotropic Effects in Human Islets
Authors: JØRGENSEN ANNE , EGESKOV MERETE , KNOP FILIP K. , STØRLING JOACHIM 
Published: June 2020

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is secreted from enteroendocrine K cells following food intake and potentiates glucose-stimulated insulin secretion from β-cells. Recent studies suggest that GIP also has glucagonotropic properties under euglycemic and hypoglycemic conditions in humans. GIP may therefore be exploited as a safeguard against hypoglycemia in patients with type 1 diabetes (T1D). However, a direct glucagonotropic effect of GIP on human α-cells has not yet been investigated. In this study, we investigated the effects of GIP on glucagon and insulin secretion in isolated human islets during 30-min exposures to low (2 mM) and high (20 mM) D-glucose concentrations. We further investigated the potency of GIP to protect human islets from cell death induced by a 48-h exposure to pro-inflammatory cytokines (50 U/mL interleukin-1β + 1,000 U/mL interferon-γ) known to contribute to islet destruction in T1D. GIP (2.5 nM) significantly (P=0.048, n=4) increased glucagon secretion by 48% at 2 mM glucose in isolated human islets as compared to 2 mM glucose alone. At 20 mM glucose, GIP did not affect glucagon secretion, but potentiated insulin secretion by 45% (P=0.015, n=4). Further, cytokine-induced human islet cell death was decreased by 50% by co-treatment with GIP (P=0.001, n=3). Our findings demonstrate that GIP protects against cytokine-induced islet death and possesses glucagonotropic and insulinotropic effects on human islets at low and high glucose, respectively. These findings support GIP as a bifunctional blood glucose-stabilizer that might be exploited to treat T1D.

Disclosure
A. Jørgensen: None. M. Egeskov: None. F.K. Knop: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk A/S, Sanofi. Consultant; Self; Carmot Therapeutics, Inc., Eli Lilly and Company, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Gubra, Novo Nordisk A/S, Sanofi, Zealand Pharma A/S. Speaker’s Bureau; Self; AstraZeneca, Lupin Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Norgine B.V., Novo Nordisk A/S. J. Størling: None.
2118-P: Regulation of Ataxia-Telangiectasia and Rad3-Related (ATR)–Dependent DNA Damage Response by Nitric Oxide in ß-Cells
Authors: YEO CHAY TENG , OLESON BRYNDON , CORBETT JOHN A. , SCHNUCK JAMIE K. 
Published: June 2020

Nitric oxide, generated in β-cells in response to cytokines, induces DNA damage that activates ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage response (DDR). While the DDR does not contribute to DNA repair in β-cells, it does regulate β-cell apoptosis when the nitric oxide production falls below a threshold level. Indeed, nitric oxide at high micromolar levels, while inducing DNA damage, also inhibits DDR and protects β-cells from apoptosis. This ability of nitric oxide, a potent inhibitor of mitochondrial oxidation, to attenuate DDR activation correlates with a 10-fold reduction in ATP and the actions are selective to β-cells. Unlike most other cell types, β-cells cannot compensate for mitochondrial inhibition by increasing glycolysis, leading to a loss in ATP levels which correlates with DDR inhibition. We now show that nitric oxide also inhibits the activation of a second DDR transducer kinase, ATR. ATR is activated in response to single-strand DNA breaks and replication stress. We showed that low levels of nitric oxide activate ATR-dependent DDR signaling by inducing replication stress. However, much like with ATM, at higher concentrations nitric oxide and other inhibitors of mitochondrial respiration that reduce ATP levels by more than 10-fold, also inhibit ATR selectively in β-cells. Nitric oxide and these mitochondrial poisons do not inhibit ATR signaling in non-β-cells as they maintain ATP levels by increasing glycolysis under mitochondrial inhibition. Importantly, when non-β-cells are forced to utilize oxidative phosphorylation for ATP generation, nitric oxide and other mitochondrial inhibitors attenuate ATR signaling in a manner similar to β-cells. Together, these studies suggest that inhibition of the DDR by nitric oxide in β-cells is dependent on its ability to inhibit mitochondrial oxidative metabolism and deplete ATP, and suggest that the inhibition of DDR signaling may be a protective response to prevent β-cell apoptosis.

