RESUMEN
Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.
Asunto(s)
Carboxiliasas/metabolismo , Metabolismo de los Lípidos , Proteínas Mitocondriales/metabolismo , Sirtuinas/metabolismo , Acetilación , Tejido Adiposo Blanco/metabolismo , Animales , Dieta , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/genética , Lípidos/biosíntesis , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Obesidad/etiología , Obesidad/metabolismo , Oxidación-Reducción , Sirtuinas/genéticaRESUMEN
AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser(485/491) of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser(485/491) phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser(485/491) phosphorylation, which was associated with a â¼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser(485/491) phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser(491) in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser(491) that plays a negative role in insulin signaling in muscle cells.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Insulina/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Animales , Línea Celular , Ratones , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Fosforilación , Serina/metabolismoRESUMEN
AMP-activated protein kinase (AMPK) is an enzyme crucial in cellular metabolism found to be inhibited in many metabolic diseases including type 2 diabetes. Thiazolidinediones (TZDs) are a class of anti-diabetic drug known to activate AMPK through increased phosphorylation at Thr172, however there has been no research to date on whether they have any effect on inhibition of AMPK's lesser known site of inhibition, Ser485/491. HepG2 cells were treated with troglitazone and phosphorylation of AMPK was found to increase at both Thr172 and Ser485 in a dose- and time-dependent manner. Treatment of HepG2 cells with insulin and PMA led to increases in p-AMPK Ser485 via Akt and PKD1 respectively; however these kinases were not found to be implicated in increases seen from troglitazone. Incubation with the other TZDs, rosiglitazone and pioglitazone, let to a minor increase in p-AMPK Ser485 phosphorylation as well as AMPK activity; however these findings were significantly less than those of troglitazone under equal conditions. These data suggest that the effects of troglitazone on AMPK are more complex than previously thought. Phosphorylation at sites of both activation and inhibition can occur in tandem, although the mechanism by which this occurs has not yet been elucidated.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Cromanos/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Activación Enzimática/efectos de los fármacos , Hipoglucemiantes/farmacología , Tiazolidinedionas/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Células Hep G2 , Humanos , Insulina/metabolismo , Fosforilación/efectos de los fármacos , Pioglitazona , Rosiglitazona , TroglitazonaRESUMEN
AIMS/HYPOTHESIS: To directly assess the role of beta cell lipolysis in insulin secretion and whole-body energy homeostasis, inducible beta cell-specific adipose triglyceride lipase (ATGL)-deficient (B-Atgl-KO) mice were studied under normal diet (ND) and high-fat diet (HFD) conditions. METHODS: Atgl flox/flox mice were cross-bred with Mip-Cre-ERT mice to generate Mip-Cre-ERT/+;Atgl flox/flox mice. At 8 weeks of age, these mice were injected with tamoxifen to induce deletion of beta cell-specific Atgl (also known as Pnpla2), and the mice were fed an ND or HFD. RESULTS: ND-fed male B-Atgl-KO mice showed decreased insulinaemia and glucose-induced insulin secretion (GSIS) in vivo. Changes in GSIS correlated with the islet content of long-chain saturated monoacylglycerol (MAG) species that have been proposed to be metabolic coupling factors for insulin secretion. Exogenous MAGs restored GSIS in B-Atgl-KO islets. B-Atgl-KO male mice fed an HFD showed reduced insulinaemia, glycaemia in the fasted and fed states and after glucose challenge, as well as enhanced insulin sensitivity. Moreover, decreased insulinaemia in B-Atgl-KO mice was associated with increased energy expenditure, and lipid metabolism in brown (BAT) and white (WAT) adipose tissues, leading to reduced fat mass and body weight. CONCLUSIONS/INTERPRETATION: ATGL in beta cells regulates insulin secretion via the production of signalling MAGs. Decreased insulinaemia due to lowered GSIS protects B-Atgl-KO mice from diet-induced obesity, improves insulin sensitivity, increases lipid mobilisation from WAT and causes BAT activation. The results support the concept that fuel excess can drive obesity and diabetes via hyperinsulinaemia, and that an islet beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion.
