RESUMEN
Four glycerol-3-phosphate acyltransferase (GPAT) isoforms, each encoded by a separate gene, catalyze the initial step in glycerolipid synthesis; in liver, the major isoforms are GPAT1 and GPAT4. To determine whether each of these hepatic isoforms performs a unique function in the metabolism of fatty acid, we measured the incorporation of de novo synthesized fatty acid or exogenous fatty acid into complex lipids in primary mouse hepatocytes from control, Gpat1(-/-), and Gpat4(-/-) mice. Although hepatocytes from each genotype incorporated a similar amount of exogenous fatty acid into triacylglycerol (TAG), only control and Gpat4(-/-) hepatocytes were able to incorporate de novo synthesized fatty acid into TAG. When compared with controls, Gpat1(-/-) hepatocytes oxidized twice as much exogenous fatty acid. To confirm these findings and to assess hepatic ß-oxidation metabolites, we measured acylcarnitines in liver from mice after a 24-h fast and after a 24-h fast followed by 48 h of refeeding with a high sucrose diet to promote lipogenesis. Confirming the in vitro findings, the hepatic content of long-chain acylcarnitine in fasted Gpat1(-/-) mice was 3-fold higher than in controls. When compared with control and Gpat4(-/-) mice, after the fasting-refeeding protocol, Gpat1(-/-) hepatic TAG was depleted, and long-chain acylcarnitine content was 3.5-fold higher. Taken together, these data demonstrate that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acids into TAG and to divert them away from oxidation.
Asunto(s)
Ácidos Grasos/biosíntesis , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Hepatocitos/enzimología , Hígado/enzimología , Triglicéridos/biosíntesis , Animales , Ácidos Grasos/genética , Glicerol-3-Fosfato O-Aciltransferasa/genética , Hepatocitos/citología , Hígado/citología , Ratones , Ratones Noqueados , Oxidación-Reducción , Triglicéridos/genéticaRESUMEN
Increased flux through the glycerolipid synthesis pathway impairs the ability of insulin to inhibit hepatic gluconeogenesis, but the exact mechanism remains unknown. To determine the mechanism by which glycerolipids impair insulin signaling, we overexpressed glycerol-3-phosphate acyltransferase-1 (GPAT1) in primary mouse hepatocytes. GPAT1 overexpression impaired insulin-stimulated phosphorylation of Akt-S473 and -T308, diminished insulin-suppression of glucose production, significantly inhibited mTOR complex 2 (mTORC2) activity and decreased the association of mTOR and rictor. Conversely, in hepatocytes from Gpat1(-/-) mice, mTOR-rictor association and mTORC2 activity were enhanced. However, this increase in mTORC2 activity in Gpat1(-/-) hepatocytes was ablated when rictor was knocked down. To determine which lipid intermediate was responsible for inactivating mTORC2, we overexpressed GPAT1, AGPAT, or lipin to increase the cellular content of lysophosphatidic acid (LPA), phosphatidic acid (PA), or diacylglycerol (DAG), respectively. The inhibition of mTOR/rictor binding and mTORC2 activity coincided with the levels of PA and DAG species that contained 16:0, the preferred substrate of GPAT1. Furthermore, di-16:0-PA strongly inhibited mTORC2 activity and disassociated mTOR/rictor in vitro. Taken together, these data reveal a signaling pathway by which phosphatidic acid synthesized via the glycerol-3-phosphate pathway inhibits mTORC2 activity by decreasing the association of rictor and mTOR, thereby down-regulating insulin action. These data demonstrate a critical link between nutrient excess, TAG synthesis, and hepatic insulin resistance.
