RESUMO
Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes are interacting comorbidities of obesity, and increased hepatic de novo lipogenesis (DNL), driven by hyperinsulinemia and carbohydrate overload, contributes to their pathogenesis. Fatty acid synthase (FASN), a key enzyme of hepatic DNL, is upregulated in association with insulin resistance. However, the therapeutic potential of targeting FASN in hepatocytes for obesity-associated metabolic diseases is unknown. Here, we show that hepatic FASN deficiency differentially affects NAFLD and diabetes depending on the etiology of obesity. Hepatocyte-specific ablation of FASN ameliorated NAFLD and diabetes in melanocortin 4 receptor-deficient mice but not in mice with diet-induced obesity. In leptin-deficient mice, FASN ablation alleviated hepatic steatosis and improved glucose tolerance but exacerbated fed hyperglycemia and liver dysfunction. The beneficial effects of hepatic FASN deficiency on NAFLD and glucose metabolism were associated with suppression of DNL and attenuation of gluconeogenesis and fatty acid oxidation, respectively. The exacerbation of fed hyperglycemia by FASN ablation in leptin-deficient mice appeared attributable to impairment of hepatic glucose uptake triggered by glycogen accumulation and citrate-mediated inhibition of glycolysis. Further investigation of the therapeutic potential of hepatic FASN inhibition for NAFLD and diabetes in humans should thus consider the etiology of obesity.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Camundongos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintases , Hiperglicemia/complicações , Leptina , Óxido Nítrico Sintase , Obesidade/complicações , Obesidade/genéticaRESUMO
Pancreatic beta cell failure is thought to underlie the progression from glucose intolerance to overt diabetes, and ER stress is implicated in such beta cell dysfunction. We have now shown that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) accumulated in the islets of diabetic animal models as a result of ER stress before the onset of hyperglycemia. Transgenic overexpression of C/EBPbeta specifically in beta cells of mice reduced beta cell mass and lowered plasma insulin levels, resulting in the development of diabetes. Conversely, genetic ablation of C/EBPbeta in the beta cells of mouse models of diabetes, including Akita mice, which harbor a heterozygous mutation in Ins2 (Ins2WT/C96Y), and leptin receptor-deficient (Lepr-/-) mice, resulted in an increase in beta cell mass and ameliorated hyperglycemia. The accumulation of C/EBPbeta in pancreatic beta cells reduced the abundance of the molecular chaperone glucose-regulated protein of 78 kDa (GRP78) as a result of suppression of the transactivation activity of the transcription factor ATF6alpha, thereby increasing the vulnerability of these cells to excess ER stress. Our results thus indicate that the accumulation of C/EBPbeta in pancreatic beta cells contributes to beta cell failure in mice by enhancing susceptibility to ER stress.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/fisiologia , Células Secretoras de Insulina/metabolismo , Fator 6 Ativador da Transcrição , Animais , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Insulina/metabolismo , Secreção de Insulina , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Receptores para Leptina/fisiologia , Transativadores/fisiologiaRESUMO
The total pancreatic beta cell mass is reduced in individuals with type 2 diabetes. We analyzed the islets of leptin receptor-deficient (Lepr-/-) mice, a model animal for type 2 diabetes with obesity. The plasma insulin levels in Lepr-/- mice peaked at approximately 7 weeks, an age at which the animals manifest normoglycemia to moderate hyperglycemia. Consistent with this, the beta cell mass was enlarged as compared with Lepr+/- mice, and it decreased thereafter. Thus, we focused on the islets of Lepr-/- mice at 7 weeks to elucidate the mechanism underlying beta cell failure. Endoplasmic reticulum (ER) stress was enhanced in beta cells of Lepr-/- mice at 7 weeks, as indicated by the increase in c-Jun and eIF2 alpha phosphorylation. Lepr-/- mice also exhibited a reduction in insulin signaling in beta cells at 7 weeks, as indicated by the decrease in Akt phosphorylation. These results indicate that both augmented ER stress and reduced insulin signaling occur before the onset of frank diabetes. Next, to examine the mutual effect of ER stress and insulin signaling in beta cells in vitro, we used MIN6 insulinoma cells. Tunicamycin induced ER stress as well as inhibited insulin signaling. Conversely, the PI-3 kinase inhibitor, LY294002, enhanced ER stress. Furthermore, the reduction in insulin signaling by LY294002 facilitated the induction of ER stress with tunicamycin. Taken together, we concluded that both ER stress and reduced insulin signaling might synergistically affect pancreatic beta cell dysfunction.
