RESUMO
Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Gluconeogênese , Hepatócitos/metabolismo , Piruvato Quinase/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Células Cultivadas , Dieta Hiperlipídica , Glucose/metabolismo , Resistência à Insulina , Masculino , Fosforilação , Piruvato Quinase/química , Ratos , Ratos Sprague-DawleyRESUMO
The increased hepatic gluconeogenesis in type 2 diabetes mellitus has often been ascribed to increased transcription of phosphoenolpyruvate carboxykinase 1, cystolic form (PEPCK1), although recent evidence has questioned this attribution. To assess the metabolic role of PEPCK1, we treated regular chow fed and high-fat fed (HFF) male Sprague-Dawley rats with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) against PEPCK1 and compared them with control ASO-treated rats. PEPCK1 ASO effectively decreased PEPCK1 expression in the liver and white adipose tissue. In chow fed rats, PEPCK1 ASO did not alter adiposity, plasma glucose, or insulin. In contrast, PEPCK1 ASO decreased the white adipose tissue mass in HFF rats but without altering basal rates of lipolysis, de novo lipogenesis, or glyceroneogenesis in vivo. Despite the protection from adiposity, hepatic insulin sensitivity was impaired in HFF PEPCK1 ASO-treated rats. PEPCK1 ASO worsened hepatic steatosis, although without additional impairments in hepatic insulin signaling or activation of inflammatory signals in the liver. Instead, the development of hepatic insulin resistance and the decrease in hepatic glycogen synthesis during a hyperglycemic clamp was attributed to a decrease in hepatic glucokinase (GCK) expression and decreased synthesis of glycogen via the direct pathway. The decrease in GCK expression was associated with increased expression of activating transcription factor 3, a negative regulator of GCK transcription. These studies have demonstrated that PEPCK1 is integral to coordinating cellular metabolism in the liver and adipose tissue, although it does not directly effect hepatic glucose production or adipose glyceroneogenesis.
Assuntos
Adiposidade , Diabetes Mellitus Tipo 2/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicogênio Hepático/biossíntese , Fígado/metabolismo , Oligonucleotídeos Antissenso/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Tecido Adiposo Branco/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Glucoquinase/genética , Glucoquinase/metabolismo , Humanos , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat-fed, and high-fat-fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina/fisiologia , Insulina/metabolismo , Proteína Quinase C-épsilon/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Animais , Animais Geneticamente Modificados , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Fosforilação , Proteína Quinase C-épsilon/genética , Proteômica/métodos , RNA Interferente Pequeno/metabolismo , Ratos , Receptor de Insulina/metabolismo , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais/fisiologiaRESUMO
AIMS/HYPOTHESIS: Targeting regulators of adipose tissue lipoprotein lipase could enhance adipose lipid clearance, prevent ectopic lipid accumulation and consequently ameliorate insulin resistance and type 2 diabetes. Angiopoietin-like 8 (ANGPTL8) is an insulin-regulated lipoprotein lipase inhibitor strongly expressed in murine adipose tissue. However, Angptl8 knockout mice do not have improved insulin resistance. We hypothesised that pharmacological inhibition, using a second-generation antisense oligonucleotide (ASO) against Angptl8 in adult high-fat-fed rodents, would prevent ectopic lipid accumulation and insulin resistance by promoting adipose lipid uptake. METHODS: ANGPTL8 expression was assessed by quantitative PCR in omental adipose tissue of bariatric surgery patients. High-fat-fed Sprague Dawley rats and C57BL/6 mice were treated with ASO against Angptl8 and insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamps in rats and glucose tolerance tests in mice. Factors mediating lipid-induced hepatic insulin resistance were assessed, including lipid content, protein kinase Cε (PKCε) activation and insulin-stimulated Akt phosphorylation. Rat adipose lipid uptake was assessed by mixed meal tolerance tests. Murine energy balance was assessed by indirect calorimetry. RESULTS: Omental fat ANGPTL8 mRNA expression is higher in obese individuals with fatty liver and insulin resistance compared with BMI-matched insulin-sensitive individuals. Angptl8 ASO prevented hepatic steatosis, PKCε activation and hepatic insulin resistance in high-fat-fed rats. Postprandial triacylglycerol uptake in white adipose tissue was increased in Angptl8 ASO-treated rats. Angptl8 ASO protected high-fat-fed mice from glucose intolerance. Although there was no change in net energy balance, Angptl8 ASO increased fat mass in high-fat-fed mice. CONCLUSIONS/INTERPRETATION: Disinhibition of adipose tissue lipoprotein lipase is a novel therapeutic modality to enhance adipose lipid uptake and treat non-alcoholic fatty liver disease and insulin resistance. In line with this, adipose ANGPTL8 is a candidate therapeutic target for these conditions.
