Mechanism Study On Skeletal Muscle Fatty Acid Transport And Insulin Resistance And Therapy In High-fat-fed Aged Rats

A significant increase in type 2 diabetes mellitus with increasing age can be demonstrated from all epidemiological studies. Insulin resistance and dysfunction of beta cell of islet are the main pathogenesis in the type 2 diabetes mellitus. Insulin resistance brings a varied of clinical manifestation because of the lower reactivity to the action of insulin on muscle, liver and fat. Insulin resistance is also the initiating agent for majority of type 2 diabetics. The incidence of insulin resistance is increasing with increasing age on a global scale, but the precise mechanism is not fully elucidated yet.Genetic and environmental factors play an important role in insulin resistance state, studies demonstrated that the incidence of type 2 diabetics and cadiovascular diseases is increasing for the increased internal fat, diets is one of precipitating factor. The people with high-fat diet habits have higher incidence of type 2 diabetics and insulin resistance than those with normal diet habits, animal experiments also proved that high-fat exposure on impaired insulin sensitivity in several insulin target organs, including skeletal muscles, liver, adipocytes etc, so high-fat diet is a single risk factor leading to insulin resistance.The skeletal muscle plays a primary role in plasma glucose and lipid regulation. The lipid milieu of skeletal muscle has been associated with skeletal muscle insulin resistance in animals and humans. Several studies suggest that a reduced capacity to oxidize fat or/and an increased capacity to uptake fat may contribute to fat accumulation. Fatty acid translocase (FAT/CD36) is important membrane protein to mediate the uptake of long-chain fatty acid in skeletal muscle, the expression of FAT/CD36 affect fatty acid transport and secondary lipid deposition and insulin resistance.Aging is associated with reduced fat oxidation.The age-related reduction in fat oxidation, therefore, may promote the lipid accumulation in skeletal muscle. Carnitine palmitoyl transferase 1β(CPTⅠβ) is the rate-limiting enzyme of intracellular fatty acid transport in the mitochondrial outer membrane. The activity of CPTⅠβdecides if long-chain fatty acid can be tansfered into mitochondria to participate in beta-oxidation, so the express and activity of CPTⅠβis the key factor to the accumulation of long-chain fatty acid in kytoplasm. It is not clear why the lipid deposition of skeletal muscle increase with age, whether it is associated with the impaired fatty acid transport, now there are few studies in the field.In recently published statements , PI3-K/PKB pathway played an important role in insulin-signaling transduction. The effects of insulin on glucose uptake are mediated via the insulin receptor - insulin receptor substrate-1-phosphatidylinositol-3-kinase (PI3-K)-protein kinase B(PKB), and results in the translocation of intracellular glucose transporter 4 (GLUT4) to the cell surface. It is the increased amount of GLUT4 on the cell plasma membrane that results in an increased rate of glucose transport into the cell. According the present study,impairment of the insulin-signaling pathway at any step may lead to the insulin resistant. Recent evidence has demonstrated that lipid metabolic intermediate impaired the insulin-signaling pathway at some link from experiments results of the young rats. But, there was few investigate the relationship between the lipid deposition of skeletal muscle and abnormal insulin-signaling transduction in elderly people.Thiazolidinediones (TZDs) are a new class of insulin-sensitizing agents for the oral treatment of type 2 diabetes. TZDs are agonists of the peroxisome proliferator-activated receptorγ(PPARγ). PPARγ, a nuclear hormone receptor, is predominantly expressed in adipocytes but also, although at lower levels, in skeletal muscle and heart. TZDs have been proven to influence intramuscular long-chain FA uptake by changing the mRNA expression levels of several key players in the protein-mediated FA uptake process, but the precise mechanism need to further study.In the present study, we explore the possible mechanisms about high incidence of insulin resistance in aged rats through investigating the fatty acid metabolism of skeletal muscle including the key enzyme expression at protein and mRNA levels. Given a high fat diets and treatment with roglitazone in the aged rats to learn about the reactive potency to intervention factors, providing a new study direction to improve insulin resistance and delay senility. The paper contains four parts below:Part one: The effects of high fat diets on fatty acid metabolism of skeletal muscle and insulin sensitivity in the aged rats and the study of rosiglitazone treatment.Objective: To investigate the effects of high fat diets and rosiglitazone treatment on fatty acid metabolism of skeletal muscle and insulin sensitivity in the aged rats and to analyze the correlation between insulin resistance and lipid of skeletal muscle.Methods: Male Wistar rats aged 22-24 months were divided into old control (OC) group (n=16), high-fat diet (HF) group (n=24), Wistar rats aged 4-5 months were selected as young control (YC) group (n=16), the rats in control group were fed with a regular low fatty acids diet containing 10.3% fat, 24.2% protein, and 65.5% carbohydrate as percentage of total calories. The rats in high-fat diet group were fed regular diets mixed with 30% lard, containing 59.8% fat, 20.1% protein and 20.1% carbohydrate as a percentage of total calories. The rats in every group were fed equal calories every day. The body weights were determined weekly for 8 weeks. The blood sample was collected by cardiac puncture after rats were anesthetized with diethyl ether for the biochemical analysis, insulin resistance was evaluated by glucose infusion rate (GIR) of hyperinsulinemic euglycemic clamp technique and oral glucose tolerance test was done at the end of 4 week(eight rats in each group). After 4 weeks, rats were randomly divided into high fat diets group and high-fat + rosiglitazone group (RSG), the rats were given rosiglitazone (GlaxoSmithKline, UK) 3 mg per kg body weight through intragastric administration once a day for 4 weeks in RSG group(eight rats in each group). At the end of 8 week, the rats were killed with phenobarbital sodium after hyperinsulinemic euglycemic clamp test and OGTT, and skeletal muscles and fat tissues were taken out freeze-clamped with copper clamps precooled in liquid N2 and were stored in -70℃refrigerator. Muscle triglyceride(TGm) was measured by automatic biochemistry analyzer after extracting by methanol and chloroform, muscle long-chain fatty acyl coenzyme A(LCACoA)was measured by fluorospectro- photometer.Results:1 The body weight in OC group rats were higher than that in YC group rats. Compared with OC group, the body weight in HF group and RSG group didn't increase significantly(p>0.05).Compared with YC group, the fasting insulin(FINS), fasting blood glucose(FBG) and free fatty acid(FFA) were higher in the OC group at the end of 8 week; Compared with OC group, FINS, FBG, FFA, total cholesterol(TC) and triglyceride(TG) were higher in the HF group(p<0.05 or p<0.01). FINS, FBG, FFA, TC and TG were lower in the RSG group than those in the HFgroup(p<0.05 or p<0.01). 2 At the end of 4 week and 8 week, compared with YC group, TGm and LCACoA were higher in OC group(p<0.05); TGm and LCACoA were significantly higher in HF group than those in OC group(p<0.05 or p<0.01); TGm and LCACoA were higher at 8 week than those at 4 week in HF group(p<0.01); TGm and LCACoA were lower in the RSG group than those in the HF group(p<0.05 or p<0.01). 3 Compared with YC group at the end of 4 week and 8 week, GIR was lower in OC group, GIR was more lower in HF group than that in OC group(p<0.01); Compared with HF group at 4 week, GIR was significantly lower in HF group at 8 week(p<0.01). GIR were higher in the RSG group than that in HF group(p<0.01)4 The results of oral glucose tolerance test. At the end of 4 week and 8 week, the glucose level of the rats in OC group was much higher than that of rats in YC group at the time of 0′and 60′, 120′after glucose loading and area under curve(AUC) significantly increased (p<0.05 or p<0.01). The glucose level at every time point and AUC of the rats in HF group increased compared with those of rats in OC group (P<0.01). The glucose level in RSG group decreased at every time point and AUC (p<0.05 or p<0.01). 5 GIR has a negative correlation with INS, FFA, TGm and LCACoA (p<0.05 or p<0.01).Conclusions: TGm and LCACoA were higher and GIR was lower in OC group than those in YC group. It demonstrated that insulin resistance and lipid deposition of skeletal muscle always exist in aged rats. High fat diets in aged rats induced a significant increase in plasma lipid and intramusculary lipid paralleled impaired GIR, the results showed that insulin resistance and lipid metabolism were more serious. GIR has a negative correlation with INS, FFA, TGm and LCACoA. It suggested that lipid disorder is associated with insulin resistance. TGm and LCACoA decreased by rosiglitazone treatment and GIR and OGTT improved in aged rats induced by high fat diets, that maybe one of the mechanisms of rosiglitazone to improve insulin rasistance. Part two: Effects of high-fat diets and rosiglitazone treatment on expression of FAT/CD36 and CPTⅠβin aged rat skeletal muscles.Objective: To observe the effects of aging, high-fat diets and rosiglitazone treatment on the expression at protein and mRNA level of FAT/CD36 and mRNA level of CPTⅠβand to analyze the correlation with lipid in muscle tissue.Methods: Animal grouping and the samples acquirement were the same as those in part one. The expression at protein level of FAT/CD36 in skeletal muscle was measured by Western-blot method and the expression at mRNA level of FAT/CD36 in skeletal muscle was measured by real time polymerase chain reaction (PCR) method; the expression of CPTⅠβmRNA was measured by semiquantitative PCR method. TRIZOL Reagent was used to extract RNA from the tissues, integrity of RNA was evaluated by formaldehyde degeneration caraphoresis and optical density (OD) was got at 260nm and 280nm. RT-PCR was carried out with better integrity and ratio of OD 260nm and 280nm in 1.8～2.0 of RNA.Results: 1 Compared with YC group, the expression of FAT/CD36 mRNA and FAT/CD36 protein in skeletal muscle were higher in the OC group(p<0.05 or p<0.01); Compared with OC group, the expression of FAT/CD36 mRNA and FAT/CD36 protein were even higher in HF group rats(p<0.05 or p<0.01); the expression of FAT/CD36 mRNA and FAT/CD36 protein were lower in RSG group rats than those of rats in HF group(p<0.05 or p<0.01). 