Effect Of Insulin Receptor Substrate-1 On The Development Of Type 2 Diabetes Mellitus

Type 2 diabetes (12DM) is one of the most common metabolic disorder affecting of the world population. The association between T2DM diabetes and micro - and macrovascular complication, such as premature atherosclerosis, ne-phropathy, retinopathy, and neuropathy, has vast public implications. Consequently , intensive investigation the causes of the disease to prevent its onset and optimize treatment after its diagnosis is required.Type 2 diabetes is characterized by disturbances in insulin action and insulin secretion, and heredity and environment play the significant role in the development of the disease. Some studies have demonstrated the impaired insulin signal transduction associated with the disease. Insulin action is initiates through binding to cell surface insulin receptor, which triggers a cascade of intracellular signaling events, including receptor autophosphorylation and subsequent phos-phorylation of downstream target molecules, such as insulin receptor substrate -1 (IRS - 1) , phosphatidylinositol 3 - kinase ( PD - K) and protein kinase B (PKB). In the study we expored the congenital and acquired effect of insulin receptor substrate -1 and another key signaling transducers on the development of type 2 diabetes mellitus.Section1. The expression and phosphorylation of insulin receptorsubstrates -1, phosphatidylinositol 3 - kinase and protein kinase B inskeletal muscle of type 2 diabetic miceObjectiveMany studies reveal that insulin resistance in skeletal muscle is one of the first measurable defects associated with type 2 diabetes. The molecular basis for the development of whole - body insulin resistance remains unclear, but studiers have observed decreased insulin - stimulated glucose transport activity in isolated skeletal muscle from type 2 diabetic patient. So an understanding of the mechanisms that control glucose transport into insulin - sensitive tissues is essential to develop strategies for reestablishing normal glucose homeostasis in people with type 2 diabetes. Insulin signaling transduction system plays an important role in mediating the metabolic effects of insulin such as glucose utilization.In the study we investigated the expression and phosphorylation of a few post - receptor key transduces regulated by insulin — stimulated: insulin receptor substrates - 1 (IRS - 1) , phosphatidylinositol 3 - kinase ( PD - K) and protein kinase B (PKB) , to verify whether there are quantitative or function changes in these proteins in isolated skeletal muscle from type 2 diabetic, which contribute to block in insulin signaling in insulin - resistance state.Subjects and MethodsThirty - four female C57BL/6J mice as animals model were divided into three groups, control group were fed on normal commercial diet, type 2 diabetes group were given high fat and sugar diet (fat : 355g/kg, amount for 58% , primarily lard; carbohydrate ;36.6 g/kg, primarily sucrose) , after 16 weeks fasting blood glucose was 7. 3mmol/L. Type 1 diabetes group were injected 40 mmol/ kg STZ, once a day for consecutive 5 days, 4 weeks later, non -fasting blood glucose was above 15mmol/L. After an overnight fast, the mice had abdominal anesthesia induced with phenobarbitone, intact skeletal muscle strips and biopsies specimen were obtained. These strips were incubation in medium containing insulin (10~7M) respectively for 0, 2, 4, 15, or 30 min, then the muscle strips were rapidly frozen in liquid nitrogen. The protein expression of IRS -1 ,PD - K ,PKB and PKB serine -473 phosphorylation were measured by Western blot analysis f the phosphorylation of IRS -1 were measured by immu-noprecipitate procedures. The fasting plasma glucose, serum insulin and bloodlipid were also detected.ResultsThere are significant increase in level of fasting blood glucose ( FBG) , fasting insulin (Fins) , total cholesterol ( CHOL) , triglyceride (TG) and significant decrease in level of insulin - sensitivity index (ISI) from 12 DM group compared to that from control group, and in T1DM group had the significant increased FBG following remarkable reduce in Fins , it suggested mice model of T2DM and T1DM were successful duplicated. The expression amount of IRS - 1 and PKB protein in skeletal muscle cell was not found different among the control group, T2DM group and T1DM group. For the expression of PI3 - K protein, the same result also was found between control group and T1DM group, but in T2DM group, the amount of PI3 - K was significant lower than in control group and T1DM group( 19% and 23% ) (p < 0.05). T2DM mice had similar level of phosphoproteins in based state and considerable decreased response curves which represented insulin - stimulated IRS - 1 tyrosine phosphorylation and PKB serine -473 phosphorylation as compared with the control group.ConclusionThis study showing decreased insulin - stimulated GLUT4 translocation, IRS -1 tyrosine phosphorylation, expression of PI3 - Kp85 and PKB serine -473 phosphorylation in vitro incubated mice intact skeletal muscle strips. Strongly suggests that impairment of post - receptor steps in insulin signaling contributes to the defect in insulin - stimulated skeletal muscle glucose uptake in type 2 diabetes mice. First, the defect is associated with reduced activation of the key steps such as IRS -1 and PKB, and then it is associated with low expression of PI3 - K protein, these