RESUMO
AGPAT2 (1-acyl-sn-glycerol-3-phosphate-acyltransferase-2) converts lysophosphatidic acid (LPA) into phosphatidic acid (PA), and mutations of the AGPAT2 gene cause the most common form of congenital generalized lipodystrophy which leads to steatohepatitis. The underlying mechanism by which AGPAT2 deficiency leads to lipodystrophy and steatohepatitis has not been elucidated. We addressed this question using an antisense oligonucleotide (ASO) to knockdown expression of Agpat2 in the liver and white adipose tissue (WAT) of adult male Sprague-Dawley rats. Agpat2 ASO treatment induced lipodystrophy and inflammation in WAT and the liver, which was associated with increased LPA content in both tissues, whereas PA content was unchanged. We found that a controlled-release mitochondrial protonophore (CRMP) prevented LPA accumulation and inflammation in WAT whereas an ASO against glycerol-3-phosphate acyltransferase, mitochondrial (Gpam) prevented LPA content and inflammation in the liver in Agpat2 ASO-treated rats. In addition, we show that overnutrition, due to high sucrose feeding, resulted in increased hepatic LPA content and increased activated macrophage content which were both abrogated with Gpam ASO treatment. Taken together, these data identify LPA as a key mediator of liver and WAT inflammation and lipodystrophy due to AGPAT2 deficiency as well as liver inflammation due to overnutrition and identify LPA as a potential therapeutic target to ameliorate these conditions.
Assuntos
Fígado Gorduroso , Lipodistrofia , Hipernutrição , Masculino , Ratos , Animais , Aciltransferases/metabolismo , Glicerol , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Ratos Sprague-Dawley , Lipodistrofia/genética , Tecido Adiposo Branco/metabolismo , Ácidos Fosfatídicos , Inflamação , FosfatosRESUMO
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, in which prognosis is determined by liver fibrosis. A common variant in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13, rs72613567-A) is associated with a reduced risk of fibrosis in NAFLD, but the underlying mechanism(s) remains unclear. We investigated the effects of this variant in the human liver and in Hsd17b13 knockdown in mice by using a state-of-the-art metabolomics approach. We demonstrate that protection against liver fibrosis conferred by the HSD17B13 rs72613567-A variant in humans and by the Hsd17b13 knockdown in mice is associated with decreased pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase. Furthermore, we show that hepatic pyrimidines are depleted in two distinct mouse models of NAFLD and that inhibition of pyrimidine catabolism by gimeracil phenocopies the HSD17B13-induced protection against liver fibrosis. Our data suggest pyrimidine catabolism as a therapeutic target against the development of liver fibrosis in NAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Camundongos , Fígado/metabolismo , Cirrose Hepática/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Pirimidinas/farmacologia , Pirimidinas/metabolismoRESUMO
AIMS/HYPOTHESIS: Athletes exhibit increased muscle insulin sensitivity, despite increased intramuscular triacylglycerol content. This phenomenon has been coined the 'athlete's paradox' and is poorly understood. Recent findings suggest that the subcellular distribution of sn-1,2-diacylglycerols (DAGs) in the plasma membrane leading to activation of novel protein kinase Cs (PKCs) is a crucial pathway to inducing insulin resistance. Here, we hypothesised that regular aerobic exercise would preserve muscle insulin sensitivity by preventing increases in plasma membrane sn-1,2-DAGs and activation of PKCε and PKCθ despite promoting increases in muscle triacylglycerol content. METHODS: C57BL/6J mice were allocated to three groups (regular chow feeding [RC]; high-fat diet feeding [HFD]; RC feeding and running wheel exercise [RC-EXE]). We used a novel LC-MS/MS/cellular fractionation method to assess DAG stereoisomers in five subcellular compartments (plasma membrane [PM], endoplasmic reticulum, mitochondria, lipid droplets and cytosol) in the skeletal muscle. RESULTS: We found that the HFD group had a greater content of sn-DAGs and ceramides in multiple subcellular compartments compared with the RC mice, which was associated with an increase in PKCε and PKCθ translocation. However, the RC-EXE mice showed, of particular note, a reduction in PM sn-1,2-DAG and ceramide content when compared with HFD mice. Consistent with the PM sn-1,2-DAG-novel PKC hypothesis, we observed an increase in phosphorylation of threonine1150 on the insulin receptor kinase (IRKT1150), and reductions in insulin-stimulated IRKY1162 phosphorylation and IRS-1-associated phosphoinositide 3-kinase activity in HFD compared with RC and RC-EXE mice, which are sites of PKCε and PKCθ action, respectively. CONCLUSIONS/INTERPRETATION: These results demonstrate that lower PKCθ/PKCε activity and sn-1,2-DAG content, especially in the PM compartment, can explain the preserved muscle insulin sensitivity in RC-EXE mice.
