Expression Of PKB,GSK,P70S6K In Rat Of Insulin Resistance

Insulin is a kind of poly-physiological functional protein hormone,which plays significant role in substance metabolism in organism.The physiological functional process of insulin includes procedures below,(1)the specific binding of insulin and insulin receptor of target cell;(2)a series of biochemical events transduce and amplify the biologic signals after the binding of insulin and receptor;(3)production of a series of biologic effect;Actually,the function of insulin is processes of evolution,transduction and implementation.In above-mentioned processes,the abnormalities of one or some links make the abnormality of insulin function,insulin resistance as well.there are a great deal of causes of insulin resistance,roughly including:(1) the intrinsic defections of target cell;(2) the secondary cause that influence the function of target cell;(3) the metabolic factors;(4) insulin receptor defects and post receptor defects.Serine/threonine kinase protein kinase B(PKB/Akt) plays critical roles in important cell function including the control of cell survival,glucose metabolism (the synthesis of glycogen,glycolysis,uptaking of glucose) and protein synthesis. PKB mediated signal transduction system participate in insulin mediated glucose transduction regulation,and intracellular insulin resistance mechanism includes defection of activation of PKB.Akt as a insulin receptor with PI-3K and downstream molecules make important effect on insulin metabolism,signal transduction and glucose transport.The degression of Aktase activity under activation of insulin induces translocation of GLUT4,defection of glucose transport,which is the important cause of insulin resistance.But the direct relationship of defection of Akt activation and insulin resistance is under discussion.The target protein of PKB,the direct substrate of PKB,includes phosphofructokinase 2 which relates regulation of glycolysis,and glycogen synthase kinase 3,after PKB phosphorylation,the action of GSK3 depresses,and when decreasing of phosphorylation of glycogen synthase,the action of GSK3 increases,and then induce increasing glycogen synthesis.In addition,GSK3 relates initiation of transcription of mRNA.It is showed that the action of GSK3 in the type 2 diabetic patients doubled in indiabtic patients(including obesity without type 2 diabetic patients).Hyperglycemia and hyperinsulinemia can incluse increasing of GSK3,and high GSK3 precipitates serine phosphorylation of IRS-1 and inhibits insulin signal transduction in skeletal tissue,inducing further glucose utilization disturbance,and ultimately type 2 diabetes occurs.Another target of PKB is ribosomal protein S6 kinase,(P70S6K),P70S6K promote the synthesis of protein kinase,but at present it isn't confirmed that P70S6K is direct phosphorylation of PKB or not.At present,the research of PKB is frequent,but the research of downstream signals GSK and P70S6K is infrequent.Especially,it isn't reported that whether insulin resistance inducing hyperlipemia and hyperglycemia has a coincident signal transduction or not.Our study plans to detect above-mentioned index on the tissue of liver,muscle,fatty tissue of insulin-resistance rat using detection methods including nor-blood glucose-hyperinsulinmia-ring clamp technology, histopathology,Immunoprecipitation,reflective immuno-technology,and further discuss the molecular biologic mechanism of insulin resistance.Subjects and Methods 一,Experimental animal40 Wistar male rats(China Medical University Experimental Animal Center supplied) four-week-age randomly divided into four groups per group has four rats,that is,common diet group(C group),high sugar diet group(T group),high fat diet group(Z group),high sugar high fat group(G group).After 8 weeks of every group diet feeding,we executed rats,got blood and obtained livers,skeletal muscles are adipose tissue,then made that quick freezing and keeping in -70℃refrigerator二The main experimental method1.The evaluation of insulin resistance by hyperinsulinism-nor blood sugar ring clamp experiment.We evaluated rats whether or not induced insulin resistance and evaluated the contents of insulin resistance2.The detection of serum TC,TG,HDL-C and LDL-C:We utilized American Abbortt Arrosset pan-automatic biochemistry analyzer3.The detection of serum hsCRP,FFA,INS:We utilized enzyme linked immunosorbent assay4.The expression of AKT in rat