bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glucose Oxidation Depletes Cytosolic Citrate and Glutamate in the Amplifying Pathways of Insulin Secretion
Authors: LEWANDOWSKI SOPHIE L. , MERRINS MATTHEW J. 
Published: June 2018

In pancreatic β-cells, glucose metabolism generates signals that trigger and amplify insulin secretion. We generated β-cell specific FRET biosensors for the real-time measurement of cytosolic citrate and glutamate, two putative metabolic coupling factors with an undefined causal relationship to the signaling process. In islet β-cells, glucose elevation from 2.5 to 10 mM was accompanied by a sharp decrease in cytosolic citrate and glutamate. Steady-state oscillations in these intermediates induced by 10 mM glucose reveal that cytosolic citrate and glutamate levels decrease as calcium levels increase, precisely when the TCA cycle is most active. Subsequent membrane depolarization with 30 mM KCl resulted in a further decrease in citrate and glutamate, suggesting that mitochondria respond to work in the form of ATP hydrolysis by importing citrate and glutamate into the TCA cycle. Indeed, parallel measurements with Perceval-HR biosensors show that cytosolic ADP/ATP rises with calcium, and corresponds to a burst of mitochondrial NADH fluorescence. These data are inconsistent with existing models of the amplifying pathway in which mitochondrial glucose oxidation results in the export of TCA intermediates to the cytosol to generate NADPH and GSH/GSSG. The observed increase in cytosolic GSH/GSSG in response to glucose, shown with a FRET reporter targeted to β-cells, implies that GSH is generated from other sources such as the pentose phosphate pathway. Our results support the strong prediction that glucose oxidation in the mitochondria is inversely proportional to insulin secretion via the NADPH-generating citrate and glutamate cycles in the cytosol, since these pathways can never occur simultaneously.

Disclosure
S.L. Lewandowski: None. M.J. Merrins: None.
Insulin and Glucagon Interaction Studied by Separate and Combined Receptor Antagonism
Authors: GALSGAARD KATRINE D. , HOLST JENS J. , WINTHER-SOERENSEN MARIE , WEWER ALBRECHTSEN NICOLAI J. , PEDERSEN JENS 
Published: June 2018

Insulin and glucagon are mutually influencing not only the actions of each other but also the secretion, which makes it very complicated to unravel their roles in integrated metabolism. Having studied in detail the actions of glucagon receptor antagonism in mice, we decided also to study the effects of insulin receptor antagonism and their combination. Female C57BL/6J mice (n=72) were administered an insulin receptor antagonist (IRA) (S961, 30 nmol/kg), a glucagon receptor antagonist (GRA) (25-2648, 100 mg/kg), or both IRA and GRA (IRA+GRA) three hours before intravenous administration of saline, glucose (0.5g/kg) or amino acids (1 µmol/g) (both antagonists were generous gifts from Novo Nordisk A/S). As expected, IRA caused hyperglycemia (15.6±1.0 mM vs. 8.4±0.3mM, P&lt;0.0001) and hyperinsulinemia (51.07±3.44ng/mL vs. 0.72±1.19ng/mL, P&lt;0.0001) at fasting, but reduced glucose-induced inhibition of glucagon secretion (dAUC0-20 min; -114.0±24.0min × pM vs. dAUC0-20 min; -211.8±10.4 min × pM, P&lt;0.0001). GRA treatment resulted in mild hypoglycemia (P=0.03), hyperglucagonemia (P=0.04), and did not influence insulin secretion (P=0.9) at fasting. However, after administration of both IRA and GRA, insulin secretion was significantly reduced (31.36±3.0 ng/mL vs. 51.07±3.44 ng/mL, P&lt;0.0001). Finally, GRA+IRA administration resulted in blood glucose concentrations equal to those of vehicle treated mice upon a glucose challenge (tAUC0-20 min; 342.7±29.3 mmol × min vs. 353.1±39.1 mmol × min, P=0.6).
In conclusion, using both glucagon and insulin receptor antagonists, it can be demonstrated in mice that both hormones are essential for the maintenance of fasting glucose metabolism and that the secretion of both is regulated by powerful negative feedback mechanisms. Insulin contributes to glucose-induced inhibition of glucagon secretion, while glucagon has a positive effect on insulin secretion.