Assuntos
Resistência à Insulina , Camundongos , Animais , Resistência à Insulina/fisiologia , Proteína Quinase C-theta/metabolismo , Proteína Quinase C-épsilon/metabolismo , Cromatografia Líquida , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos Endogâmicos C57BL , Espectrometria de Massas em Tandem , Insulina/metabolismo , Músculo Esquelético/metabolismo , Triglicerídeos/metabolismo , Ceramidas/metabolismoRESUMO
Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp-/-) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp-/- mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp-/- mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp-/- mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp-/- mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp-/- mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp-/- mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp-/- mice.
Assuntos
Proteínas de Transporte/genética , Membrana Celular/metabolismo , Diglicerídeos/metabolismo , Técnicas de Inativação de Genes , Resistência à Insulina , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
Exposure to the toxins methylene cyclopropyl acetic acid (MCPA) and methylene cyclopropyl glycine (MCPG) of unripe ackee and litchi fruit can lead to hypoglycemia and death; however, the molecular mechanisms by which MCPA and MCPG cause hypoglycemia have not been established in vivo To determine the in vivo mechanisms of action of these toxins, we infused them into conscious rodents and assessed rates of hepatic gluconeogenesis and ketogenesis, hepatic acyl-CoA and hepatic acetyl-CoA content, and hepatocellular energy charge. MCPG suppressed rates of hepatic ß-oxidation as reflected by reductions in hepatic ketogenesis, reducing both short- and medium-chain hepatic acyl-CoA concentrations. Hepatic acetyl-CoA content decreased, and hepatic glucose production was inhibited. MCPA also suppressed ß-oxidation of short-chain acyl-CoAs, rapidly inhibiting hepatic ketogenesis and hepatic glucose production, depleting hepatic acetyl-CoA content and ATP content, while increasing other short-chain acyl-CoAs. Utilizing a recently developed positional isotopomer NMR tracer analysis method, we demonstrated that MCPA-induced reductions in hepatic acetyl-CoA content were associated with a marked reduction of hepatic pyruvate carboxylase (PC) flux. Taken together, these data reveal the in vivo mechanisms of action of MCPA and MCPG: the hypoglycemia associated with ingestion of these toxins can be ascribed mostly to MCPA- or MCPG-induced reductions in hepatic PC flux due to inhibition of ß-oxidation of short-chain acyl-CoAs by MCPA or inhibition of both short- and medium-chain acyl-CoAs by MCPG with resultant reductions in hepatic acetyl-CoA content, with an additional contribution to hypoglycemia through reduced hepatic ATP stores by MCPA.