Assuntos
Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/fisiologia , Insulina/fisiologia , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Humanos , Células Secretoras de Insulina/patologia , Insulinoma/patologia , Insulinoma/fisiopatologia , Camundongos , Camundongos Knockout , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Receptores para Leptina/fisiologiaRESUMO
Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic beta-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on beta-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic beta cells (betaTSC2(-/-) mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual beta cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the betaTSC2(-/-) mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of beta cells. These results thus indicate that TSC2 regulates pancreatic beta-cell mass in a biphasic manner.
Assuntos
Células Secretoras de Insulina/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Envelhecimento/fisiologia , Animais , Glicemia/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos , Hiperinsulinismo/metabolismo , Insulina/sangue , Fator de Crescimento Insulin-Like I/fisiologia , Camundongos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases/fisiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaAssuntos
Ciclo Celular/genética , Diabetes Mellitus/etiologia , Células Secretoras de Insulina/citologia , Animais , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Resistência à Insulina , Fator de Crescimento Insulin-Like I/fisiologia , Células Secretoras de Insulina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The total mass of islets of Langerhans is reduced in individuals with type 2 diabetes, possibly contributing to the pathogenesis of this condition. Although the regulation of islet mass is complex, recent studies have suggested the importance of a signaling pathway that includes the insulin or insulin-like growth factor-1 receptors, insulin receptor substrate and phosphatidylinositol (PI) 3-kinase. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a serine-threonine kinase that mediates signaling downstream of PI 3-kinase. Here we show that mice that lack PDK1 specifically in pancreatic beta cells (betaPdk1-/- mice) develop progressive hyperglycemia as a result of a loss of islet mass. The mice show reductions in islet density as well as in the number and size of cells. Haploinsufficiency of the gene for the transcription factor Foxo1 resulted in a marked increase in the number, but not the size, of cells and resulted in the restoration of glucose homeostasis in betaPdk1-/- mice. These results suggest that PDK1 is important in maintenance of pancreatic cell mass and glucose homeostasis.
Assuntos
Diabetes Mellitus Experimental/genética , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
The protein p27(Kip1) regulates cell cycle progression in mammals by inhibiting the activity of cyclin-dependent kinases (CDKs). Here we show that p27(Kip1) progressively accumulates in the nucleus of pancreatic beta cells in mice that lack either insulin receptor substrate 2 (Irs2(-/-)) or the long form of the leptin receptor (Lepr(-/-) or db/db). Deletion of the gene encoding p27(Kip1) (Cdkn1b) ameliorated hyperglycemia in these animal models of type 2 diabetes mellitus by increasing islet mass and maintaining compensatory hyperinsulinemia, effects that were attributable predominantly to stimulation of pancreatic beta-cell proliferation. Thus, p27(Kip1) contributes to beta-cell failure during the development of type 2 diabetes in Irs2(-/-) and Lepr(-/-) mice and represents a potential new target for the treatment of this condition.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Inibidores Enzimáticos/metabolismo , Hiperinsulinismo/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Modelos Animais de Doenças , Hiperglicemia/metabolismo , Proteínas Substratos do Receptor de Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Leptina/genética , Leptina/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores para Leptina , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.
