| Modern life style has precipitated a steep rise in obesity throughout the world. Thus there is thus an urgent need for ideal weight-loss treatments. One appealing new target for treatment is the activation of brown adipose tissue (BAT), an organ inducing thermogenesis, increasing energy consumption and improving insulin resistance. Recently this organ was found to be functional in adult humans. After years of research, we have eventually recognized plenty of features of BAT, including definition, physiological characteristics, development, the impact on energy balancing and metabolism and related molecular mechanisms and signaling pathway. Unfortunately, nonspecific drugs cannot be found to stimulate human BAT effectively without marked effects.Liver X receptors (LXRs), including LXRα and LXRβ, are nuclear receptors that play central roles in regulating lipid metabolism and glucose homeostasis through transcriptional regulation of their target genes. Recently several studies showed that genetic knockout of LXRs was able to activate adipose browning in vitro and in vivo. 22-S-hydroxycholesterol (22-S-HC), behaves like an LXRs antagonist. It can reduce lipid formation and increase glucose metabolism. However, the relationship between 22-S-HC and adipose browning is unknown. Recognizing the role of LXRs depression in adipose browning would help to further understand the effect and mechanisms of LXRs and their antagonist 22-S-HC in glucose and lipid metabolism, and would helped to explore the prospect of them as a new target for prevention and treatment of diabetes.In this study, MEFs were differentiated into adipocytes with brown-like characteristics by using the browning differentiation protocol. On this basis, we observed the effects of 22-S-HC in activating adipose browning. The results showed that 22-S-HC promoted MEFs express the characteristics of brown fat cells including significant increasing in the expression of BAT-specific genes like UCP1 and PGCla, and enhanced fluorescence effect of mitochondria-UCP1 immunofluorescence. Consistent with the in vitro results, we observed enhancive expression of UCP1 in brown adipose tissue(BAT) and inguinal white adipose tissue (iWAT) in high fat diet (HFD) fed mice treated with 22-S-HC. We also observed less lipid droplets in BAT and smaller lipid droplets in iWAT and improved glucose metabolism. In the research of molecular mechanisms of activating adipose browning by LXRs inhibition, we found that both 2-S-HC and LXRs knockdown could enhance the activity of UCP1, PPARy, and PGCla promoters. Through the above research, we have revealed the potential therapeutic value of 22-S-HC in obesity and type 2 diabetes mellitus and have discovered a part of molecular mechanisms involved.Our research was divided into three parts:Part Ⅰ:The effects of LXRs antagonist 22-S-HC in adipose browning and metabolism;Part Ⅱ:The molecular mechanisms of activating adipose browning by LXRs inhibition.Objectives:The LXRs are important transcription factors belonging to the metabolic nuclear receptor superfamily. In recent years, several studies found that LXRs knockout in vitro and in vivo could promote adipose browning, increase the expression of the specific functional protein, uncoupling protein 1 (uncoupling protein-1, UCP1). In this part of the study, we aimed to demonstrate that the LXRs antagonist 22-S-HC has a promoting effect on adipose browning in vitro and in vivo.Methods:MEFs were differentiated into adipocytes with brown-like characteristics by using the browning differentiation protocol. To examine the role of 22-HC in adipose browning, MEFs were treated with 22-S-HC throughout the whole differentiation. We identified the safe concentration range of 22-S-HC using MTS. We observed the expression of brown adipocytes characteristics of MEFs treated with different concentrations of 22-S-HC and during the differentiation program by oil red, mitochondria-UCPl immunofluorescence, Western blot and Real-time RT-PCR. The ability of glucose uptake was tested through detection of 2NBDG uptake. HFD C57BL/6J mice were administrated with 22-S-HC (22-S-HC group) or vehicle (control group). We observed the weight change, assessed insulin sensitivity through IPGTT and ITT, and examined the effects of 22-S-HC on lipid droplets, UCP1 and metabolism related genes expression in deferent depots