Assuntos
Ciclopropanos , Glicina/análogos & derivados , Hipoglicemia/induzido quimicamente , Animais , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemia/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Hepatic insulin resistance is a principal component of type 2 diabetes, but the cellular and molecular mechanisms responsible for its pathogenesis remain unknown. Recent studies have suggested that saturated fatty acids induce hepatic insulin resistance through activation of the toll-like receptor 4 (TLR-4) receptor in the liver, which in turn transcriptionally activates hepatic ceramide synthesis leading to inhibition of insulin signaling. In this study, we demonstrate that TLR-4 receptor signaling is not directly required for saturated or unsaturated fat-induced hepatic insulin resistance in both TLR-4 antisense oligonucleotide treated and TLR-4 knockout mice, and that ceramide accumulation is not dependent on TLR-4 signaling or a primary event in hepatic steatosis and impairment of insulin signaling. Further, we show that both saturated and unsaturated fats lead to hepatic accumulation of diacylglycerols, activation of PKCε, and impairment of insulin-stimulated IRS-2 signaling. These data demonstrate that saturated fat-induced insulin resistance is independent of TLR-4 activation and ceramides.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Fígado Gorduroso/metabolismo , Resistência à Insulina , Fígado/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diglicerídeos/metabolismo , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/patologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Fígado/patologia , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Comparative gene identification 58 (CGI-58) is a lipid droplet-associated protein that promotes the hydrolysis of triglyceride by activating adipose triglyceride lipase. Loss-of-function mutations in CGI-58 in humans lead to Chanarin-Dorfman syndrome, a condition in which triglyceride accumulates in various tissues, including the skin, liver, muscle, and intestines. Therefore, without adequate CGI-58 expression, lipids are stored rather than used for fuel, signaling intermediates, and membrane biosynthesis. CGI-58 knockdown in mice using antisense oligonucleotide (ASO) treatment also leads to severe hepatic steatosis as well as increased hepatocellular diacylglycerol (DAG) content, a well-documented trigger of insulin resistance. Surprisingly, CGI-58 knockdown mice remain insulin-sensitive, seemingly dissociating DAG from the development of insulin resistance. Therefore, we sought to determine the mechanism responsible for this paradox. Hyperinsulinemic-euglycemic clamp studies reveal that the maintenance of insulin sensitivity with CGI-58 ASO treatment could entirely be attributed to protection from lipid-induced hepatic insulin resistance, despite the apparent lipotoxic conditions. Analysis of the cellular compartmentation of DAG revealed that DAG increased in the membrane fraction of high fat-fed mice, leading to PKCε activation and hepatic insulin resistance. However, DAG increased in lipid droplets or lipid-associated endoplasmic reticulum rather than the membrane of CGI-58 ASO-treated mice, and thus prevented PKCε translocation to the plasma membrane and induction of insulin resistance. Taken together, these results explain the disassociation of hepatic steatosis and DAG accumulation from hepatic insulin resistance in CGI-58 ASO-treated mice, and highlight the importance of intracellular compartmentation of DAG in causing lipotoxicity and hepatic insulin resistance.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Diglicerídeos/metabolismo , Retículo Endoplasmático/metabolismo , Resistência à Insulina , Lipídeos/química , Fígado/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dieta Hiperlipídica , Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Proteína Quinase C-épsilon/metabolismo , Transporte Proteico/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fatty acid amide hydrolase (FAAH) knockout mice are prone to excess energy storage and adiposity, whereas mutations in FAAH are associated with obesity in humans. However, the molecular mechanism by which FAAH affects energy expenditure (EE) remains unknown. Here we show that reduced energy expenditure in FAAH(-/-) mice could be attributed to decreased circulating triiodothyronine and thyroxine concentrations secondary to reduced mRNA expression of both pituitary thyroid-stimulating hormone and hypothalamic thyrotropin-releasing hormone. These reductions in the hypothalamic-pituitary-thyroid axis were associated with activation of hypothalamic peroxisome proliferating-activated receptor γ (PPARγ), and increased hypothalamic deiodinase 2 expression. Infusion of NAEs (anandamide and palmitoylethanolamide) recapitulated increases in PPARγ-mediated decreases in EE. FAAH(-/-) mice were also prone to diet-induced hepatic insulin resistance, which could be attributed to increased hepatic diacylglycerol content and protein kinase Cε activation. Our data indicate that FAAH deletion, and the resulting increases in NAEs, predispose mice to ectopic lipid storage and hepatic insulin resistance by promoting centrally mediated hypothyroidism.