Asunto(s)
Insulina/metabolismo , Metabolismo de los Lípidos , Complejos Multiproteicos/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transactivadores/metabolismo , Animales , Glicerol-3-Fosfato O-Aciltransferasa/genética , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transactivadores/genética , Factores de TranscripciónRESUMEN
OBJECTIVE: Hepatic steatosis is strongly associated with insulin resistance, but a causal role has not been established. In ob/ob mice, sterol regulatory element binding protein 1 (SREBP1) mediates the induction of steatosis by upregulating target genes, including glycerol-3-phosphate acyltransferase-1 (Gpat1), which catalyzes the first and committed step in the pathway of glycerolipid synthesis. We asked whether ob/ob mice lacking Gpat1 would have reduced hepatic steatosis and improved insulin sensitivity. RESEARCH DESIGN AND METHODS: Hepatic lipids, insulin sensitivity, and hepatic insulin signaling were compared in lean (Lep(+/?)), lean-Gpat1(-/-), ob/ob (Lep(ob/ob)), and ob/ob-Gpat1(-/-) mice. RESULTS Compared with ob/ob mice, the lack of Gpat1 in ob/ob mice reduced hepatic triacylglycerol (TAG) and diacylglycerol (DAG) content 59 and 74%, respectively, but increased acyl-CoA levels. Despite the reduction in hepatic lipids, fasting glucose and insulin concentrations did not improve, and insulin tolerance remained impaired. In both ob/ob and ob/ob-Gpat1(-/-) mice, insulin resistance was accompanied by elevated hepatic protein kinase C-epsilon activation and blunted insulin-stimulated Akt activation. CONCLUSIONS: These results suggest that decreasing hepatic steatosis alone does not improve insulin resistance, and that factors other than increased hepatic DAG and TAG contribute to hepatic insulin resistance in this genetically obese model. They also show that the SREBP1-mediated induction of hepatic steatosis in ob/ob mice requires Gpat1.
Asunto(s)
Hígado Graso/prevención & control , Glicerol-3-Fosfato O-Aciltransferasa/deficiencia , Glicerol-3-Fosfato O-Aciltransferasa/genética , Resistencia a la Insulina/genética , Obesidad/genética , Animales , Cruzamientos Genéticos , Hígado Graso/epidemiología , Heterocigoto , Humanos , Leptina/deficiencia , Lípidos/fisiología , Ratones , Ratones Obesos/genética , Músculo Esquelético/metabolismo , Obesidad/complicaciones , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
Cancer cachexia is a syndrome of unintentional weight loss that is characterized by wasting of both skeletal muscle and adipose tissue. Glucose intolerance and insulin resistance have been associated with cancer cachexia. However, it is unknown whether resistance to insulin has a role in the development of cachexia. In the present study, male CD2F1 mice with colon-26 adenocarcinoma tumors underwent an insulin tolerance test before the onset of weight loss. Compared to mice without tumors, mice with tumors had a profoundly blunted blood glucose response to insulin. Corroborating these findings, mice with tumors had decreased phosphorylation of Akt in quadriceps muscle and epididymal adipose tissue at the end of the study. Expression of Akt-regulated genes Atrogin-1, MuRF-1, and Bnip3 was increased in muscle, suggesting a role for decreased insulin signaling in the induction of both proteasomal proteolysis and autophagy in cachectic muscle. Rosiglitazone treatment increased serum adiponectin, insulin sensitivity, and body weight, and decreased Atrogin-1 and MuRF-1 expression in the skeletal muscle of tumor-bearing mice. In conclusion, insulin resistance is an early event in mice with cachexia induced by colon-26 tumors. Rosiglitazone improves insulin sensitivity and decreases early markers of cachexia. These data provide evidence that insulin resistance is not only present in cachexia, but also has a role in cachexia pathogenesis. Correction of insulin resistance may be a novel therapeutic target for the treatment of cancer cachexia.
