| Objective To investigate the effect of chronic high-fat fed on insulin sensitivity of rats and examine the expressions of phosphorylation of protein kinase B and AS160,body fat and plasma fat level.To explore the effects of PPAR-γactivator and AMPK-αactivator respectively on expression of phosphoration of protein kinase B,phosphoration of AS 160 and GLUT4,as well as insulin sensitivity in insulin resistant rat muscle induced by high-fat fed.Methods Male Wistar rats were divided randomly into two groups according to their body weights:control group,high-fat group(HF).The control group was provided with standard diet of rat,the other group was given a high-fat diet,in which 59%carolie was provided by saturated fat.After feeding 12 weeks,high-fat group(HF)were divided randomly into three groups:high-fat group(HF),HF+ROS group and HF+MET group.Rosiglitazone and metformin were separately administrated in HF+ROS group and HF+MET group with daily dose of 3mg/kg and 300mg/kg for 8 weeks.Evaluation of insulin sensitivity:Rat's body weights and fasting plasma glucose levels were monitored every two weeks.At last fasting serum insulin levels,plasma triglyceride and cholesterol levels,visceral fat contents and oral glucose tolerance test as well as insulin sensitivity were measured.To further explore insulin sensitivity of rats' skeletal muscle,the ability of insulin-stimulated glucose uptake in rat muscle was detected.Activities of Akt Ser473,AS160 and GLUT4 were evaluated by measuring proteins levels using Western blotting analysis.To explore the role of PPAR-γand AMPK-αin lipotoxicity,the effect of rosiglitazone and metformin,on high-fat fed induced insulin resistance were evaluated.Results(1)Compared with the control,high-fat-fed rats showed significantly increased levels of body weight(49.6%,P＜0.05);fasting plasma glucose(41.4%,P＜0.05)and serum insulin(181%,P＜0.01).The second hour glucose disposal were impaired by high-fat fed during glucose tolerance test(P＜0.01).Plasma triglyceride and cholesterol levels also increased 172% and 101.6%(both P＜0.01),accompanied with elevated visceral fat content(132.9%,P＜0.01)and decreased ISI(P＜0.01),showing the existance of insulin resistance in high-fat -fed rats.Both basal(30.8%,P＜0.05)and insulin-stimulated(61.3%,P＜0.01)glucose uptake in isolated rat skeletal muscle decreased by high-fat fed,so that indicating onset of insulin resistance. (2)Protein levels of phosphorylated Akt Ser473 significantly decreased after high-fat fed (19.8%,P＜0.01),indicating impaired Akt activity.However,protein levels of phosphorylated AS160 significantly elevated(33.9%,P＜0.05),showing increased activity of AS160 after high-fat fed.Compared with the control,protein levels of GLUT4 significantly decreased after high-fat fed(27.8%,P＜0.01).(3)Compared with the high-fat fed group,activating PPAR-γand AMPK-αrespectively by rosiglitazone and metformin brought decreases of plasma level of glyceride(both P＜0.01), visceral fat content(39%,P＜0.05;41%,P＜0.05)and ameroliated insulin resistance(14.1%,P＜0.05;16.5%,P＜0.05).Protein levels of phosphorylated Akt Ser473 significantly increased after treating rosiglitazone and metformin(47%,P＜0.01 and 37.7%,P＜0.01),the protein level of phosphorylated AS160 significantly decreased after treating rosiglitazone(26.5%,P＜0.05),the protein levels of phosphorylated AS160 decreased 20.6%after treating metformin.After treating rosiglitazone and metformin,the protein levels of GLUT4 increased(31%,P＜0.01,14.6% P＜0.01)compared with the high-fat fed group.(4)In comparison with the high-fat fed group,by treating with rosiglitazone basal(26.7%,P＜0.05)and insulin-stimulated glucose uptake(75.2%,P＜0.01)or by treating with metformin basal(25.4%,P＜0.05)and insulin-stimulated(78.9%,P＜0.01)glucose uptake in isolated skeletal muscle apparently increased,which showed improved insulin sensitivity.Conclusions:Insulin resistance could be induced by prolonged high-fat fed.High-fat fed increased body fat especially in skeletal muscle which impaired the muscular expressions and activities of both phosphorylated Akt Ser473and phosphorylated AS160,at the same time the protein level of GLUT4 on cell membrane decreased.Rosiglitazone and metformin by separately activating PPAR-γand AMPK-αapparently ameliorated high-fat induced insulin resistance by direct or indirect adjusting the muscular expressions and activities of both phosphorylated Akt and phosphorylated AS160,which showed the possible roles of Akt and AS160 in high-fat-fed induced insulin resistance. Objective To investigate whether AS160 interact physiologically with 14-3-3 in skeletal muscle of insulin resistant rat induced by high-fat-diet feeding during insulin regulated GLUT4 translocation.Methods Male Wistar rats were fed 14 weeks a high-fat diet,in which 59%carolie was provided by saturated fatty acid.At last fasting serum insulin levels,plasma cholesterol and triglyceride levels,visceral fat contents,oral glucose tolerance test as well as insulin sensitivity were measured by the ability of insulin-stimulated glucose uptake in the rat muscle,which finally indicate the onset of insulin resistant animal model.After extracted proteins from skeletal muscle stimulated or not by insulin,with ordinary control mice IgG we incubated the extraction with agarose conjugated beads pre-absorbed anti-pAkt substrate antibodies(AS160)to acquire AS160.By washing with