Disclosure
K.D. Galsgaard: None. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Zealand Pharma A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp., AstraZeneca. Research Support; Self; Danish Diabetes Academy, Novo Nordisk Foundation. Other Relationship; Self; Antag Therapeutics. Other Relationship; Spouse/Partner; Antag Therapeutics. M. Winther-Soerensen: None. N.J. Wewer Albrechtsen: Speaker's Bureau; Self; Merck Sharp & Dohme Corp.. Other Relationship; Self; Mercodia, Alpco. J. Pedersen: None.
Mechanisms Underlying the Progressive Loss of Beta-Cell Function and Mass in Neonatal Diabetes Are Reverted by Dapagliflozin Therapy
Authors: SHYR ZEENAT A. , YAN ZIHAN , USTIONE ALESSANDRO , REMEDI MARIA S. 
Published: June 2018

Progressive loss of β-cell function and mass are classic findings in diabetes, and are frequently attributed to membrane hyperexcitability, insulin hypersecretion and β-cell death. However, we demonstrated that in an insulin secretory-deficient mouse model of human neonatal diabetes (KATP-GOF), loss of β-cell mass still occurs in the absence of these factors. To study the underlying mechanisms of it, we performed experiments on day 7, 15, 22 and 30 post-disease induction. While KATP-GOF mice are hyperglycemic by day 7, insulin content is maintained at control levels. Surprisingly however, there is a dramatic decrease in insulin content at day 15, accompanied by augmented proinsulin/insulin ratio, altered calcium dynamics and increased oxidative and ER stress. We hypothesized that this is induced by hyperglycemia, and not by lack of insulin; and that lowering blood glucose alone will be sufficient to prevent loss of β-cell mass and improve function. KATP-GOF mice were treated with dapagliflozin, a sodium-glucose transporter-2 (SGLT2) inhibitor clinically used to reduce blood glucose by preventing renal glucose reabsorption. Dapagliflozin significantly reduced blood glucose by increasing urinary glucose excretion without changing circulating insulin levels. Dapagliflozin therapy (10 days) markedly improved insulin content, reduced proinsulin/insulin ratio and enhanced β-cell function by alleviating oxidative and ER stress. Together, we demonstrate that β-cell failure and loss of β-cell mass is induced by increased oxidative and ER stress in a reversible manner, and that normalization of blood glucose alone is sufficient to prevent/revert the progressive β-cell dysfunction and failure, opening the exciting possibility of prevention of diabetes progression.

Disclosure
Z.A. Shyr: None. Z. Yan: None. A. Ustione: None. M.S. Remedi: None.
Diminished Ca2+ Signaling, CaV1 Channel Expression, and Activity in Pancreatic Beta Cells Expressing Mutated DISC1 (Disrupted in Schizophrenia 1)
Authors: LU PING , SHARMA ROHIT B. , ALONSO LAURA C. , ZHUGE RONGHUA , RITTENHOUSE ANN R. , JURCZYK AGATA 
Published: June 2018

In addition to cognitive impairment, schizophrenics often suffer from non-obese T2D. Here, we examined whether similar cellular changes occur in pancreatic beta-cells as found in neurons from a genetic model of schizophrenia. Specifically we tested whether beta-cells exhibit disrupted excitation-secretion coupling when a truncated human DISC1 (thDISC1) gene, originally discovered in a Scottish family having high penetrance for schizophrenia, is expressed selectively in mouse beta-cells upon ingesting doxycycline (+DOX). Expression of thDISC1 significantly decreases blood insulin levels and GSIS from isolated mouse islets, revealing an independent role for DISC1 in beta-cells. In central neurons, DISC1 regulates Ca2+ CaV2 channel expression, Ca2+ influx and transmitter release. To determine whether DISC1 similarly regulates Ca2+ physiology in beta-cells, we tested thDISC1 DOX+ vs. DOX- beta-cells for changes in: i) intracellular Ca2+signaling using Fluo 3-AM; ii) CaV1 channel expression, the dominant channel class controlling GSIS; iii) and CaV1 channel activity using whole-cell recording methods. We found increases in [Ca2+]i following 20 mM glucose were delayed &gt;2 min and diminished ∼50% in dissociated beta-cells expressing thDISC1. When sections of pancreas were stained with an anti-CaV1.2/1.3 antibody, thDISC1 DOX+ vs. DOX- insulin-positive cells exhibited decreased staining. Lastly, both peak inward Ba2+ current and FPL 64176 induced long-lasting L-type tail current decreased significantly in thDISC1 DOX+ vs. DOX- beta-cells. No obvious change in activation kinetics was observed. These changes in excitation-secretion coupling parallel those in neurons expressing truncated DISC1. Thus at the cellular level, truncated DISC1 precipitates disorders of secretion such as schizophrenia and T2D.