tissues:We utilized immunity histoehemistry method5.The expression of PI3K and GLUT4 in rat tissue:We utilized hybridization in situ method6.The expression of IRS-1 GSK P70S6K in rat tissues:We utilized immunity imprinting method7.The expression of CSK3 P70S6K in rat tissue:We utilized PT-PCR methodResults1,The body weight changes and epididymis fat pad weights in every group' s rats,the body weight increased most is Group G,by turns,Group Z,Group T. Group C increased least.The weight of epididymis fat pat gained most is Group G, by turns Group Z,Group T and Group C,especially.Group T is more than Group C,but have no statistic difference(P=0.02),other groups all have statistic significance.2,The serum insulin detection:The level of serum insulin of Group G and Group Z is obviously higher than Group C and Group T(P＜0.01).Group C and Group T have no statistic difference Group G and Group Z is neither.Respectively p is 0.421 and 0.594.The blood sugar levels in every group have no obvious difference.The results of GIR.The level of Group G and Group Z are obviously higher than Group C and Group T(P＜0.01,but Group D and Group T have no statistic difference p=0.194)3,The serum blood fat series detection the main detecctive index is trig Chol, HDL,LDL(1) The contents of serum trig in Group G and Group Z are obvious higher than that of Group C(P＜0.01).Group G is obvious higher than other groups, Group Z is higher than Group T,Group T is higher than Group C,but two groups have no statistic difference(P＞0.05).Group Z is higher than Group C,two groups have obvious statistic difference(P＜0.01).(2) The contents of serum Chol in Group G and Group Z are obvious higher than Group C(P＜0.01).Group G is obviously higher than other groups.Group Z is higher than Group T.Group T is higher than Group C,but two groups have no statistic difference.(P＞0.05) Group Z is higher than Group C.Two groups have obvious statistic difference.(P＜0.01).(3) The contents of serum LDL-C of Group G and Group T are obviously higher than Group C and Group(P＜0.05) T.Group G and Group Z has no obvious difference.Group C and Group T have no statistic difference(P＞0.05).(4) The contents of serum HDL-C are obvious higher than Group C and Group T(P＜0.05).Group G and Group Z have no obvious difference.Group C and Group T have no statistic difference(P＞0.05).The contents of serum HDL-C in Group G and Group Z are lower than Group C and Group T(P＜0.05).Group G and Group T has no statistic difference.Group C and Group T are neither (P＞0.05).4,FFA,no-specificity inflammation index super sensitivity CRP detection.(1) The contents of serum FFA in Group G and Group Z are higher than Group D and Group(P＜0.05) T.Group G and Group Z have no statistic difference.Group D and Group T are neither(P＞0.05).(2) The contents of serum hs-CRP in Group G and Group Z are higher than Group D and Group T(P＜0.05).Group G and Group Z has no statistic difference. Group D and Group T are neither(P＞0.05).5,FFA is obvious positive correlation with body weight,epididymis fat pads weight and obviously negative correlation with GIR.6,The expression of IRS-1 PI3K P70S6K in livers muscles fat of insulin resistance rats.The expression of P70S6K is obviously higher than that of normal rats.7,The expression of AKT GLUT4 GSK-3 found in livers muscles fats of insulin resistance rats.The expression of AKT GLUTt4 in liver muscles fats of insulin resistance is obvious lower than normal rats.The expression of GSK is obvious higher than that of normal rats.Conclusions1,After high sugar high fat diet feeding for 8 weeks,it could successfully induce Wistar rats to have insulin resistance 2,The weight of high sugar high fat group high fat group FINS,FFA,TC, TG,the epididymis fat pads weight hSCRP were all higher than normal control.The changes of high sugar high fat group are higher than that of high fat.The body weight is higher than that of normal control,but other index with normal control have no statistic difference.3,The form of insulin resistance is relationship with high-level FFA,further is relationship with inflammation4,The expression of IRS-1 PKB GLUT4 PI3K in livers muscles fats of insulin resistance decreased or disappearred,further the decreased extent is relationship with the extent of IR.5,The expression of GSK-3,P70S6K in livers muscles fats of insulin resistance rats increased That is positive correlation with FFA,but negative correlation with GIR.