Asunto(s)
Tejido Adiposo/metabolismo , Peso Corporal/fisiología , Metabolismo Energético/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Dieta Alta en Grasa/efectos adversos , Femenino , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Lipasa/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Lipólisis/efectos de los fármacos , Lipólisis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tamoxifeno/farmacología , Espectrometría de Masas en TándemRESUMEN
Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Ácidos Grasos/metabolismo , Mitocondrias/enzimología , Mitocondrias/metabolismo , Sirtuina 3/metabolismo , Acetilación , Acil-CoA Deshidrogenasa de Cadena Larga/química , Adenosina Trifosfato/biosíntesis , Adenosina Trifosfato/metabolismo , Tejido Adiposo Pardo/enzimología , Tejido Adiposo Pardo/metabolismo , Animales , Regulación de la Temperatura Corporal , Restricción Calórica , Carnitina/análogos & derivados , Carnitina/metabolismo , Línea Celular , Frío , Ayuno/metabolismo , Humanos , Hipoglucemia/metabolismo , Hígado/enzimología , Hígado/metabolismo , Masculino , Espectrometría de Masas , Ratones , Oxidación-Reducción , Sirtuina 3/deficiencia , Sirtuina 3/genética , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
Dysregulated autophagy and decreased AMP-activated protein kinase (AMPK) activity are each associated with atherogenesis. Atherogenesis is preceded by high circulating concentrations of glucose and fatty acids, yet the mechanism by which these nutrients regulate autophagy in human aortic endothelial cells (HAECs) is not known. Furthermore, whereas AMPK is recognized as an activator of autophagy in cells with few nutrients, its effects on autophagy in nutrient-rich HAECs has not been investigated. We maintained and passaged primary HAECs in media containing 25 mM glucose and incubated them subsequently with 0.4 mM palmitate. These conditions impaired basal autophagy and rendered HAECs more susceptible to apoptosis and adhesion of monocytes, outcomes attenuated by the autophagy activator rapamycin. Glucose and palmitate diminished AMPK activity and phosphorylation of the uncoordinated-51-like kinase 1 (ULK1) at Ser555, an autophagy-activating site targeted by AMPK. 5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR)-mediated activation of AMPK phosphorylated acetyl-CoA carboxylase, but treatment with AICAR or other AMPK activators (A769662, phenformin) did not restore ULK1 phosphorylation or autophagosome formation. To determine whether palmitate-induced ceramide accumulation contributed to this finding, we overexpressed a ceramide-metabolizing enzyme, acid ceramidase. The increase in acid ceramidase expression ameliorated the effects of excess nutrients on ULK1 phosphorylation, without altering the effects of the AMPK activators. Thus, unlike low nutrient conditions, AMPK becomes uncoupled from autophagy in HAECs in a nutrient-rich environment, such as that found in patients with increased cardiovascular risk. These findings suggest that combinations of AMPK-independent and AMPK-dependent therapies may be more effective alternatives than either therapy alone for treating nutrient-induced cellular dysfunction.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aorta/fisiología , Autofagia/fisiología , Endotelio Vascular/fisiología , Glucosa/administración & dosificación , Ácido Palmítico/administración & dosificación , Aorta/efectos de los fármacos , Autofagia/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Desacopladores/administración & dosificaciónRESUMEN
Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser(485/491), on the α-subunit of AMP-activated protein kinase (AMPK); however, little is known about the regulation of this site in liver and skeletal muscle. We examined whether the inhibitory effects of insulin on AMPK activity may be mediated through the phosphorylation of this inhibitory Ser(485/491) site in hepatocytes, myotubes and incubated skeletal muscle. HepG2 and C2C12 cells were stimulated with or without insulin for 15-min. Similarly, rat extensor digitorum longus (EDL) muscles were treated +/- insulin for 10-min. Insulin significantly increased Ser(485/491) p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40% to 70%, despite no change in p-AMPK Thr(172). Akt inhibition both attenuated the increase in Ser(485/491) p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser(485/491) AMPK phosphorylation, and this too was blunted by inhibition of Akt. Inhibition of the mTOR pathway with rapamycin, however, had no effect on insulin-stimulated Ser(485/491) p-AMPK. These data suggest that insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser(485/491) on its α-subunit.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Hepatocitos/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Serina/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Línea Celular , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Fosforilación , Ratas , Ratas Sprague-Dawley , Sirolimus/farmacologíaRESUMEN
