RESUMO
AIMS/HYPOTHESIS: Insufficient insulin secretion and reduced pancreatic beta cell mass are hallmarks of type 2 diabetes. Here, we focused on a family of serine-threonine kinases known as homeodomain-interacting protein kinases (HIPKs). HIPKs are implicated in the modulation of Wnt signalling, which plays a crucial role in transcriptional activity, and in pancreas development and maintenance. The aim of the present study was to characterise the role of HIPKs in glucose metabolism. METHODS: We used RNA interference to characterise the role of HIPKs in regulating insulin secretion and transcription activity. We conducted RT-PCR and western blot analyses to analyse the expression and abundance of HIPK genes and proteins in the islets of high-fat diet-fed mice. Glucose-induced insulin secretion and beta cell proliferation were measured in islets from Hipk3 ( -/- ) mice, which have impaired glucose tolerance owing to an insulin secretion deficiency. The abundance of pancreatic duodenal homeobox (PDX)-1 and glycogen synthase kinase (GSK)-3ß phosphorylation in Hipk3 ( -/- ) islets was determined by immunohistology and western blot analyses. RESULTS: We found that HIPKs regulate insulin secretion and transcription activity. Hipk3 expression was most significantly increased in the islets of high-fat diet-fed mice. Furthermore, glucose-induced insulin secretion and beta cell proliferation were decreased in the islets of Hipk3 ( -/- ) mice. Levels of PDX1 and GSK-3ß phosphorylation were significantly decreased in Hipk3 ( -/- ) islets. CONCLUSIONS/INTERPRETATION: Depletion of HIPK3 impairs insulin secretion and glucose tolerance. Decreased levels of HIPK3 may play a substantial role in the pathogenesis of type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica , Feminino , Teste de Tolerância a Glucose , Secreção de Insulina , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Interferência de RNARESUMO
Previous clinical studies showed an apparent correlation between hypertension and insulin resistance, and patients with diabetes are known to have increased blood pressure responsiveness to salt loading. To investigate the effect of high salt intake on insulin sensitivity and the insulin signaling pathway, a high-salt diet (8% NaCl) or a normal diet was given to 7-week-old SD rats for 2 weeks. High salt-fed rats developed slightly but significantly higher systolic blood pressure than controls (133 +/- 2 vs. 117 +/- 2 mmHg, P < 0.001), with no change in food intake or body weight. High salt-fed rats were slightly hyperglycemic (108.5 +/- 2.8 vs. 97.8 +/- 2.5 mg/dl, P = 0.01) and slightly hyperinsulinemic (0.86 +/- 0.07 vs. 0.61 +/- 0.06 ng/ml, P = 0.026) in the fasting condition, as compared with controls. Hyperinsulinemic-euglycemic clamp study revealed a 52.7% decrease in the glucose infusion rate and a 196% increase in hepatic glucose production in high salt-fed rats, which also showed a 66.4% decrease in 2-deoxyglucose uptake into isolated skeletal muscle and a 44.5% decrease in insulin-induced glycogen synthase activation in liver, as compared with controls. Interestingly, despite the presence of insulin resistance, high salt-fed rats showed enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, IRS-2 (liver and muscle), and IRS-3 (liver only). Phosphatidylinositol (PI) 3-kinase activities associated with IRS and phosphotyrosine in the insulin-stimulated condition increased 2.1- to 4.1-fold, as compared with controls. Insulin-induced phosphorylation of Ser-473 of Akt and Ser-21 of glycogen synthase kinase-3 also increased 2.9- and 2-fold, respectively, in the liver of the high salt-fed rats. Therefore, in both the liver and muscle of high salt-fed rats, intracellular insulin signaling leading to PI 3-kinase activation is enhanced and insulin action is attenuated. The hyperinsulinemic-euglycemic clamp study showed that decreased insulin sensitivity induced with a high-salt diet was not reversed by administration of pioglitazone. The following can be concluded: 1) a high-salt diet may be a factor promoting insulin resistance, 2) the insulin-signaling step impaired by high salt intake is likely to be downstream from PI 3-kinase or Akt activation, and 3) this unique insulin resistance mechanism may contribute to the development of diabetes in patients with hypertension.
