| In the recent years, the prevalence of type 2 diabetes is increasing quickly, estimated at 3 5000 billion suffers worldwide by the year 2030.The death toll of diabetes just follows cardiovascular disease and malignant tumor, thus called"the third killer". The number of diabetics in China ranks No. 2 in the world. Epidemiologic data indicate that the rate of type-2 diabetes increases sharply with aging, being one of the main diseases affecting the health of the middle-aged and the old. Some chronic complications related to diabetes, such as cardiovascular disease, diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy are main reasons causing death or deformity, seriously affecting the health of mankind. Insulin resistance (IR), defined as the decreased reactivity of the target tissues such as liver, fat, and muscles to the bio-effect of insulin, is the early-stage and important mechanism of obesity and type 2 diabetes. However, the specific mechanism of IR is not clear. Yet we do know that skeletal muscles (SM) are the place where glucose and lipid are taken in and utilized, and where insulin plays its important role.Genetic and environmental factors both can induce IR. The human and animal experiments all indicate that the increased free fatty acids (FFA), caused either by lipid perfusion or by chronic high-lipid diet, can result in IR, the functions of the fatty acids differ, with the saturated fatty acids the most important, and high lipid is the independent risk factor triggering IR. Long time high-lipid diet and transient fatty acids input both can cause the changed expression and dysfunction of the mitochondria-related proteins of the skeletal muscles in human and animals. In the old, type 2 diabetes, and their first-degree relatives, the metabolic capacity of the SM mitochondria declines, the expression of the landmark, indicating insulin resisting individual mitochondria metabolism, also has changed, with decreased mitochondria function, which leads to obesity and fat accumulating. Accordingly, the landmark of mitochondria metabolism makes a decisive difference in IR and in the pathogenesis of type-2 diabetes. Considering this, the dysfunction of SM mitochondria is the main reason of IR, but the mechanism is not clear yet. In the old, IR is probably associated with decreased function or defect of the mitochondria in the muscle cells.Peroxisome proliferator-activated receptorγco-activator 1α(PGC-1α), a nuclear transcription co-activator, may regulate many bioprocesses, such as mitochondria biogenesis, glycometabolism, fatty acid oxidation, and insulin secretion etc. Mitofusin 2 (Mfn2) can not only promote mitochondria fusion, sustain the completeness of mitochondria,but also be involved in the metabolic regulation. Nuclear respiratory factor-1 (NRF-1), a nuclear transcription factor coded by nuclear genome, regulating the expression of respiratory chain subunits and the transcription and duplication of mitochondria DNA, serves to regulate the key enzymes and protein factors in the process of duplication, transcription, and translation of the mitochondria genome expression; meanwhile, NRF-1 plays an important role in sustaining mitochondria morphologically and functionally. As the transcription co-activator, PGC-1αcan regulate Mfn2 expression and NRF-1 transcription. Therefore, the abnormal expression of the landmarks of mitochondria metabolism such as SM PGC-1α, Mfn2 and NRF-1 can cause the change of SM mitochondria morphologically and functionally, of which the roles of PGC-1αin the mitochondria function and IR has become the focus of study in the recent years.Some researches found out that high lipid can induce decreased expression of SM mitochondria-related proteins, such as PGC-1α, Mfn2, and NRF-1, meanwhile accompanied by abnormal expression of the insulin signal pathway-related proteins and IR, demonstrating that SM mitochondria function and IR are closely related and PGC-1αmay play an important role in regulating the functions of mitochondria and IR. The current hypothesis is that the increased FFAs in the blood or the dysfunctions of mitochondria, caused by any of the above mentioned reasons, affect the oxidation process of fatty acid entering mitochondria, and as a result, cause accumulated long-chain fatty acyl coenzyme A (LCACoA), increased genesis of diacylglycerol (DAG), and inactivated protein kinase C(PKC),and then restrain the activity of insulin receptor substrate-1 (IRS-1), finally interfere with the insulin signal conduction system, reduce the transportation of glucose, and lastly lead to IR. As to the functions of PGC-1α, the important nuclear transcription co-activator, in the changed function of mitochondria caused by high lipid and in IR, and as to the mechanism, there is no definite answer and the relative reports are rarely seen.After establishing IR models by feeding the old-aged rats with high lipid diet, we aim to analyze the expression changes of SM mitochondria-related proteins, such as PGC-1α, Mfn2, and NRF-1, and the correlation in the condition of IR and after being interfered with Rosiglitazone(RSG), and