Study On The Mechanism Of Insulin Resistance Induced By Hight Fat And The Preventive Effect Of Arachidonic Acid

It is well-known that insulin resistance is an important characteristic of Type 2 diabetes. Different free fatty acid play different role in the occurrence of insulin resistance. It is reported that saturated-fatty acid can induce insulin resistance, but unsaturated-fatty acid can improve insulin resistance. However, the mechanism is not fully understood. And liver is one of the most important insulin target tissues, the onset of hepatic insulin resistance typically precedes the appearance of peripheral insulin resistance in human. The 1st part of the present study demonstrates role of PKC in insulin resistance induced by palmitate in HepG2 cells. The 2nd part of the present study demonstrates preventing effect of arachidonic acid, a kind of unsaturated-fatty acid on HepG2 cell.Previous researches reported that only AA had no therapeutic effect in genetically diabetic animal models. AA decrease in blood is the earlier event than hyperglycemia and hyperinsulinemia during the insulin resistance development induced by diet. plasma AA concentrations in human and animal models with typeⅡdiabete are lower than in normal subjects. Thus we hypothesized that optimal AA dose administration could prevent high-fat diet induced insulin resistance by preventing the AA decrease on membranes, maintaining the integrity of membranes and cell normal signal transduction and by other mechanism. To test this hypothesis, the effect of AA on the preventing the insulin resistance was determined in Wistar rats. The 3rd part of the present study demonstrates oral AA administration prevents early whole-body insulin resistance induced by high-fat diet significantly. And the study also demonstrates oral AA administration affects hepatic glycogen storage and relative HGO. The 4th part of the present study demonstrates preventive effect of arachidonic acid on hepatic insulin signaling in high-fat-diet fed rats.Part 1 Role of PKC in insulin resistance induced by palmitate in HepG2 cellsObjective To study on the mechanism of insulin resistance (IR) for HepG2 cells induced by high concentrations of palmitate (PA) through protein kinase C (PKC) signaling pathway analysis. Methods The model of hepatic insulin resistance was established induced by PA.HepG2 cells were randomly didided into control group,PA group (250μM PA) and HI group (5×10-7 M insulin) and treated for 24 hours. Then both of control group and PA group were redivded into subgroups: control(-),control(+),PA(-),PA(+). Only control(+) and PA(+) groups were pretreated by 5μM chelerythrine chloride (CC) of PKC inhibitor for lh before insulin stimulation. Insulin-stimulated glucose consumption was measured using the glucose oxidase method at 12-hour timepoint,cell glycogen was measured with anthrone method at 3-hour timepoint and protein levels of phosphate-protein kinase B (P-Ser473 PKB) and phosphate-glycogen synthase kinase (P-Ser21/9 GSK-3α/β) at 15-min timepoint were determined in total cell lysates by Western-blotting. Results There was no significant difference in glucose consumption between PA(-) group and HI group(P>0.05). All levels of glucose consumption,glycogen conten,P-Ser473 PKB,P-Ser21 GSK-3αand P-Ser9 GSK-3βin PA(-) group were reduced significantly(P<0.05)compared with control(-) group, and were increased significantly(P<0.05)in PA(+) group compared with PA(-) group. Conclusions The model of hepatic insulin resistance was established successfully. Overaction of PKC may play an important role in inducing insulin resistance in HepG2 cells.Part 2 Preventive effect of arachidonic acid on HepG2 cell insulin resistance induced by palmitateObjective To study the effect of arachidonic acid (AA) on preventing HepG2 cell insulin resistance induced by palmitate (PA) and the possible mechanism. Methods The model of hepatic insulin resistance was established induced by PA.HepG2 cells were randomly divided into control group,PA group(250μM PA),PA+AA group (250μM PA plus 20μM AA) and HI group (5×10-7 M insulin) and cultured for 24 hours. Then insulin-stimulated glucose consumption was measured using the glucose oxidase method at 12 hours, cell glycogen was measured with anthrone method at 3 hours and protein levels