RESUMO
AIM/HYPOTHESIS: AMP-activated protein kinase (AMPK), encoded by Prkaa genes, is emerging as a key regulator of overall energy homeostasis and the control of insulin secretion and action. We sought here to investigate the role of AMPK in controlling glucagon secretion from pancreatic islet alpha cells. METHODS: AMPK activity was modulated in vitro in clonal alphaTC1-9 cells and isolated mouse pancreatic islets using pharmacological agents and adenoviruses encoding constitutively active or dominant negative forms of AMPK. Glucagon secretion was measured during static incubation by radioimmunoassay. AMPK activity was assessed by both direct phosphotransfer assay and by western (immuno-)blotting of the phosphorylated AMPK α subunits and the downstream target acetyl-CoA carboxylase 1. Intracellular free [Ca²(+)] was measured using Fura-Red. RESULTS: Increasing glucose concentrations strongly inhibited AMPK activity in clonal pancreatic alpha cells. Forced increases in AMPK activity in alphaTC1-9 cells, achieved through the use of pharmacological agents including metformin, phenformin and A-769662, or via adenoviral transduction, resulted in stimulation of glucagon secretion at both low and high glucose concentrations, whereas AMPK inactivation inhibited both [Ca²(+)](i) increases and glucagon secretion at low glucose. Transduction of isolated mouse islets with an adenovirus encoding AMPK-CA under the control of the preproglucagon promoter increased glucagon secretion selectively at elevated glucose concentrations. CONCLUSIONS/INTERPRETATION: AMPK is strongly regulated by glucose in pancreatic alpha cells, and increases in AMPK activity are sufficient and necessary for the stimulation of glucagon release in vitro. Modulation of AMPK activity in alpha cells may therefore provide a novel approach to controlling blood glucose concentrations.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Secretoras de Glucagon/enzimologia , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Acetiltransferases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Compostos de Bifenilo , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Feminino , Células Secretoras de Glucagon/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Imuno-Histoquímica , Metformina/farmacologia , Camundongos , Fenformin/farmacologia , Fosforilação/efeitos dos fármacos , Pironas/farmacologia , Tiofenos/farmacologiaRESUMO
AIMS/HYPOTHESIS: AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain. METHODS: The catalytic alpha1 and alpha2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkalpha1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkalpha2 [also known as Prkaa2] alleles (Ampkalpha1 ( -/- ),alpha2( fl/fl ),), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca(2+) were measured with standard techniques. RESULTS: Trigenic Ampkalpha1 ( -/- ),alpha2( fl/fl ) expressing Cre recombinase and lacking both AMPKalpha subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca(2+), while granule docking and insulin secretion were enhanced. Conversely, betaAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion. CONCLUSIONS/INTERPRETATION: Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Hipotálamo/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Neurônios/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Análise de Variância , Animais , Glicemia/metabolismo , Peso Corporal/genética , Gorduras na Dieta , Ingestão de Alimentos/genética , Eletrofisiologia , Imunofluorescência , Teste de Tolerância a Glucose , Hiperglicemia/genética , Hiperglicemia/metabolismo , Insulina/genética , Secreção de Insulina , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , RatosRESUMO
Treatment of type 1 diabetes by islet transplantation is currently limited by loss of functional beta-cell mass after transplantation. We investigated here whether adenovirus-mediated changes in AMP-activated protein kinase (AMPK) activity, previously shown to affect insulin secretion in vitro, might affect islet graft function in vivo. In isolated mouse and rat islets, insulin secretion stimulated by 17 (vs 3) mmol/l glucose was inhibited by 36.5% (P<0.01) and 43% (P<0.02) respectively after over-expression of constitutively-active AMPK- (AMPK CA) versus null (eGFP-expressing) viruses, and glucose oxidation was decreased by 38% (P<0.05) and 26.6% (P<0.05) respectively. Increases in apoptotic index (terminal deoxynucleotide transferase-mediated deoxyuridine trisphosphate biotin nick end-labelling) (TUNEL)) were also observed in AMPK CA- (22.8 +/- 3.6% TUNEL-positive cells, P<0.001), but not AMPK DN- (2.72 +/- 3.9%, positive cells, P=0.05) infected islets, versus null adenovirus-treated islets (0.68 +/- 0.36% positive cells). Correspondingly, transplantation of islets expressing AMPK CA into streptozotocin-diabetic C57 BL/6 mice improved glycaemic control less effectively than transplantation with either null (P<0.02) or AMPK-DN-infected (P<0.01) islets. We conclude that activation of AMPK inhibits beta-cell function in vivo and may represent a target for therapeutic intervention during islet transplantation.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Adenoviridae/genética , Animais , Células Cultivadas , Diabetes Mellitus Experimental/fisiopatologia , Ativação Enzimática , Expressão Gênica , Vetores Genéticos/farmacologia , Teste de Tolerância a Glucose , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/farmacologiaRESUMO
AMP-activated protein kinase (AMPK) is a regulator of cellular metabolism in response to changes in the energy status of the cells. AMPK was known to shut down energy-consuming pathways in response to a fall in the ATP/AMP ratio by phosphorylating key enzymes of intermediate metabolism. Here we will discuss the recent evidence implicating AMPK in the regulation of gene expression in mammals, mainly in the liver and in the pancreatic beta-cells.
