Publications by authors named "Benoit Hastoy"

20 Publications

  • Page 1 of 1

Chromatin 3D interaction analysis of the STARD10 locus unveils FCHSD2 as a regulator of insulin secretion.

Cell Rep 2021 02;34(5):108703

Section of Cell Biology and Functional Genomics, Department of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK; Lee Kong Chian School of Medicine, Nan Yang Technological University, Singapore, Singapore. Electronic address:

Using chromatin conformation capture, we show that an enhancer cluster in the STARD10 type 2 diabetes (T2D) locus forms a defined 3-dimensional (3D) chromatin domain. A 4.1-kb region within this locus, carrying 5 T2D-associated variants, physically interacts with CTCF-binding regions and with an enhancer possessing strong transcriptional activity. Analysis of human islet 3D chromatin interaction maps identifies the FCHSD2 gene as an additional target of the enhancer cluster. CRISPR-Cas9-mediated deletion of the variant region, or of the associated enhancer, from human pancreas-derived EndoC-βH1 cells impairs glucose-stimulated insulin secretion. Expression of both STARD10 and FCHSD2 is reduced in cells harboring CRISPR deletions, and lower expression of STARD10 and FCHSD2 is associated, the latter nominally, with the possession of risk variant alleles in human islets. Finally, CRISPR-Cas9-mediated loss of STARD10 or FCHSD2, but not ARAP1, impairs regulated insulin secretion. Thus, multiple genes at the STARD10 locus influence β cell function.
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http://dx.doi.org/10.1016/j.celrep.2021.108703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856552PMC
February 2021

A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function.

Wellcome Open Res 2019 29;4:150. Epub 2020 Apr 29.

Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, OX3 7LE, UK.

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes ( , , ) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for and with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. KO cells were not viable and displayed elevated markers for ER stress and apoptosis. -KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, -KO cells demonstrated no reduction in K channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.
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http://dx.doi.org/10.12688/wellcomeopenres.15447.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961417PMC
April 2020

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

Nat Commun 2020 01 24;11(1):467. Epub 2020 Jan 24.

Institute of Metabolism and Systems Research (IMSR), and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
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http://dx.doi.org/10.1038/s41467-020-14309-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981144PMC
January 2020

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion.

Nat Genet 2019 11 1;51(11):1596-1606. Epub 2019 Nov 1.

Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Oxford, UK.

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived β-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human β cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced K channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.
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http://dx.doi.org/10.1038/s41588-019-0513-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858874PMC
November 2019

Insulin inhibits glucagon release by SGLT2-induced stimulation of somatostatin secretion.

Nat Commun 2019 01 11;10(1):139. Epub 2019 Jan 11.

Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, OX3 7LE, UK.

Hypoglycaemia (low plasma glucose) is a serious and potentially fatal complication of insulin-treated diabetes. In healthy individuals, hypoglycaemia triggers glucagon secretion, which restores normal plasma glucose levels by stimulation of hepatic glucose production. This counterregulatory mechanism is impaired in diabetes. Here we show in mice that therapeutic concentrations of insulin inhibit glucagon secretion by an indirect (paracrine) mechanism mediated by stimulation of intra-islet somatostatin release. Insulin's capacity to inhibit glucagon secretion is lost following genetic ablation of insulin receptors in the somatostatin-secreting δ-cells, when insulin-induced somatostatin secretion is suppressed by dapagliflozin (an inhibitor of sodium-glucose co-tranporter-2; SGLT2) or when the action of secreted somatostatin is prevented by somatostatin receptor (SSTR) antagonists. Administration of these compounds in vivo antagonises insulin's hypoglycaemic effect. We extend these data to isolated human islets. We propose that SSTR or SGLT2 antagonists should be considered as adjuncts to insulin in diabetes therapy.
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http://dx.doi.org/10.1038/s41467-018-08193-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329806PMC
January 2019

The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment.

Biochim Biophys Acta Biomembr 2019 03 20;1861(3):670-676. Epub 2018 Dec 20.

Laboratoire de Chimie et Biologie des Membranes et Nano-objets, CNRS UMR 5248, Université de Bordeaux, Allée Geoffroy St Hilaire, F-33600 Pessac, France. Electronic address:

Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2, or a peptide mutated at the central residues G/C (VAMP2VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2, in terms of α-helices and β-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G/C within the transmembrane domain during lipid rearrangements in membrane fusion.
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http://dx.doi.org/10.1016/j.bbamem.2018.12.011DOI Listing
March 2019

Electrophysiological properties of human beta-cell lines EndoC-βH1 and -βH2 conform with human beta-cells.

Sci Rep 2018 11 19;8(1):16994. Epub 2018 Nov 19.

Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

Limited access to human islets has prompted the development of human beta cell models. The human beta cell lines EndoC-βH1 and EndoC-βH2 are increasingly used by the research community. However, little is known of their electrophysiological and secretory properties. Here, we monitored parameters that constitute the glucose-triggering pathway of insulin release. Both cell lines respond to glucose (6 and 20 mM) with 2- to 3-fold stimulation of insulin secretion which correlated with an elevation of [Ca], membrane depolarisation and increased action potential firing. Similar to human primary beta cells, K channel activity is low at 1 mM glucose and is further reduced upon increasing glucose concentration; an effect that was mimicked by the K channel blocker tolbutamide. The upstroke of the action potentials reflects the activation of Ca channels with some small contribution of TTX-sensitive Na channels. The repolarisation involves activation of voltage-gated Kv2.2 channels and large-conductance Ca-activated K channels. Exocytosis presented a similar kinetics to human primary beta cells. The ultrastructure of these cells shows insulin vesicles composed of an electron-dense core surrounded by a thin clear halo. We conclude that the EndoC-βH1 and -βH2 cells share many features of primary human β-cells and thus represent a useful experimental model.
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http://dx.doi.org/10.1038/s41598-018-34743-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242937PMC
November 2018

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells.

Nat Genet 2018 08 27;50(8):1122-1131. Epub 2018 Jul 27.

Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, UK.

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human β-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in β-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.
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http://dx.doi.org/10.1038/s41588-018-0173-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237273PMC
August 2018

NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations.

Stem Cell Res 2018 05 23;29:220-231. Epub 2018 Apr 23.

Department of Stem Cell Biology, Novo Nordisk A/S, DK-2760 Måløv, Denmark. Electronic address:

Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.
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http://dx.doi.org/10.1016/j.scr.2018.04.010DOI Listing
May 2018

AP2σ Mutations Impair Calcium-Sensing Receptor Trafficking and Signaling, and Show an Endosomal Pathway to Spatially Direct G-Protein Selectivity.

Cell Rep 2018 01 28;22(4):1054-1066. Epub 2018 Jan 28.

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. Electronic address:

Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gα and Gα pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gα. Thus, compartmental bias for CaSR-mediated Gα endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.
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http://dx.doi.org/10.1016/j.celrep.2017.12.089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792449PMC
January 2018

Fusion pore in exocytosis: More than an exit gate? A β-cell perspective.

Cell Calcium 2017 12 7;68:45-61. Epub 2017 Nov 7.

Laboratoire de Chimie et Biologie des Membranes et Nano-objets (CBMN), CNRS UMR 5248, Université de Bordeaux, Allée de Geoffrey St Hilaire, 33600 Pessac, France. Electronic address:

Secretory vesicle exocytosis is a fundamental biological event and the process by which hormones (like insulin) are released into the blood. Considerable progress has been made in understanding this precisely orchestrated sequence of events from secretory vesicle docked at the cell membrane, hemifusion, to the opening of a membrane fusion pore. The exact biophysical and physiological regulation of these events implies a close interaction between membrane proteins and lipids in a confined space and constrained geometry to ensure appropriate delivery of cargo. We consider some of the still open questions such as the nature of the initiation of the fusion pore, the structure and the role of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (SNARE) transmembrane domains and their influence on the dynamics and regulation of exocytosis. We discuss how the membrane composition and protein-lipid interactions influence the likelihood of the nascent fusion pore forming. We relate these factors to the hypothesis that fusion pore expansion could be affected in type-2 diabetes via changes in disease-related gene transcription and alterations in the circulating lipid profile. Detailed characterisation of the dynamics of the fusion pore in vitro will contribute to understanding the larger issue of insulin secretory defects in diabetes.
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http://dx.doi.org/10.1016/j.ceca.2017.10.005DOI Listing
December 2017

The β-cell assassin: IAPP cytotoxicity.

J Mol Endocrinol 2017 10 15;59(3):R121-R140. Epub 2017 Aug 15.

Oxford Centre for Diabetes Endocrinology and MetabolismUniversity of Oxford, Oxford, UK

Islet amyloid polypeptide (IAPP) forms cytotoxic oligomers and amyloid fibrils in islets in type 2 diabetes (T2DM). The causal factors for amyloid formation are largely unknown. Mechanisms of molecular folding and assembly of human IAPP (hIAPP) into β-sheets, oligomers and fibrils have been assessed by detailed biophysical studies of hIAPP and non-fibrillogenic, rodent IAPP (rIAPP); cytotoxicity is associated with the early phases (oligomers/multimers) of fibrillogenesis. Interaction with synthetic membranes promotes β-sheet assembly possibly via a transient α-helical molecular conformation. Cellular hIAPP cytotoxicity can be activated from intracellular or extracellular sites. In transgenic rodents overexpressing hIAPP, intracellular pro-apoptotic signals can be generated at different points in β-cell protein synthesis. Increased cellular trafficking of proIAPP, failure of the unfolded protein response (UPR) or excess trafficking of misfolded peptide via the degradation pathways can induce apoptosis; these data indicate that defects in intracellular handling of hIAPP can induce cytotoxicity. However, there is no evidence for IAPP overexpression in T2DM. Extracellular amyloidosis is directly related to the degree of β-cell apoptosis in islets in T2DM. IAPP fragments, fibrils and multimers interact with membranes causing disruption and These findings support a role for extracellular IAPP in β-sheet conformation in cytotoxicity. Inhibitors of fibrillogenesis are useful tools to determine the aberrant mechanisms that result in hIAPP molecular refolding and islet amyloidosis. However, currently, their role as therapeutic agents remains uncertain.
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http://dx.doi.org/10.1530/JME-17-0105DOI Listing
October 2017

A Central Small Amino Acid in the VAMP2 Transmembrane Domain Regulates the Fusion Pore in Exocytosis.

