Magalie A. Ravier

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated β-cells. Among the potential miR-124a gene targets identified by biocomputation, Foxa2 is known to play a role in β-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 β-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K+ (KATP) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose- or KCl-stimulated intracellular free Ca2+ concentrations in single MIN6 and INS-1 (832/13) β-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the β-cell exocytotic machinery to Ca2+. In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in β-cells responsible for modulating intracellular signaling.

The mitochondrial Ca2+ uniporter MCU is essential for glucose-induced ATP increases in pancreatic β-cells.

Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca2+ influx. As well as activating ATP-consuming processes, cytosolic Ca2+ increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca2+ transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca2+ uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca2+ and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca2+ uniporter, MCU, is required for depolarisation-induced mitochondrial Ca2+ increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na+-Ca2+ exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca2+ uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca2+ accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.

Frequency-dependent mitochondrial Ca2+ accumulation regulates ATP synthesis in pancreatic β cells

Pancreatic β cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K+ channels and electrical spiking. The latter results in oscillatory Ca2+ influx through voltage-gated Ca2+ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca2+]cyt and [Ca2+]mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca2+-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca2+ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary β cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca2+ and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca2+ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca2+ oscillation frequency as stable increases in [Ca2+]mit and cytosolic ATP/ADP; (3) mitochondrial Ca2+ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca2+]cyt and [Ca2+]mit is essentially unaffected by changes in endoplasmic reticulum Ca2+ ([Ca2+]ER). Our findings thus highlight new aspects of Ca2+ signalling in β cells of relevance to the actions of both glucose and sulphonylureas.

Isolation and Culture of Mouse Pancreatic Islets for Ex Vivo Imaging Studies with Trappable or Recombinant Fluorescent Probes

The endocrine pancreas contains small clusters of 1,000-2,000 neuroendocrine cells termed islets of Langerhans. By secreting insulin, glucagon, or other hormones as circumstances dictate, islets play a central role in the control of glucose homeostasis in mammals. Islets are dispersed throughout the exocrine tissue and comprise only 1-2% of the volume of the whole organ; human pancreas contains about 10(6) islets whereas rodents have approximately 2 x 10(3) islets. The isolation of islets from the exocrine tissue usually begins with digestion of the pancreas with collagenase. Collagenase-containing medium is either injected into the pancreatic duct, and the organ left to digest in situ, or added after isolation of the pancreas and its dissection into small pieces ex vivo. Islets can then be separated from the exocrine tissue by gradient density or by handpicking. The islets obtained can either be used intact, for example, to measure insulin or glucagon secretion or be dispersed into single cells with a Ca(2+)-free medium or with trypsin/dispase. The latter facilitates the introduction of recombinant or trappable probes and microimaging studies of, for example, changes in cytosolic-free Ca(2+) concentration or the dynamics of individual organelles or proteins.

Emerging roles for β-arrestin-1 in the control of the pancreatic β-cell function and mass: new therapeutic strategies and consequences for drug screening.

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase in β-cell mass, decreased β-cell survival and impaired glucose-dependent insulin release. Pancreatic β-cell proliferation, survival and secretion are thought to be regulated by signalling pathways linked to G-protein coupled receptors (GPCRs), such as the glucagon-like peptide-1 (GLP-1) and the pituitary adenylate cyclase-activating polypeptide (PACAP) receptors. β-arrestin-1 serves as a multifunctional adaptor protein that mediates receptor desensitization, receptor internalization, and links GPCRs to downstream pathways such as tyrosine kinase Src, ERK1/2 or Akt/PKB. Importantly, recent studies found that β-arrestin-1 mediates GLP-1 signalling to insulin secretion, GLP-1 antiapoptotic effect by phosphorylating the proapoptotic protein Bad through ERK1/2 activation, and PACAP potentiation of glucose-induced long-lasting ERK1/2 activation controlling IRS-2 expression. Together, these novel findings reveal an important functional role for β-arrestin-1 in the regulation of insulin secretion and β-cell survival by GPCRs.

Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4.

Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells.

Proteasomal degradation of the histone acetyl transferase p300 contributes to beta-cell injury in a diabetes environment

In type 2 diabetes, amyloid oligomers, chronic hyperglycemia, lipotoxicity, and pro-inflammatory cytokines are detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The histone acetyl transferase p300, involved in remodeling of chromatin structure by epigenetic mechanisms, is a key ubiquitous activator of the transcriptional machinery. In this study, we report that loss of p300 acetyl transferase activity and expression leads to beta-cell apoptosis, and most importantly, that stress situations known to be associated with diabetes alter p300 levels and functional integrity. We found that proteasomal degradation is the mechanism subserving p300 loss in beta-cells exposed to hyperglycemia or pro-inflammatory cytokines. We also report that melatonin, a hormone produced in the pineal gland and known to play key roles in beta-cell health, preserves p300 levels altered by these toxic conditions. Collectively, these data imply an important role for p300 in the pathophysiology of diabetes.

O51 La beta-arrestine 2 joue un rôle clé dans l’expansion compensatoire de la masse des cellules beta-pancréatiques

Introduction La resistance a l’insuline au cours de l’obesite induit une augmentation compensatoire de la masse des cellules β pancreatiques. Nous avons recemment montre que la β-arrestine-2 (β-arr2), proteine d’echafaudage, est requise dans la signalisation insulinique connue pour reguler la masse fonctionnelle des cellules β. Notre but est d’evaluer le role de la β-arr2 dans l’expansion de la masse des cellules β induite par un regime riche en graisses (HFD), en utilisant des souris invalidees pour la b-arr2 (β-arr2-/-). Materiels et methodes Les experiences ont ete realisees sur des souris mâles β-arr2+/+ et β-arr2-/-. Les animaux ont ete nourris par un regime standard (5 % kcal % graisse) ou par un HFD (45 % kcal % graisse) pendant 19 semaines. La proliferation (anti-Ki67) et la masse des cellules β (anti-insuline) ont ete evaluees par immunofluorescence. Resultats Sous regime standard, les souris b-arr2-/- presentent une architecture normale des ilots mais une masse reduite des cellules β (40 %). Sous HFD, les souris β-arr2-/- montrent une surcharge ponderale comparable aux β-arr2+/+ mais une hyperinsulinemie compensatoire deficiente. De plus, alors que les souris β-arr2+/+ presentent un doublement de la masse des cellules β associee a une augmentation de la proliferation, les souris β-arr2-/- sont incapables de presenter cette adaptation compensatrice. De maniere interessante, l’expression de la β-arr2 dans les cellules β est reduite dans des conditions d’insulino-resistance induites par le palmitate (in vitro) et l’obesite (in vivo). Conclusion La β-arr2 est impliquee dans la plasticite de la masse des cellules β, non seulement en condition normale, mais egalement dans l’expansion compensatoire en reponse au regime gras. Un defaut d’expression de cette proteine dans les etats d’insulino-resistance pourrait participer au deficit de l’expansion compensatoire de la masse des cellules β survenant au cours du diabete de type 2.