| It is well established that obese individuals display a characteristic imbalance of the adipokine profile, manifested as low Adiponectin in serum and increased inflammatory factors, such as TNF-a and IL-6. The altered adipokine profile leads to insulin resistance and increases the risk of metabolic syndrome (MS), type 2 diabetes mellitus and cardiovascular diseases. At present, adipokine is often used as the starting point to study the relationship between obesity and MS.Peroxisome proliferator activated receptor gamma (PPARy) is highly expressed in adipose tissue. TZDs, a class of PPARy agonists, were confirmed to improve insulin sensitivity mainly by regulating the secretion of adipokines. Liver X receptors (LXRs) are also greatly expressed in adipose tissue. A somewhat similar function of LXRs in the glucose and lipid metabolism to that of PPARy was reported. However, it has been shown that the activation of LXRs suppresses the PPARa signaling in hepatocytes. Although PPARy is known to regulate the expression and secretion of many adipokines, there have been no systematic studies about the effect of LXRs activation on the expression of adipokines which were transcriptional regulated by PPARy, which is one of the most important aspects to evaluate the role of LXRs in insulin sensitivity. Part one:The Effect of LXRs Activation on the Expression of Adipokines Transcriptional Regulated by PPARyObjective:To observe the effect of LXRs activation or LXRa silencing on the expression of adipokines transcriptional regulated by PPARy.Methods:Differentiation of 3T3-L1 preadipocyte was induced by a standard "Cocktail" Firstly, we observed the expression patterns of LXRs and PPARy during the differentiation program by Real-time RT-PCR and Western blot. Then, mature adipocytes were treated with T0901317 (LXRs agonist,0,0.1,1.0 and 10.0?M respectively), with or without the presence of Piogliazone (PPARy agonist,3?M); The expression of Adiponectin and Visfatin, as well as inflammatory cytokines. TNF-a and IL-6 stimulated by LPS 2?g/ml, were determined by Real-time RT-PCR, Western blot and ELISA. Change of the expression of adipokines mentioned above was also observed by LXRa silencing with the retroviral shRNA-LXRa transfection. Finally, the effect of LXRs activation on the expression of PPARy was also investigated by Real-time RT-PCR and Western blot.Results:(1) In the process of adipocyte differentiation, the mRNA and protein levels of PPARy and LXRa increased gradually, significantly higher by the 4th day of induction (DAY 4) than those of before induction (P<0.05 VS DAY 0), and reached the peak at DAY 8-10. but the expression of LXR(3 did not change.(2) T0901317 dose-dependently suppressed the mRNA and protein levels of Adiponectin and Visfatin, and also dose-dependently reversed the increase in Adiponectin and Visfatin expressions highly induced by Piogliazone (P<0.05 VS DMSO/Pioglitazone). As for the expression of inflammatory cytokines. T0901317 dose-dependently suppressed the expression of TNF-a and IL-6 stimulated by LPS, and dose-dependently strengthened the inhibitory effect of Pioglitazone on the inflammatory cytokines (P<0.05 VS LPS/LPS+Pioglitazone).(3) Expressions of Adiponectin and Visfatin up-regulated by Pioglitazone were further increased (P<0.05 VS group of shRNA-Control+Pioglitazone). while TNF-?and IL-6 expressions down-regulated by Pioglitazone were also increased by LXRa silencing (P<0.05 VS group of shRNA-Control+ Pioglitazone+LPS)(4) LXRs activation increased the mRNA and protein levels of PPARy (P<0.05 VS DMSO), significantly higher PPARy mRNA and protein levels were detected at the concentrations of 1.0 and 10.0?M of T0901317.Conclusion:In mature adipocytes, down-regulation of adipokines transcriptional activated by PPARy, such as Adiponectin and Visfatin, by LXRs activation, might have a negative effect on the insulin sensitivity; while suppressing the secretion of inflammatory cytokines synergistically with PPARy might improve insulin sensitivity. For LXRs agonist, how to maximize its advantage of insulin sensitivity is a pressing issue. Further studies are needed to identify the possible mechanisms how the activation of LXRs regulate the expression of adipokines transcriptional regulated by PPARy. Part two:Possible Mechanisms Regulating the Expression of Adipokines Transcriptional Regulated by PPARy Due to LXRs ActivationObjective:To investigate the possible mechanisms regulating the expression of adipokines transcriptional regulated by PPARy due to LXRs activation.Methods:Promoter reporter genes. PPRE-Luc and?B-Luc, respectively representing the transcriptional activation of PPARy and NF-?B, were introduced. By multiple plasmid co-transfections with PPRE-Luc or?B-Luc, plasmids of LXRa and PPARy, we observed the effect of T0901317 on the activity of PPRE-Luc and?B-Luc with or without the presence of Pioglitazone in HEK293. EMS A was used to assess the PPARy or NF-?B binding activity to PPRE or special DNA sequence for NF-?B in mature adipocytes. Furthermore, plasmid of RXRa was co-transfected to HEK293 or transfected to mature adipocyte, to investigate whether the change of transcriptional activation of PPARy and following adipokines expressions due to LXRs activation could be reversed by RXRa supplementation. The effect of LXRs and PPARy activation on the NF-?B pathway in adipocytes was also investigated by Western blot.Results:(1) In HEK293, LXRs agonist, T0901317 dose-dependently suppressed the PPRE-Luc activity induced by PPARy and its agonist, Pioglitazone (P<0.05 VS DMSO/ Pioglitazone), but dose-dependently strengthened the inhibition of?B-Luc activity induced by Pioglitazone (P<0.05 VS LPS+Pioglitazone).(2) In adipocytes, T0901317 antagonized the increase in PPAR?binding activity to PPRE which was highly induced by Pioglitazone; While, T0901317 strengthened the inhibition of NF-?B binding activity to special DNA sequence induced by Pioglitazone.(3) The inhibition of PPRE-Luc activity due to T0901317 treatment could be reversed by supplementation with RXRa in HEK293, and the expression of Adiponectin and Visfatin was almost restored by RXRa supplementation in adipocytes (P<0.05 VS pcDNA3.1).(4) In adipocytes,T0901317 and Pioglitazone could independently or synergistically suppress the phosphorylation of I?B?and the nuclear translocation of phosphorylated NF-KBp65 induced by LPS (P<0.05 VS LPS). The activity of NF-KB pathway was increased by LXRa silencing despite the presence of Pioglitazone and T0901317 ((P<0.05 VS shRNA-Control).Conclusion:In adipocytes, LXRs activation could antagonize the transcriptional activation of PPARy through competition for RXR?, which then suppressed the expression of adipokines transcriptional activated by PPARy, and this effect could be almost reversed by supplementation of RXRa. Activation of PPARy and LXRs might synergistically suppress the activity of NF-?B pathway and then the expression of inflammatory adipokines transcriptional inhibited by PPARy. Taken together with the data abovementioned. adding RXRa or activating RXRa might maximize the effect of LXRs agonists on insulin sensitivity. |
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