Sci Rep 2017 06 6;7(1):2835. Epub 2017 Jun 6.

Laboratory of Membrane Chemistry and Biology (CBMN), UMR CNRS 5248, Université de Bordeaux, Allée de Geoffroy St Hilaire, 33600, Pessac, France.

Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial. The TMD of the SNARE protein synaptobrevin2/VAMP2 contains two highly conserved small amino acids, G and C, in its central portion. Substituting G and/or C with the β-branched amino acid valine impairs the structural flexibility of the TMD in terms of α-helix/β-sheet transitions in model membranes (measured by infrared reflection-absorption or evanescent wave spectroscopy) during increase in protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs at fusion sites. This structural change is accompanied by reduced membrane fluidity (measured by infrared ellipsometry). The GV/CV mutation nearly abolishes depolarization-evoked exocytosis (measured by membrane capacitance) and hormone secretion (measured biochemically). Single-vesicle optical (by TIRF microscopy) and biophysical measurements of ATP release indicate that GV/CV retards initial fusion-pore opening, hinders its expansion and leads to premature closure in most instances. We conclude that the TMD of VAMP2 plays a critical role in membrane fusion and that the structural mobility provided by the central small amino acids is crucial for exocytosis by influencing the molecular re-arrangements of the lipid membrane that are necessary for fusion pore opening and expansion.
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http://dx.doi.org/10.1038/s41598-017-03013-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460238PMC
June 2017

Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion.

Diabetes 2016 07 18;65(7):1952-61. Epub 2016 Mar 18.

Oxford Centre for Diabetes, Endocrinology & Metabolism, Radcliffe Department of Medicine, Oxford, U.K. Department of Neuroscience and Physiology, University of Göteborg, Göteborg, Sweden Oxford National Institute of Health Research, Biomedical Research Centre, Churchill Hospital, Oxford, U.K.

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.
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http://dx.doi.org/10.2337/db15-1489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996324PMC
July 2016

GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation.

J Clin Invest 2015 Dec 16;125(12):4714-28. Epub 2015 Nov 16.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.
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http://dx.doi.org/10.1172/JCI81975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665783PMC
December 2015

RFX6 regulates insulin secretion by modulating Ca2+ homeostasis in human β cells.

Cell Rep 2014 Dec 11;9(6):2206-18. Epub 2014 Dec 11.

INSERM, U1016, Institut Cochin, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Paris 75014, France. Electronic address:

Development and function of pancreatic β cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse β cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human β cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human β cell line EndoC-βH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca(2+)-channel genes resulting in the reduction in L-type Ca(2+)-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G) that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca(2+)-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes.
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http://dx.doi.org/10.1016/j.celrep.2014.11.010DOI Listing
December 2014

Glutamate acts as a key signal linking glucose metabolism to incretin/cAMP action to amplify insulin secretion.

Cell Rep 2014 Oct 16;9(2):661-73. Epub 2014 Oct 16.

Division of Molecular and Metabolic Medicine, Kobe University Graduate School of Medicine, Chuo-ku, Kobe 650-0017, Japan; The Integrated Center for Mass Spectrometry, Kobe University Graduate School of Medicine, Chuo-ku, Kobe 650-0017, Japan. Electronic address:

Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.
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http://dx.doi.org/10.1016/j.celrep.2014.09.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536302PMC
October 2014

MicroRNA-7a regulates pancreatic β cell function.

J Clin Invest 2014 Jun 1;124(6):2722-35. Epub 2014 May 1.

Dysfunctional microRNA (miRNA) networks contribute to inappropriate responses following pathological stress and are the underlying cause of several disease conditions. In pancreatic β cells, miRNAs have been largely unstudied and little is known about how specific miRNAs regulate glucose-stimulated insulin secretion (GSIS) or impact the adaptation of β cell function to metabolic stress. In this study, we determined that miR-7 is a negative regulator of GSIS in β cells. Using Mir7a2 deficient mice, we revealed that miR-7a2 regulates β cell function by directly regulating genes that control late stages of insulin granule fusion with the plasma membrane and ternary SNARE complex activity. Transgenic mice overexpressing miR-7a in β cells developed diabetes due to impaired insulin secretion and β cell dedifferentiation. Interestingly, perturbation of miR-7a expression in β cells did not affect proliferation and apoptosis, indicating that miR-7 is dispensable for the maintenance of endocrine β cell mass. Furthermore, we found that miR-7a levels are decreased in obese/diabetic mouse models and human islets from obese and moderately diabetic individuals with compensated β cell function. Our results reveal an interconnecting miR-7 genomic circuit that regulates insulin granule exocytosis in pancreatic β cells and support a role for miR-7 in the adaptation of pancreatic β cell function in obesity and type 2 diabetes.
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http://dx.doi.org/10.1172/JCI73066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038573PMC
June 2014
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