Benoit Hastoy

MicroRNA-7a regulates pancreatic β cell function

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.

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

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.

RFX6 Regulates Insulin Secretion by Modulating Ca2+ Homeostasis in Human β Cells

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.

Increased expression of the diabetes gene SOX4 reduces insulin secretion by impaired fusion pore expansion

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 Ca2+ 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.

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

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, G100 and C103, in its central portion. Substituting G100 and/or C103 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 G100V/C103V 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 G100V/C103V 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.

The β-cell assassin: IAPP cytotoxicity

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 in vivo and in vitro 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.

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

Summary 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-2 σ 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 Gαq/11 and Gαi/o 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 Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.

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

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.

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion

A rare loss-of-function variant p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8) 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, especially compared with individuals matched for the genotype of a common T2D risk variant in SLC30A8, p.Arg325. In genome-edited human IPS-derived β-like cells, we establish that the p.Arg138* variant results in reduced SLC30A8 expression due to haploinsufficiency. In human β-cells loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function, which was also seen in isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aiming at maintaining insulin secretion capacity in T2D.

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

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.