Assuntos
Amidoidrolases/genética , Metabolismo Energético/fisiologia , Hipotireoidismo/complicações , Hipotireoidismo/genética , Resistência à Insulina/fisiologia , Amidas , Amidoidrolases/deficiência , Análise de Variância , Animais , Ácidos Araquidônicos/administração & dosagem , Cromatografia Líquida , Endocanabinoides/administração & dosagem , Metabolismo Energético/genética , Etanolaminas/administração & dosagem , Hipotireoidismo/enzimologia , Immunoblotting , Camundongos , Camundongos Knockout , PPAR gama , Ácidos Palmíticos/administração & dosagem , Reação em Cadeia da Polimerase , Alcamidas Poli-Insaturadas/administração & dosagem , Espectrometria de Massas em Tandem , Tireotropina/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
AIMS/HYPOTHESIS: Aerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca(2+)/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo. METHODS: The effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4([Symbol: see text])) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic-euglycaemic clamp and isotopic measurements of glucose metabolism. RESULTS: The rate of insulin-stimulated whole-body glucose uptake was increased by â¼25% in CAMK4([Symbol: see text]) mice. This was largely attributed to an increase of â¼60% in insulin-stimulated glucose uptake in the quadriceps, the largest hindlimb muscle. These changes were associated with improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4([Symbol: see text]) mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4([Symbol: see text]) skeletal muscle. CONCLUSIONS/INTERPRETATION: Activation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Músculo Esquelético/enzimologia , Animais , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Feminino , Masculino , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/genéticaRESUMO
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
Insulin resistance is associated with nonalcoholic fatty liver disease (NAFLD) and is a major factor in the pathogenesis of type 2 diabetes. The development of hepatic insulin resistance has been ascribed to multiple causes, including inflammation, endoplasmic reticulum (ER) stress, and accumulation of hepatocellular lipids in animal models of NAFLD. However, it is unknown whether these same cellular mechanisms link insulin resistance to hepatic steatosis in humans. To examine the cellular mechanisms that link hepatic steatosis to insulin resistance, we comprehensively assessed each of these pathways by using flash-frozen liver biopsies obtained from 37 obese, nondiabetic individuals and correlating key hepatic and plasma markers of inflammation, ER stress, and lipids with the homeostatic model assessment of insulin resistance index. We found that hepatic diacylglycerol (DAG) content in cytoplasmic lipid droplets was the best predictor of insulin resistance (R = 0.80, P < 0.001), and it was responsible for 64% of the variability in insulin sensitivity. Hepatic DAG content was also strongly correlated with activation of hepatic PKCε (R = 0.67, P < 0.001), which impairs insulin signaling. In contrast, there was no significant association between insulin resistance and other putative lipid metabolites or plasma or hepatic markers of inflammation. ER stress markers were only partly correlated with insulin resistance. In conclusion, these data show that hepatic DAG content in lipid droplets is the best predictor of insulin resistance in humans, and they support the hypothesis that NAFLD-associated hepatic insulin resistance is caused by an increase in hepatic DAG content, which results in activation of PKCε.
Assuntos
Fígado Gorduroso/fisiopatologia , Resistência à Insulina , Adulto , Diglicerídeos/metabolismo , Ativação Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase C-épsilon/metabolismoRESUMO
To examine the roles of mitochondrial calcium Ca2+ ([Ca2+]mt) and cytosolic Ca2+ ([Ca2+]cyt) in the regulation of hepatic mitochondrial fat oxidation, we studied a liver-specific mitochondrial calcium uniporter knockout (MCU KO) mouse model with reduced [Ca2+]mt and increased [Ca2+]cyt content. Despite decreased [Ca2+]mt, deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux, α-ketoglutarate dehydrogenase flux, and succinate dehydrogenase flux in vivo. Rates of [14C16]palmitate oxidation and intrahepatic lipolysis were increased in MCU KO liver slices, which led to decreased hepatic triacylglycerol content. These effects were recapitulated with activation of CAMKII and abrogated with CAMKII knockdown, demonstrating that [Ca2+]cyt activation of CAMKII may be the primary mechanism by which MCU deletion promotes increased hepatic mitochondrial oxidation. Together, these data demonstrate that hepatic mitochondrial oxidation can be dissociated from [Ca2+]mt and reveal a key role for [Ca2+]cyt in the regulation of hepatic fat mitochondrial oxidation, intrahepatic lipolysis, gluconeogenesis, and lipid accumulation.
