| Mitofusin 2 (Mfn2) was first found by professor Kuang-Hueih Chen in 1997. Mfn2 gene is highly expressed in vascular smooth muscle cells (VSMCs) of normal rat, and is markedly reduced in the arteries of spontaneously hypertensive rat. Thus, it is named as hypertension related gene (HRG). Later it is found that HRG inhibits the Ras–raf–ERK-MAPK signaling pathway, induces cell cycle arrest and exhibits anti-proliferating effects, so it is renamed as hyperplasia suppressor gene (HSG). The other researchers find that HSG is homologous with Mfn2 which can promote mitochondrial fusion. Mfn2 is not only expressed in VSMCs, but also highly expressed in heart, brain, lung, kidney, liver, skeletal muscle and brown adipose tissue.Mfn2 localizes at the mitochondrial outer membrane and plays an essential role in mitochondrial fusion, and participates in mitochondrial morphology regulation. Some evidences support that Mfn2 participates in mitochondrial metabolism regulation. Repression of Mfn2 reduces glucose oxidation, mitochondrial membrane potential, and cell respiration. The change of Mfn2 expression is paralleled to respiratory chain protein subunits expression. Peroxisome proliferators-activated receptorγcoactivator-1α(PGC-1α) is a novel co-activator of a number of nuclear receptors. PGC-1αcan bind to the cis-elements in the promoter region of a lot of genes, and regulates target gene transcription, inducing diverse biological effects. As known by so far, PGC-1αpromotes coordinately the expression of genes involved in the mitochondrial biogenesis, adaptive thermogenesis, glucose metabolism regulation, fatty acids oxidation regulation, muscle fiber type regulation, breathe, insulin secretion and glyconeogenesis. Recently, binding site of PGC-1αwas found in Mfn2 gene promoter region, and the expression of Mfn2 was induced by PGC-1α. In the first part of this study, rat insulin resistance model was set up by high fat diets. The expression of PGC-1αand Mfn2 was detected. The changes of mitochondrial morphology were measured. The aim of this part was try to find the relationship of PGC-1α, Mfn2 and mitochondria with the development of insulin resistance.In the various pathogenesis of metabolic syndrome, oxidative stress is a crucial pathogenic factor. It has important effect on apoptosis. The development and complication appearance of many metabolic syndrome have close relationship with apoptosis which is induced by oxidative stress. The reactive oxygen species are produced in mitochondria metabolism, and are main pro-apoptotic factor. Some evidences have suggested that Mfn2 participates in the mitochondria pathway mediating apoptosis, but there were arguments about its function in apoptosis. Some scholar considered that Mfn2, as an apoptotic factor, promotes apoptosis. However, the other researchers considered Mfn2, as an anti-apoptosis factor, was highly expressed to protect cells against apoptosis under apoptotic stimulus. The second part of this study tries to clarify this hypothesis.At the same time, because metabolic syndrome has the character of inheritance tendency, the third part of this study analyzed the relationship of Mfn2 gene polymorphism with insulin resistance.Part one: The expression changes of PGC-1αand Mfn2 in insulin resistance and rosiglitazone interference rat skeletal muscleObjective: To investigate the expression of PGC-1αand Mfn2 and the changes of mitochondrial morphology in high fat diets induced insulin resistance rat skeletal muscle. To explore the relationship of PGC-1αand Mfn2 with insulin resistance. To investigate the rosiglitazone interference on expression of PGC-1αand Mfn2 and the mitochondrial morphology in rat skeletal muscle. To explore the mechanism of rosiglitazone interference on insulin resistance.Methods: Adult Wistar rats were randomly divided into normal control (NC) group, high-fat diet (HF) group, and high-fat plus rosiglitazone interference (HF+RSG) group. The rats in NC group were fed with a regular low fatty acids diet. The rats in the other two groups were fed with high fat diets. At the fourth weekend, the blood sample was collected by cardiac puncture for the biochemical analysis. Insulin resistance in each group was evaluated by glucose infusion rate (GIR) of hyperinsulinemic euglycemic clamp technique under conscious state. Insulin resistance was set up in the two high fat diet groups. The HF+RSG group added rosiglitazone by gastric gavage,mice in the other two groups were administered with physiologic saline of the same volume. At the eighth weekend, the blood sample was collected by cardiac puncture for the biochemical analysis again. Insulin resistance in each group was evaluated again by glucose infusion rate (GIR) of hyperinsulinemic euglycemic clamp technique under conscious state. Then the animals were killed by bloodletting. The skeletal muscles were collected and quickly frozen in liquid nitrogen, and stored in -70℃refrigerator. The expression level of PGC-1αand Mfn2 was detected by real time PCR and Western blot. The changes of mitochondrial morphology were observed by transmission electron microscope (TEM). Mitochondrial morphological parameters in the TEM photograph were analyzed by Image-Pro Plus 6 software.Results:1. After four weeks high fat diets, glucose infusion rate was decreased, so insulin resistance rat model was duplicated successfully. After next four weeks rosiglitazone interference, insulin resistance induced by high fat diets was improved. 