RESUMEN
Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C epsilon activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.
Asunto(s)
Acetiltransferasas/metabolismo , Dieta Aterogénica , Grasas de la Dieta/farmacología , Resistencia a la Insulina , Obesidad/metabolismo , Acetiltransferasas/deficiencia , Acetiltransferasas/genética , Animales , Peso Corporal/efectos de los fármacos , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Grasas de la Dieta/administración & dosificación , Elongasas de Ácidos Grasos , Eliminación de Gen , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/genética , Fosfoproteínas/fisiología , Fosforilación , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here, we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and MafA, a beta cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviral-mediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had been knocked down). SREBP-1c and granuphilin were activated in islets from beta cell-specific SREBP-1c transgenic mice, as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential mechanism for impaired insulin secretion in diabetes, contributing to beta cell lipotoxicity.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental/genética , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Palmitatos/farmacología , Palmitatos/toxicidad , Potasio/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic beta-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, BetaEpsilonTauAlpha2, MafA, and IRS-2 were suppressed, partially explaining the loss and dysfunction of beta-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous beta-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts beta-cell mass and function.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Insulina/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/fisiología , Animales , Núcleo Celular/metabolismo , Colesterol/genética , Colesterol/metabolismo , Perfilación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Secreción de Insulina , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Transactivadores/genética , Transactivadores/metabolismo , Triglicéridos/genética , Triglicéridos/metabolismoRESUMEN
The Kaifukuki-Rehabilitation Ward (KRW) is a type of inpatient rehabilitation facility in Japan. In the KRW of our institute, mortality and frequency of emergency referrals in 2013 were rather high, 2.6% and 4.3%, respectively. We aimed to investigate the usefulness of an original gatekeeping system to reduce mortality and morbidity from cardiac complications, and to improve the quality of medical care in the KRW. A total of 370 consecutive patients admitted to the KRW of Kobayashi Memorial Hospital between 1 May 2015 and 31 March 2016 were enrolled in this prospective observational study. All patients underwent a screening evaluation in which we defined patients as being screen positive (SC-positive) if they had at least one of 20 diagnostic ECG codes and/or BNP level over 140 pg/dL at admission. A cardiologist provided weekly interventions to those among SC-positive patients who needed cardiac disease treatment during hospitalization. In all, 129 patients were classified as SC-positive (mean age 80 years, 124 [32%] male), and weekly intervention was needed in 28 patients, including start of cardiac medication in 17 cases. Mortality and frequency of emergency transfer due to cardiac disease during hospital stay were 0.3% and 0.3%, respectively. Our gatekeeping system involving a screening evaluation at admission and weekly intervention in selected patients by a cardiologist may be useful in reducing mortality and rate of transfer due to cardiac disease and may improve quality of medical care in KRWs.
Asunto(s)
Cardiopatías/rehabilitación , Anciano , Anciano de 80 o más Años , Electrocardiografía , Femenino , Cardiopatías/diagnóstico , Cardiopatías/mortalidad , Humanos , Masculino , Péptido Natriurético Encefálico/análisis , Estudios ProspectivosRESUMEN
Elovl-6, a long fatty acid elongase, contributes to de novo synthesis of fatty acids and regulates hepatic insulin sensitivity. Hepatic regulation of Elovl-6 gene expression in various nutritional conditions suggested that, like other lipogenic enzyme genes, Elovl-6 is a target of SREBP-1, a transcription factor governing fatty acid synthesis. Supportively, adenoviral RNAi knockdown of SREBP-1 in mouse liver suppressed Elovl-6 mRNA and fatty acid synthase levels. Therefore, we analyzed mouse Elovl-6 gene promoter to determine its role as an SREBP-1 target. Luciferase reporter assays of 1.4-kb 5' flanking region of mouse Elovl-6 gene in HepG2 cells demonstrated that nuclear SREBPs activated the Elovl-6 promoter, highlighting two SREBP binding sites: proximal SRE-1 and distal SRE-2. EMSA indicated that SRE-1 had higher affinity than SRE-2 for SREBP. ChIP assays confirmed in vivo binding of hepatic nuclear SREBP-1c protein. These data demonstrated that Elovl-6 is regulated directly and primarily by SREBP-1c.