to analyze the changes of mitochondria functions and insulin sensitivity in the old-aged rats fed with high lipid diet. At the same time, muscle cells C2C12 were incubated with fatty acids to comprehend the effects of different types of acids on the expression of PGC-1αin skeletal muscles, with the purpose of finding the model of studying PGC-1αmechanism on cellular level. Meanwhile, after regulating the expression of PGC-1αthrough drugs and small interference RNA (siRNA) technique, we are to analyze the expression changes of mitochondria-related proteins and insulin signal pathway-related proteins, with the aim to explore the action mechanism of PGC-1αin the condition of high lipid, mitochondria function, and IR, and finally to provide novel target in developing drugs used to prevent IR and type 2 diabetes and ultimately to offer theoretical foundation. This experiment consists of the following 4 parts.Part I The expression of mitochondria-related proteins in skeletal muscles and of in the old-aged insulin resistant rats with high-fat-fedObjectives: To assay insulin insensitivity and the expression changes of PGC-1α, Mfn2 and NRF-1 in the skeletal muscles of the rats fed with high lipid and interfered with RSG, and to explore the correlation among high lipid, mitochondria function, and IR.Methods: Forty Wistar rats, male, ages 22-24 months, were divided into 2 groups: Old Control (OC) with 16 and Old Fat (OF) with 24; another 16 young rats ages 4-5 months were used as Young Controls (YC). The controls were given basal feed, with the calories composition of carbohydrate 65.5%, fat 10.3%, and protein 24.2%, totaling 348kcal/100g. OF members were given high-lipid feed, with the calories composition of carbohydrate 20.1%, fat 59.8 %, and protein 20.1%, totaling 501kcal/100g. The two old groups were given the feed with the same amount of calories daily lasting 8 weeks, and weighed once per week. At the end of week 4, blood samples from heart were collected to determine the amount of fasting blood-glucose (FBG), insulin (INS), serum triglyceride (TG), serum total cholesterol (TC), and FFA. Meanwhile, 8 rats were randomly selected from each group to be given the oral glucose tolerance test(OGTT)to evaluate the glucose tolerance, and then to be administered hyperinsulinemic euglycemic clamp technique (HGCT) to evaluate the condition of IR. After the models were established, HF members were divided into 2 subgroups: high fat and Rosiglitazone interference( OR group), with 8 in each. In addition to high lipid feed, Rosiglitazone interference members were given RSG 3mg/kg through intragastric administration, while OF done with the same amount of physiological saline. After another 4 weeks'feeding, at the end of week 8, blood samples were collected again to determine the serum indexes, and OGTT and HGCT were done to evaluate IR in each group. Afterwards, the animals were executed at the carotid. Quadriceps femoris and fatty tissues were taken out to be frozen in the liquid nitrogen and preserved in the cryogenic refrigerator of -70℃. The expression of PGC-1α, Mfn2 and NRF-1 mRNA and protein was measured by semiquantitative PCR and Western blot method.Results: 1. The comparison of the general items in each group. Compared with those in YC, fasting insulin (FINS), FBG, and FFA were higher in the old groups. Compared with OC group, FINS, FBG, FFA, TG, and TG in OF group increased obviously, and those in OR group decreased compared with those in OF, with statistical significance in both (p<0.05 or p<0.01).2. The amount changes of SM TG and the glucose infusion rate. Compared with YC, at week 4 and 8, the amount of SM TG in OC increased and the glucose infusion rate decreased, with statistical significance (P<0.05). Compared with OC, in OF group, SM TG began to increase after 4 week feeding, and that in week 8 was apparently higher than that at week 4, and the infusion rate decreased markedly compared with that in week 4, with statistical significance (P<0.01). Compared with OF, SM TG in OR decreased and infusion rate increased, with statistical significance (P<0.01).3. The expression of SM PGC-1αand Mfn2. Compared with YC, the expression of SM PGC-1αand Mfn2 in OC decreased, with statistical significance (P<0.01), and those in OF decreased than those in OC, with statistical significance (P<0.01). The expression in OR was higher than that in OF, but still lower than that in OC, with statistical significance between groups (P<0.01)4. The expression of SM PGC-1α, Mfn2 and NRF-1 mRNA of Compared with YC, the expression of SM PGC-1α, Mfn2 and NRF-1 mRNA in OC decreased, with statistical significance (P<0.01); and those in OF decreased than those in OC, with statistical significance (P<0.01). The expressions in OR were higher than those in OF, but still lower than those in OC, with statistical significance between groups (P<0.01).5. Results indication: The results demonstrated that glucose infusion rate showed obvious negative correlation with INS, FFA, and SMTG (γ=-0.4931, -0.5825, -0.4270, respectively; and P=0.0376, 0.0112, 0.0398, respectively) SM PGC-1αshowed positive correlation with Mfn2 and NRF-1 (γ=0.4931,0.532; P=0.0076, 0.002 respectively).Conclusions: 1. IR animal models are successfully made after feeding the old-aged rats with high-lipid diet, followed by FFA increase in the blood, SM TG increase, and glucose infusion rate decrease. 