A Study Of High-fat And Palmitate-induced Insulin Resistance In Adipose Tissue And3T3-L1Adipocytes And Mechanisms Of Resveratrol Intervention

Objective:1. To investigate the mechanisms of high-calorie and high-cholesterol diet (HCD) induced insulin resistance in C57BL/6mice, and to observe whether the protective effects of resveratrol (RES) with adipose tissues were related with reduced endoplasmic reticulum stress (ERS), inflammatory activation and expression of adipocytokines.2. To observe whether palmitate (PA) could induce ERS and insulin resistance in mature3T3-L1adipocytes, and to further explore whether the protective effects of resveratrol (RES) was connected with decreased ERS and relevant signaling pathways.Methods:1. In-vivo experiments:The8-week-old C57BL/6mice were divided into standard chow diet (SCD) group,high-calorie and high-cholesterol diet(HCD) group and RES treatment group(HCD+RES) randomly.The C57BL/6mice were fed with HCD for3months as hyperlipidemic models, the HCD+RES mice were treated with resveratrol by gavage in the meantime. The improvement on insulin resistance were performed by Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT).The levels of serum lipid indices including TG, TC, HDL-C and LDL-C were detected. Inflammation indices such as TNF-α, IL-6and MCP-1in serum were determined by ELISA assay. The body mass, subcutaneous adipose tissues (SAT) and visceral adipose tissues (VAT) were weighed respectively.The pathological changes of two adipose tissues were analyzed with HE staining.The protein distribution and expression levels of leptin, TNF-a and GRP78were measured with immunohistochemical assay. The mRNA expressions of leptin, resistin, adiponectin,TNF-α, IL-1β,GRP78, CHOP, PERK, IRE1, ATF6, PPARa, SIRT1and SREBPlc were assessed by Real Time RT-PCR.The protein expressions of GRP78, CHOP, JNK, pJNK and SIRT1were evaluated by Western blot.2. In-vitro experiments:The3T3-L1pre-adipocytes were induced maturation by the methods of classical cocktails, and3T3-L1mature adipocytes were treated with different concentrations of palmitic acid (PA) to determine the optimal concentration.Tunicamycin (TM) was used as a positive control of ERS.The cells were divided into normal control (NC) group, palmitate group(PA) and PA+RES group. The gene expression of leptin, resistin, TNF-a, GRP78, CHOP, PERK, IRE1, ATF6, SREBPlc, IL-1β, adiponection and SRIT1were assessed by Real Time RT-PCR,and Western blot was used to detect the protein expression of GRP78, CHOP, JNK, pJNK and SIRT1.Results:1. In-vivo experiments:①The body mass and weight of SAT and VAT were all obviously increased in HCD group as compared with SCD group after three months of HCD feeding.②The serum levels of TG, TC and LDL-C were all elevated obviously, while level of HDL-C was decreased in HCD group. The results of GTT and ITT indicated that the HCD-fed mice had showed obvious insulin resistance.③Serum inflammatory markers TNF-α、IL-6and MCP-1were significantly increased.④The cell volumes of adipocytes were increased significantly in SAT and VAT respectively.⑤The protein expressions of leptin, TNF-α and GRP78were elevated obviously and mainly located at cytoplasm in2adipose tissues of the HCD-fed mice.⑥The mRNA expressions of genes leptin, resistin, TNF-α, IL-1β, GRP78, CHOP, PERK, IRE1, ATF6, SREBPlc in adipose tissues of the HCD-fed mice were significantly elevated, while the mRNA expression of SIRT1were decreased obviously.⑦The protein expressions of GRP78, CHOP, JNK and pJNK were significantly increased, while the protein expression of SIRT1were decreased obviously.⑧Resveratrol could improve insulin resistance,decrease body weight and serum lipid indices.The volumes of adipocytes and mRNA expressions of leptin, resistin, TNF-α, IL-1β,GRP78, CHOP, PERK, IRE1, ATF6, SREBP1c were decreased but SIRT1, PPARα, adiponectin were elevated significantly in VAT and SAT in HCD+RES group. The protein expressions of GRP78, CHOP, JNK pJNK were decreased significantly but the protein expression of SIRT1was increased obviously in HCD+RES group.2. In vitro experiments:①Compared with the NC group, the mRNA expressions of leptin, TNF-a, GRP78, CHOP, PERK, IRE1, ATF6, IL-1β, SREBP1c were increased obviously but the mRNA expression of PPARa was decreased in PA group.②The protein expressions of GRP78, CHOP, JNK and pJNK were increased but SIRT1was decreased significantly in PA group.③Compared with the PA group, the mRNA expressions of leptin, resistin, TNF-α, IL-1β, GRP78, CHOP, PERK, IRE1, ATF6were all decreased but adiponection, PPARa, SIRT1was increased obviously in PA+RES group.②The protein expressions of GRP78, CHOP, JNK, pJNK were decreased but SIRT1was increased in PA+RES group.Conclusions:1. Resveratrol treatment could obviously decrease the body weight and adipocyte size induced by high-calorie and high-cholesterol diet and improve insulin resistance, the mechanism were possibly related with anti-inflmmation, decreased adipocytokines and attenuated ERS.2. PA could induce ER stress and insulin resistance in3T3-L1mature adipocytes. Resveratrol improved insulin resistance probably by attenuating endoplasmic reticulum stress and reduced expression of inflammatory factors and adipocytokines.