of phosphate-protein kinase B(P-Ser473 PKB) and phosphate-glycogen synthase kinase (P-Ser21/9 GSK-3α/β) at 15 minutes were determined in total cell lysates by Western-blotting. Results There was no significant difference in glucose consumption between PA group and HI group. All levels of glucose consumption,glycogen conten,P-Ser473 PKB,P-Ser21 GSK-3αand P-Ser9 GSK-3βin PA group were reduced significantly(P<0.05)compared with control group, and were increased significantly (P<0.05) in PA+AA group compared with PA group. Conclusions AA can prevent HepG2 cell insulin resistance induced by palmitate significantly, keeping insulin-stimulated P-Ser473 PKB and P-Ser21/9 GSK-3α/βlevels. Part 3 Preventive effect of arachidonic acid on early insulin resistance induced by high-fat dietObjective To investigate whole-body metabolic disorder and hepatic glucose output(HGO) disturbance in rats with insulin resistance induced by short-term high-fat diet, and the effect of arachidonic acid(AA). Methods Twenty-four normal male Wistar rats (230-250g) were randomly divided into 3 groups according to their weight and fed for 12 weeks: control group, n=8, to be fed with standard chow diet; high-fat (HF) group, n=8, to be fed with high-fat diet; HF+AA group, n=8, to be fed with high-fat diet and administered orally 3mg.kg-1.d-1AA. By the end of experiment, an oral glucose tolerance test (OGTT) was carried out. At the end of the treatment period, every group was measured in weight and decapitated after an overnight fast. Blood samples were collected and plasma was prepared for the determination of plasma blood glucose (BG), triglyceride (TG), cholesterin (CH) and insulin. Livers were separated and frozen for futher analysis of glycogen content, PEPCK activity, PEPCK, GS and G-6-Pase mRNA levels. HE staining was used to observe lipid deposit in hepatic cells. Results Early insulin resistance was successfully induced in HF rats with hyperinsulinemia (P<0.05), higher plasma TG (P<0.05), higher fasting liver glycogen content (P<0.01) and higher glucose-insulin index (P<0.01) during OGTT. The treatment significantly decreased glucose-insulin index (P<0.01), blood TG (P<0.05) and glycogen content (P<0.05) in liver. Significantly decreased mRNA levels and activity of PEPCK (P<0.05) and lowered G-6-Pase mRNA levels (P<0.01) in liver were also observed. But there were significant differences in glucose-insulin index (P<0.01) during OGTT, and glycogen content (P<0.01) between HF+AA group and control group. There was no significant differences in GS mRNA levels among the three group. No lipid deposit was observed in hepatic cells of control group, but a large quantity of lipid deposit was observed in HF group, and a small quantity was observed in HF+AA group. Conclusions AA can significantly prevent whole-body insulin resistance induced by high-fat diet, as well as accompanied HGO disturbance in overnight fasting state, but not thoroughly.Part 4 Preventive effect of arachidonic acid on hepatic insulin signaling in high-fat-diet fed ratsObjective To investigate P-Ser473 PKB,P-Ser21/9 GSK-3α/βlevels in rats with insulin resistance induced by short-term high-fat diet, and the effect of arachidonic acid(AA). Methods Twenty-four normal male Wistar rats (230-250g) were randomly divided into 3 groups according to their weight and fed for 12 weeks: control group, n=8, to be fed with standard chow diet; high-fat (HF) group, n=8, to be fed with high-fat diet; HF+AA group, n=8, to be fed with high-fat diet and administered orally 3mg.kg-1.d-1AA. By the end of experiment, every group was decapitate 15 minutes afte intraperitoneal injection of insulin (6U/kg). Livers were separated and frozen for futher analysis of P-Ser473 PKB,P-Ser21/9 GSK-3α/βlevels. Results P-Ser473 PKB and P-Ser21/9 GSK-3α/βlevels were significantly decreased in HF rats compared with control rats(P<0.01). The treatment (3mg.kg-1.d-1AA) significantly increased P-Ser473 PKB(P<0.05),P-Ser21 GSK-3α(P<0.01)and P-Ser9 GSK-3β(P<0.05) levels. But there were significant differences in P-Ser473 PKB(P<0.05)and P-Ser21 GSK-3α(P<0.01) between HF+AA group and control group. Conclusions AA can significantly improve hepatic insulin signaling in high-fat-diet fed rats through increasing P-Ser473 PKB,P-Ser21/9 GSK-3α/βlevels, but not thoroughly.