PURPOSE OF REVIEW: Despite a strong correlation between obesity and insulin resistance, 25% of severely obese (BMI >40) individuals are insulin sensitive. In this review, we will examine the factors in adipose tissue that distinguish the two groups, as well as reasons for believing the insulin-sensitive group will be less disease prone. RECENT FINDINGS: Obesity has been linked to the metabolic syndrome with an increase in visceral (intra-abdominal) compared to subcutaneous fat. Recent studies in which adipose tissue of insulin-sensitive and insulin-resistant patients with severe obesity were compared indicate that the insulin-resistant group is also distinguished by increases in oxidative stress and decreases in AMP-activated protein kinase (AMPK) activity. In contrast, changes in the expression of genes for SIRT1, inflammatory cytokines, mitochondrial biogenesis and function, and the two α-isoforms of AMPK showed more depot variation. Studies of how these and other changes in adipose tissue respond to bariatric surgery are still in their infancy. SUMMARY: Available data suggest that increases in oxidative stress, decreases in AMPK activity and SIRT1 gene expression, depot-specific changes in inflammatory, mitochondrial and other genes distinguish adipose tissue of insulin resistant from insulin-sensitive individuals with severe obesity.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Tejido Adiposo/patología , Resistencia a la Insulina , Obesidad Mórbida/patología , Proteínas Quinasas Activadas por AMP/genética , Tejido Adiposo/enzimología , Tejido Adiposo/metabolismo , Animales , Cirugía Bariátrica , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Activación Enzimática , Humanos , Inflamación/patología , Insulina/metabolismo , Síndrome Metabólico/metabolismo , Síndrome Metabólico/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Obesidad Mórbida/enzimología , Obesidad Mórbida/metabolismo , Obesidad Mórbida/cirugía , Estrés Oxidativo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
AMP-activated protein kinase (AMPK) and the NAD(+)-dependent histone/protein deacetylase sirtuin 1 (SIRT1) are metabolic sensors that can increase each other's activity. They are also both activated by the antidiabetic drug metformin and downregulated in the liver under conditions of nutrient excess (e.g., hyperglycemia, high-fat diet, obesity). In these situations, the abundance of the tumor suppressor p53 is increased; however, the relevance of this to the changes in AMPK and SIRT1 is not known. In the present study we investigated this question in HepG2 cells under high glucose conditions. Metformin induced activation of AMPK and SIRT1 and decreased p53 protein abundance. It also decreased triglyceride accumulation and cytosolic oxidative stress (a trigger for p53 accumulation) and increased the deacetylation of p53 at a SIRT1-targeted site. The decrease in p53 abundance caused by metformin was abolished by inhibition of murine double minute 2 (MDM2), a ubiquitin ligase that mediates p53 degradation, as well as by overexpression of a dominant-negative AMPK or a shRNA-mediated knockdown of SIRT1. In addition, overexpression of p53 decreased SIRT1 gene expression and protein abundance, as well as AMPK activity in metformin-treated cells. It also diminished the triglyceride-lowering action of metformin, an effect that was rescued by incubation with the SIRT1 activator SRT2183. Collectively, these findings suggest the existence of a novel reciprocal interaction between AMPK/SIRT1 and p53 that may have implications for the pathogenesis and treatment of metabolic diseases.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/farmacología , Metformina/farmacología , Sirtuina 1/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Glucosa/metabolismo , Células Hep G2 , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño , Sirtuina 1/genética , Triglicéridos/biosíntesisRESUMEN
