RESUMO
OBJECTIVE: Unexplained changes in regulation of branched chain amino acids (BCAA) during diabetes therapy with metformin have been known for years. Here we have investigated mechanisms underlying this effect. METHODS: We used cellular approaches, including single gene/protein measurements, as well as systems-level proteomics. Findings were then cross-validated with electronic health records and other data from human material. RESULTS: In cell studies, we observed diminished uptake/incorporation of amino acids following metformin treatment of liver cells and cardiac myocytes. Supplementation of media with amino acids attenuated known effects of the drug, including on glucose production, providing a possible explanation for discrepancies between effective doses in vivo and in vitro observed in most studies. Data-Independent Acquisition proteomics identified that SNAT2, which mediates tertiary control of BCAA uptake, was the most strongly suppressed amino acid transporter in liver cells following metformin treatment. Other transporters were affected to a lesser extent. In humans, metformin attenuated increased risk of left ventricular hypertrophy due to the AA allele of KLF15, which is an inducer of BCAA catabolism. In plasma from a double-blind placebo-controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin caused selective accumulation of plasma BCAA and glutamine, consistent with the effects in cells. CONCLUSIONS: Metformin restricts tertiary control of BCAA cellular uptake. We conclude that modulation of amino acid homeostasis contributes to therapeutic actions of the drug.
Assuntos
Metformina , Humanos , Metformina/farmacologia , Metformina/uso terapêutico , Aminoácidos de Cadeia Ramificada/metabolismo , Aminoácidos/metabolismo , Glucose , HomeostaseRESUMO
The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.
Assuntos
Proteínas de Transporte/metabolismo , Obesidade/metabolismo , Receptores para Leptina/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Peso Corporal/fisiologia , Proteínas de Transporte/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leptina/metabolismo , Leptina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Receptores para Leptina/fisiologia , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Although considerable efforts have been made since the 1950s to better understand the action of metformin, the first line therapeutic for type 2 diabetes, its mechanisms of action has not been fully elucidated. The main antidiabetic effect of this drug is to decrease hepatic glucose production. A plausible molecular mechanism of action now emerges from recent breakthroughs that place metformin at the control of energy homeostasis. Metformin was shown to induce a mild and transient inhibition of the mitochondrial respiratory chain complex 1. The resulting decrease in hepatic energy state activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, and provided a generally accepted mechanism for metformin action on hepatic gluconeogenic program. However, the role of AMPK activation in metformin action has recently been challenged by loss-of-function experiments. Recent evidence showed that metformin-induced inhibition of hepatic glucose output is mediated by reducing cellular energy charge rather than direct inhibition of gluconeogenic gene expression. Furthermore, recent data support a novel mechanism of action for metformin involving antagonism of glucagon signaling pathways by inducing the accumulation of AMP, which inhibits adenylate cyclase and reduced levels of cAMP.
Assuntos
Hipoglicemiantes/farmacologia , Fígado/efeitos dos fármacos , Metformina/farmacologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Galega/química , História do Século XX , História do Século XXI , História Medieval , Humanos , Hipoglicemiantes/farmacocinética , Fígado/metabolismo , Fígado/fisiologia , Metformina/farmacocinética , Fitoterapia/históriaRESUMO
AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been proposed to function as a fuel gauge to monitor cellular energy status in response to nutritional environmental variations. AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. Numerous observations obtained with pharmacological activators and agents that deplete intracellular ATP have been supportive of AMPK playing a role in the control of energy metabolism but none of these studies have provided conclusive evidence. Relatively recent developments in our understanding of precisely how AMPK complexes might operate to control energy metabolism is due in part to the development of transgenic and knockout mouse models. Although there are inevitable caveats with genetic models, some important findings have emerged. In the present review, we discuss recent findings obtained from animal models with inhibition or activation of AMPK signaling pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/química , Tecido Adiposo/enzimologia , Animais , Animais Geneticamente Modificados , Vasos Sanguíneos/enzimologia , Vasos Sanguíneos/fisiologia , Metabolismo Energético , Técnicas de Inativação de Genes , Humanos , Hipoglicemiantes/farmacologia , Hipotálamo/enzimologia , Resistência à Insulina , Fígado/enzimologia , Modelos Animais , Músculo Esquelético/enzimologia , Miocárdio/enzimologia , Conformação ProteicaRESUMO
Activation of the peroxisome proliferator-activated receptor (PPAR)-alpha increases lipid catabolism and lowers the concentration of circulating lipid, but its role in the control of glucose metabolism is not as clearly established. Here we compared PPARalpha knockout mice with wild type and confirmed that the former developed hypoglycemia during fasting. This was associated with only a slight increase in insulin sensitivity but a dramatic increase in whole-body and adipose tissue glucose use rates in the fasting state. The white sc and visceral fat depots were larger due to an increase in the size and number of adipocytes, and their level of GLUT4 expression was higher and no longer regulated by the fed-to-fast transition. To evaluate whether these adipocyte deregulations were secondary to the absence of PPARalpha from liver, we reexpresssed this transcription factor in the liver of knockout mice using recombinant adenoviruses. Whereas more than 90% of the hepatocytes were infected and PPARalpha expression was restored to normal levels, the whole-body glucose use rate remained elevated. Next, to evaluate whether brain PPARalpha could affect glucose homeostasis, we activated brain PPARalpha in wild-type mice by infusing WY14643 into the lateral ventricle and showed that whole-body glucose use was reduced. Hence, our data show that PPARalpha is involved in the regulation of glucose homeostasis, insulin sensitivity, fat accumulation, and adipose tissue glucose use by a mechanism that does not require PPARalpha expression in the liver. By contrast, activation of PPARalpha in the brain stimulates peripheral glucose use. This suggests that the alteration in adipocyte glucose metabolism in the knockout mice may result from the absence of PPARalpha in the brain.