RESUMO
Hepatic insulin resistance has been attributed to both increased endoplasmic reticulum (ER) stress and accumulation of intracellular lipids, specifically diacylglycerol (DAG). The ER stress response protein, X-box-binding protein-1 (XBP1), was recently shown to regulate hepatic lipogenesis, suggesting that hepatic insulin resistance in models of ER stress may result from defective lipid storage, as opposed to ER-specific stress signals. Studies were designed to dissociate liver lipid accumulation and activation of ER stress signaling pathways, which would allow us to delineate the individual contributions of ER stress and hepatic lipid content to the pathogenesis of hepatic insulin resistance. Conditional XBP1 knock-out (XBP1Δ) and control mice were fed fructose chow for 1 week. Determinants of whole-body energy balance, weight, and composition were determined. Hepatic lipids including triglyceride, DAGs, and ceramide were measured, alongside markers of ER stress. Whole-body and tissue-specific insulin sensitivity were determined by hyperinsulinemic-euglycemic clamp studies. Hepatic ER stress signaling was increased in fructose chow-fed XBP1Δ mice as reflected by increased phosphorylated eIF2α, HSPA5 mRNA, and a 2-fold increase in hepatic JNK activity. Despite JNK activation, XBP1Δ displayed increased hepatic insulin sensitivity during hyperinsulinemic-euglycemic clamp studies, which was associated with increased insulin-stimulated IRS2 tyrosine phosphorylation, reduced hepatic DAG content, and reduced PKCε activity. These studies demonstrate that ER stress and IRE1α-mediated JNK activation can be disassociated from hepatic insulin resistance and support the hypothesis that hepatic insulin resistance in models of ER stress may be secondary to ER stress modulation of hepatic lipogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Resistência à Insulina , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-BoxRESUMO
The mammalian succinate dehydrogenase (SDH) complex has recently been shown as capable of operating bidirectionally. Here, we develop a method (Q-Flux) capable of measuring absolute rates of both forward (VSDH(F)) and reverse (VSDH(R)) flux through SDH in vivo while also deconvoluting the amount of glucose derived from four discreet carbon sources in the liver. In validation studies, a mitochondrial uncoupler increased net SDH flux by >100% in awake rodents but also increased SDH cycling. During hyperglucagonemia, attenuated pyruvate cycling enhances phosphoenolpyruvate carboxykinase efficiency to drive increased gluconeogenesis, which is complemented by increased glutaminase (GLS) flux, methylmalonyl-CoA mutase (MUT) flux, and glycerol conversion to glucose. During hyperinsulinemic-euglycemic clamp, both pyruvate carboxylase and GLS are suppressed, while VSDH(R) is increased. Unstimulated MUT is a minor anaplerotic reaction but is readily induced by small amounts of propionate, which elicits glucagon-like metabolic rewiring. Taken together, Q-Flux yields a comprehensive picture of hepatic mitochondrial metabolism and should be broadly useful to researchers.