Disclosure
P. Lu: None. R.B. Sharma: None. L.C. Alonso: None. R. Zhuge: None. A.R. Rittenhouse: None. A. Jurczyk: None.
Generation of Insulin Expressing Cells in Mouse Small Intestine by Pdx1, MafA, and BETA2/NeuroD
Authors: HYUN LEE SO , KIM JIWON , YOON KUN-HO 
Published: June 2018

To develop the beta-like cells for diabetes therapy, adult stem cells have been identified in various tissues and studied for their conversion into beta cells. Pancreatic progenitor cells are derived from the endodermal epithelium and formed in a similar manner as gut progenitor cells. Here, we generated insulin-expressing cells from the intestinal epithelial cells that induced many of the specific pancreatic transcription factors using adenoviral vectors carrying three genes: Pdx1, MafA, and BETA2/NeuroD (PMB). By direct injection into the whole intestine, adenoviruses (Ad) were successfully delivered to the intestine. After virus injection, we could confirm that the small intestine of the mouse was appropriately overexpressed with the Ad-Pdx1 and triple Ad-PMB. Four weeks after the injection, insulin mRNA was expressed in the small intestine, and the insulin gene expression was induced in Ad-Pdx1 as well as Ad-PMB compared to control Ad-GFP. In addition, the conversion of intestinal cells into insulin-expressing cells was detected in parts of the crypts and villi located in the small intestine.
In conclusion, these data indicated that Pdx1, MafA, and BETA2/NeuroD facilitate the differentiation of mouse intestinal cells into insulin-expressing cells. The small intestine is an accessible and abundant source of surrogate insulin-producing cells.

Disclosure
L. So Hyun: None. J. Kim: None. K. Yoon: None.
Evaluation of CFTR Expression and Localisation in Human Pancreas
Authors: MAHESHWARI RASHMI R. , JONES CLAIRE J. , SHAW JAMES A.M. , WHITE MICHAEL G. 
Published: June 2018

Introduction: Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity in people with CF, occurring in 40-50% of adults. Whilst it is established that β-cell dysfunction in cystic fibrosis (CF) leads to diabetes, the mechanism by which the CF transmembrane conductance regulator (CFTR) channel influences insulin secretion remains debated. Currently, three major hypotheses have been proposed: 1. Intrinsic CFTR-dependent pathways of insulin secretion 2. Pancreas-extrinsic CFTR defects 3. Remodeling of islets following loss of exocrine tissue due to inflammation. Since the contribution of each to the pathogenesis of CFRD remains largely unknown, we sought to determine CFTR localisation within human pancreas using novel and highly sensitive approaches.
Methods: Expression of chromogranin A (ChrA), CFTR and keratin 19 (K19) was assessed by immunofluorescence (IF) staining of tissue obtained from deceased donors without diabetes (n=10, age: 23-71 years). Two CFTR antibodies (ab576 and ab590) obtained from the CF Foundation were used to confirm specificity, with ductal and endocrine cells determined by K19 and ChrA respectively. CFTR RNA expression was determined by RNAscope® in situ hybridisation (ISH) and combined with either ChrA or insulin immunohistochemistry (IHC).
Results: IF staining of pancreatic tissues indicated co-localisation of CFTR in K19+ ductal cells, but not in ChrA+ endocrine cells. These observations were confirmed by combined RNAscope® ISH and IHC (ChrA and insulin), which demonstrated the absence of CFTR RNA in human islets.
Conclusion: Employment of these highly sensitive techniques has demonstrated absence of CFTR within normal β-cells or any other islet endocrine cell types. This is in line with recent observations in isolated human islets. We conclude that CFTR abnormalities do not directly impact beta-cell function and that CFRD is mediated by factors extrinsic to the pancreatic endocrine compartment.

