M19 Modulates Skeletal Muscle Differentiation and Insulin Secretion in Pancreatic β-Cells through Modulation of Respiratory Chain Activity

Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion

Pancreatic and muscular neuronal NO synthases in the pathogenesis of prediabetic states

Le diabète de type 2, défini par une hyperglycémie chronique, résulte d'un déficit de la sécrétion d'insuline et d'une insulinorésistance. Durant le prédiabète qui précède la maladie, la cellule ß pancréatique est capable d'établir une hyperactivité sécrétoire compensatrice de l'insulinorésistance. Les NO synthases neuronales (nNOS) pancréatique et musculaire contrôlent respectivement la sécrétion d'insuline induite par le glucose dans la cellule ß et la force contractile, la captation et l'utilisation du glucose dans les myocytes. Dans le modèle génétique du rat obèse Zucker fa/fa mimant l'état prédiabétique associant un hyperinsulinisme et une insulinorésistance, nous avons retrouvé au niveau de la cellule ß une forte augmentation du complexe entre la nNOS et son inhibiteur endogène PIN (Protein Inhibitor of Neuronal NOS) au niveau des granules de sécrétion d'insuline. Ce complexe, grâce à une interaction accrue avec la myosine V, participe à l'hyperactivité sécrétoire de la cellule ß pancréatique. En effet, des molécules inhibant spécifiquement l'interaction nNOS-PIN permettent de rétablir, chez le rat fa/fa, une sécrétion d'insuline normale. Au niveau musculaire, nous avons observé, dans ce modèle animal, une diminution d'expression de la nNOS sans variation du taux d'ARNm, traduisant une protéolyse accrue de la protéine. L'inhibition de la dégradation protéasomale permet de restaurer l'expression et l'activité catalytique de la nNOS dans le muscle squelettique. Cette perte de fonctionnalité de l'enzyme participerait à l'installation de l'insulinorésistance. Ces travaux ont permis de valider la nNOS comme une cible potentielle pour la prévention du diabète de type 2.Type 2 diabetes is a chronic disorder defined by chronic hyperglycemia resulting from a deficiency of insulin secretion and an insulin resistance in peripheral tissues and liver. A long lasting silent phase, called prediabetes, precedes the disease and in which pancreatic ß cell is able to improve insulin secretion to compensate for the insulin resistance. The pancreatic and muscular neuronal nitric oxide synthases (nNOS) control respectively glucose-induced insulin secretion in pancreatic ß cell and glucose uptake and utilization in myocytes. In the genetic model of obese Zucker fa/fa rat mimicking the prediabetic state characterized by hyperinsulinemia and insulin resistance, we found a high increase in the amount of the complex between nNOS and its endogenous inhibitor PIN (Protein Inhibitor of Neuronal NOS) at the level of insulin secretory granules within the ß cell. This complex, through an increased interaction with myosin V, participates in the secretory hyperactivity of the pancreatic ß cell, observed in this model of prediabetes. Indeed, molecules that specifically inhibit nNOS-PIN interaction allow to restore a normal insulin secretion in fa/fa rat. In skeletal muscle of this model, we observed a decreased expression of nNOS protein with no change in mRNA levels, suggesting an increased proteolysis of the protein. Inhibition of proteasomal degradation restores the expression and the catalytic activity of nNOS in skeletal muscle. Thus, this loss of functionality of the enzyme could participate in the installation of insulin resistance. This work therefore validated nNOS as a potential target for the prevention of type 2 diabetes

Differential regulation of mouse pancreatic islet insulin secretion and Smad proteins by activin ligands

International audienceGlucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is regulated by paracrine factors, the identity and mechanisms of action of which are incompletely understood. Activins are expressed in pancreatic islets and have been implicated in the regulation of GSIS. Activins A and B signal through a common set of intracellular components, but it is unclear whether they display similar or distinct functions in glucose homeostasis

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

International audienceInsulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect

S-Ethyl-Isothiocitrullin-Based Dipeptides and 1,2,4-Oxadiazole Pseudo-Dipeptides: Solid Phase Synthesis and Evaluation as NO Synthase Inhibitors

International audienceWe previously reported dipeptidomimetic compounds as inhibitors of neuronal and/or inducible NO synthases (n/iNOS) with significant selectivity against endothelial NOS (eNOS). They were composed of an S-ethylisothiocitrullin-like moiety linked to an extension through a peptide bond or a 1,2,4-oxadiazole link. Here, we developed two further series where the extension size was increased to establish more favorable interactions in the NOS substrate access channel. The extension was introduced on the solid phase by the reductive alkylation of an amino-piperidine moiety or an aminoethyl segment in the case of dipeptide-like and 1,2,4-oxadiazole compounds, respectively, with various benzaldehydes. Compared to the previous series, more potent inhibitors were identified with IC 50 in the micromolar to the submicromolar range, with significant selectivity toward nNOS. As expected, most compounds did not inhibit eNOS, and molecular modeling was carried out to characterize the reasons for the selectivity toward nNOS over eNOS. Spectral studies showed that compounds were interacting at the heme active site. Finally, selected inhibitors were found to inhibit intra-cellular iNOS and nNOS expressed in RAW264.7 and INS-1 cells, respectively

Deregulation of hepatic insulin sensitivity induced by central lipid infusion in rats is mediated by nitric oxide.