Assuntos
Regulação da Expressão Gênica , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Ativação Enzimática , Humanos , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Complexos Multienzimáticos/genética , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Mutations in the HNF4alpha gene are responsible for type 1 maturity-onset diabetes of the young (MODY1), which is characterized by a defect in insulin secretion. Hepatocyte nuclear factor (HNF)-4alpha is a transcription factor that plays a critical role in the transcriptional regulation of genes involved in glucose metabolism in both hepatocytes and pancreatic beta-cells. Recent evidence has implicated AMP-activated protein kinase (AMPK) in the modulation of both insulin secretion by pancreatic beta-cells and the control of glucose-dependent gene expression in both hepatocytes and beta-cells. Therefore, the question could be raised as to whether AMPK plays a role in these processes by modulating HNF-4alpha function. In this study, we show that activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) in hepatocytes greatly diminished HNF-4alpha protein levels and consequently downregulates the expression of HNF-4alpha target genes. Quantitative evaluation of HNF-4alpha target gene expression revealed diminished mRNA levels for HNF-1alpha, GLUT2, L-type pyruvate kinase, aldolase B, apolipoprotein (apo)-B, and apoCIII. Our data clearly demonstrate that the MODY1/HNF-4alpha transcription factor is a novel target of AMPK in hepatocytes. Accordingly, it can be suggested that in pancreatic beta-cells, AMPK also acts by decreasing HNF-4alpha protein level, and therefore insulin secretion. Hence, the possible role of AMPK in the physiopathology of type 2 diabetes should be considered.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 1/metabolismo , Complexos Multienzimáticos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/farmacologia , Animais , Apolipoproteína C-III , Apolipoproteínas B/biossíntese , Apolipoproteínas B/genética , Apolipoproteínas C/biossíntese , Apolipoproteínas C/genética , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Regulação para Baixo , Ativação Enzimática , Frutose-Bifosfato Aldolase/biossíntese , Frutose-Bifosfato Aldolase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 2 , Fator 4 Nuclear de Hepatócito , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Fígado/enzimologia , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Piruvato Quinase/biossíntese , Piruvato Quinase/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/farmacologia , Fatores de Tempo , Transcrição GênicaRESUMO
Elevated glucose concentrations stimulate the transcription of the pre-proinsulin (PPI), L-type pyruvate kinase (L-PK), and other genes in islet beta cells. In liver cells, pharmacological activation by 5-amino-4-imidazolecarboxamide riboside (AICAR) of AMP-activated protein kinase (AMPK), the mammalian homologue of the yeast SNF1 kinase complex, inhibits the effects of glucose, suggesting a key signaling role for this kinase. Here, we demonstrate that AMPK activity is inhibited by elevated glucose concentrations in MIN6 beta cells and that activation of the enzyme with AICAR prevents the activation of the L-PK gene by elevated glucose. Furthermore, microinjection of antibodies to the alpha2- (catalytic) or beta2-subunits of AMPK complex, but not to the alpha1-subunit or extracellular stimulus-regulated kinase, mimics the effects of elevated glucose on the L-PK and PPI promoter activities as assessed by single-cell imaging of promoter luciferase constructs. In each case, injection of antibodies into the nucleus and cytosol, but not the nucleus alone, was necessary, indicating the importance of either a cytosolic phosphorylation event or the subcellular localization of the alpha2-subunits. Incubation with AICAR diminished, but did not abolish, the effect of glucose on PPI transcription. These data suggest that glucose-induced changes in AMPK activity are necessary and sufficient for the regulation of the L-PK gene by the sugar and also play an important role in the regulation of the PPI promoter.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Ilhotas Pancreáticas/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Quinase/genética , Proteínas Quinases Ativadas por AMP , Sequência de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Anticorpos/imunologia , Linhagem Celular , Hipoglicemiantes/farmacologia , Insulina/genética , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/enzimologia , Dados de Sequência Molecular , Complexos Multienzimáticos/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Ribonucleotídeos/farmacologia , Frações Subcelulares/enzimologiaRESUMO
This review describes the latest approaches towards using gene therapy as a treatment for non-insulin dependent diabetes mellitus (NIDDM; Type 2 diabetes). We examine attempts to directly deliver the insulin gene to non-beta-cells, to improve insulin secretion from existing beta-cells and to develop ex vivo approaches to implanting genetically modified cells. Future research into the pathology of non-insulin dependent diabetes, combined with the latest developments in gene delivery systems, may potentially make gene therapy an attractive alternative NIDDM treatment in the future.