Asunto(s)
Adenocarcinoma/fisiopatología , Caquexia/fisiopatología , Neoplasias del Colon/fisiopatología , Resistencia a la Insulina , Adenocarcinoma/complicaciones , Adenocarcinoma/patología , Adiponectina/sangre , Animales , Autofagia , Glucemia/análisis , Caquexia/etiología , Caquexia/prevención & control , Neoplasias del Colon/patología , Cruzamientos Genéticos , Insulina , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/etiología , Atrofia Muscular/fisiopatología , Proteínas de Neoplasias/análisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-akt/análisis , Distribución Aleatoria , Rosiglitazona , Tiazolidinedionas/uso terapéutico , Pérdida de PesoRESUMEN
Conjugated linoleic acid (CLA) reduces body weight and adipose mass in a variety of species. The mechanisms by which CLA depletes adipose mass are unclear, but two independent microarray analyses indicate that in white adipose tissue (WAT), uncoupling protein 1 (UCP1) was among genes most changed by CLA. The objective of this study was to determine whether CLA induces ectopic expression of UCP1 in WAT, which may contribute to increased energy expenditure and weight loss. Six-week old, male ob/ob mice were fed either a control diet (CON) or a diet supplemented with 1.5% mixed isomer CLA (CLA) for 4 weeks. A third group of mice (LEPTIN) was fed the control diet and received daily injections of recombinant leptin as a positive control for adipose depletion in ob/ob mice. CLA did not alter several mRNA markers of lipid oxidation in epididymal white adipose tissue (eWAT) , but significantly increased carnitine palmitoyltransferase-1b (CPT1b) and PPAR gamma coactivator-1alpha (PGC1alpha) expression. Notably, CLA increased both mRNA and protein expression of uncoupling protein-1 (UCP1). beta3-adrenoceptor mRNA and phosphorylated-p38 mitogen activated protein kinase (MAPK) protein levels were not affected by CLA, but were upregulated by LEPTIN. These data suggest the increased CPT1b, PGC1alpha, and UCP1, in WAT of CLA-fed mice may contribute to the depletion of adipose, and CLA does not appear to increase UCP1 through beta3-adrenergic signaling. Future studies will focus on understanding how CLA increases mitochondrial oxidation and energy dissipation in white adipose tissue.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Canales Iónicos/genética , Ácidos Linoleicos Conjugados/farmacología , Proteínas Mitocondriales/genética , Tejido Adiposo Blanco/efectos de los fármacos , Animales , Peso Corporal , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Metabolismo Energético , Canales Iónicos/metabolismo , Leptina/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Obesidad/genética , Obesidad/metabolismo , ARN Mensajero/metabolismo , Proteína Desacopladora 1RESUMEN
Four homologous isoforms of glycerol-3-phosphate acyltransferase (GPAT), each the product of a separate gene, catalyze the synthesis of lysophosphatidic acid from glycerol-3-phosphate and long-chain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-of-function and loss-of-function studies in mice and in cell culture strongly suggests that each isoform contributes to the synthesis of triacylglycerol. Much work remains to fully delineate the regulation of each GPAT isoform and its individual role in triacylglycerol synthesis.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Lipogénesis , Triglicéridos/biosíntesis , Acilcoenzima A/metabolismo , Animales , Ácidos Grasos/metabolismo , Glicerofosfatos/metabolismo , Humanos , Isoenzimas , Cinética , Lisofosfolípidos/metabolismo , Ratones , Microsomas/enzimología , Membranas Mitocondriales/enzimología , Orgánulos/enzimologíaRESUMEN
Conjugated linoleic acid (CLA) induces insulin resistance preceded by rapid depletion of the adipokines leptin and adiponectin, increased inflammation, and hepatic steatosis in mice. To determine the role of leptin in CLA-mediated insulin resistance and hepatic steatosis, recombinant leptin was coadministered with dietary CLA in ob/ob mice to control leptin levels and to, in effect, negate the leptin depletion effect of CLA. In a 2 x 2 factorial design, 6 week old male ob/ob mice were fed either a control diet or a diet supplemented with CLA and received daily intraperitoneal injections of either leptin or vehicle for 4 weeks. In the absence of leptin, CLA significantly depleted adiponectin and induced insulin resistance, but it did not increase hepatic triglyceride concentrations or adipose inflammation, marked by interleukin-6 and tumor necrosis factor-alpha mRNA expression. Insulin resistance, however, was accompanied by increased macrophage infiltration (F4/80 mRNA) in adipose tissue. In the presence of leptin, CLA depleted adiponectin but did not induce insulin resistance or macrophage infiltration. Despite this, CLA induced hepatic steatosis. In summary, CLA worsened insulin resistance without evidence of inflammation or hepatic steatosis in mice after 4 weeks. In the presence of leptin, CLA failed to worsen insulin resistance but induced hepatic steatosis in ob/ob mice.