of adipose tissue through HE staining, Western blot and Real-time RT-PCR.Results:1) We established the browning differentiation protocol to differentiate MEFs into adipocytes with brown-like characteristics and found increased expression of UCP1through Western blot and mitochondria-UCP1 immunofluorescence in this differentiation protocol.2) First of all, we identified that 22-S-HC safety range was less than 10μM, and then found that 22-S-HC at the concentration of 0.5, 1μM could promote the browning differentiation better than without 22-S-HC, which was presented with significantly increased UCP1 expression through Western blot and Real-time RT-PCR (P<0.05), the more obvious dyeing of mitochondrial-UCP1 immunofluorescence. In the differentiation process, the expression of UCP1 and several other brown adipose related genes gradually increased from day 4 onwards, generally reached peak on day 14.3) Western blot and Real-time RT-PCR results suggested that 22-S-HC could increase UCP1 and PGC1α levels (P<0.05) in vitro. We measured metabolism related genes through Real-time RT-PCR and found AP2、PPARγ、Adiponectin expression significantly increased and SREBP1c expression significantly decreased (P<0.05). 2NBDG uptake experiments suggested that 22-S-HC could improve the ability of glucose uptake of the differentiated MEFs (P<0.05).4) The mice in 22-S-HC group showed a decreasing trend in body weight compared with the mice, but there was no statistical difference. The Area Under Curve(AUC) of IPGTT and ITT was significantly reduced in 22-S-HC group (P<0.05). We also We also observed less lipid droplets in BAT and smaller lipid droplets in iWAT in 22-S-HC group. Western blot and Real-time RT-PCR results showed UCP1 expression significantly increased (P<0.05) in 22-S-HC group. The variation trend of AP2, PPARy and SREBPlc and Adiponectin was different in different depots of adipose tissue.Conclusions:MEFs can be differentiated to adipocytes with brown-like characteristics; 22-S-HC can promote adipose browning in vitro and in vivo; 22-S-HC can improve glucose and lipid metabolism in vitro and in vivo; 22-S-HC can affect several important genes in glucose and lipid metabolism; 22-S-HC has different effect on different depots of adipose tissue.Objectives:The aim was to detect whether the effect of LXRs knockdown on adipose browning was consistent with the LXRs antagonist 22-S-HC. Furthermore, we investigated whether 22-S-HC and LXRs knockdown could activate the promoters of UCP1, PPARy, PGCla, COX2 which are all important factors affecting adipose browning.Methods:LXRs shRNA plasmids were pursued from Origen Company. Stable LXRα knockdown MEF cell lines were generated using retroviral-mediated infection. And then we observed the expression of brown adipocytes characteristics of LXRa knockdown MEFs by Western blot and Real-time RT-PCR compared with wild MEFs. The ability of glucose uptake was tested through detection of 2NBDG uptake. We constructed reporter gene plasmids containing UCP1, PPARy, PGC1α, COX2 promoters respectively. We aimed to figure out whether LXRa knockdown or 22-S-HC could enhance the activity of promoters of UCP1, PPARy, PGC1α through Dual-Luciferase reporter assays.Results:1) The retro viral vectors containing LXRs-shRNA sequences were successfully constructed. LXRa shRNA3 was screened out because of its most effective knockdown. And the stable LXRa knockdown cell line was successfully obtained.2) The stable LXRa knockdown MEFs were induced by browning differentiation protocol. We observed that lipid droplets had no obvious change through oil red staining. Western blot and Real-time RT-PCR results showed UCP1, PPARy, PGCla, COX2 expression levels were significantly increased (P<0.05). The glucose uptake increased significantly in the stable knockdown MEFs compared to the wild MEFs (P<0.05).3) The reporter gene plasmids containing UCP1, PPARγ, PGC1α, COX2 promoters were successfully constructed. We figured out LXRa knockdown and 22-S-HC both could enhance the activity of promoters of UCP1, PPARγ, PGC1α through Dual-Luciferase reporter assays.Conclusions:LXRa knockdown has a similar effect on adipose browning as 22-S-HC.22-S-HC and LXRa knockdown can enhance the activity of promoters of UCP1, PPARy, PGC1α which are key genes in adipose browning. |
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