Assuntos
Antígenos de Neoplasias , Biomarcadores Tumorais , Diabetes Mellitus Tipo 2/terapia , Terapia Genética , Proteínas de Homeodomínio , Insulina/genética , Lectinas Tipo C , Adulto , Animais , Glicemia/análise , Transplante de Células , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Regulação da Expressão Gênica , Genes Sintéticos , Vetores Genéticos , Glucagon/genética , Glucagon/fisiologia , Peptídeo 1 Semelhante ao Glucagon , Glucoquinase/genética , Glucoquinase/fisiologia , Transportador de Glucose Tipo 2 , Humanos , Hiperinsulinismo/etiologia , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Camundongos , Pessoa de Meia-Idade , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/fisiologia , Contração Muscular , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo I , Proteínas Associadas a Pancreatite , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Precursores de Proteínas/fisiologia , Proteínas/genética , Proteínas/fisiologia , Ratos , Transativadores/genética , Transativadores/fisiologiaRESUMO
5-Amino-4-imidazolecarboxamide riboside (AICAR) is known to stimulate rat liver 5'-AMP-activated protein kinase (AMPK). AMPK is the mammalian homologue of Snf1p in yeast, involved in derepression of glucose-repressed genes. We used AICAR to test if AMPK could also play a role in the regulation of glucose-dependent genes in mammalian cells. At a concentration which induces phosphorylation-dependent inactivation of HMG-CoA reductase, AICAR blocked glucose activation of three glucose responsive genes, namely L-type pyruvate kinase (L-PK), Spot 14 and fatty acid synthase genes in primary cultured hepatocytes, but was without any action on glucose phosphorylation to glucose 6-phosphate and on expression of PEPCK, albumin and beta-actin genes. AICAR was also found to inhibit activation of the L-PK gene promoter by glucose in transiently transfected hepatoma cells. Therefore our results suggest that AMPK is probably involved in the glucose signal pathway regulating gene expression in the liver.
Assuntos
Glucose/metabolismo , Fígado/enzimologia , Complexos Multienzimáticos/metabolismo , Proteínas Quinases/metabolismo , Ativação Transcricional , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Carcinoma Hepatocelular , Células Cultivadas , Ativação Enzimática , Ácido Graxo Sintases/efeitos dos fármacos , Ácido Graxo Sintases/genética , Glucose/antagonistas & inibidores , Glucose-6-Fosfato/metabolismo , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Nucleares , Fosforilação , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/efeitos dos fármacos , Proteínas/genética , Piruvato Quinase/genética , RNA Mensageiro/metabolismo , Ribonucleotídeos/farmacologia , Fatores de Transcrição , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
Possible changes in glucose tolerance and substrate oxidation after a high-carbohydrate, low-fat diet were studied in seven healthy volunteers. Each subject consumed two experimental diets for 1 week after 1 week on a stabilization diet; diet no. 1 11% fat and 64% carbohydrates, and diet no. 2 30% fat and 45% carbohydrates. At the end of each experimental week, plasma levels of glucose, insulin, and free fatty acids were measured before and every 30 minutes for 6 hours after a 75-g oral glucose challenge. At the same time, energy expenditure and substrate oxidation were measured by indirect calorimetry. Plasma lipid and lipoprotein levels were measured at the end of one stabilization period and at the end of each diet. Plasma glucose concentrations and areas under the curve of glucose concentrations were identical after the two experimental periods; the means +/- standard deviation for the values at 120 minutes were 6.4 +/- 0.3 and 6.4 +/- 0.6 mmol/L after diets no. 1 and 2, respectively, and areas under the curve were 1,853 +/- 115 and 1,862 +/- 211 mmol.min/L after diets no. 1 and 2, respectively. Similarly, plasma concentrations of insulin and free fatty acids after glucose ingestion were unaffected by the dietary changes. Energy expenditure increased after glucose administration, and this thermic effect of glucose was identical after the two experimental diets at 4.2% +/- 1.4%, and 3.9% +/- 1.4% of ingested energy for diets no. 1 and 2, respectively. Substrate oxidation rates were also identical for both the fasted and post-glucose periods after the two diets.(ABSTRACT TRUNCATED AT 250 WORDS)