Asunto(s)
Adiponectina/sangre , Grasas Insaturadas en la Dieta/administración & dosificación , Hígado Graso/inducido químicamente , Resistencia a la Insulina , Leptina/metabolismo , Ácidos Linoleicos Conjugados/administración & dosificación , Animales , Peso Corporal , Hígado Graso/metabolismo , Glucosa/análisis , Insulina/sangre , Interleucina-6/sangre , Leptina/administración & dosificación , Metabolismo de los Lípidos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Mutantes , Ratones Obesos , Obesidad/metabolismo , Proteínas Recombinantes/administración & dosificación , Factor de Necrosis Tumoral alfa/sangreRESUMEN
The dietary fatty acid conjugated linoleic acid (CLA) reduces hepatic lipid accumulation in some rodent models for obesity and hepatic steatosis. However, these effects are variable and complex due to differences in isomer responses and degree and sensitivity to changes in adiposity. Here, we hypothesized that CLA decreases hepatic steatosis in a diet-induced model of obesity in rats which are resistant to the adipose-lowering effects of CLA. To investigate this, we fed male Wistar rats a high-fat (20%) diet for 4 weeks to induce obesity and hepatic steatosis followed by low-fat (6.5%) experimental diets containing either 6.5% soybean oil (CON) or 1.5% CLA triglyceride mix plus 5% soybean oil (CLA). Dietary CLA significantly lowered hepatic triglycerides without changing weight, adiposity or adipokines, and was associated with significantly lower hepatic fatty acid synthase and stearoyl CoA desaturase-1 (SCD-1) mRNA levels and SCD-1 index along with significantly lower sterol regulatory element binding protein-1 mRNA, a transcription factor that regulates lipogenesis. Furthermore, the lower lipogenesis was associated with significantly higher mRNA expression of lipid oxidation gene peroxisome proliferator-activated receptor-alpha and acetyl CoA oxidase in the livers of rats fed dietary CLA. The lipid-lowering effects of CLA in the liver were observed in the absence of changes in adipose tissue and body weight. Thus, we conclude that in the Wistar rat model, where adipose levels remain static after feeding dietary CLA, hepatic lipid accumulation is reduced and these effects are not due to an improvement in overall adiposity.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Grasas Insaturadas en la Dieta/farmacología , Hígado Graso/prevención & control , Ácidos Linoleicos Conjugados/farmacología , Ácidos Linoleicos Conjugados/uso terapéutico , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Estearoil-CoA Desaturasa/metabolismo , Triglicéridos/metabolismoRESUMEN
Dysfunctional cross talk between adipose tissue and liver tissue results in metabolic and inflammatory disorders. As an insulin sensitizer, rosiglitazone (Rosi) improves insulin resistance yet causes increased adipose mass and weight gain in mice and humans. Conjugated linoleic acid (CLA) reduces adipose mass and body weight gain but induces hepatic steatosis in mice. We examined the combined effects of Rosi and CLA on adiposity, insulin sensitivity, and hepatic steatosis in high-fat-fed male C57Bl/6 mice. CLA alone suppressed weight gain and adipose mass but caused hepatic steatosis. Addition of Rosi attenuated CLA-induced insulin resistance and dysregulation of adipocytokines. In adipose, CLA significantly suppressed lipoprotein lipase and fatty acid translocase (FAT/CD36) mRNA, suggesting inhibition of fatty acid uptake into adipose; addition of Rosi completely rescued this effect. In addition, CLA alone increased markers of macrophage infiltration, F4/80, and CD68 mRNA levels, without inducing TNF-alpha in epididymal adipose tissue. The ratio of Bax to Bcl2, a marker of apoptosis, was significantly increased in adipose of the CLA-alone group and was partially prevented by treatment of Rosi. Immunohistochemistry of F4/80 demonstrates a proinflammatory response induced by CLA in epididymal adipose. In the liver, CLA alone induced microsteatotic liver but surprisingly increased the rate of very-low-density lipoprotein-triglyceride production without inducing inflammatory mediator-TNF-alpha and markers of macrophage infiltration. These changes were accompanied by significantly increased mRNA levels of stearoyl-CoA desaturase, FAT/CD36, and fatty acid synthase. The combined administration of CLA and Rosi reduced hepatic liver triglyceride content as well as lipogenic gene expression compared with CLA alone. In summary, dietary CLA prevented weight gain in Rosi-treated mice without attenuating the beneficial effects of Rosi on insulin sensitivity. Rosi ameliorated CLA-induced lipodystrophic disorders that occurred in parallel with rescued expression of adipocytokine and adipocytes-abundant genes.