Disclosure
R.R. Maheshwari: None. C.J. Jones: None. J.A.M. Shaw: Advisory Panel; Self; Novo Nordisk A/S. M.G. White: None.
Quantitative Proteomic Analysis on Human Islets—New Markers of Cellular and Metabolic Dysfunction
Authors: ASSUNTA CEFALO CHIARA MARIA , MEZZA TERESA , KULKARNI ROHIT , GIACCARI ANDREA 
Published: June 2018

The pathogenesis of type 2 diabetes is characterized by a progressive β-cell dysfunction resulting in both quantitative and qualitative loss of insulin secretion. However, the molecular mechanisms underlying this progressive functional loss are still unknown. The aim of the study is to highlight changes in the proteome of pancreatic human islets that can anticipate and eventually predict diabetes’ onset. High performance liquid chromatography mass spectrometry (HPLC-MS) analysis was applied to islets isolated by laser capture microdissection (LCM) from human samples of both diabetic and no diabetic subjects, underwent to duodeno-cefalopancreatectomy for extra-pancreatic, low grading tumors. The subjects were classified on the basis of glucose tolerance assessed by an OGTT before surgery in normal tolerant (NGT, n:7), glucose intolerant (IGT, n:5) and diabetic subjects (T2DM, n:2). Qualitative and quantitative analysis were performed to detect differential protein expression among the 3 study’s groups. Sixty-seven proteins were found to be differentially regulated in diabetic subjects compare to NGT, with 29 upregulated and 38 downregulated proteins, while 95 proteins were differentially expressed in IGT compare to NGT with 49 upregulated and 46 downregulated. These proteins are mainly involved in cellular and metabolic processes. In particular IGT and DM, compared to NGT, showed a lower expression of proteins involved in cell proliferation such as PURA and NAP1L1, as well as proteins involved in the insulin cleavage process, like ERO1B. While other proteins involved in endoplasmic reticulum stress, CASP14, ERP27 and PDIA3, resulted upregulated in both IGT and DM compare to NGT. Our data suggest that metabolic and cellular processes of islet cells are already dysregulated in patients with impaired glucose tolerance, highlighting some proteins as potential early markers of β-cell dysfunction and novel therapeutic targets to slow the progression to type 2 diabetes.

Disclosure
C. Cefalo: None. T. Mezza: None. R. Kulkarni: None. A. Giaccari: Advisory Panel; Self; Sanofi-Aventis. Speaker's Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp., Amgen Inc..
Type 1 Diabetes Development Is Modulated by Select Lipid Signaling Derived from Beta Cells and Immune Cells
Authors: NELSON ALEXANDER , PARK MARGARET A. , CHALFANT CHARLES , RAMANADHAM SASANKA 
Published: June 2018

Type 1 diabetes (T1D) is a consequence of autoimmune destruction of β-cells and we find that the Ca2+-independent phospholipase A2β (iPLA2β) is induced in β-cells and immune cells and mitigation of iPLA2β attenuates β-cell death and T1D incidence. Activation of iPLA2β leads to generation of pro-inflammatory or anti-inflammatory bioactive lipids and we previously reported that iPLA2β activation favors M1 pro-inflammatory macrophage phenotype. Here, we utilized UPLC ESI-MS/MS protocols to assess the lipid profile generated by macrophages as a means to identify select lipids that are associated with T1D development. Macrophages were treated with DMSO (vehicle), IFNγ+LPS (to induce M1 polarization), or IL-4 (to induce M2 polarization) and at 16h, the media was collected and processed for assessment of eicosanoid class of bioactive lipids. Comparison of spontaneous diabetes-resistant C57BL/6J and spontaneous diabetes-prone NOD mice revealed that nearly all pro-inflammatory eicosanoids were elevated in NOD during the prediabetic phase, relative to C57. Theabundances increased in the NOD between 4-8 and decreased by 15 weeks, correlating with onset of insulitis. These findings are consistent with a higher inflammatory status in the NOD vs. C57. To assess contribution of iPLA2β to the lipid profile, macrophage responses in NOD were compared with those in NOD.iPLA2β-/+. Such analyses revealed that production of select pro-inflammatory and anti-inflammatory lipids were decreased and increased, respectively, from macrophages and islets of NOD.iPLA2β-/+, relative to age-matched NOD-WT, and this correlated with reduced T1D incidence in the NOD.iPLA2β-/+. We further find that iPLA2β-derived lipids (iDLs) play a critical role in alternative splicing events that favor generation of pro-apoptotic variants of several factors (Bcl-x, capsase-9, and RAGE). These findings raise the possibility that iDLs are candidates for targeting to counter T1D development.

