Study Of High-fat Diets Induced Insulin Resistance And The Mechanism In C57/BL6Mice

Objective:Obesity is a potential risk factor for insulin resistance and associated with the development of Type2Diabetes. Macrophage infiltration and related local inflammation in obese adipose tissue is the main reason. Hypoxia in excessive expance adipose tissue plays a role. To detect macrophage infiltration in skeletal muscle and adipose tissue, hypoxia of adipose tissue and insulin signal in obese mice, we established the mice model of insulin resistance with high fat diet。Methods:In part1, C57/BL6mice were fed with chow diet (CHOW) as control or with high fat diet (HFD) from age4weeks to16weeks to establish insulin resistance.Body weight of mice at0th w,4th w,8th w,12th w,16th w were measured. Insulin resistance was detected by intraperitoneal glucose tolerance test (IPGTT) at the same time points. Mice were killed at0th w or16th w and quadriceps femoris muscle, gastrocnemius muscle and epididymal fat pads were taken for experiments.In part2, paraffin embedding and tissue section were made in gastrocnemius muscle and epididymal fat pads. The macrophage infiltration in skeletal muscle and adipose tissue was determined by immunohistochemistry (IHC) using mac-2antibody. Hypoxia of adipose tissue was detected by IHC, using HIF-1α antibody.In part3, we extract protein from quadriceps femoris muscle and epididymal fat pads. The phosphorylation of Akt was determined by Western Blot.In part4, abdominal adipose tissues of16non-obese females (BMI<28kg/m2) and18obese females (BMI>28kg/m2) were collected by surgery. The mRNA expression of HIF-1α was detected by real-time PCR. The expression of HIF-1α protein was determined by Western Blot. The concentrations of FBG and FINS in peripheral blood were measured and the HOMA index was calculated to determine whole body insulin resistance.Results:In the first part of this study, results show that compared with chow group, body weight was higher in high fat diet group from the8th week (p<0.005,p<0.001, p<0.001). IPGTT data shows that at the16th week, the glucose level of HFD group increase markedly at five different time points (Omin,30min,60min,90min,120min after injecting glucose) compared with chow group.In the second part of this study, results show that there is no difference between HFD group and chow group in mcrophage infiltration in adipose tissue and muscle at week0. After16weeks, the adipocytes in adipose tissue of HFD group were bigger than that in chow group, more macrophages can be detected and the level of HIF-1α was higher. Moreover, macrophages in skeletal muscle were detected.In the third part of this study, the increase of phosphorylation of Akt in adipose tissue and skeletal muscle after insulin treatment has no difference between two groups. While after16weeks, there is no increase of phosphorylation of Akt after insulin treatment in adipose tissue and skeletal muscle in HFD group.In the forth part of this study, mRNA and protein expressions of HIF-1α and HOMA index were higher in obese adipose tissue than that in non-obese subjects (p<0.004,p<0.028,p<0.001, respectively).Conclusion:1. High fat diet induces body weight gain and insulin resistance of C57/BL6J, suggesting mice model of insulin resistance is established.2. Macrophage infiltration in adipose tissue and skeletal muscle increased markly after high fat diet.3. High fat diet reduces insulin-stimulated phosphorylation of Akt in adipose tissue and skeletal muscle.4. Obese subjects have hypoxic adipose tissues and systemic insulin resistance.