2 There were no significant difference of the expression of CPTⅠβmRNA between the YC group and OC group rats(P>0.05). Compared with OC group, the expression of CPTⅠβmRNA was lower in HF group rats(p<0.01). In response to the rosiglitazone treatment , the expression of CPTⅠβmRNA was significantly higher compared with HF group. 3 LCACoA has a positive correlation with the expression of FAT/CD36mRNA and has a negative correlation with the expression of CPTⅠβmRNA.Conclusions: 1 It is possible reason for aged rats to produce more lipid deposition in skeletal muscles than young rats for the higher expression of FAT/CD36 mRNA and protein. 2 The abnormal expression of FAT/CD36 and CPTⅠβof skeletal muscle in the aged rats induced by high fat diets may be one of the mechanisms of lipid deposition. 3 Correlation analyses suggested that lipid accumulation in muscles was associated with the abnormal expression of key enzyme to fatty acid transport. The expression of CPTⅠβmRNA increased and the expression of FAT/CD36mRNA and protein decreased in high-fat diets rats treated with rosiglitazone maybe associate with reduced lipid deposition and improve insulin resistance.Part three: Effects of high-fat diets on expression of GLUT4 and PKB in aged rat skeletal muscles and rosiglitazone treatment.Objective: To observe the effects of aging and high-fat diets on the expression at protein and mRNA level of PKB and GLUT4 and to analyze the correlation with lipid in muscle tissue.Methods: Animal grouping and the samples acquirement were the same as those in the part one. The expression at protein and mRNA level of PKB and GLUT4 in skeletal muscle was measured by Western blot method and real time PCR method respectively.Results: 1 Compared with YC group, the expression of PKBmRNA and PKB protein was lower in the OC group(P<0.05); Compared with OC group, the expression of PKBmRNA and protein was significantly lower in HF group rats (P<0.05); the expression of PKBmRNA and protein was higher in RSG group than that in HF group(p<0.05). 2 Compared with YC group, the expression of GLUT4mRNA and protein was lower in OC group (P<0.05 or P<0.01); Compared with OC group, and the expression of GLUT4mRNA and protein were significantly lower in HF group (P<0.05 or P<0.01); the expression of GLUT4mRNA and protein was higher in RSG group than that in HF group(p<0.01).3 The expression of PKBmRNA and GLUT4mRNA have possitive correlation with GIR and the expression of GLUT4mRNA has a negative correlation with LCACoA(P <0.01).Conclusions: 1 It is possible reason for the high incidence of insulin resistance in the aged rats because the impaired insulin-signaling transduction accounting for the abnormal expression of PKB and GLUT4. 2That insulin signal transduction impaired because of the abnormal expression of PKB and GLUT4 induced by high fat diets may be one of the mechnisms of insulin resistance. 3 Correlation analyses suggested that lipid accumulation associated with skeletal muscle insulin resistance through the effect on the key enzyme of insulin signal transduction. 4 The expression of PKB and GLUT4 increased in high-fat diets rats treated with rosiglitazone maybe one of the mechanisms to improve insulin resistance.Part four: Effects of high-fat diets and rosiglitazone treatment on the expression of PPARγin fat and skeletal muscle tissues in aged rat Objective: To observe the effects of aging and high-fat diets on the expression at protein and mRNA level of PPARγin fat and skeletal muscle tissues and rosiglitazone interference study.Methods: Animal grouping and the samples acquirement were the same as part one. The expression at protein and mRNA level of PPARγwas measured by Western-blot method and real time PCR method respectively.Results: 1 Compared with YC group, the expression of PPARγmRNA and PPARγprotein in fat tissue were lower in the old control group rats(P<0.05); Compared with OC group, the expression of PPARγmRNA and PPARγprotein in fat tissue were even lower in HF group (P<0.05); the expression of PPARγwas higher in RSG group than that of rats in HF group(p<0.01). 2 There were no significant differerce of the PPARγmRNA and PPARγprotein in sheletal muscle between YC group and OC group (p>0.05). Compared with OC group, the expression of PPARγmRNA and protein in skeletal muscle were lower in HF group (P<0.05 or P<0.01); the expression of PPARγmRNA and protein in skeletal muscle were higher in RSG group than those of rats in HF group (P<0.01).Conclusions: 1 It is possible for lipid metabolic disorder in aged rats because the lower expression of PPARγin fat tisssues than that of in young rats.2 That the lower expression of PPARγin fat and skeletal muscle tissues induced by high-fat diets maybe assosiated with abnormal lipid metabolism and insulin resistance. 3 Rosiglitazone treatment maybe affect adipocyte differentiation and improve insulin resistance through increasing the expression of PPARγin fat and skeletal muscle tissues.