Assuntos
Regulação da Expressão Gênica , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Proteína Quinase C/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Gorduras na Dieta/farmacologia , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Fator 3-beta Nuclear de Hepatócito , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Isoenzimas , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Interleukins 9 (IL-9) and 4 are cytokines within the IL-2 receptor gamma chain (IL-2R gamma) superfamily that possess similar and unique biological functions. The signaling mechanisms, which may determine cytokine specificity and redundancy, are not well understood. IRS proteins are tyrosine-phosphorylated following IL-9 and IL-4 stimulation, a process in part mediated by JAK tyrosine kinases (Yin, T. G., Keller, S. R., Quelle, F. W., Witthuhn, B. A., Tsang, M. L., Lienhard, G. E., Ihle, J. N., and Yang, Y. C. (1995) J. Biol. Chem. 270, 20497--20502). In the present study, we used 32D cells stably transfected with insulin receptor (32D(IR)), which do not express any IRS proteins, as a model system to study the requirement of different structural domains of IRS proteins in IL-9- and IL-4-mediated functions. Overexpression of IRS-1 and IRS-2, but not IRS-4, induced proliferation of 32D(IR) cells in response to IL-9. The pleckstrin homology (PH) domain of IRS proteins is required for IRS-mediated proliferation stimulated by IL-9. The phosphotyrosine binding and Shc and IRS-1 NPXY binding domains are interchangeable for IRS to transduce the proliferative effect of IL-4. Therefore, the PH domain plays different roles in coupling IRS proteins to activated IL-9 and IL-4 receptors. The role of IRS proteins in determining cytokine specificity was corroborated by their ability to interact with different downstream signaling molecules. Although phosphatidylinositol 3' -kinase (PI3K) and Grb-2 interact with tyrosine-phosphorylated IRS proteins, Shp-2 only binds to IRS proteins following IL-4, but not IL-9, stimulation. Although PI3K activity is necessary for the IRS-1/2-mediated proliferative effect of IL-9 and IL-4, Akt activation is only required for cell proliferation induced by IL-4, but not IL-9. These data suggest that IRS-dependent signaling pathways work by recruiting different signaling molecules to determine specificity of IL-2R gamma superfamily cytokines.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Citocinas/metabolismo , Interleucina-4/metabolismo , Interleucina-9/metabolismo , Fosfoproteínas/metabolismo , Receptores de Interleucina-2/química , Receptores de Interleucina-2/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Divisão Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Proteína Adaptadora GRB2 , Proteínas Substratos do Receptor de Insulina , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Transfecção , Tirosina/metabolismoRESUMO
Stat6 and IRS-2 are two important signaling proteins that associate with the cytoplasmic tail of the interleukin 4 (IL-4) receptor. Data from numerous in vitro experiments have led to a model for IL-4 signal transduction in which the Stat6 signaling pathway is responsible for the IL-4 induced changes in gene expression and differentiation events, while the IRS-2 signaling pathway provides mitogenic and antiapoptotic signals. In order to determine the relative contributions of these signaling molecules in primary lymphocytes, we have examined IL-4 responses in T cells from mice deficient for either Stat6 or IRS-2 as well as from mice doubly deficient for both genes. Both IRS-2 and, especially, Stat6 are shown to be critically involved in IL-4-induced proliferation of T cells, presumably through the cooperative regulation of the Cdk inhibitor p27kip1. Like Stat6-deficient Th cells, IRS-2-deficient cells are also compromised in their ability to secrete Th2 cytokines, revealing a previously unrecognized role for IRS-2 in Th2 cell development. Although Stat6 and/or IRS-2 expression is required for IL-4-induced proliferative and differentiative responses, both signaling proteins are dispensable for the antiapoptotic effect of IL-4. However, treatment of lymphocytes with a protein tyrosine phosphatase inhibitor is able to block the antiapoptotic effect of IL-4 specifically in Stat6- or IRS-2-deficient cells and not in wild-type cells. Our results suggest that Stat6 and IRS-2 cooperate in promoting both IL-4-induced proliferative and differentiating responses, while an additional signaling mediator that depends on protein tyrosine phosphatase activity contributes to the antiapoptotic activities of IL-4 in primary T cells.