2. The results of real time PCR demonstrated the mRNA copies of PGC-1αhave no significant differences in NC group and HF+RSG group. But the copies in HF group are reduced obviously. The differences have statistical significance compared with NC and HF+RSG group (P<0.05). The mRNA copies of Mfn2 are the most in NC group, and are the least in HF group, and are intermediate in HF+RSG group. The differences among three groups have statistical significance (P<0.05). 3. The results of Western blot showed the expression levels of PGC-1αprotein have no significant differences in NC group and HF+RSG group. But the expression levels in HF group are reduced obviously. The differences have statistical significance compared with NC and HF+RSG group (P<0.05). The expression levels of Mfn2 protein are the most in NC group, and are the least in HF group, and are intermediate in HF+RSG group. The differences among three groups have statistical significance (P<0.05). 4. Observed by TEM, the mitochondrial morphology in HF group change apparently compared with normal control group. The intermyofibrillar mitochondria have lessening figure,and more trivial and smashed. Nevertheless the subsarcolemmal mitochondria are huge and cluster distributed. Often found myelin figure, pyknosis and swelling mitochondria, which were characters of metabolism dysfunction. According to statistics of Image-Pro Plus 6 software, the mitochondrial area and diameter in HF group are reduced obviously. The volume percentage of mitochondria to myofibrils is also reduced significantly (P<0.05). While each index in HF+RSG group has no statistically significant difference compared with normal control group.Summaries: 1.After the mice long-term high-fat feeding, the expression activity of PGC-1αand Mfn2 gene are reduced, and mitochondrial morphology are abnormal, and these changes participate in the formation of insulin resistance. 2. The mechanism of rosiglitazone interference on insulin resistance may involve in its regulation to the expression of PGC-1αand Mfn2 gene, and maintaining normal mitochondrial morphology and function.Part two: The influence of Mfn2 on oxidative stress induced apoptosisObjective: The aim of this part is to investigate the influence of Mfn2 to mitochondrial redox function. By using the wild type (pEGFP-Mfn2 plasmid) and dominant activated mutation (pEGFP-Mfn2G12V plasmid) plasmid to transfect HEK293 cells, and then treated the cells by semi lethal dose H2O2, the mechanism of Mfn2 and its mutation on oxidative stress induced apoptosis was observed.Methods: 1.The influence of H2O2 in different concentrations on apoptosis. HEK293 cells were cultivated by DMEM medium with 10% new born calf serum, and inoculated in 96 holes plates. After 24 hours growth, H2O2 in different concentrations were added to the cells for 2 hours, then change H2O2 with fresh medium. Continuing incubation for another 24 hours, 10μl CCK-8 solution was added to each hole for cell viability detection。After 4 hours incubation, the absorbance in 450nm was detected, and the optimal H2O2 concentration N was determined. 2. The influence of H2O2 in different concentrations on Mfn2 expression. Basic procedures as former, cells were inoculated in 6 holes plates. The H2O2 in different concentrations (0,1/2N,N,2Nμmol/L) was added to the cells, and gross proteins were extracted for Western blot analysis. 3. Cell transfection rate observation. The plasmid transform, extraction and identification by restriction enzyme digestion were carried out as routine methods. Cells were inoculated in 6 holes plates. Cell transfection was undertaken by SofastTM Transfection reagents at different plasmid concentrations. After 24 hours incubation, the green fluorescent proteins carried by the plasmid were observed under fluorescence microscope to calculate the transfection rate. 4. CCK-8 assay. Cells were divided into four groups: untreated group (transfected with empty vector pEGFP-C1 plasmid, no H2O2 treatment), empty plasmid group (transfected with empty vector pEGFP-C1 plasmid, added H2O2 treatment), Mfn2 group (transfected with pEGFP-Mfn2 plasmid, added H2O2 treatment) and Mfn2G12V group (transfected with Mfn2G12V pEGFP-C1 plasmid, added H2O2 treatment). Cell viability was detected by CCK-8 assay. 5. Hoechst staining. The procedures and groups as former, the ratio of cell apoptosis was estimated with Hoechst 33258 staining, observed and photographed under fluorescence microscope. 6. Flow cytometry detection by AnnexinⅤ-FITC/PI staining. The procedures and groups as former, the ratio of cell apoptosis was estimated by flow cytometry after AnnexinⅤ-FITC/PI staining. 7. Mitochondrial membrane potential detection. The procedures and groups as former, the ratio of red to green fluorescence were detected by flow cytometry after JC-1 staining. 8. Caspase-3 activity detection. The procedures and groups as former, AC-DEVD-pNA the substrate of caspase-3 was added to each group, to the terminal concentrations of 50μmol/L. The absorbance in 405nm was detected by enzyme calibration system, and to indicate the relative activity of caspase-3. 