the detergent,the proteins got off after using the antibodies of 14-3-3 protein and pAkt substrate(AS160)to screen and isolate protein in the deposition portion.Results(1)Compared with the control,high-fat-fed rats showed significantly increased levels of body weight(49.6%,P＜0.05);fasting plasma glucose(41.4%,P＜0.05)and serum insulin(181%,P＜0.01).The second hour glucose disposal were impaired by high-fat-fed during glucose tolerance test(P＜0.01).Plasma triglyceride and cholesterol levels also increased 172% and 101.6%(both P＜0.01),accompanied with elevated visceral fat content 132.9%(P＜0.01)and decreased ISI(P＜0.01),showing the existance of insulin resistance in high-fat -fed rats.Both basal(30.8%,P＜0.05)and insulin-stimulated(61.3%,P＜0.01)glucose uptake in isolated rat skeletal muscle decreased by high-fat fed,so that indicating onset of insulin resistance.(2)The 14-3-3 protein can be co-immunoprecipitated with AS160 in extracts from skeletal muscle of insulin resistant rat induced by high-fat fed.Both AS160 and 14-3-3 protein could be shown in beads by immunoblotting in contrast to the negative results in ordinary control mice IgG beads.The expression levels of both AS160 and 14-3-3 protein ascended by insulin stimulation,it showed insulin enhanced their interaction.Conclusions As insulin regulate GLUT4 translocation,there is a physiological interaction between 14-3-3 protein and AS160 in skeletal muscle of insulin resistant rat induced by chronically high-fat fed.Insulin may elevate the interaction betweenl 4-3-3 protein and AS160. Objective To investigate the genes expressions of UCP-3mRNA and GLUT4mRNA in IR rats'muscle induced by high-fat-fed.Observe the effect of PPAR-αactivator and AMPK-αactivator respectively on the high-fat fed rats 'gene levels of UCP-3mRNA and GLUT4m RNA as well as insulin sensitivity.Methods Male Wistar rats were divided randomly into two groups according to their body weights:control group,high-fat group(HF).The control group was provided with standard diet of rat,the other group was given a high-fat diet,in which 59%carolie was provided by fatty acid.After feeding 12 weeks,high-fat group(HF)were divided randomly into three groups: high-fat group(HF),HF+ROS group and HF+MET group.Rosiglitazone and metformin were separately administrated in HF+ROS group and HF+MET group with daily dose of 3mg/kg and 300mg/kg for 8 weeks.Evaluation of insulin sensitivity:Rat's body weights and fasting plasma glucose levels were monitored every two weeks.At last fasting serum insulin levels,plasma triglyceride and cholesterol levels,visceral fat contents and oral glucose tolerance test as well as insulin sensitivity were measured.To further explore insulin sensitivity of rats'skeletal muscle,the ability of insulin-stimulated glucose uptake in rat muscle was detected.The gene levels of UCP-3mRNA and GLUT4mRNA were respectively measured using RT-PCR.Evaluation of insulin sensitivity:Rat's body weights and glucose levels were monitored monthly.At last fasting serum insulin levels,visceral fat contents,skeletal muscle intake of glucose were measured.The content of muscular triglyceride(TG)was measured by GPO- PAP,the muscles were stained by oil red.To further explore the role of UCP-3 and GLUT4 in lipotoxity,the effects of PPAR-γactivator rosiglitazone and AMPK-αactivator metformin,on high-fat fed induced insulin resistance were evaluated by examine gene levels of UCP-3mRNA and GLUT4mRNA.Results(1)Compared with the control,high-fat-fed rats showed significantly increased levels of body weight(49.6%,P＜0.05);fasting plasma glucose(41.4%,P＜0.05)and serum insulin (181%,P＜0.01).The second hour glucose disposal were impaired by high-fat-fed during glucose tolerance test(P＜0.01).Plasma triglyceride and cholesterol levels also increased 172%and 101.6%(both P＜0.01),accompanied with elevated visceral fat content 132.9%(P＜0.01)and decreased ISI(P＜0.01),showing the existance of insulin resistance in high-fat -fed rats.Both basal(30.8%,P＜0.05)and insulin-stimulated(61.3%,P＜0.01)glucose uptake in isolated rat skeletal muscle decreased by high-fat fed,so that indicating onset of insulin resistance.(2)Compared with the control,gene expression of UCP-3 significantly decreased after high-fat fed48.2%(P＜0.01),indicating impaired UCP-3 activity.However,gene expression level of GLUT4 significantly dropped 33.9%(P＜0.05)significantly decreased after high-fat feeding,showing decreased glucose transportation.(3)Gene expression of UCP-3 significantly mounted 107.2%(P＜0.01)and 97.5%(P＜0.01) compared with control after PPAR-γactivator rosiglitazone and AMPK-αactivator metformin treatments.Meanwhile gene expression of UCP-3 significantly mounted 125.1%(P＜0.01)and 149.7%(P＜0.01)compared with the control after PPAR-γactivator rosiglitazone and AMPK-αactivator metformin treatments.(4)Muscular triglyceride content significantly increased in HF than the control(P＜0.01), after treating rosiglitazone and metformin both of the content separately increased(P＜0.05).Conclusions:High-fat fed increased body fat especially in muscle which impaired the muscular gene expressions of UCP-3mRNA and GLUT4mRNA.Through activating PPAR-γand AMPK-α,rosiglitazone and metformin increased UCP-3 rnRNA and GLUT4mRNA levels and lessen the content of TG in rat muscle.Insulin sensitivity and metabolic flexibility also improved.
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