Assuntos
Dieta Hipossódica , Resistência à Insulina , Insulina/fisiologia , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologia , Tiazolidinedionas , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Desoxiglucose/farmacocinética , Ativação Enzimática , Técnica Clamp de Glucose , Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Técnicas In Vitro , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/enzimologia , Masculino , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/metabolismo , Tiazóis/farmacologia , Tirosina/metabolismoAssuntos
Acidose Láctica/induzido quimicamente , Overdose de Drogas , Metformina/intoxicação , Tentativa de Suicídio , Adulto , Diabetes Mellitus Tipo 2/tratamento farmacológico , Overdose de Drogas/complicações , Humanos , Hipoglicemiantes/intoxicação , Hipoglicemiantes/uso terapêutico , Masculino , Metformina/uso terapêuticoRESUMO
AIMS/HYPOTHESIS: Recently, several groups have carried out whole-genome association studies in European and European-origin populations and found novel type 2 diabetes-susceptibility genes, fat mass and obesity associated (FTO), solute carrier family 30 (zinc transporter), member 8 (SLC30A8), haematopoietically expressed homeobox (HHEX), exostoses (multiple) 2 (EXT2), CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1), cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), which had not been in the list of functional candidates. The aim of this study was to determine the association between single nucleotide polymorphisms (SNPs) in these genes and type 2 diabetes in participants from the Japanese population. METHODS: Sixteen previously reported SNPs were genotyped in 864 Japanese type 2 diabetes individuals (535 men and 329 women; age 63.1 +/- 9.5 years (mean+/-SD), BMI 24.3 +/- 3.9 kg/m(2)) and 864 Japanese control individuals (386 men and 478 women; age 69.5 +/- 6.8 years, BMI 23.8 +/- 3.7 kg/m(2)). RESULTS: The SNPs rs5015480 [odds ratio (OR) = 1.46 (95% CI 1.20-1.77), p = 2.0 x 10(-4)], rs7923837 [OR = 1.40 (95% CI 1.17-1.68), p = 2.0 x 10(-4)] and rs1111875 [OR = 1.30 (95% CI 1.11-1.52), p = 0.0013] in HHEX were significantly associated with type 2 diabetes with the same direction as previously reported. SNP rs8050136 in FTO was nominally associated with type 2 diabetes [OR = 1.22 (95% CI 1.03-1.46), p = 0.025]. SNPs in other genes such as rs7756992 in CDKAL1, rs10811661 in CDKN2B and rs13266634 in SLC30A8 showed nominal association with type 2 diabetes. rs7756992 in CDKAL1 and rs10811661 in CDKN2B were correlated with impaired pancreatic beta cell function as estimated by the homeostasis model assessment beta index (p = 0.023, p = 0.0083, respectively). CONCLUSIONS/INTERPRETATION: HHEX is a common type 2 diabetes-susceptibility gene across different ethnic groups.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodomínio/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Índice de Massa Corporal , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/etnologia , Frequência do Gene , Ligação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Japão , Fatores de RiscoRESUMO
AIMS/HYPOTHESIS: Resistin and the resistin-like molecules (RELMs) comprise a novel class of cysteine-rich proteins. Among the RELMs, RELMbeta and RELMgamma are produced in non-adipocyte tissues, but the regulation of their expression and their physiological roles are largely unknown. We investigated in mice the tissue distribution and dimer formation of RELMbeta and RELMgamma and then examined whether their serum concentrations and tissue expression levels are related to insulin resistance. METHODS: Specific antibodies against RELMbeta and RELMgamma were generated. Dimer formation was examined using COS cells and the colon. RELMbeta and RELMgamma tissue localisation and expression levels were analysed by an RNase protection assay, immunoblotting and immunohistochemical study. Serum concentrations in high-fat-fed and db/db mice were also measured using the specific antibodies. RESULTS: The intestinal tract produces RELMbeta and RELMgamma, and colonic epithelial cells in particular express both RELMbeta and RELMgamma. In addition, RELMbeta and RELMgamma were shown to form a homodimer and a heterodimer with each other, in an overexpression system using cultured cells, and in mouse colon and serum. Serum RELMbeta and RELMgamma levels in high-fat-fed mice were markedly higher than those in mice fed normal chow. Serum RELMbeta and RELMgamma concentrations were also clearly higher in db/db mice than in lean littermates. Tissue expression levels revealed that elevated serum concentrations of RELMbeta and RELMgamma are attributable to increased production in the colon and bone marrow. CONCLUSIONS/INTERPRETATION: RELMbeta and RELMgamma form homo/heterodimers, which are secreted into the circulation. Serum concentrations of RELMbeta and RELMgamma may be a novel intestinal-tract-mediating regulator of insulin sensitivity, possibly involved in insulin resistance induced by obesity and a high-fat diet.
Assuntos
Células da Medula Óssea/citologia , Gorduras na Dieta/farmacologia , Hormônios Ectópicos/genética , Intestinos/citologia , Camundongos Obesos/sangue , Proteínas/genética , Animais , Glicemia/metabolismo , Peso Corporal , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , Hormônios Ectópicos/sangue , Hormônios Ectópicos/metabolismo , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intercelular , Intestinos/fisiologia , Camundongos , Fator de Crescimento Neural/sangue , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Proteínas/metabolismo , Proteínas Recombinantes/sangue , Análise de RegressãoRESUMO
AIMS/HYPOTHESIS: Oxidative stress is associated with diabetes, hypertension and atherosclerosis. Insulin resistance is implicated in the development of these disorders. We tested the hypothesis that oxidative stress induces insulin resistance in rats, and endeavoured to identify mechanisms linking the two. METHODS: Buthionine sulfoximine (BSO), an inhibitor of glutathione synthase, was administered to Sprague-Dawley rats and 3T3-L1 adipocytes. Glucose metabolism and insulin signalling both in vivo and in 3T3-L1 adipocytes were examined. In 3T3-L1 adipocytes, the effects of overexpression of a dominant negative mutant of inhibitory kappa B (I kappa B), one role of which is to block oxidative-stress-induced nuclear factor (NF)-kappa B activation, were investigated. RESULTS: In rats given BSO for 2 weeks, the plasma lipid hydroperoxide level doubled, indicating increased oxidative stress. A hyperinsulinaemic-euglycaemic clamp study and a glucose transport assay using isolated muscle and adipocytes revealed insulin resistance in BSO-treated rats. BSO treatment also impaired insulin-induced glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. In BSO-treated rat muscle, adipose tissue and 3T3-L1 adipocytes, insulin-induced IRS-1 phosphorylation in the low-density microsome (LDM) fraction was specifically decreased, while that in whole cell lysates was not altered, and subsequent translocation of phosphatidylinositol (PI) 3-kinase from the cytosol and the LDM fraction was disrupted. BSO-induced impairments of insulin action and insulin signalling were reversed by overexpressing the dominant negative mutant of I kappa B, thereby suppressing NF-kappa B activation. CONCLUSIONS/INTERPRETATION: Oxidative stress induces insulin resistance by impairing IRS-1 phosphorylation and PI 3-kinase activation in the LDM fraction, and NF-kappa B activation is likely to be involved in this process.