2. The expressions of SM PGC-1αand mitochondria-related factors Mfn2 and NRF-1 decrease after the old-aged rats being fed with high-lipid diet. 3. There exists positive correlation between SM PGC-1αand mitochondria-related factors Mfn2 and NRF-1. 4. The INS sensitivity heightens in the rats interfered with RSG, and the expression of SM PGC-1αand mitochondria-related factors Mfn2 and NRF-1, shows up-regulation, testifying the close relationship between PGC-1αand mitochondria function and IR.Part II The effects of fatty adics on the expression of PGC-1αin muscle cell C2C12Objectives: To explore on cellular level the effects of different FAs on the expression SM PGC-1α, after the muscle cells C2C12 of the mice were incubated with different types of fatty adics and the expressions of PGC-1αmRNA and proteins assayed; and ultimately to lay groundwork for studying the relation between PGC-1αand high lipid, mitochondria function and IR.Methods: DMEM in the New-born calf's serum was used to incubate muscle cell C2C12 in the 6-well plates. After 24 hours, the cells were incubated in the serum free medium, and grouped into palmitic acid (C16:0), stearic acid (C18:0), palmic acid(C16:1), oleic acid(C18:1), and linoleic acid (C18:2) after the related acids were added in, with 3 concentration gradients of 0.25mM,0.5mM and 0.75mM. After another 24 hours'incubation, the expression of PGC-1αwas determined by real-time fluorescent quantitative PCR and Western-blot.Results: 1. The effects of fatty adics with different concentrations on the expression of muscle cell PGC-1αmRNA and proteins. At concentration 0.25mM of FA, compared with the control group, there showed no statistical significance of the expression of muscle cell PGC-1αmRNA and proteins in the groups of palmitic acid, stearic acid, palmic acid, oleic acid, and linoleic acid (P> 0.05). At concentration 0.5mM and 0.75mM, the expressions in the palmitic acid group were lower than those in the control group(P <0.05), while the expressions in stearic acid ,palmic acid, oleic acid, and linoleic acid groups and those in the control group showed no statistical significance (P>0.05). 2. The expressions PGC-1αmRNA and proteins at different times in the muscle cells incubated with palmitic acid. As to the expressions, there showed no statistical significance in 0h and in 1h(P>0.05), while the expressions were lower at 6h, 12h, and 24h, compared with those in 0h and 1h, with the expression decreased most apparently in 24h, with statistical significance(P <0.01).Conclusions: The expression level of PGC-1αin the muscle cell C2C12 incubated with saturate fatty acids decreases, the level depending on concentration and time. 2. The muscle cell C2C12 incubated with monounsaturated and polyunsaturated fatty acids has no distinct effect on the expression level of PGC-1α.Part III The effects of SM PGC-1αexpression on mitochondria protein and insulin signal pathway in C2C12 cellObjectives: To analyze the functions of PGC-1αin mitochondria dysfunction and in IR induced by high lipid, using metformin, RSG, and agonist AMPK to up-regulate the expression level of PGC-1αin muscle cell C2C12, and using siRNA to restrain.Methods: Culturing and sub-culturing methods are the same as used in Part II. After C2C12 was incubated in the culture of C16:0 palmitic acid, with the concentration of 0.5 mM, 2mmol/l metformin, 2mmol/l RSG, and 10μmmol/l AMPK agonist (AICAR) were administered respectively to incubate the cells for another 24 hours, then PGC-1αexpression level was assayed. Then C2C12 was transfected with PGC1-α-siRNA or NS-siRNA. Six hours later, the cells were incubated with 10μmmol/l AICAR for 48 hours, followed by assay of the expression levels of PGC-1α, Mfn2, NFR-1, IRS-1 and GLUT4 mRNA and proteins. The cells interfered by AICAR were divided into groups of control, palmitic acid, and AICAR; and the siRNA group was subdivided into groups of control, AICAR (AICAR), AICAR+NS-siRNA (NS-siRNA), and AICAR+PGC1α-siRNA (PGC1α-siRNA).Results: 1. The effects of drugs on the expression of PGC-1αin muscle cell C2C12 incubated with palmitic acid. Metformin, RSG and AICAR all up-regulated the expressions of PGC-1αprotein and mRNA, compared with the condition in control group, with statistical significance (P <0.01).2. The effects of muscle cells incubated with palmitic acid and interfered with AICAR on the expression of mitochondria-related proteins. The expression levels of PGC-1α, Mfn2, NRF-1 mRNA and proteins decreased in the palmitic acid group compared with those in the controls, the difference with statistical significance (P <0.01). In addition, the above mentioned factors'mRNA and proteins in the AICAR group showed higher levels than those in the control and palmitic acid groups, also with statistical significance (P <0.01).3. The effects of muscle