Disclosure
A. Nelson: None. M.A. Park: None. C. Chalfant: None. S. Ramanadham: None.
Increased Obesity Is Causal for Increased Inflammation—A Mendelian Randomisation Study
Authors: ZUYDAM NATALIE VAN , WIELSCHER MATTHIAS , MCCARTHY MARK , JARVELIN MARJO-RIITTA 
Published: June 2018

Inflammation is endemic to obesity and type 2 diabetes (T2D) but it is unclear if it is a cause or a consequence. If the latter, then inflammation may increase the risk of obesity and diabetes complications. Large genome-wide association studies (GWAS) of inflammation and cardiometabolic traits can be used to investigate causal mechanisms. Summary statistics were assembled from GWAS of 49 cytokine (N=840-80,000) and 20 cardiometabolic traits (N=2,447-465,333). Shared genetic background was measured using LD score regression. Polygenic scores (PGS) were generated from GWAS data for 49 cytokines (representing inflammation) and 20 cardiometabolic traits and used in two-way Mendelian Randomisation (MR) analyses. Causal relationships were identified using summary statistics for all traits, and individual data (UK Biobank, N=465,333) to test the association of cytokine PGS with body mass index (BMI) and T2D. LD score regression analysis demonstrated that BMI and T2D shared a genetic background with acute phase cytokines. However, MR analyses showed that genetically-driven variation in cytokine levels were not associated with increased BMI or T2D risk. This argues against a causal role for inflammation (as represented by these cytokines) in the development of obesity and T2D. In contrast, genetically driven obesity was associated with increased inflammation: a PGS for increased BMI was associated with increased C-reactive protein (Beta [95% CI], 0.40 [0.34,0.46], p=1.3×10-36) and activated plasminogen inhibitor (Beta [95% CI], 0.39 [0.19, 0.59], p=7.8×10-5) levels. Genetically-driven variation in cytokine levels were causally linked with chronic kidney disease in subjects with T2D: fibroblast growth factor 2 (OR[95% CI], 0.58[0.46,0.72], p=1.8×10-6) and interleukin 13 (OR[95% CI], 1.28[1.15,1.43], p=7.0×10-6) We conclude that chronic inflammation is a consequence, rather than a cause, of T2D and obesity, and may contribute to some of their complications.

Disclosure
N. van Zuydam: None. M. Wielscher: None. M. McCarthy: Research Support; Self; Eli Lilly and Company, Takeda Pharmaceuticals U.S.A., Inc., Novo Nordisk A/S. Advisory Panel; Self; Novo Nordisk A/S, Pfizer Inc.. Research Support; Self; Pfizer Inc., Sanofi, Boehringer Ingelheim GmbH, AstraZeneca, Merck Sharp & Dohme Corp.. M. Jarvelin: None.
AER Is a More Sensitive Predictive Factor than eGFR for CVD in Patients with Type 2 Diabetes—Results from a Perspective Study in Beijing Communities
Authors: ZHANG XUE LIAN , YUAN SHEN YUAN ,   BCDS GROUP
Published: June 2018

Objective: The predictive values of AER and eGFR for CVD outcomes in T2DM patients have not been well documented. In this study, the effects of AER and eGFR on outcomes of CVD were analyzed in a population of T2DM patients.
Methods: 1914 T2DM patients were enrolled in this 8 -year prospective study in Beijing Communities. The patients’ serum creatinine levels were lower than 176.8μmol/l. The risk of CVD onset was assessed according to CKD staging, which was categorized by urine albumin excretion (AER; mg/d) and estimated GFR (eGFR; ml min −11.73 m−2). The effects of AER and eGFR on risk of cardiovascular disease onset in type 2 diabetes after eight years’ prevention were also analyzed.
Results: The mean age and BMI of patients were 66.4 ± 10.1 years and 25.5 ± 3.5 kg/m2, respectively; the duration of diabetes was 5.7 to 17.8 years. The proportion of patients with hypertension and smokers were 71.11% and 16.84%. During the follow-up period (median 6.8 years), 71 CVD events occurred. At baseline, those with AER≥300 mg/d and co-existing eGFR 60-89 ml min −11.73 m−2 or &lt;60 ml min −1/1.73 m−2 showed increased risk for CVD onset when compared with ’no CKD’. No increased risk was observed for patients with AER &lt;30 mg/d and eGFR &lt;60 ml min −11.73 m−2. The increased CVD risk was observed in patients progressed to AER ≥30mg/d during follow-up period, whereas patients who progressed to eGFR &lt;90 ml min −11.73 m−2 alone showed no increased CVD risk. During the follow-up period, 8.7% patients with microalbuminuria (n=166) and 1.8% patients with overt nephropathy (n=34) reversed to normoalbuminuria or microalbuminuria.
Conclusions: AER is a more sensitive predictor than eGFR for CVD onset in T2DM patients. DKD, even patients with overt nephropathy can be reversed after multi-factorial intervention in a part of patients.

Disclosure
X. Zhang: None. S. Yuan: None.