Assuntos
Tecido Adiposo/metabolismo , Encéfalo/fisiologia , Transportador de Glucose Tipo 4/análise , Glucose/metabolismo , Fígado/fisiologia , PPAR alfa/deficiência , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/química , Animais , Glicemia/análise , Composição Corporal , Encéfalo/efeitos dos fármacos , Tamanho Celular , Jejum , Feminino , Hepatócitos/metabolismo , Hipotálamo/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/genética , PPAR alfa/fisiologia , Proliferadores de Peroxissomos/administração & dosagem , Pirimidinas/administração & dosagem , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The 5' AMP-activated protein kinase (AMPK) is a sensor of cellular energy homeostasis well conserved in all eukaryotic cells. AMPK is activated by rising AMP and falling ATP, either by inhibiting ATP production or by accelerating ATP consumption, by a complex mechanism that results in an ultrasensitive response. AMPK is a heterotrimeric enzyme complex consisting of a catalytic subunit alpha and two regulatory subunits beta and gamma. AMP activates the system by binding to the gamma subunit that triggers phosphorylation of the catalytic alpha subunit by the upstream kinases LKB1 and CaMKKbeta. Once activated, it switches on catabolic pathways (such as fatty acid oxidation and glycolysis) and switches off ATP-consuming pathways (such as lipogenesis) both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. Dominant mutations in the regulatory gamma subunit isoforms cause hypertrophy of cardiac and skeletal muscle providing a link in human diseases caused by defects in energy metabolism. As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of adipokines such as leptin and adiponectin. Moreover, the AMPK system is one of the probable target for the anti-diabetic drug metformin and rosiglitazone. The relationship between AMPK activation and beneficial metabolic effects provides the rationale for the development of new therapeutic strategies. Thus, pharmacological AMPK activation may, through signaling, metabolic and gene expression effects, reduce the risk of Type 2 diabetes, metabolic syndrome and cardiac diseases.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Glucose/metabolismo , Lipogênese/fisiologia , Complexos Multienzimáticos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Quinases Ativadas por AMP , Monofosfato de Adenosina/fisiologia , Trifosfato de Adenosina/metabolismo , Adipogenia/efeitos dos fármacos , Regulação Alostérica , Animais , Doenças Cardiovasculares/enzimologia , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Desenho de Fármacos , Ingestão de Energia , Ativação Enzimática , Ácidos Graxos/biossíntese , Homeostase/fisiologia , Humanos , Hipotálamo/fisiologia , Lipogênese/efeitos dos fármacos , Mamíferos/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Modelos Biológicos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Miocárdio/metabolismo , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Subunidades Proteicas , Rosiglitazona , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêuticoRESUMO
A role for glucose in the control of feeding has been proposed, but its precise physiological importance is unknown. Here, we evaluated feeding behavior in glut2-null mice, which express a transgenic glucose transporter in their beta-cells to rescue insulin secretion (ripglut1;glut2-/- mice). We showed that in the absence of GLUT2, daily food intake was increased and feeding initiation and termination following a fasting period were abnormal. This was accompanied by suppressed regulation of hypothalamic orexigenic and anorexigenic neuropeptides expression during the fast-to-refed transition. In these conditions, however, there was normal regulation of the circulating levels of insulin, leptin, or glucose but a loss of regulation of plasma ghrelin concentrations. To evaluate whether the abnormal feeding behavior was due to suppressed glucose sensing, we evaluated feeding in response to intraperitoneal or intracerebroventricular glucose or 2-deoxy-D-glucose injections. We showed that in GLUT2-null mice, feeding was no longer inhibited by glucose or activated by 2-deoxy-D-glucose injections and the regulation of hypothalamic neuropeptide expression by intracerebroventricular glucose administration was lost. Together, these data demonstrate that absence of GLUT2 suppressed the function of central glucose sensors, which control feeding probably by regulating the hypothalamic melanocortin pathway. Furthermore, inactivation of these glucose sensors causes overeating.