cells incubated with palmitic acid and interfered with AICAR on the expression of insulin signal pathway-related proteins. The expression levels of IRS-1 and GLUT4 mRNA and proteins in the palmitic acid group decreased compared with those in the control group, with statistical significance (P <0.01). Furthermore, IRS-1 and GLUT4 mRNA and proteins in the AICAR group showed higher expression levels than those in the control and palmitic acid groups, also with statistical significance (P <0.01).4. The effects of down-regulating PGC-1αexpression level in muscle cell C2C12 by using siRNA on the expression of mitochondria–related proteins. The expression levels of PGC-1α, Mfn2, NRF-1 mRNA and proteins increased in the AICAR group compared with those in the controls, the difference with statistical significance (P <0.01). The above mentioned factors'mRNA and proteins in the NS-siRNA group showed higher levels than those in the controls, also with statistical significance (P <0.01), no obvious difference with those in AICAR group (P >0.05). The indicators used above in PGC-1α-siRNA group showed distinct decrease than those in the other 3 groups, with statistical significance (P <0.01).5. The effects of down-regulating PGC-1αexpression level in muscle cell C2C12 by using siRNA on the expression of insulin signal pathway–related proteins. The expression levels of IRS-1 and GLUT4 mRNA and proteins increased in the AICAR group compared with those in the controls, the difference with statistical significance (P <0.01). Meanwhile, the expression levels of IRS-1 mRNA and proteins increased in the NS-siRNA group compared with those in the controls, the difference with statistical significance (P <0.01), no obvious difference with those in AICAR group (P >0.05). Additionally, the expression levels of IRS-1 and GLUT4 mRNA and proteins in PGC-1α-siRNA group showed apparent decrease compared with the other 3 groups, with statistical significance (P <0.01).Conclusions: 1. Metformin, RSG, and AICAR interfering can all up-regulate PGC-1αexpressions in C2C12 incubated with high-concentrated palmitic acid , indicating all the three drugs can reverse the function of palmitic acid on C2C12. 2. Up-regulating PGC-1αexpression level in C2C12 with drugs or down-regulating with siRNA will be accompanied by the changes of mitochondria–related and insulin signal pathway–related proteins, suggesting PGC-1αdecrease can cause mitochondria dysfunction and IR. 3. Mitochondria dysfunction and IR induced by high-concentrated palmitic acid are mediated by PGC-1α.Part IV The regulation of MAPKs pathway on PGC-1αexpression in C2C12 cellObjectives: To reveal the relation between MAPKs signal transduction pathway and PGC-1αexpression, and to investigate the up-stream regulatory pathway of the changed PGC-1αexpression in muscle cells induced by palmitic acid, by assaying the expression levels of Erk, p38, and JNK in palmitic acid-incubated C2C12.Methods: Culturing and sub-culturing methods were the same as used in Part II. After C2C12 was incubated in the culture of palmitic acid, with the concentration of 0.5 mM, the cells were collected at 0h, 0.5h, 1h, 6h, and 12h to determine the expression levels of PGC-1α, ERK, JNK, p38, and the phosphorylased proteins. After being interfered with inhibitor p38MAPK, the expression levels of PGC-1α, P38, and p-P38 were assayed, and the cells were divided into groups of control, palmitic acid, inhibitor p38MAPK, and palmitic acid + inhibitor p38MAPK.Results: 1. The expressions of ERK and JNK in muscle cells C2C12 incubated with palmitic acid. There showed no obvious change of ERK, P-ERK, JNK, and P-JNK at 0h, 1h, 6h, 12h, and 24h, with no statistical significance (P >0.05). 2. The p38MAPK expression in muscle cells incubated with palmitic acid. There showed no marked change of p38MAPK at 0h, 1h, 6h, 12h, and 24h, with no statistical significance (P >0.05). However, the expression of p38MAPK increased gradually at 0h, 1h, 6h, 12h, and 24h, all with statistical significance in any two groups (P <0.051). 3. The PGC-1αexpression in muscle cells C2C12 interfered with inhibitor p38MAPK. The expression level in the p38MAPK group was the highest, with statistical significance compared with the other 3 groups (P <0.05 or P <0.01). There showed increased expression level in group palmitic acid + inhibitor p38MAPK than those in control and palmitic acid groups, with statistical significance (P <0.05), with higher level in control group than that in palmitic acid group, but without statistical significance (P>0.05).Conclusions: 1. The expression levels of p38MAPK and P- p38MAPK in muscle cells increase gradually in the different time span incubated with palmitic acid. 2. There show no obvious changes of the expression of ERK, P-ERK, JNK, and P-JNK. 3. After the muscle cells incubated with palmitic acid are interfered with inhibitor p38MAPK, the expression level increases markedly compared with that before inhibitor adding, indicating the changed expression of PGC-1αinduced by palmitic acid may be regulated by p38MAPK.
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