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The Expression of Mfn2and Mitochondrial Function in Liver in Insulin Resistance Rats Induced by High Fat Feeding

Author: ZhuChunJing
Tutor: SongGuangYao
School: Hebei Medical University
Course: Internal Medicine
Keywords: Mfn2 mitochondrial structure mitochondrial function insulin resistance high-fat-diet
CLC: R587.1
Type: Master's thesis
Year: 2012
Downloads: 127
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Abstract


Objective: To observe the change in expression of Mitofusin2(Mfn2),mitochondrial structure and function in liver in insulin resistant rats inducedby high fat feeding. To identify the relationshop of between Mfn2withmitochondrial function and to investigate the molecular mechanism of insulinresistance.Methods: After adaptable feeding for one week, four-week aged SD ratswere randomly divided into two groups according to randomization method:normal diet group (N, n=20) and high-fat diet group (F, n=20).1Establishment of insulin resistant SD rats model by high-fat feeding.Normal diet group rats were fed a standard diet (calory composition: fat10.3%,carbohydrate65.5%, protein24.2%, total calories348kcal/100g);high-fat diet group rats were fed high-fat diet (calory composition:carbohydrate59.8%, fat20.1%,protein20.1%,total calories501kcal/100g).2After4-week feeding,10rats were randomly selected from each group.Hyperinsulinemic-euglycemic clamp study were applied to evaluate thedegree of insulin resistance.2.1Routine index detection: fasting glucose were detected from tailblood by glucose oxidase method; fasting insulin was detected byeuzyme-linked immunosorbent assay; total triglycerides and total cholesterolwere detected by biochemistry assay.2.2Mitochondrial struction and function detection: Liver was stained byHE method to observe adipose degeneration in liver tissue under lightmicroscopy;the change in mitochondrial structure was observed in liver underelectron microscope. The activity of succinic dehydrogenase and cytochrome c oxidase which in liver mitochondrial suspension were detected bycolorimetric.2.3The mRNA expression and protein content of Mitofusin2in liverwere analyzed by Real-Time Reverse Transcription polymerase chain reactionand western blot.3After8-week feeding, Hyperinsulinemic--euglycemic clamp techniquewere applied to evaluate the degree of insulin resistance. Remanent rats werekilled and the same index were detected by the same methods as those in weekfour.Results:1At the end of4-week feeding, glucose infusion rate was lower inhigh-fat diet group compared with normal diet group (P<0.05); there were nosignificant difference in body weight, fasting blood glucose, total triglyceridesand total cholesterol between two groups(P>0.05).Fasting insulin level washigher in high-fat diet group compared with that in normal diet group(P<0.05).2At the end of8-week feeding, glucose infusion rate was lower inhigh-fat diet group compared with that in normal diet group (P<0.01). Therewas no significant difference in body weight in both groups(P>0.05). Fastingblood glucose, total triglycerides and total cholesterol were higher in high-fatdiet group compared with those normal diet group (P<0.01). Fasting insulinlevel was higher in high-fat diet group compared with that in normal dietgroup (P<0.01).3Observation of morphology of liver cells by HE staining: at the4thweek, the morphology of liver cells is normal and the volume of liver cells isnormal. Steatosis, swelling cells and lipid droplet vacuole in liver wereobserved in high-fat diet group. At the8th week, morphology and volume ofliver cells are normal, there were no inflammation cellular infiltration innormal diet group were observed. Diffuse steatosis especially in areasurrounding central veins were present in liver in high-fat diet group. Cellswere significantly swelling,massive lipid droplet vacuole and inflammatorycell infiltration in lobula of liver were observed in high-fat diet group. 4Observation of mitochondria ultrastructure under transmission electronmicroscope: at the4th week, in liver cells in normal diet group, mitochondrialinner-outer menbrance were clearly seen. The morphology of mitochondrialcristae was normal; the cristae arrange was regular, and there was no lipiddroplet in intracytoplasm. In high-fat diet group, there was a small quantity oflipid droplet in hepatocytes, mitochondrial inner-outer menbrance is normalwhile mitochondrial cristae partly disappeared. At the8th week, mitochondrialinner-outer menbrance were clear in livers in normal diet group. Themorphology of mitochondrial cristae was normal and cristae arrangement wasregular in hepatocytes. In high-fat diet group, there was major volume lipiddroplet in hyalomitome in hepatocytes; meromixis were observed inmitochondrial inner-outer menbrance; mitochondrial crista totally disappearedand vacuolization were observed.5Determine results of two key enzyme activity which reflectingmitochondrial function: at the end of4weeks feeding, the activity of succinicdehydrogenase was lower in high-fat diet group compared with that in normaldiet group (P<0.01). The activity of cytochrome c oxidase was not statisticallydifferent in both groups. At the end of8feeding weeks, the activity of succinicdehydrogenase was lower in high-fat diet group compared with that in normaldiet group (P<0.01).The activity of cytochrome c oxidase was also lower inhigh-fat diet group compared with that in normal diet group (P<0.01).6Detection of the expression of Mfn2by Real-Time ReverseTranscription polymerase chain reaction: after4weeks, compared with normaldiet group, mRNA expression of Mfn2in high-fat diet group was suppressed(P<0.05). After8weeks, compared with normal diet group, mRNA expressionof Mfn2in high-fat diet group was also suppressed (P<0.01).7Detection of the protein content of Mfn2by western blot. After4weeks,compared with normal diet group, the protein content of Mfn2in high-fat dietgroup was decreased (P<0.05). After8weeks, compared with normal dietgroup, the protein content of Mfn2in high-fat diet group was decreased(P<0.01) Conclusion:1Companying with the worsening of insulin resistance induced by highfat feeding in SD rats, adipose degeneration significantly increased in livercells and mitochondrial structure were seriously impaired in hepatocytes.2Along with the worsening of insulin resistance induced by high fatfeeding, the activity of succinic dehydrogenase and cytochrome c oxidasewhich reflect hepatocyte mitochondrial function are gradually decreased.3The protein content and gene expression of hepatocyte mitofusin2were reduced in high-fat-diet-induced insulin resistant rats.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetes
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