Proteomic Study On White Fat And Brown Adipose Tissue In C57BL / 6J Mice

Objective:We aimed to identify the protein expression profile of white adipose tissue (WAT) and brown adipose tissue (BAT) in normal C57BL/6J mice. To understand the functions of WAT and BAT in obesity, we performed comparative proteome analysis of WAT and BAT between normal and diet-induced obese mice. Furthermore, we examined the effects of metformin and liraglutide on the regulation of protein of WAT and BAT in obesity and explored the underlying mechanisms.Methods:Six-week-old female C57BL/6J mice were fed a standard chow (SC; 10% lipids) diet (NOR) or a high-fat (HF; 45% lipids) diet for 22 weeks. High-fat diet-induced obese mice were randomly divided into three groups, i.e. model group (HFD, ddH2O 200 mg/kg/d, i.g.), metformin group (MET,200 mg/kg/d, i.g.) and liraglutide group (GLP-1,200μg/kg/d, i.h.). Food intake for 24 hours and body weight (once per week) were dynamically monitored. After treatment for 8 weeks, insulin tolerance test (ITT), oral glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp were performed to evaluate the improvement of glucose tolerance and insulin resistance. The plasma levels of total cholesterol and triglyceride (TG) were tested by enzymic method. Moreover, the concentration of leptin, resistin and adiponectin in serum were measured by euzymelinked immunosorbent assay (ELISA) kits. Gonadal WAT and interscapular BAT of mice in NOR group (n=6), HFD group (n=6), MET group (n=6) and GLP-1 (n=6) group were collected. Total protein of WAT and BAT was extracted and homogeneous samples were selected. Then we performed label-free quantitative technology based on liquid chromatography tandem mass spectrometry (Label-free LC-MS/MS) to identify and quantify the protein expression profile of WAT and BAT in normal C57BL/6J mice. Moreover, we identified and quantified the protein expression of WAT and BAT in mice of NOR, HFD, MET and GLP-1 group, using iTRAQ-coupled 2D LC-MS/MS. MS). The results were analyzed by MASCOT, Scaffold and IPA (Ingenuity Pathway Analysis Software).Results:Through three repeated experiments,5847 proteins in WAT and 4706 proteins were identified in normal C57BL/6J mice. Among them 5408 and 4260 proteins were quantified in WAT and BAT, respectively. As compared WAT to BAT, we obtained 1440 differentially expressed proteins, which were up-regulated or down-regulated greater than or equal to four times.After 22 weeks, as compared with the NOR group, the body weight of mice fed HFD was increased significantly (25.1±0.9 g vs.41.6±0.8 g, p=0.000). Treated with metformin or liraglutide for 8 weeks, as compared with the HFD group, the body weight of obesity mice was decreased significantly(41.1±1.6 g vs. 36.6±0.9 g vs.32.0±0.9 g, p<0.05).Gonadal WAT weight of HFD group per body weight was significantly higher than that of NOR group (4.74±0.57% vs.2.16±0.38%, p=0.004). Liraglutide reduced gonadal WAT weight of mice significantly (2.93±0.42% vs.4.74±0.57% p=0.045), while metformin had no significant effect on WAT. The interscapular BAT weight of HFD group per body weight was significantly lower than that of the NOR group. However, liraglutide and metformin had no significant effect on BAT.As measured by hyperinsulinemic-euglycemic clamp, Glucose infusion rate (GIR) in HFD group was lower than the NOR group (14.748±2.089 mg·kg-1·min-1 vs.52.332±8.056 mg·kg-1·min-1, p=0.001). GIR of MET and GLP-1 group was increased significantly in contrast to HFD group (38.285±5.729 mg·kg-1-min-1 vs. 29.111±8.445 mg·kg-1·min-1 vs.14.748±2.089 mg·kg-1·min-1, P<0.05), and there was no considerable difference between liraglutide group and metformin group (P=0.22).As compared with the NOR group, the plasma levels of total cholesterol (TC) and triglyceride (TG) was strongly higher in HFD group (p<0.05), while both metformin and liraglutide decreased the plasma levels of TG, but only liraglutide decreased the plasma levels of TC (p<0.05).In comparison with NOR group, the serum level of leptin in the HFD group increased significantly (p=0.000), while metformin and liraglutide decreased it significantly (P<0.05). Adiponectin level in serum was decreased in HFD group, as compared to the NOR group, and metformin and liraglutide increased it significantly (P<0.05). The change of resistin was not obviously.Using iTRAQ-coupled LC-MS/MS, we identified 4388 and 3486 proteins in WAT and BAT, respectively. Among them, we quantified 4061 and 3212 proteins in WAT and BAT, respectively. With the NOR group was considered as a control, in WAT, we obtained 529,363,240 differentially expressed proteins in HFD, MET and GLP-1 group, since the fold change≥1.5 (fold change is the ratio of intensity of protein expression in HFD (MET or GLP-1) to NOR adipose tissue) was considered as a threshold to minimize biological and technical errors. Likewise, in BAT, we obtained 727,240,217 differentially expressed proteins in HFD, MET and GLP-1 group.Functional analysis found that those differentially expressed proteins, we found that proteins were mainly assigned to the biological function of lipid metabolism, molecular transport, small molecule biochemistry, cardiovascular disease and carbohydrate metabolism. Metformin and liraglutide improved carbohydrate and lipid metabolism in mice.Differentially expressed proteins in WAT were mainly assigned to the pathway of EIF2 signaling, LXR/RXR activation and acute phase response signaling. Differentially expressed proteins in WAT were mainly assigned to the pathway of mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling.Conclusions:The protein expression profile of WAT and BAT was highly related to cancer, and the most important related signal pathway was EIF2 signaling.The differential proteins between WAT and BAT were mainly assigned to the biological function of inflammation response, and those proteins were expressed higher in WAT. Those differential proteins were mainly assigned to the pathway of mitochondrial dysfunction, and they were expressed higher in BAT. Moreover, the differential proteins were mostly associated with energy production and lipid metabolism.HFD might induce protein synthesis and cell growth of WAT through EIF2 signaling. HFD also inhibited glucose metabolism and lipid synthesis, while activated compensatory lipolysis in WAT and BAT. Moreover, HFD might lead to the down-regulation of free radical scavenger in brown adipocytes and vulnerable to oxidative stress, and then induce the cell apoptosis. However, HFD had no similar effect on WAT. Interestingly, we found that HFD might promote the browning of white adipocytes.Metformin and liraglutide improved the body weight and insulin sensitivity of mice. Meanwhile, they had positive effect on EIF2 signaling, mitochondrial dysfunction and lipid metabolism in WAT and BAT. Metformin is more of benefit to lipid metabolism and inflammation in WAT, and protein synthesis in BAT. Besides, liraglutide is more of benefit to energy production in BAT, and cell growth and protein synthesis in WAT.