Assuntos
Tecido Adiposo/metabolismo , Proteínas Semelhantes a Angiopoietina/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Oligonucleotídeos Antissenso/genética , Hormônios Peptídicos/genética , Proteína 8 Semelhante a Angiopoietina , Animais , Composição Corporal , Calorimetria Indireta , Dieta Hiperlipídica , Teste de Tolerância a Glucose , Resistência à Insulina , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
NAFLD is closely linked with hepatic insulin resistance. Accumulation of hepatic diacylglycerol activates PKC-ε, impairing insulin receptor activation and insulin-stimulated glycogen synthesis. Peripheral insulin resistance indirectly influences hepatic glucose and lipid metabolism by increasing flux of substrates that promote lipogenesis (glucose and fatty acids) and gluconeogenesis (glycerol and fatty acid-derived acetyl-CoA, an allosteric activator of pyruvate carboxylase). Weight loss with diet or bariatric surgery effectively treats NAFLD, but drugs specifically approved for NAFLD are not available. Some new pharmacological strategies act broadly to alter energy balance or influence pathways that contribute to NAFLD (e.g., agonists for PPAR γ, PPAR α/δ, FXR and analogs for FGF-21, and GLP-1). Others specifically inhibit key enzymes involved in lipid synthesis (e.g., mitochondrial pyruvate carrier, acetyl-CoA carboxylase, stearoyl-CoA desaturase, and monoacyl- and diacyl-glycerol transferases). Finally, a novel class of liver-targeted mitochondrial uncoupling agents increases hepatocellular energy expenditure, reversing the metabolic and hepatic complications of NAFLD.
Assuntos
Doenças Metabólicas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Glucose/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos , Hepatopatia Gordurosa não Alcoólica/terapiaRESUMO
In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake.
Assuntos
Proteínas de Transporte/metabolismo , Glucose/metabolismo , Músculo Esquelético/metabolismo , Vasopressinas/metabolismo , Células 3T3-L1 , Animais , Transporte Biológico , Cistinil Aminopeptidase/metabolismo , Exocitose , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Rodents are commonly used in food restriction refeeding studies to investigate weight regain. Mice that are rationed food every 24 h may consume all allocated food in a short time (gorge) and therefore undergo a brief well-fed period followed by an extended fasted period until the next day's food allotment. These exaggerated metabolic states are not typical in mice fed ad libitum (nibbling). The aim of the current study was to elucidate the intraday and cumulative metabolic consequences of gorging (induced by food restriction) in mice during controlled refeeding. Accordingly, following a temporary food restriction, mice were fed rations similar to intakes of controls fed ad libitum. Temporary food restriction initiated gorging behavior that persisted during refeeding; consequently, metabolism-related measurements were obtained in the gorging mice during their daily fed and fasted metabolic states. Robust differences in adipose tissue lipogenic and inflammatory gene expression were found in the gorging mice by metabolic state (fed versus fasted). Additionally, despite a reduced cumulative food intake compared to mice fed ad libitum, restriction-induced gorging mice had increased intraabdominal fat accumulation, diminished hepatic and peripheral insulin sensitivity, and a gene expression profile favoring lipid deposition. Our findings highlight the intraday differences in gene expression in gorging mice before and after feeding that confound comparisons with mice fed ad libitum, or nibbling. The present study also provides evidence that weight regain following food restriction is associated with cumulative metabolic and behavioral abnormalities in mice.
