RESUMO
Tight coupling between cytosolic and mitochondrial metabolism is key for GSIS (glucose-stimulated insulin secretion). In the present study we examined the regulatory contribution of PDH (pyruvate dehydrogenase) kinase 1, a negative regulator of PDH, to metabolic coupling in 832/13 clonal beta-cells. Knockdown of PDH kinase 1 with siRNA (small interfering RNA) reduced its mRNA (>80%) and protein level (>40%) after 72 h. PDH activity, glucose-stimulated cellular oxygen consumption and pyruvate-stimulated mitochondrial oxygen consumption increased 1.7- (P<0.05), 1.6- (P<0.05) and 1.6-fold (P<0.05) respectively. Gas chromatography/MS revealed an altered metabolite profile upon silencing of PDH kinase 1, determined by increased levels of the tricarboxylic acid cycle intermediates malate, fumarate and alpha-ketoglutarate. These metabolic alterations were associated with exaggerated GSIS (5-fold compared with 3.1-fold in control cells; P<0.01). Insulin secretion, provoked by leucine and dimethylsuccinate, which feed into the tricarboxylic acid cycle bypassing PDH, was unaffected. The oxygen consumption and metabolic data strongly suggest that knockdown of PDH kinase 1 in beta-cells permits increased metabolic flux of glucose-derived carbons into the tricarboxylic acid cycle via PDH. Enhanced insulin secretion is probably caused by increased generation of tricarboxylic acid cycle-derived reducing equivalents for mitochondrial electron transport to generate ATP and/or stimulatory metabolic intermediates. On the basis of these findings, we suggest that PDH kinase 1 is an important regulator of PDH in clonal beta-cells and that PDH kinase 1 and PDH are important for efficient metabolic coupling. Maintaining low PDH kinase 1 expression/activity, keeping PDH in a dephosphorylated and active state, may be important for beta-cells to achieve the metabolic flux rates necessary for maximal GSIS.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Linhagem Celular , Células Clonais , Humanos , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Mitocôndrias/enzimologia , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/fisiologia , Piruvato Desidrogenase Quinase de Transferência de Acetil , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Metabolites generated from fuel metabolism in pancreatic beta-cells control exocytosis of insulin, a process which fails in type 2 diabetes. To identify and quantify these metabolites, global and unbiased analysis of cellular metabolism is required. To this end, polar metabolites, extracted from the clonal 832/13 beta-cell line cultured at 2.8 and 16.7 mM glucose for 48 h, were derivatized followed by identification and quantification, using gas chromatography (GC) and mass spectrometry (MS). After culture at 16.7 mM glucose for 48 h, 832/13 beta-cells exhibited a phenotype reminiscent of glucotoxicity with decreased content and secretion of insulin. The metabolomic analysis revealed alterations in the levels of 7 metabolites derived from glycolysis, the TCA cycle and pentose phosphate shunt, and 4 amino acids. Principal component analysis of the metabolite data showed two clusters, corresponding to the cells cultured at 2.8 and 16.7 mM glucose, respectively. Concurrent changes in protein expression were analyzed by 2-D gel electrophoresis followed by LC-MS/MS. The identities of 86 spots corresponding to 75 unique proteins that were significantly different in 832/13 beta-cells cultured at 16.7 mM glucose were established. Only 5 of these were found to be metabolic enzymes that could be involved in the metabolomic alterations observed. Anticipated changes in metabolite levels in cells exposed to increased glucose were observed, while changes in enzyme levels were much less profound. This suggests that substrate availability, allosteric regulation, and/or post-translational modifications are more important determinants of metabolite levels than enzyme expression at the protein level.