Disclosure
C. Yeo: None. B. Oleson: None. J.A. Corbett: None. J.K. Schnuck: None.

Funding
National Institutes of Health (DK052194, AI-44458)
2124-P: Raptor Levels Are Critical in the Adaptation of Beta Cells to High-Fat Diet
Authors: BLANDINO-ROSANO MANUEL , BERNAL-MIZRACHI ERNESTO 
Published: June 2020

mTORC1 signaling is a central regulator of autophagy and altered autophagic activity has been implicated in the beta-cells of patients with type 2 diabetes, and in the beta-cells of obese mice. The importance of mTORC1 signaling and Raptor in beta-cells has been recently demonstrated using raptor knock-out models (βraKO mice). These studies have shown that complete deletion of Raptor impairs beta-cell proliferation and survival of the beta-cell, and both in function and insulin processing. However, nothing is known whether reduced mTORC1 levels impair beta-cell adaptation to diabetogenic conditions. Therefore, we decided to study mice with haploinsufficiency of Raptor in beta-cells (βraHET). Mice with heterozygous deletion of Raptor in beta-cells were generated by crossing raptorf/f with Rip-Cre mice (βraHET). βraHET and control mice were fed regular chow (RC) or high-fat diet (HFD). Mice fed with RC showed no difference at the metabolic level, islets morphology, or beta-cell function. βraHET mice fed HFD for eight weeks showed an impaired Intraperitoneal Glucose Tolerance Test, fed insulin secretion and a decreased beta-cell mass, compared to control mice. Isolated islets from βraHET mice fed HFD for eight weeks showed impaired insulin secretion either measured by perifusion or static incubation compared to control islets. Next, we tested whether isolated islets from βraHET mice are more susceptible to beta-cell stressors. Beta-cell death determined by TUNEL and cleavage Caspase 3 was increased (50 and 60%) in the βraHET islets after 24 h exposure to palmitate. In addition, autophagy markers such as LC3 and p62 were increased in islets from βraHET treated with palmitate. The increase in cell death and autophagy is specific to lipotoxicity since there βraHET islets exhibited no increase in cleavage Caspase 3 in TUNEL when they were treated with proinflammatory cytokines or ER Stress inducers.
In conclusion, we demonstrate that adequate levels of Raptor are critical in the adaptation of the beta-cell to HFD.

Disclosure
M. Blandino-Rosano: None. E. Bernal-Mizrachi: None.
2132-P: Single-Cell RNA-Sequencing of Mouse Islets after Inflammatory Cytokine Exposure
Authors: STANCILL JENNIFER , KHATUN ACHIA , KASMANI MOUJTABA , CUI WEIGUO , CORBETT JOHN A. 
Published: June 2020

Type 1 diabetes is an autoimmune disease caused by destruction of pancreatic β-cells. During disease progression, β-cells are exposed to proinflammatory cytokines like interleukin-1β (IL-1β) and interferon-γ (IFN-γ), stimulating them to express inducible nitric oxide synthase (iNOS) and produce micromolar levels of nitric oxide. While some cytokine-mediated changes in islet gene expression are known, we do not have a global view of the genes induced by cytokines nor do we know how non-β-cells respond to cytokines. Following 6-hr treatment with IL-1β and IFN-γ, adult C57BL/6 mouse islets were subjected to single-cell RNA-sequencing using the 10x Genomics Chromium platform. iNOS mRNA (Nos2) was induced in 73% of β-cells in response to both cytokines. Unexpectedly, 72% of Δ-cells and 44% of α-cells expressed Nos2 at levels similar to that found in β-cells. This is surprising since β-cells are thought to be the primary islet source of nitric oxide. Of the 241 genes enriched in Nos2-expressing β-cells, 70% were also enriched in Nos2-expressing Δ- and α-cells, suggesting that these cells initially respond to cytokine exposure by activating similar pathways. Among the enriched genes were those indicative of anti-viral defenses, suggesting induction of protective responses. Nos2 expression was negatively correlated with expression of genes encoding heat shock proteins, such as Hsp70, consistent with previous reports that heat shock inhibits cytokine-induced iNOS expression. Finally, we observed IL-1β-induced repression of β-cell identity genes, including Mafa, Pdx1, Slc2a2, and Ucn3. Repression of these identity genes was negatively correlated with Hsp70 expression, suggesting that cellular stress prevents this dedifferentiation. While these studies are ongoing, they suggest that (1) Δ- and α-cells respond to cytokines by upregulating Nos2 mRNA, (2) cytokines repress β-cell identity genes, and (3) both of these outcomes can be inhibited by induction of cellular stress.