Assuntos
Adiposidade , Restrição Calórica/efeitos adversos , Comportamento Alimentar , Hiperfagia/etiologia , Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Fígado/metabolismo , Adipocinas/sangue , Adipocinas/genética , Adipocinas/metabolismo , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Animais , Comportamento Animal , Cruzamentos Genéticos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hiperfagia/sangue , Hiperfagia/metabolismo , Hiperfagia/patologia , Mediadores da Inflamação/metabolismo , Gordura Intra-Abdominal/enzimologia , Gordura Intra-Abdominal/patologia , Fígado/enzimologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Distribuição Aleatória , Aumento de PesoRESUMO
ApoA5 has a critical role in the regulation of plasma TG concentrations. In order to determine whether ApoA5 also impacts ectopic lipid deposition in liver and skeletal muscle, as well as tissue insulin sensitivity, we treated mice with an antisense oligonucleotide (ASO) to decrease hepatic expression of ApoA5. ASO treatment reduced ApoA5 protein expression in liver by 60-70%. ApoA5 ASO-treated mice displayed approximately 3-fold higher plasma TG concentrations, which were associated with decreased plasma TG clearance. Furthermore, ApoA5 ASO-treated mice fed a high-fat diet (HFD) exhibited reduced liver and skeletal muscle TG uptake and reduced liver and muscle TG and diacylglycerol (DAG) content. HFD-fed ApoA5 ASO-treated mice were protected from HFD-induced insulin resistance, as assessed by hyperinsulinemic-euglycemic clamps. This protection could be attributed to increases in both hepatic and peripheral insulin responsiveness associated with decreased DAG activation of protein kinase C (PKC)-ε and PKCθ in liver and muscle, respectively, and increased insulin-stimulated AKT2 pho-sphory-lation in these tissues. In summary, these studies demonstrate a novel role for ApoA5 as a modulator of susceptibility to diet-induced liver and muscle insulin resistance through regulation of ectopic lipid accumulation in liver and skeletal muscle.
Assuntos
Apolipoproteínas/metabolismo , Gorduras na Dieta/farmacologia , Resistência à Insulina , Fígado/metabolismo , Músculo Esquelético/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteína A-V , Apolipoproteínas/genética , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triglicerídeos/genéticaRESUMO
Coordinating energy supply with use is critical for survival and is tightly regulated. There is growing evidence that the oestrogen-related receptors (ERRs), a family of orphan nuclear receptors, play key roles in this regulation, ERRγ being the most recently discovered member of this family. Hepatic expression and activity of ERRγ is induced by fasting and repressed in response to feeding. Work reported by Kim and colleagues (DOI: 10.1007/s00125-014-3366-x ) dissects the mechanism for the latter observation. Taken together with prior studies by this group and others, there is emerging evidence that this protein helps coordinate metabolism and may be a potential therapeutic target.
Assuntos
Gluconeogênese/efeitos dos fármacos , Insulina/farmacologia , Fígado/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , AnimaisRESUMO
In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation.
Assuntos
Resistência à Insulina , Fígado/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Ativação Enzimática , Fatores de Transcrição Forkhead/metabolismo , Gluconeogênese , Glicogênio/biossíntese , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteína Fosfatase 2/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt-ß-catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt-ß-catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.
Assuntos
Adipogenia , Adiposidade , Densidade Óssea , Proteínas do Olho/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteínas do Olho/genética , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Serpinas/genética , Proteínas Wnt/metabolismo , Via de Sinalização WntRESUMO
The term "fat" may refer to lipids as well as the cells and tissue that store lipid (ie, adipocytes and adipose tissue). "Lipid" is derived from "lipos," which refers to animal fat or vegetable oil. Adiposity refers to body fat and is derived from "adipo," referring to fat. Adipocytes and adipose tissue store the greatest amount of body lipids, including triglycerides and free cholesterol. Adipocytes and adipose tissue are active from an endocrine and immune standpoint. Adipocyte hypertrophy and excessive adipose tissue accumulation can promote pathogenic adipocyte and adipose tissue effects (adiposopathy), resulting in abnormal levels of circulating lipids, with dyslipidemia being a major atherosclerotic coronary heart disease risk factor. It is therefore incumbent upon lipidologists to be among the most knowledgeable in the understanding of the relationship between excessive body fat and dyslipidemia. On September 16, 2012, the National Lipid Association held a Consensus Conference with the goal of better defining the effect of adiposity on lipoproteins, how the pathos of excessive body fat (adiposopathy) contributes to dyslipidemia, and how therapies such as appropriate nutrition, increased physical activity, weight-management drugs, and bariatric surgery might be expected to impact dyslipidemia. It is hoped that the information derived from these proceedings will promote a greater appreciation among clinicians of the impact of excess adiposity and its treatment on dyslipidemia and prompt more research on the effects of interventions for improving dyslipidemia and reducing cardiovascular disease risk in overweight and obese patients.