We previously reported that adenosine monophosphate-activated protein kinase (AMPK) activity is lower in adipose tissue of morbidly obese individuals who are insulin resistant than in comparably obese people who are insulin sensitive. However, the number of patients and parameters studied were small. Here, we compared abdominal subcutaneous, epiploic, and omental fat from 16 morbidly obese individuals classified as insulin sensitive or insulin resistant based on the homeostatic model assessment of insulin resistance. We confirmed that AMPK activity is diminished in the insulin resistant group. A custom PCR array revealed increases in mRNA levels of a wide variety of genes associated with inflammation and decreases in PGC-1α and Nampt in omental fat of the insulin resistant group. In contrast, subcutaneous abdominal fat of the same patients showed increases in PTP-1b, VEGFa, IFNγ, PAI-1, and NOS-2 not observed in omental fat. Only angiotensinogen and CD4(+) mRNA levels were increased in both depots. Surprisingly, TNFα was only increased in epiploic fat, which otherwise showed very few changes. Protein carbonyl levels, a measure of oxidative stress, were increased in all depots. Thus, adipose tissues of markedly obese insulin resistant individuals uniformly show decreased AMPK activity and increased oxidative stress compared with insulin sensitive patients. However, most changes in gene expression appear to be depot-specific.
Asunto(s)
Adenilato Quinasa/metabolismo , Tejido Adiposo/patología , Regulación Enzimológica de la Expresión Génica , Resistencia a la Insulina , Obesidad Mórbida/genética , Estrés Oxidativo , Adenilato Quinasa/genética , Tejido Adiposo/metabolismo , Adulto , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Índice de Masa Corporal , Activación Enzimática , Femenino , Homeostasis , Humanos , Inflamación/genética , Inflamación/metabolismo , Insulina/genética , Insulina/metabolismo , Masculino , Persona de Mediana Edad , Obesidad Mórbida/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart. METHODS AND RESULTS: Mice harboring 1 functional allele of ABC-me (ABC-me(+/-)) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me(+/-) mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me(+/-) hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me(+/-) hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion. CONCLUSIONS: Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Mitocondrias/fisiología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Estrés Oxidativo/genética , Recuperación de la Función/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Volumen Cardíaco/fisiología , Catalasa/metabolismo , Femenino , Predisposición Genética a la Enfermedad/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mitocondrias/efectos de los fármacos , Mutagénesis Insercional , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Compuestos Organometálicos/farmacología , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Presión Ventricular/fisiologíaRESUMEN
Exercise can prevent endothelial cell (EC) dysfunction and atherosclerosis even in the absence of improvements in plasma lipids. However, the mechanisms responsible for these effects are incompletely understood. In this study we examined in mice whether an acute bout of exercise activates enzymes that could prevent EC dysfunction, such as AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). We also examined whether exercise alters known regulators of these enzymes. C57BL/6 mice underwent a single bout of exhaustive treadmill exercise after which their aortas were analyzed for activation of AMPK, AMPK regulatory proteins, eNOS, and various enzymes that, like AMPK, activate eNOS. We found that such exercise acutely activates both AMPK and eNOS in the whole aorta and that the magnitude of these effects correlated with both the distance run and activation of the AMPK regulatory proteins silent information regulator-1 (SIRT1)-LKB1 and CaMKKß. In contrast, Akt, PKA, PKG, and Src, other kinases known to activate eNOS, were unaffected. Immunohistochemical analysis revealed that AMPK and eNOS were both activated in the ECs of the aorta. This study provides the first evidence that an acute bout of exercise activates AMPK and eNOS in the endothelium of the aorta. The results also suggest that AMPK likely is the principal activator of eNOS in this setting and that its own activation may be mediated by both SIRT1-LKB1 and CaMKKß.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aorta Torácica/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Western Blotting , Células Endoteliales/enzimología , Células Endoteliales/fisiología , Endotelio Vascular/enzimología , Endotelio Vascular/fisiología , Activación Enzimática/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/genéticaRESUMEN
Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser(473)) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3ß (Ser(9)) and AS160 (Thr(642)) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.