9. Western blot analysis. The procedures and groups as former, total protein was extracted for Western blot analysis. The protein expression of Mfn2, Cyt c, Bcl-2, and Bax was detected.Results:1. The apoptosis rate of HEK293 cells has positive linear correlation with the concentration of H2O2 when in the range of 40100μmol/L. The 50% apoptosis concentration is 80μmol/L. 2. The expression of Mfn2 has dose dependent effects with the concentration of H2O2. The expression of Mfn2 increased along with the concentration increasing of H2O2 on the four dose ( 0,40,80,160μmol/L). 3. The transfection rates of HEK293 cells were high by using SofastTM Transfection reagents. They were above 85%, and had no significant difference among the three plasmid concentration (1,1.5,2μg). 4. Detected by CCK-8 assay, Hoechst staining and flow cytometry after AnnexinⅤ-FITC/PI staining, and the ratio of cell apoptosis were lowest in untreated group, and highest in empty plasmid group. The ratio of cell apoptosis in Mfn2 group and Mfn2 G12V group were greatly reduced, and Mfn2 G12V group was lower. 5. The difference of cell apoptosis ratio among four groups correlated with the change of mitochondrial membrane potential. 6. Caspase-3 activity was lowest in untreated group, and highest in empty plasmid group. Compared with empty plasmid group, Caspase-3 activity in Mfn2 group and Mfn2 G12V group were greatly reduced. 7. The increased expression band of Mfn2 could be detected in Mfn2 group and Mfn2 G12V group, so it demonstrates transfection by wild type and mutation Mfn2 plasmid were successful. The expressions of Bcl-2 were lowest in empty plasmid group, and were higher in other groups. There were no significant differences among other groups. The expressions of Bax were highest in empty plasmid group, and were lower in other groups. The expressions of Bax in Mfn2 group were slightly higher than Mfn2 G12V group. The expressions of Cyt c were highest in empty plasmid group, and were lower in other groups. There were no significant differences among other groups.Summaries: Under the chosen H2O2 concentration, Mfn2 can protect HEK293 cells avoid of apoptosis. The mechanism involves in the decrease of mitochondrial membrane potential and Caspase-3 activity. It is also relevant with Bcl-2 expression increasing, Bax and Cyt c expression decreasing.Part three: Correlation between the PGC-1αand ERRαcis-acting element polymorphism in Mfn2 promotor and type 2 diabetes mellitusObjective: In order to investigate the correlation between Mfn2 and diabetes, the polymorphisms in the Mfn2 promotor near the PGC-1αand ERRαcis-acting element was screened, and the relationship of polymorphism sites and type 2 diabetes mellitus was studied.Methods: 100 in patients which were diagnosed type 2 diabetes mellitus were selected, and 112 healthy normal individuals with age and sex matched were selected too. 4mL EDTA-K2 anticoagulant blood sample was collected. Some of them for routine biochemical indexes analysis. The others used for genome DNA extraction. Primers near the PGC-1αand ERRαcis-acting element in the Mfn2 promotor were designed and used for PCR amplification. The different individuals were screened by single strand conformation polymorphism (sscp) analysis. The PCR products were sequenced. The sequencing results were aligned by DNAMAN software with the promotor sequences of Mfn2 (NM014874) published in GenBank. The polymorphism sites were found out. Gene counting methods were used to calculate the gene frequency and allele frequency. Hardy-Weinberg equilibrium tests were used to affirm the population representativeness.χ2 tests were used to analysis if there were some differences between healthy individuals and type 2 diabetes mellitus about the distribution of the polymorphism sites.Results:1. Three polymorphism sites were found in Mfn2 promotor, downstream of PGC-1αand ERRαcis-acting element, up stream of the transcription initiation site. They are -166A/G, -45A/G and +110T/C polymorphism sites respectively. 2. There are a certain degree gene linkages among this three polymorphism sites. The basic law is AAT/GGC. Among them the former two polymorphism sites are totally linked (100%). While the third site's linkage rate with the former two is 87.7%. 3. According toχ2 test, the gene frequency and allele frequency of these three polymorphism sites have no significant differences between type 2 diabetes mellitus and healthy individuals.Summaries: Three polymorphism sites are first found in Mfn2 promotor. The gene frequency and allele frequency of the three polymorphism sites have no significant differences between type 2 diabetes mellitus and healthy individuals.Conclusions:1 After the mice long-term high-fat feeding, the expression activity of PGC-1αand Mfn2 gene are reduced, and mitochondrial morphology are abnormal, and these changes participate in the formation of insulin resistance. The mechanism of rosiglitazone interference on insulin resistance may involve in its regulation to the expression of PGC-1αand Mfn2 gene, and maintaining normal mitochondrial morphology and function.2 Under the chosen H2O2 concentration, Mfn2 can protect HEK293 cells avoid of apoptosis. The mechanism involves in the decrease of mitochondrial membrane potential and Caspase-3 activity. It is also relevant with Bcl-2 expression increasing, Bax and Cyt c expression decreasing.3 Three polymorphism sites are first found in Mfn2 promotor. The gene frequency and allele frequency of the three polymorphism sites have no significant differences between type 2 diabetes mellitus and healthy individuals. |
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