Assuntos
Adiposidade/fisiologia , Dislipidemias/fisiopatologia , Obesidade/fisiopatologia , Adiposidade/efeitos dos fármacos , Conferências de Consenso como Assunto , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Dislipidemias/sangue , Dislipidemias/tratamento farmacológico , Humanos , Resistência à Insulina/fisiologia , Obesidade/sangue , Obesidade/tratamento farmacológico , Triglicerídeos/sangueRESUMO
We measured the mRNA and protein expression of the key gluconeogenic enzymes in human liver biopsy specimens and found that only hepatic pyruvate carboxylase protein levels related strongly with glycemia. We assessed the role of pyruvate carboxylase in regulating glucose and lipid metabolism in rats through a loss-of-function approach using a specific antisense oligonucleotide (ASO) to decrease expression predominantly in liver and adipose tissue. Pyruvate carboxylase ASO reduced plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also reduced adiposity, plasma lipid concentrations, and hepatic steatosis in high fat-fed rats and improved hepatic insulin sensitivity. Pyruvate carboxylase ASO had similar effects in Zucker Diabetic Fatty rats. Pyruvate carboxylase ASO did not alter de novo fatty acid synthesis, lipolysis, or hepatocyte fatty acid oxidation. In contrast, the lipid phenotype was attributed to a decrease in hepatic and adipose glycerol synthesis, which is important for fatty acid esterification when dietary fat is in excess. Tissue-specific inhibition of pyruvate carboxylase is a potential therapeutic approach for nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes.
Assuntos
Adiposidade/fisiologia , Gluconeogênese/fisiologia , Resistência à Insulina/fisiologia , Fígado/enzimologia , Piruvato Carboxilase/metabolismo , Tecido Adiposo/enzimologia , Adulto , Animais , Fígado Gorduroso/enzimologia , Feminino , Glicerol/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Ratos ZuckerRESUMO
Estrogen replacement therapy reduces the incidence of type 2 diabetes in postmenopausal women; however, the mechanism is unknown. Therefore, the aim of this study was to evaluate the metabolic effects of estrogen replacement therapy in an experimental model of menopause. At 8 weeks of age, female mice were ovariectomized (OVX) or sham (SHAM) operated, and OVX mice were treated with vehicle (OVX) or estradiol (E2) (OVX+E2). After 4 weeks of high-fat diet feeding, OVX mice had increased body weight and fat mass compared with SHAM and OVX+E2 mice. OVX mice displayed reduced whole-body energy expenditure, as well as impaired glucose tolerance and whole-body insulin resistance. Differences in whole-body insulin sensitivity in OVX compared with SHAM mice were accounted for by impaired muscle insulin sensitivity, whereas both hepatic and muscle insulin sensitivity were impaired in OVX compared with OVX+E2 mice. Muscle diacylglycerol (DAG), content in OVX mice was increased relative to SHAM and OVX+E2 mice. In contrast, E2 treatment prevented the increase in hepatic DAG content observed in both SHAM and OVX mice. Increases in tissue DAG content were associated with increased protein kinase Cε activation in liver of SHAM and OVX mice compared with OVX+E2 and protein kinase Cθ activation in skeletal muscle of OVX mice compared with SHAM and OVX+E2. Taken together, these data demonstrate that E2 plays a pivotal role in the regulation of whole-body energy homeostasis, increasing O(2) consumption and energy expenditure in OVX mice, and in turn preventing diet-induced ectopic lipid (DAG) deposition and hepatic and muscle insulin resistance.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Estradiol/metabolismo , Estradiol/farmacologia , Resistência à Insulina/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Estradiol/deficiência , Terapia de Reposição de Estrogênios , Feminino , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Menopausa/metabolismo , Camundongos , Modelos Animais , Ovariectomia , Proteína Quinase C/metabolismoRESUMO