Disclosure
J. Stancill: None. A. Khatun: None. M. Kasmani: None. W. Cui: None. J.A. Corbett: None.
2139-PUB: Antihypertensive Effect of Azilsartan in Newly Diagnosed Stage 2 Diabetic Hypertensives
Authors: MAHAPATRA HARIBALLAV , KHUNTIA MONALISA , MISHRA SUSANT , MISHRA SANAT K. , PADHI RAJESH K. , JENA BISWARANJAN , DAS SREEYA , KHATUA RANJIT K. , JENA APURBA R. , MAHAPATRA RAMABALLAV , MAHAPATRA LAXMINARAYAN , SAHOO ABHAY K. 
Published: June 2020

Background and Aim: Coexistence of hypertension (HTN) and type 2 diabetes (T2DM) multiplies the risk of cardiovascular events. Early identification and prompt management of elevated blood pressure in T2DM has shown to improve the quality of life and to reduce the economic burden to the patients. We studied blood pressure (BP) lowering effect of Azilsartan (AZST) monotherapy in newly diagnosed stage 2 hypertensives with a history of T2DM. We also analysed its effects on blood glucose and renal indices.
Methods: T2DM Subjects attending a specialized diabetes clinic, who were diagnosed with HTN for the first time, were invited to participate. Mean of 3 BP readings was considered for analysis after due consent. Subjects with stage 2 hypertension were administered AZST 40mg once daily. After 3 months they had a repeat BP recording. Subjects underwent serum creatinine, Na+, K+, fasting (FPG) and postprandrial (PPG) plasma glucose measurements at baseline and after 3moths’ of enrolment. We compared BP lowering efficacy and changes in plasma glucose and renal parameters at baseline and after 3months of AZST therapy. Diabetic, dyslipidemic and other chronic medicines were continued unchanged during the study period.
Results: Inclusion criteria were met by 474 subjects (117, 25% females). Mean age and duration of diabetes were 53.3 (95% CI 52.3-54.3) and 7.03 (CI 6.6-7.5) years respectively. With AZST therapy mean SBP dropped from 152.7 (CI 151.1-154.3) mm Hg to 136.4 (CI 134.9-138.0) mm Hg (p &lt;0.05) and the post treatment DBP declined to 79.6 (CI 78.8-80.4) mm Hg from its baseline value of 90.5 (CI 89.6-91.4) mm Hg (p &lt;0.05). The mean baseline FPG and PPG were 158.8 (CI 154.0-163.4) mg/dL and 237.3 (CI 231.9-242.7) mg/dL in order. There was a non-significant decline in plasma glucose, but no changes were seen in eGFR or electrolytes. No adverse event was reported.
Conclusion: Azilsartan monotherapy was found to be safe and effective in managing newly diagnosed stage 2 hypertension in type 2 diabetes without hampering renal function.

Disclosure
H. Mahapatra: None. M. Khuntia: None. S. Mishra: None. S.K. Mishra: None. R.K. Padhi: None. B. Jena: None. S. Das: None. R.K. Khatua: None. A.R. Jena: None. R. Mahapatra: None. L. Mahapatra: None. A.K. Sahoo: None.

Funding
Jyoti Diabetes Research Foundation