Asunto(s)
Glucosa/metabolismo , Resistencia a la Insulina , Músculos/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas Activadoras de GTPasa/metabolismo , Glucosa/administración & dosificación , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Masculino , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas WistarRESUMEN
We studied the extraction and analysis of integral membrane proteins possessing hydrophobic and hydrophilic domains and found that a nonionic detergent called MEGA-10, used in lysis buffers, had a superior extraction effect compared to most conventional detergents. A sodium dodecyl sulfate (SDS) concentration of >0.4% (w/v) in the sample buffer was crucial for those proteins to be clearly analyzed by electrophoresis and Western blotting. Furthermore, MEGA-10 had the tendency to maximally extract proteins around its critical micelle concentration (CMC) of 0.24% (w/v). These solutions can greatly assist functional investigations of membrane proteins in the proteomics era.
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Ácidos Grasos/química , Glucosamina/análogos & derivados , Proteínas de la Membrana/análisis , Proteínas de la Membrana/aislamiento & purificación , Dodecil Sulfato de Sodio/química , Tensoactivos/química , Western Blotting/métodos , Tampones (Química) , Electroforesis/métodos , Glucosamina/química , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Micelas , SolubilidadRESUMEN
In recent years, it has become widely accepted that obesity is characterized by a chronic low-grade inflammation of adipose tissue that predisposes affected individuals to insulin resistance, Type 2 diabetes and other disorders associated with the metabolic syndrome. On the other hand, a subset of obese individuals appears to be protected against insulin resistance and the disorders to which it predisposes. The comparison between such insulin-sensitive and insulin-resistant obese individuals offers a unique opportunity to identify key factors that either contribute to or prevent the development of insulin resistance in humans, without the confounding effect of a major difference in fat mass. In the previous issue of the Biochemical Journal, Barbarroja et al. reported that insulin-sensitive obese individuals show less inflammation in their visceral adipose tissue than a group of insulin-resistant subjects matched for BMI (body mass index). This finding reinforces the concept that inflammation in adipose tissue may be a cause of insulin resistance in most obese individuals, although it does not prove it. Further studies will be required for this purpose, as well as to identify the pathogenetic factors that determine whether or not adipose tissue of an obese individual becomes inflamed.
Asunto(s)
Resistencia a la Insulina , Grasa Intraabdominal/inmunología , Obesidad/inmunología , Humanos , Grasa Intraabdominal/metabolismo , Obesidad/metabolismoRESUMEN
Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.
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Hidrolasas de Éster Carboxílico/metabolismo , Insulina/metabolismo , Animales , Secuencia de Bases , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/genética , Línea Celular , Ayuno/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Glucosa/farmacología , Técnica de Clampeo de la Glucosa , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Lipasa/antagonistas & inhibidores , Lipasa/genética , Lipasa/metabolismo , Lipólisis , Masculino , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , Ratas , Transducción de Señal , Triglicéridos/metabolismoRESUMEN
AMP-activated protein kinase (AMPK) and the histone/protein deacetylase SIRT1 are fuel-sensing molecules that have coexisted in cells throughout evolution. When a cell's energy state is diminished, AMPK activation restores energy balance by stimulating catabolic processes that generate ATP and downregulating anabolic processes that consume ATP but are not acutely needed for survival. SIRT1 in turn is best known historically for producing genetic changes that mediate the increase in longevity caused by calorie restriction. Although the two molecules have been studied intensively for many years, only recently has it become apparent that they have similar effects on diverse processes such as cellular fuel metabolism, inflammation, and mitochondrial function. In this review we will examine the evidence that these similarities occur because AMPK and SIRT1 both regulate each other and share many common target molecules. In addition, we will discuss the clinical relevance of these interactions and in particular the possibility that their dysregulation predisposes to disorders such as type 2 diabetes and atherosclerotic cardiovascular disease and is a target for their therapy.