By 2030, nearly half of Americans will have nonalcoholic fatty liver disease. In part, this epidemic is fueled by the increasing consumption of caloric sweeteners coupled with an innate capacity to convert sugar into fat via hepatic de novo lipogenesis. In addition to serving as substrates, monosaccharides also increase the expression of key enzymes involved in de novo lipogenesis via the carbohydrate response element-binding protein (ChREBP). To determine whether ChREBP is a potential therapeutic target, we decreased hepatic expression of ChREBP with a specific antisense oligonucleotide (ASO) in male Sprague-Dawley rats fed either a high-fructose or high-fat diet. ChREBP ASO treatment decreased plasma triglyceride concentrations compared with control ASO treatment in both diet groups. The reduction was more pronounced in the fructose-fed group and attributed to decreased hepatic expression of ACC2, FAS, SCD1, and MTTP and a decrease in the rate of hepatic triglyceride secretion. This was associated with an increase in insulin-stimulated peripheral glucose uptake, as assessed by the hyperinsulinemic-euglycemic clamp. In contrast, ChREBP ASO did not alter hepatic lipid content or hepatic insulin sensitivity. Interestingly, fructose-fed rats treated with ChREBP ASO had increased plasma uric acid, alanine transaminase, and aspartate aminotransferase concentrations. This was associated with decreased expression of fructose aldolase and fructokinase, reminiscent of inherited disorders of fructose metabolism. In summary, these studies suggest that targeting ChREBP may prevent fructose-induced hypertriglyceridemia but without the improvements in hepatic steatosis and hepatic insulin responsiveness.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Frutose/farmacologia , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Oligonucleotídeos Antissenso/genética , Ratos , Ratos Sprague-Dawley , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMO
UNLABELLED: Genome-wide array studies have associated the patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms with hepatic steatosis. However, it is unclear whether PNPLA3 functions as a lipase or a lipogenic enzyme and whether PNPLA3 is involved in the pathogenesis of hepatic insulin resistance. To address these questions we treated high-fat-fed rats with specific antisense oligonucleotides to decrease hepatic and adipose pnpla3 expression. Reducing pnpla3 expression prevented hepatic steatosis, which could be attributed to decreased fatty acid esterification measured by the incorporation of [U-(13) C]-palmitate into hepatic triglyceride. While the precursors for phosphatidic acid (PA) (long-chain fatty acyl-CoAs and lysophosphatidic acid [LPA]) were not decreased, we did observe an â¼20% reduction in the hepatic PA content, â¼35% reduction in the PA/LPA ratio, and â¼60%-70% reduction in transacylation activity at the level of acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase. These changes were associated with an â¼50% reduction in hepatic diacylglycerol (DAG) content, an â¼80% reduction in hepatic protein kinase Cε activation, and increased hepatic insulin sensitivity, as reflected by a 2-fold greater suppression of endogenous glucose production during the hyperinsulinemic-euglycemic clamp. Finally, in humans, hepatic PNPLA3 messenger RNA (mRNA) expression was strongly correlated with hepatic triglyceride and DAG content, supporting a potential lipogenic role of PNPLA3 in humans. CONCLUSION: PNPLA3 may function primarily in a lipogenic capacity and inhibition of PNPLA3 may be a novel therapeutic approach for treatment of nonalcoholic fatty liver disease-associated hepatic insulin resistance.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/fisiopatologia , Resistência à Insulina/fisiologia , Lipídeos/efeitos adversos , Proteínas de Membrana/fisiologia , Fosfolipases A2/fisiologia , Animais , Biópsia , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Oligonucleotídeos Antissenso/farmacologia , Fosfolipases A2/efeitos dos fármacos , Fosfolipases A2/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Triglicerídeos/metabolismoRESUMO
A major cause of hyperglycemia in diabetic patients is inappropriate hepatic gluconeogenesis. PGC-1α is a master regulator of gluconeogenesis, and its activity is controlled by various posttranslational modifications. A small portion of glucose metabolizes through the hexosamine biosynthetic pathway, which leads to O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins. Using a proteomic approach, we identified a broad variety of proteins associated with O-GlcNAc transferase (OGT), among which host cell factor C1 (HCF-1) is highly abundant. HCF-1 recruits OGT to O-GlcNAcylate PGC-1α, and O-GlcNAcylation facilitates the binding of the deubiquitinase BAP1, thus protecting PGC-1α from degradation and promoting gluconeogenesis. Glucose availability modulates gluconeogenesis through the regulation of PGC-1α O-GlcNAcylation and stability by the OGT/HCF-1 complex. Hepatic knockdown of OGT and HCF-1 improves glucose homeostasis in diabetic mice. These findings define the OGT/HCF-1 complex as a glucose sensor and key regulator of gluconeogenesis, shedding light on new strategies for treating diabetes.