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Adiposidad/efectos de los fármacos , Hígado Graso/prevención & control , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Ácidos Linoleicos Conjugados/farmacología , Hígado/efectos de los fármacos , Obesidad/tratamiento farmacológico , Tiazolidinedionas/farmacología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adiponectina/sangre , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Tejido Adiposo/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Grasas de la Dieta , Modelos Animales de Enfermedad , Quimioterapia Combinada , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Hígado Graso/fisiopatología , Hipoglucemiantes/efectos adversos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leptina/sangre , Ácidos Linoleicos Conjugados/efectos adversos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Hígado/patología , Hígado/fisiopatología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/metabolismo , Obesidad/fisiopatología , ARN Mensajero/metabolismo , Rosiglitazona , Tiazolidinedionas/efectos adversos , Factores de Tiempo , Triglicéridos/metabolismoRESUMEN
Conjugated linoleic acid (CLA) causes insulin resistance and hepatic steatosis in conjunction with depletion of adipokines in some rodent models. Our objective was to determine whether the maintenance of adipokines, mainly leptin and adiponectin, by either removing CLA from diets or using an adiponectin enhancer, rosiglitazone (ROSI), could attenuate CLA-induced insulin resistance. Male C57BL/6 mice were consecutively fed two experimental diets containing 1.5% CLA mixed isomer for 4 weeks followed by a diet without CLA for 4 weeks. CLA significantly depleted adiponectin but not leptin and was accompanied by hepatic steatosis and insulin resistance. These effects were attenuated after switching mice to the diet without CLA along with restoration of adiponectin. To further elucidate the role of adiponectin in CLA-mediated insulin resistance, ROSI was used in a subsequent study in male ob/ob mice fed either control (CON) or CLA diet. ROSI maintained significantly higher adiponectin levels in CON- and CLA-fed mice and prevented the depletion of epididymal adipose tissue and the development of insulin resistance. In conclusion, we show that insulin resistance induced by CLA may be related more to adiponectin depletion than to leptin and that maintaining adiponectin levels alone either by removing CLA or using ROSI can attenuate these effects.
Asunto(s)
Adiponectina/metabolismo , Grasas de la Dieta/farmacología , Resistencia a la Insulina/fisiología , Ácidos Linoleicos Conjugados/farmacología , Animales , Peso Corporal , Modelos Animales de Enfermedad , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de los ÓrganosRESUMEN
Dietary CLA has been shown to enhance glucose tolerance in several animal models, but in mice it induces insulin resistance and lipodystrophy. In this study, the effects of 2 wk of diet supplementation with either 1.5% CLA or 0.2% troglitazone (TZD), an insulin-sensitizing thiazolidinedione, on glucose tolerance, lipid accumulation, and composition of both lean and Zucker diabetic fatty (fa/fa; ZDF) rats were examined. Compared with lean rats, which maintained normal glucose tolerances after 2 wk of feeding regardless of diet, ZDF rats fed a control diet (CON) had significantly worsened glucose tolerance. ZDF rats fed CLA and TZD diets, however, maintained normal glucose tolerances. In contrast to the significantly elevated lipid levels in ZDF rats fed the CON diet, concentrations of plasma FFA and TG in ZDF rats fed CLA and TZD diets were normalized. A similar reduction of plasma lipid levels was observed in lean rats fed CLA and TZD compared with lean rats fed the CON diet. Although ZDF CON rats developed significant hepatic steatosis, both CLA- and TZD-fed rats had hepatic TG levels similar to those of lean rats. Both lean and ZDF rats fed the CLA diet had reduced adipose mass compared with respective genotype controls; however, TZD had no effect. Ratios of 16:1/16:0 and 18:1/18:0 FA, surrogate markers for stearoyl-CoA desaturase-1 (SCD-1) activity, were reduced in livers of ZDF rats fed CLA and TZD diets. These results show that, like TZD, CLA normalizes glucose tolerance and plasma lipids and also improves hepatic steatosis and FA composition in ZDF rats. The effects of CLA and TZD on hepatic lipid composition suggest that the effects of these two agents on glucose tolerance may be associated with a reduction in SCD-1.