Assuntos
Metilmalonil-CoA Mutase , Succinato Desidrogenase , Animais , Glucose/metabolismo , Glutaminase/metabolismo , Fígado/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Proteínas/metabolismo , Ácido Pirúvico/metabolismo , Succinato Desidrogenase/metabolismo , RoedoresRESUMO
Recent studies have implicated endoplasmic reticulum (ER) stress in insulin resistance associated with caloric excess. In mice placed on a 3-day high fat diet, we find augmented eIF2α signaling, together with hepatic lipid accumulation and insulin resistance. To clarify the role of the liver ER stress-dependent phospho-eIF2α (eIF2α-P) pathway in response to acute caloric excess on liver and muscle glucose and lipid metabolism, we studied transgenic mice in which the hepatic ER stress-dependent eIF2α-P pathway was inhibited by overexpressing a constitutively active C-terminal fragment of GADD34/PPP1R15a, a regulatory subunit of phosphatase that terminates ER stress signaling by phospho-eIF2α. Inhibition of the eIF2α-P signaling in liver led to a decrease in hepatic glucose production in the basal and clamped state, which could be attributed to reduced gluconeogenic gene expression, resulting in reduced basal plasma glucose concentrations. Surprisingly, hepatic eIF2α inhibition also impaired insulin-stimulated muscle and adipose tissue insulin sensitivity. This latter effect could be attributed at least in part by an increase in circulating IGFBP-3 levels in the transgenic animals. In addition, infusion of insulin during a hyperinsulinemic-euglycemic clamp induced conspicuous ER stress in the 3-day high fat diet-fed mice, which was aggravated through continuous dephosphorylation of eIF2α. Together, these data imply that the hepatic ER stress eIF2α signaling pathway affects hepatic glucose production without altering hepatic insulin sensitivity. Moreover, hepatic ER stress-dependent eIF2α-P signaling is implicated in an unanticipated cross-talk between the liver and peripheral organs to influence insulin sensitivity, probably via IGFBP-3. Finally, eIF2α is crucial for proper resolution of insulin-induced ER stress.
Assuntos
Tecido Adiposo/metabolismo , Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/genética , Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Transdução de Sinais/fisiologiaRESUMO
UNLABELLED: Pyruvate dehydrogenase plays a critical role in the regulation of hepatic glucose and fatty acid oxidation; however, surprisingly little is known about its regulation in vivo. In this study we examined the individual effects of insulin and substrate availability on the regulation of pyruvate dehydrogenase flux (V(PDH) ) to tricarboxylic acid flux (V(TCA) ) in livers of awake rats with lipid-induced hepatic insulin resistance. V(PDH) /V(TCA) flux was estimated from the [4-(13) C]glutamate/[3-(13) C]alanine enrichments in liver extracts and assessed under conditions of fasting and during a hyperinsulinemic-euglycemic clamp, whereas the effects of increased plasma glucose concentration on V(PDH) /V(TCA) flux was assessed during a hyperglycemic clamp in conjunction with infusions of somatostatin and insulin to maintain basal concentrations of insulin. The effects of increases in both glucose and insulin on V(PDH) /V(TCA) were examined during a hyperinsulinemic-hyperglycemic clamp. The effects of chronic lipid-induced hepatic insulin resistance on this flux were also examined by performing these measurements in rats fed a high-fat diet for 3 weeks. Using this approach we found that fasting V(PDH) /V(TCA) was reduced by 95% in rats with hepatic insulin resistance (from 17.2 ± 1.5% to 1.3 ± 0.7%, P < 0.00001). Surprisingly, neither hyperinsulinemia per se or hyperglycemia per se were sufficient to increase V(PDH) /V(TCA) flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did V(PDH) /V(TCA) flux increase (44.6 ± 3.2%, P < 0.0001 versus basal) in low-fat fed animals but not in rats with chronic lipid-induced hepatic insulin resistance. CONCLUSION: These studies demonstrate that the combination of both hyperinsulinemia and hyperglycemia are required to increase V(PDH) /V(TCA) flux in vivo and that this flux is severely diminished in rats with chronic lipid-induced hepatic insulin resistance.