changes is a down - regulated in insulin signaling system. So we know a few defective steps of insulin resistance in T2DM without o-besity: blood glucose, insulin, serum lipid f —* IRS - 1 tyrosine phosphorylation I -+PI3 - Kp85a i ->-PKB serine -473 phosphorylation | -+GLUT4 translo-cation I —?glucose uptake I . This study demonstrates that insulin resistance in type 2 diabetes without obesity may be caused by degradation mediation of insulin signaling transduction system, which manifests mainly the decrease in activity of transduction protein.Section2. The change of translocation of GLUT4 in skeletal muscle cell of type 2 diabetic miceObjectiveSkeletal muscle is the major site for insulin - dependent glucose disposal, and type 2diabetic patients are characterized by a marked decrease in insulin stimulated glucose utilization in muscle mainly due to reduced glucose uptake and storage. Insulin stimulates glucose uptake principally by increasing the translocation of GLUT4 - containing vesicles to the plasma membrane. In the study we investigate the change of translocation of glucose transporter protein 4 (GLUT4) in isolated skeletal muscle from type 2 diabetes mice model.MethodsTwenty - four female C57BL/6J mice were divided equally into control group and type 2 diabetes (T2DM) group, control group were fed on normal commercial diet,T2DM group were given high fat and sugar diet( fat : 355g/kg, amount for 58% , primarily lard; carbohydrate: 366 g/kg, primarily sucrose). In sixteen weeks the type 2 diabetes mice model was established. After an overnight fast, the mice had abdominal anesthesia induced with phenobarbitone, intact skeletal muscle strips and biopsies specimen were obtained, then incubated in medium containing insulin (10 ~7 M) for 30 min. Skeletal muscle plasma membranes and the intracellular membranes were separated by sucrose gradient ultracentrifugation,then the levels of GLUT4 protein were measured by Western blot analysis,meanwhile the fasting plasma glucose,serum insulin and blood lip-id were also detected.ResultsT2DM group had significantly higher FBG, Fins, CHOL, TG and significantly lower ISI than control group ( p < 0. 05 or p < 0. 01) , but body weight had not remarked change between two groups. No significant different was observed in total GLUT4 amount contained both in intracellular membranes and plasma membranes in skeletal muscle between control group and T2DM group. Insulin - stimulation induced a significant increase (1.60 fold) in GLUT4 level in skeletal muscle plasma membranes obtained from the control subjects ( p < 0.01) and also induced a significant increase (1. 29 fold) in T2DM group ( p < 0. 05). The decreased amount of GLUT4 in intracellular membranes after stimulated by insulin is similar between 12DM group and control group, whereas the added amount of GLUT4 in plasma membranes from T2DM group is only 51% of amount from control group, means that quantity of GLUT4 translocated from intracellular membranes to plasma membranes was significant decreased in skeletal muscle cell from T2DM compared to control group ( p < 0. 05) , that suggests the GLUT4 may have missed during translocation.ConclusionThese results indicate that the decrease of insulin stimulated GLUT4 trans-location , not the decrease of quantity in skeletal muscle cell from non - obesity T2DM mice, leads to degradation mediation of GLUT4. The degradation mediation may be one of the molecule mechanism that contributes to the insulin resistance in the skeletal muscle of T2DM. The decrease of insulin stimulated GLUT4 translocation may be associated with choice lose from vesicles owing to mis - targeting of GLUT4. The block in the GLUT4 translocation may be caused by a blunted insulin signaling, especially the decrease in IRS -1 tyrosine phospho-rylation, PD — K85 and PKB serine - 473 phosphorylation in type 2diabetes mice.Section3. Gene polymorphisms of insulin receptor substrate -1 in Northern Chinese Han pedigrees with type 2 diabetes mellitusObjectiveInsulin receptor substrate - 1 (IRS - 1) gene, is a important candidate gene for T2DM in white population . To understand its role in the development of the disease in Chinese population, we investigate the prevalence and clinical significance of the codon 971,804 variation of the IRS -1 gene in northern Chinese Han pedigrees with T2DM.Subjects and Methods1. Subjects The study subjects included 80 unrelated normal control subjects , 80 unrelated T2DM patients. All subjects recruited from the Han Chinese living in North China. After an overnight fast, All subjects were assessed by a 75 - g oral glucose tolerance test, meanwhile insulin, C - peptide, glycosylated haemoglobin and lipids were measured.2. Polymerase chain reaction methods Genomic DNA was isolated from human leukocyte nuclei isolated from whole blood by protein kinase K digestion followed by phenol - chloroform extraction. The primer was designed by a computer software according to the gene sequences of human skeletal muscle IRS -1 in JSNP database. Then polymerase chain reaction (PCR) was done.3. Sequence specific primers analysis Polymorphisms in codon 804 and 971 of IRS - lgene in these subjects were analyzed by PCR - sequence specific primer (PCR -SSP) technique followed by polyacrylamide gel electrophoresis (PAGE) and silver staining. Each unusual sample was directly sequenced.ResultsThe sequence variant of IRS -1 gene found among 80 normal subjects and...