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Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Metabolismo Energético/fisiología , Sirtuina 1/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Diabetes Mellitus Tipo 2/enzimología , Activación Enzimática/fisiología , Regulación Enzimológica de la Expresión Génica , Humanos , Sirtuina 1/genéticaRESUMEN
Insulin resistance in skeletal muscle in obesity and T2DM is associated with reduced muscle oxidative capacity, reduced expression in nuclear genes responsible for oxidative metabolism, and reduced activity of mitochondrial electron transport chain. The presented study was undertaken to analyze mitochondrial content and mitochondrial enzyme profile in skeletal muscle of sedentary lean individuals and to compare that with our previous data on obese or obese T2DM group. Frozen skeletal muscle biopsies obtained from lean volunteers were used to estimate cardiolipin content, mtDNA (markers of mitochondrial mass), NADH oxidase activity of mitochondrial electron transport chain (ETC), and activity of citrate synthase and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes of TCA cycle and beta-oxidation pathway, respectively. Frozen biopsies collected from obese or T2DM individuals in our previous studies were used to estimate activity of beta-HAD. The obtained data were complemented by data from our previous studies and statistically analyzed to compare mitochondrial content and mitochondrial enzyme profile in lean, obese, or T2DM cohort. The total activity of NADH oxidase was reduced significantly in obese or T2DM subjects. The cardiolipin content for lean or obese group was similar, and although for T2DM group cardiolipin showed a tendency to decline, it was statistically insignificant. The total activity of citrate synthase for lean and T2DM group was similar; however, it was increased significantly in the obese group. Activity of beta-HAD and mtDNA content was similar for all three groups. We conclude that the total activity of NADH oxidase in biopsy for lean group is significantly higher than corresponding activity for obese or T2DM cohort. The specific activity of NADH oxidase (per mg cardiolipin) and NADH oxidase/citrate synthase and NADH oxidase/beta-HAD ratios are reduced two- to threefold in both T2DM and obesity.
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Diabetes Mellitus Tipo 2/metabolismo , Transporte de Electrón/fisiología , Mitocondrias/enzimología , Obesidad/metabolismo , Fosforilación Oxidativa , Músculo Cuádriceps/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Adulto , Biopsia , Glucemia/metabolismo , Cardiolipinas/metabolismo , Citrato (si)-Sintasa/metabolismo , ADN Mitocondrial/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/fisiología , Persona de Mediana Edad , Mitocondrias/patología , Complejos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Músculo Cuádriceps/patología , Ácido Tricloroacético/metabolismoRESUMEN
AMPK (AMP-activated protein kinase) is a phylogenetically conserved fuel-sensing enzyme that is present in all mammalian cells. During exercise, it is activated in skeletal muscle in humans, and at least in rodents, also in adipose tissue, liver and perhaps other organs by events that increase the AMP/ATP ratio. When activated, AMPK stimulates energy-generating processes such as glucose uptake and fatty acid oxidation and decreases energy-consuming processes such as protein and lipid synthesis. Exercise is perhaps the most powerful physiological activator of AMPK and a unique model for studying its many physiological roles. In addition, it improves the metabolic status of rodents with a metabolic syndrome phenotype, as does treatment with AMPK-activating agents; it is therefore tempting to attribute the therapeutic benefits of regular physical activity to activation of AMPK. Here we review the acute and chronic effects of exercise on AMPK activity in skeletal muscle and other tissues. We also discuss the potential role of AMPK activation in mediating the prevention and treatment by exercise of specific disorders associated with the metabolic syndrome, including Type 2 diabetes and Alzheimer's disease.
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Proteínas Quinasas Activadas por AMP/fisiología , Enfermedad/etiología , Ejercicio Físico/fisiología , Salud , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Biomarcadores/metabolismo , Humanos , Modelos Biológicos , Actividad Motora/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Factores de TiempoRESUMEN
We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD(+)-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5mM glucose for 6h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1mM) for 2h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.