Assuntos
Gorduras/metabolismo , Glucose/metabolismo , Resistência à Insulina/fisiologia , Fígado/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Animais , Glicemia/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Técnica Clamp de Glucose , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Mutations in Dyrk1b are associated with metabolic syndrome and nonalcoholic fatty liver disease in humans. Our investigations showed that DYRK1B levels are increased in the liver of patients with nonalcoholic steatohepatitis (NASH) and in mice fed with a high-fat, high-sucrose diet. Increasing Dyrk1b levels in the mouse liver enhanced de novo lipogenesis (DNL), fatty acid uptake, and triacylglycerol secretion and caused NASH and hyperlipidemia. Conversely, knockdown of Dyrk1b was protective against high-calorie-induced hepatic steatosis and fibrosis and hyperlipidemia. Mechanistically, Dyrk1b increased DNL by activating mTORC2 in a kinase-independent fashion. Accordingly, the Dyrk1b-induced NASH was fully rescued when mTORC2 was genetically disrupted. The elevated DNL was associated with increased plasma membrane sn-1,2-diacylglyerol levels and increased PKCε-mediated IRKT1150 phosphorylation, which resulted in impaired activation of hepatic insulin signaling and reduced hepatic glycogen storage. These findings provide insights into the mechanisms that underlie Dyrk1b-induced hepatic lipogenesis and hepatic insulin resistance and identify Dyrk1b as a therapeutic target for NASH and insulin resistance in the liver.
Assuntos
Insulina/metabolismo , Lipogênese , Fígado/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Quinases DyrkRESUMO
Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance and type 2 diabetes mellitus, but the molecular signals linking hepatic fat accumulation to hepatic insulin resistance are unknown. Three days of high-fat feeding in rats results specifically in hepatic steatosis and hepatic insulin resistance. In this setting, PKCepsilon, but not other isoforms of PKC, is activated. To determine whether PKCepsilon plays a causal role in the pathogenesis of hepatic insulin resistance, we treated rats with an antisense oligonucleotide against PKCepsilon and subjected them to 3 days of high-fat feeding. Knocking down PKCepsilon expression protects rats from fat-induced hepatic insulin resistance and reverses fat-induced defects in hepatic insulin signaling. Furthermore, we show that PKCepsilon associates with the insulin receptor in vivo and impairs insulin receptor kinase activity both in vivo and in vitro. These data support the hypothesis that PKCepsilon plays a critical role in mediating fat-induced hepatic insulin resistance and represents a novel therapeutic target for type 2 diabetes.
Assuntos
Fígado Gorduroso/enzimologia , Resistência à Insulina , Insulina/metabolismo , Proteína Quinase C-épsilon/fisiologia , Animais , Insulina/sangue , Resistência à Insulina/genética , Metabolismo dos Lipídeos , Masculino , Oligonucleotídeos Antissenso/farmacologia , Proteína Quinase C-épsilon/antagonistas & inibidores , Proteína Quinase C-épsilon/genética , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/agonistas , Transdução de SinaisRESUMO
Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCtheta in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1- (IRS-1-) and IRS-2-associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCtheta activity in whole-body fat-matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus.
Assuntos
Regulação da Expressão Gênica , Resistência à Insulina , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP , Envelhecimento/fisiologia , Animais , Ativação Enzimática , Hormônios/sangue , Humanos , Insulina/sangue , Canais Iônicos/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Complexos Multienzimáticos/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Desacopladora 3 , Aumento de PesoRESUMO
Considering a system of gaussian particles confined between two hard, parallel plates, we investigate at T = 0, ordered equilibrium configurations that the system forms as the distance D between the plates gradually increases. Using a very sensitive and reliable optimization technique that is based on ideas of genetic algorithms, we are able to identify the emerging sequences of the energetically most favorable structures. Although the resulting phase diagram is rather complex, its essential features can be reduced to the discussion of two archetypes of structural transitions: (i) a continuous transformation at a fixed number of layers, leading from a square to a centered rectangular and then to a hexagonal lattice; (ii) a discontinuous transition, transforming a hexagonal to a square lattice via complex intermediate structures, i.e., the so-called buckling transition, which is encountered as the system forms a new layer. Detailed Monte Carlo simulations are able to confirm the theoretical predictions on a semiquantitative level but are not able to grasp the tiny energetic differences between competing structures.