Asunto(s)
Acetiltransferasas/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Elementos Reguladores de la Transcripción/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Activación Transcripcional/genética , Animales , Elongasas de Ácidos Grasos , Ratones , Unión ProteicaRESUMEN
Influx of excess fatty acids and the resultant accumulation of intracellular triglycerides are linked to impaired insulin secretion and action in the pathogenesis of type 2 diabetes. Sterol regulatory element-binding protein (SREBP)-1c is a transcription factor that controls cellular synthesis of fatty acids and triglycerides. SREBP-1c is highly expressed in high-energy and insulin-resistant states. To investigate effects of this synthetic lipid regulator on insulin secretion, we generated transgenic mice overexpressing nuclear SREBP-1c under the insulin promoter. beta-Cell-specific expression of SREBP-1c caused reduction in islet mass and impaired glucose-stimulated insulin secretion and was associated with accumulation of triglycerides, suppression of pancreas duodenal homeobox-1, and upregulation of uncoupling protein 2 gene expression. The mice presented with impaired glucose tolerance that was exacerbated by a high-energy diet. Taken together with enhanced insulin secretion from SREBP-1-null islets, these data suggest that SREBP-1c and endogenous lipogenesis could be involved in beta-cell dysfunction and diabetes.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Núcleo Celular/fisiología , Proteínas de Unión al ADN/genética , Intolerancia a la Glucosa/genética , Islotes Pancreáticos/fisiología , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Cartilla de ADN , ADN Complementario/genética , Proteínas de Unión al ADN/fisiología , Dieta , Metabolismo Energético , Regulación de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Proteínas de Homeodominio/genética , Humanos , Hiperinsulinismo/prevención & control , Insulina/genética , Insulina/metabolismo , Secreción de Insulina , Canales Iónicos , Islotes Pancreáticos/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Transgénicos , Proteínas Mitocondriales/genética , Regiones Promotoras Genéticas , Ratas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Transactivadores/genética , Factores de Transcripción/fisiología , Proteína Desacopladora 2RESUMEN
In addition to the prevention of cardiovascular diseases by lowering plasma LDL cholesterol, recent studies suggest that statins could have some impact on insulin action. To estimate the direct effects of statins on insulin secretion from pancreatic beta-cells, MIN6 cells were treated with pravastatin, simvastatin, or atorvastatin. Basal insulin secretion at low glucose concentration was unexpectedly increased at very high doses of simvastatin or atorvastatin after 24- and 48-hour incubation. Insulin secretion at high glucose was not significantly changed, and thus, net glucose-stimulated insulin secretion was apparently decreased by these lipophilic statins. The changes in insulin secretion were highly associated with increased endogenous SREBP activities in response to HMG-CoA inhibition as estimated by SRE-luciferase assays, and finally after 48-hour incubation, accompanied by impaired cell viability as estimated by MTT assays. In contrast, these changes were much less prominent by the addition of pravastatin. Meanwhile, glucose-stimulated insulin secretion of islets isolated from C57BL/6 mice was not significantly changed by any of the statins. Overall, taken up by beta-cells, statins can affect insulin secretion through either HMG-CoA inhibition or cytotoxicity, as observed by the addition of extraordinary high doses of lipophilic statins, but not hydrophilic statins, to the medium.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animales , Anticolesterolemiantes/farmacología , Atorvastatina , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ácidos Heptanoicos/farmacología , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Pravastatina/farmacología , Pirroles/farmacología , Simvastatina/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
To determine the role of cholesterol synthesis in pancreatic beta-cells, a transgenic model of in vivo activation of sterol-regulatory element binding protein 2 (SREBP-2) specifically in beta-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in beta-cells resulted in severe diabetes as evidenced by greater than 5-fold elevations in glycohemoglobin compared with C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose- and potassium-stimulated insulin secretion as determined by glucose tolerance test. Isolated islets of TgSREBP-2 mice were fewer in number, smaller, deformed, and had decreased insulin content. SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes involved in beta-cell differentiation, such as PDX1 and BETA2, were suppressed, explaining loss of beta-cell mass, whereas IRS2 expression was not affected. These phenotypes were dependent on the transgene expression. Taken together, these results indicate that activation of SREBP-2 in beta-cells caused severe diabetes by loss of beta-cell mass with accumulation of cholesterol, providing a new lipotoxic model and a potential link of disturbed cholesterol metabolism to impairment of beta-cell function.
Asunto(s)
Colesterol/metabolismo , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Animales , Humanos , Inmunohistoquímica , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Ratas , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
OBJECTIVE: Chronic exposure to fatty acids causes beta-cell failure, often referred to as lipotoxicity. We investigated its mechanisms, focusing on contribution of SREBP-1c, a key transcription factor for lipogenesis. RESEARCH DESIGN AND METHODS: We studied in vitro and in vivo effects of saturated and polyunsaturated acids on insulin secretion, insulin signaling, and expression of genes involved in beta-cell functions. Pancreatic islets isolated from C57BL/6 control and SREBP-1-null mice and adenoviral gene delivery or knockdown systems of related genes were used. RESULTS: Incubation of C57BL/6 islets with palmitate caused inhibition of both glucose- and potassium-stimulated insulin secretion, but addition of eicosapentaenoate (EPA) restored both inhibitions. Concomitantly, palmitate activated and EPA abolished both mRNA and nuclear protein of SREBP-1c, accompanied by reciprocal changes of SREBP-1c target genes such as insulin receptor substrate-2 (IRS-2) and granuphilin. These palmitate-EPA effects on insulin secretion were abolished in SREBP-1-null islets. Suppression of IRS-2/Akt pathway could be a part of the downstream mechanism for the SREBP-1c-mediated insulin secretion defect because adenoviral constitutively active Akt compensated it. Uncoupling protein-2 (UCP-2) also plays a crucial role in the palmitate inhibition of insulin secretion, as confirmed by knockdown experiments, but SREBP-1c contribution to UCP-2 regulation was partial. The palmitate-EPA regulation of insulin secretion was similarly observed in islets from C57BL/6 mice pretreated with dietary manipulations. Furthermore, administration of EPA to diabetic KK-Ay mice ameliorated impairment of insulin secretion in their islets. CONCLUSIONS: SREBP-1c plays a dominant role in palmitate-mediated insulin secretion defect, and EPA prevents it through SREBP-1c inhibition, implicating a therapeutic potential for treating diabetes related to lipotoxicity.
