| ObjectivesThe aim of the study was to investigate the effect of free cholesterol overload on adipokines secretion from adipocytes and explore the potential mechanisms and to investigate the effect of endoplasmic reticulum stress on adipokines secretion in obese mouse.Methods1.The Effect of Endoplasmic Reticulum Stress on3T3-LlAdipocytes Secretion Function3T3-L1adipocytes were cultured and differentiated into mature adipocytes in vitro. We observed the morphology changes by microscope and the qualitative analysis of Oil Red-O stain was carrried out. The adipocytes were intervened by ox-LDL with various concentrations (0-100ug/ml) and AC AT inhibitor Sandoz58035(10ug/ml) for48hours; ox-LDL (50ug/ml) and Sandoz58035(10ug/ml) for0-48hour or ox-LDL (50ug/ml), Sandoz58035(10ug/ml) and4-PBA with various concentrations (0-20mM) for48hours. Moreover, adipocytes was incubated by ox-LDL (50ug/ml) and Sandoz58035(10ug/ml) for46h,and then NF-ΚB inhibitor PS1145(10μmol/L) was added. The protein expressions of GRP78and CHOP in adipocytes were detected by Western Blot. The mRNA of adipokines was detected by Real-time PCR, and the levels of adipokines in supenatant were examined by ELISA.2. The Effect of Endoplasmic Reticulum Stress on Adipokines Secretion Function in vivoC57BL/6wild type mouse (n=40) were randomly divided into4groups:high-fat (HF) diet+4-PBA group (n=10) fed with a HF diet for8weeks and the same HF diet supplemented with4-PBA20mg.kg-1.d-1, TUDCA for4weeks; HF diet+TUDCA group(n=10) fed with a HF diet for8weeks and the same HF diet supplemented with TUDCA0.5g.k-1.d-1for6weeks; HF diet group fed with a HF diet for14weeks; standard diet group fed with a standard diet for the14weeks. After the indicated time, the morphological changes of adipocytes in epididymal adipose tissue from mouse were observed by H.E. staining and microscopy. Real-time-PCR was used to analyze adiponectin, resistin, TNF-a and MCP-1mRNA expression in adipose tissue; ELISA method was used to detect adiponectin, resistin, TNF-a and MCP-1protein level. Plasma levels of TG, TC, LDL-C, HDL-C and BS were analyzed. Western Blot was used to determine the expression of Grp78and CHOP and NF-kB in adipose tissue.Results1.The Effect of Endoplasmic Reticulum Stress on3T3-L1Adipocytes Secretion Function1. After the adipocytes incubated with ox-LDL and Sandoz58035, the cholesterol concentration in adipocytes was upregulate with the increase of the ox-LDL concentration in a dose-dependent manner. The cholesterol concentrations in adipocytes were higher than the control group (ox-LDL Oug/ml) and the differences were significance (P<0.05).2. After the adipocytes treated with Sandoz58035and ox-LDL at different concentrations or ox-LDL (50ug/ml) and Sandoz58035(10ug/ml) for a variety of time, the protein expressions of GRP78and CHOP protein in adipocytes were increased at a dose-dependent and time-dependent manner. The differences were significance (P<0.05).3. After the adipocytes incubated with Sandoz58035and ox-LDL at different concentrations or ox-LDL (50ug/ml) and Sandoz58035(10ug/ml) for a variety of time, and the adiponectin mRNA and protein levels in the supernatant fluid were decreased; Resisting TNF-α and MCP-1mRNA and protein secretion were upregulated. The differences were statistical significance (P<0.05).4. After the intervention with ox-LDL, Sandoz58035and different concentrations of TUDCA for48hours, the expressions of GRP78and CHOP protein expression and resistin,TNF-α and MCP-1levels in the supernatant fluid in adipocytes were decresed but the adiponectin levels increased in a dose-dependent manner (P<0.05).5. After the intervention with ox-LDL, Sandoz58035and then added with NF-κB inhibitor PS1145, resistin,TNF-α and MCP-1levels in the supernatant fluid from adipocytes were decresed but the adiponectin levels increased (P<0.05).2. The Effect of Endoplasmic Reticulum Stress on Adipokines Secretion Function in vivo1. After fed with a HF diet for14weeks, the body weight significantly increased and average (percentage mean) size of the adipocytes in the HF-diet C56BL/6mice was increased compared with the control group. Plasma TG, TC and glucose were found to elevated in HF diet group. The expression of Grp78and CHOP were found to be markedly elevated in adipose tissue from HF-diet mice compared with standard diet mice.2. C57BL/6mice fed a HF diet supplemented with4-PBA or TUDCA showed a significant reduction in weight gain and had reduced adipocyte size, as compared with the HF diet alone group. Furthermore,4-PBA or TUDCA supplementation lowered plasma TG, TC, glucose without altering food intake.3.4-PBA or TUDCA supplementation decreased GRP78and Chop expression in the adipose tissue and increased adiponectin levels lowered plasma resistin concentration. But there was no significant difference in adiponectin, resistin, TNF-α and MCP-1mRNA expression and plasma TNF-α and MCP-1secretion.4. C57BL/6mice fed a HF diet supplemented with4-PBA or TUDCA showed a significant reduction in NF-kB expression in adipose tissue compared with the HF diet alone group.Conclusions1. The exogenous ox-LDL can be uptaked by adipocytes, leading to the cholesterol overload in adipocytes.2. The cholesterol overload can promote the adipokines secretion dysfunction of adipocytes in a time-dependent and dose-dependent manner.3. Eendoplasmic reticulum stress may be, at least partly, responsible for the secretion dysfunction of adipocytes caused by cholesterol overload.4. IKK/NF-κB may paticipate in the signal transduction in the ER stress of adipocyte caused by intracellular free cholesterol overload.5. High-fat diet is an important factor for weight gain, dyslipidemia and disturbance of glucose metabolism. HF-diet contributes to strengthened ER stress in adipose tissue and lead to adipokines secretion dysfunction. IKK/NF-κB signal pathways may be involved in this process.
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