International audienceBACKGROUND: Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis. METHODOLOGY: We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats). PRINCIPAL FINDINGS: Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid). CONCLUSIONS/SIGNIFICANCE: We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production

Reduced insulin secretion in M19-deficient INS-1 cells.

<p>(<b>A</b>) Northern blot analysis of the <i>M19</i> gene in human tissues. Pa: pancreas; Ki: kidney; Sk: skeletal muscle; Li: liver; Lu: lung; Pl: placenta; Br: brain; He: heart. Molecular markers are shown on the left. (<b>B</b>) Fluorescence microscopy of INS-1 cells double-labeled with the M19-specific P70612 antibody (M19, merge; green) and the MitoTracker dye (MitoTracker, merge; red). (<b>C</b>) Cell fractionation of INS-1 cells. Proteins of the total cell lysate (Lys), the cytosolic (Cyt) and the mitochondria (Mi) fractions were subjected to Western immunobloting. The cytosolic protein tubulin, the mitochondrial protein VDAC and M19 are detected. (<b>D</b>) INS-1 cells were transfected with a control pHYPER vector (sh control) or with the pHYPER vector encoding a M19-specific shRNA (sh M19). Western immunoblot analysis of the cell extracts shows expression levels of M19 and the control protein, tubulin. ATP production was determined in these cells (<b>E</b>), and insulin secretion was measured under basal glucose conditions (<b>F</b>). Results are the mean ± SEM of five (<b>E</b>), or four (<b>F</b>) independent experiments. (*) indicates statistical significance at p<0.05.</p

Identification of a mitochondrial targeting signal.

<p>(<b>A</b>) Prediction of the secondary structure of mouse M19 (<i>Mus musculus</i> NM026063) using 4 different algorithms: phyre, PSIPRED, SAM and jufo. The predicted α-helices are indicated by black lines along the amino-acid sequence. (<b>B</b>) Helical wheel presentation of the N-terminal α-helix of mouse M19, from amino acid 1 to 13. Hydrophobic residues are indicated in black circles while the positively charged amino acids are mentioned with a “+”. The first methionine (amino acid 1), at the top of the figure, is considered as a positively charged residue. (<b>C</b>, <b>D</b>) C2C12 myoblasts were transfected with the pQETriSystem vector encoding histidine-tagged M19 (<b>C</b>) or a histidine-tagged M19 mutant lacking amino acids 1 to 12 (<b>D</b>). Indirect immunofluorescence was performed using an anti-histidine antibody. (<b>E</b>–<b>J</b>) C2C12 myoblasts were transfected with the pEGFP-N1 vector encoding GFP alone (<b>E</b>, <b>F</b>), the pEGFP-N1 vector encoding the N-terminal M19 α-helix fused to the N-terminal end of GFP (<b>G</b>, <b>H</b>), and the PEGFP-C3 vector encoding the N-terminal M19 α-helix coupled to the C-terminal end of GFP (<b>I</b>, <b>J</b>). Fluorescence microscopy allows the direct detection of GFP constructs (<b>E</b>, <b>G</b>, <b>I; green</b>) and the indirect detection of cytochrome c using an anti-cytochrome c antibody (<b>F</b>, <b>H</b>, <b>J; red</b>).</p

Expression of late muscle differentiation markers is affected in differentiated M19-deficient C2C12 cells.

<p>(<b>A</b>, <b>B</b>) C2C12 myoblasts were transfected with a control siRNA (si control) or a M19-specific siRNA (si M19) and were then placed in differentiation medium for 7 days. Protein extracts from transfected cells grown in proliferation medium (d0) or in differentiation-promoting conditions for 3, 5 and 7 days (d3, d5, d7) were analyzed by Western immunobloting using the M19-specific P70612 antibody (<b>A</b>) and a MHCII antibody (<b>B</b>). Densitometry analysis of the detected bands is presented as the relative expression of M19 (<b>A</b>) and MHCII (<b>B</b>) normalized to tubulin. Results are the mean ± SEM of three independent experiments. (*) and (**) indicate statistical significance at p<0.05 and at p<0.01. In a similar experiment, C2C12 myoblasts were transfected with the M19-specific shRNA vector allowing the expression of the specific shRNA with GFP. Cells were placed in differentiation medium for 7 days. Fluorescence microscopy allows the direct visualization of GFP-labeled cells expressing the M19-specific shRNA (<b>C, merge; green</b>) and the detection of MHCII using an anti-MHCII antibody (<b>D, merge; red</b>). (<b>E</b>) Protein extracts from control shRNA-transfected C2C12 cells (sh control) and M19-specific shRNA-transfected C2C12 cells (sh M19) grown in differentiation-promoting conditions for 7 days were analyzed by Western immunoblotting. The expression of the late muscle differentiation markers α-actinin 2, troponin T, MHCI and MHCII is shown, as well as the control protein tubulin.</p