Asunto(s)
Ácido Eicosapentaenoico/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Palmitatos/toxicidad , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Adenoviridae/genética , Animales , Ácido Eicosapentaenoico/metabolismo , Expresión Génica/efectos de los fármacos , Silenciador del Gen , Proteínas Sustrato del Receptor de Insulina , Secreción de Insulina , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Lipogénesis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Técnicas de Cultivo de Órganos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína Desacopladora 2RESUMEN
The absorption of glycerol was examined using the closed loop of the rat small intestine in situ to clarify the transport mechanism. The absorption of glycerol, evaluated by its disappearance from the intestinal lumen, was saturable and reduced under the Na(+)-free conditions, suggesting the involvement of an Na(+)-dependent carrier-mediated transport system. Furthermore, glycerol absorption was selectively inhibited by several alcohols, among which 1,3-propanediol caused the greatest inhibition, and also by glycerol-3-phosphate and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol absorption included D-glucose and L-ascorbate, which are known to be transported by specific carriers. Therefore, the carriers for these two compounds do not seem to be involved in glycerol absorption. It is likely that the carrier-mediated transport system involved in glycerol absorption is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. Thus, the present study has provided in situ evidence for the presence of an Na(+)-dependent carrier-mediated transport system for glycerol in the rat small intestine. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system could be involved in drug absorption and also that it could be used for oral drug delivery.
Asunto(s)
Proteínas Portadoras/fisiología , Glicerol/metabolismo , Absorción Intestinal/fisiología , Intestino Delgado/metabolismo , Sodio/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Absorción Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Masculino , Ratas , Ratas WistarRESUMEN
The mechanism of intestinal glycerol transport was investigated by using the in vitro everted sac method involving the rat small intestine. The uptake of glycerol into everted sacs was saturable with a Michaelis constant (K(m)) of 0.77 mM and a maximum transport rate (J(max)) of 11.5 nmol/min/100 mg wet tissue weight (wtw), suggesting the involvement of carrier-mediated transport, and was accompanied by unsaturable transport (passive transport) with a membrane permeability clearance (CL(m,d)) of 4.9 microl/min/100 mg wtw. The carrier-mediated uptake of glycerol was inhibited by the removal of Na(+) and also by the addition of 2,4-dinitrophenol (DNP) and sodium azide (NaN(3)), which are metabolic inhibitors. These results suggest that the carrier-mediated glycerol transport is Na(+)-dependent and secondary active. Since glycerol uptake was also inhibited by p-chloromercuribenzene sulfonate (pCMBS), a thiol-modifying reagent, cysteine residues, which have a thiol group, seem to play an important role in the function of the carrier. We further found that glycerol uptake was selectively inhibited by glycerol-3-phosphate, chloramphenicol and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol uptake included D-glucose and 5-fluorouracil, which are known to be transported by specific carriers, and none of the selective inhibitors of glycerol uptake inhibited the uptake of D-glucose and 5-fluorouracil. Therefore, the carriers for these two compounds do not seem to be involved in glycerol uptake. It is likely that the carrier-mediated transport system involved in glycerol uptake is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system might be involved in drug absorption and also that it might be used for oral drug delivery.
Asunto(s)
Glicerol/farmacocinética , Inositol/análogos & derivados , Absorción Intestinal , Intestino Delgado/metabolismo , Alcoholes/farmacología , Animales , Transporte Biológico Activo , Cloranfenicol/farmacología , Colon/metabolismo , Portadores de Fármacos , Glicerol/antagonistas & inhibidores , Técnicas In Vitro , Inositol/farmacología , Masculino , Ratas , Ratas Wistar , Sodio/metabolismo , Factores de TiempoRESUMEN
The permeability of glycerol, a small hydrophilic solute, across the intestinal membrane would be low, if passive diffusion restricted to the paracellular route is the principal transport mechanism as generally assumed for this class of solutes. However, in the present study using a closed loop of rat small intestine in situ, we found that the absorption of glycerol was faster than that of urea, a probe solute widely assumed to permeate exclusively via the paracellular route. This finding is inconsistent with the paracellular permeation hypothesis, which predicts that the absorption of glycerol, which is larger than urea in terms of molecular size, could not be faster than that of urea. We also found that glycerol absorption was saturable. These findings suggest the involvement of carrier-mediated transport in intestinal glycerol absorption. Glycerol absorption in the colon was also saturable, suggesting the involvement of carrier-mediated transport, although it was much slower than that in the small intestine. Carrier-mediated glycerol transport might play an important role in absorbing glycerol liberated from dietary triglyceride. It would be interesting to further examine the possibility that a carrier-mediated glycerol transport system (or systems) might be involved in drug absorption and also that it might be utilized for oral drug delivery.