Assuntos
Gluconeogênese/fisiologia , Proteínas de Choque Térmico/metabolismo , Fator C1 de Célula Hospedeira/metabolismo , Hiperglicemia/fisiopatologia , Complexos Multiproteicos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Análise de Variância , Animais , Western Blotting , Imunoprecipitação da Cromatina , Cromatografia Líquida de Alta Pressão , Células HEK293 , Células Hep G2 , Humanos , Imunoprecipitação , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em TandemRESUMO
Recent studies reveal a strong relationship between reduced mitochondrial content and insulin resistance in human skeletal muscle, although the underlying factors responsible for this association remain unknown. To address this question, we analyzed muscle biopsy samples from young, lean, insulin resistant (IR) offspring of parents with type 2 diabetes and control subjects by microarray analyses and found significant differences in expression of ~512 probe pairs. We then screened these genes for their potential involvement in the regulation of mitochondrial biogenesis using RNA interference and found that mRNA and protein expression of lipoprotein lipase (LPL) in skeletal muscle was significantly decreased in the IR offspring and was associated with decreased mitochondrial density. Furthermore, we show that LPL knockdown in muscle cells decreased mitochondrial content by effectively decreasing fatty acid delivery and subsequent activation of peroxisome proliferator-activated receptor (PPAR)-δ. Taken together, these data suggest that decreased mitochondrial content in muscle of IR offspring may be due in part to reductions in LPL expression in skeletal muscle resulting in decreased PPAR-δ activation.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina/fisiologia , Lipase Lipoproteica/metabolismo , Mitocôndrias/fisiologia , Músculo Esquelético/metabolismo , Linhagem Celular , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Lipase Lipoproteica/genética , Análise de Sequência com Séries de Oligonucleotídeos , PPAR delta/genética , PPAR delta/metabolismo , Interferência de RNARESUMO
Body fat, insulin resistance, and type 2 diabetes are often linked together, but the molecular mechanisms that unify their association are poorly understood. Wnt signaling regulates adipogenesis, and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. LRP6(+/-) mice on a high fat diet were protected against diet-induced obesity and hepatic and adipose tissue insulin resistance compared with their wild-type (WT) littermates. Brown adipose tissue insulin sensitivity and reduced adiposity of LRP6(+/-) mice were accounted for by diminished Wnt-dependent mTORC1 activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6(+/-) mice also exhibited reduced endogenous hepatic glucose output, which was due to diminished FoxO1-dependent expression of the key gluconeogenic enzyme glucose-6-phosphatase (G6pase). In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with increased leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6(+/-) mice. Our study identifies LRP6 as a nutrient-sensitive regulator of body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.
Assuntos
Tecido Adiposo Marrom/metabolismo , Metabolismo Energético/fisiologia , Glucose/metabolismo , Homeostase/fisiologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mitocôndrias/metabolismo , Adiposidade/fisiologia , Alelos , Animais , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Metabolismo Energético/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Gluconeogênese/fisiologia , Glucose/genética , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Homeostase/efeitos dos fármacos , Resistência à Insulina/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Complexos Multiproteicos , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Serina-Treonina Quinases TOR , Via de Sinalização Wnt/fisiologiaRESUMO
Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in the United States and is strongly associated with hepatic insulin resistance. We examined whether the thyroid hormone receptor-α (Thra) would be a potential therapeutic target to prevent diet-induced NAFLD and insulin resistance. For that purpose, we assessed insulin action in high-fat diet-fed Thra gene knockout (Thra-0/0) and wild-type mice using hyperinsulinemic-euglycemic clamps combined with (3)H/(14)C-labeled glucose to assess basal and insulin-stimulated rates of glucose and fat metabolism. Body composition was assessed by (1)H magnetic resonance spectroscopy and energy expenditure by indirect calorimetry. Relative rates of hepatic glucose and fat oxidation were assessed in vivo using a novel proton-observed carbon-edited nuclear magnetic resonance technique. Thra-0/0 were lighter, leaner, and manifested greater whole-body insulin sensitivity than wild-type mice during the clamp, which could be attributed to increased insulin sensitivity both in liver and peripheral tissues. Increased hepatic insulin sensitivity could be attributed to decreased hepatic diacylglycerol content, resulting in decreased activation of protein kinase Cε and increased insulin signaling. In conclusion, loss of Thra protects mice from high-fat diet-induced hepatic steatosis and hepatic and peripheral insulin resistance. Therefore, thyroid receptor-α inhibition represents a novel pharmacologic target for the treatment of NAFLD, obesity, and type 2 diabetes.