| The excessive deposition of body fat resulted in decreased meat quality of livestock and human metabolic disease. From the cellular level point of view, body fat content is a macro expression of TG accumulation in adipocytes, and the lipolysis rate and the proliferation/differentiation degree of adipocytes together contribute to fat deposit in single adipocyte. For the last decate, multiple signaling pathways and nuclear transcriptional factors have been demonstrated to be involved in fat cell TG accumulation process. Although the adipose tissue deposition in estrogen receptor-related receptorα(Estrogen-related receptorα, ERRα) knockout mice was significantly reduced by 50% or more when compared with wild type, its express pattern and role in adipose tissue is unknown currently.In present work, 2 or 4 weeks old SD rats were used as our experimental animals. We firstly cloned the CDS regions of rat and pig ERRαgenes with Touch-down PCR. Western blot and cell immol/Lunofluorescence method were used to investigate the expression pattern of ERRαprotein in white adipose tissue (WAT) and white adipocytes. Then, rat primary preadipocytes and differentiated adipocytes were treated by a specific inverse agonist XCT790 or infected with adenoviral vector expressed ERRα(Ad-ERRα) to study the function of ERRαin adipogenesis. Oil red O staining and TG measure kit were used to determine the cellular TG level; Gene expression related to adipocytes differentiation including PPARγ, C/EBPα, SREBP-1C, aP2, FAS and SCD-1 were analyzed with Real time PCR; Proteins involved in signal pathway, nuclear transcriptional factors and differentiation markers includingβ-catenin, p38 MAPK, pp38MAPK, ERK1/2, pERK1/2, PPARγ, C/EBPα, SREBP-1C, PGC-1βand aP2 were analyzed with Western blot; Co-IP was adopted to investigate the cross-regulation effect of ERRαon the interaction between PGC-1βwith PPARγand SREBP-1C; the transcriptional activation of ERRαon PGC-1βpromoter was measured with a luciferase reporter plasmid. Finally, PD98059 or H89 pretreated adipocytes were infected with Ad-ERRα, then Glycerol determine kit was used to measure the glycerol release; key proteins involved in lipolysis including perilipin A, p-perilipin A, HSL, ATGL were analyzed with Western blot. The results are as follow:1.Compared with high energy demand tissues, ERRαis highly expressed in WAT and the difference between genders is not significantly. ERRαprotein expression is increased as preadipocytes differentiation and prior to some key adipogenesis factors. Besides, we observed a translocation of ERRαfrom cytoplasm into nucleus in differentiated adipocytes.2.Compared with ERK1/2 and P38 MAPK pathways, we found that Wnt/β-catenin pathway was selectively inhibited by ERRαin a way of reducingβ-catenin protein. Thereby, adipogenesis in preadipocytes was increased by increasing the expression of PPARγand C/EBPα.3.ERRαcould directly utilize PGC-1βas its co-regulator to up-regulate PGC-1βexpression in mRNA and protein level. As a consequence, the interactions between PGC-1βwith PPARγand SREBP-1c were enhanced. Thereby, FAS and SCD-1 was up-regulated to increase the adipogenesis ability of adipocytes.4.ERRαcould increase the glycerol release of adipocytes via up-regulating the expression of HSL and ATGL. Besides, PKA and ERK1/2 pathways were not involved in the ERRαstimulated lipolysis. To sum up, ERRαis highly expressed in adipose tissue without gender differences.ERRαpromotes adipogenesis via at least two ways: in preadipocytes, ERRαselectively reduces the expression ofβ-catenin protein, thereby inhibiting the negative effect of Wnt /β-catenin signaling pathway on adipogenesis; in differentiated adipocytes, ERRαtranscriptional activates PGC-1βpromoter and to promote its mRNA and protein expression. The increased PGC-1βcould augment the adipogenesis abilities of PPARγand SREBP-1c. ERRαis a stimulator for lipolysis via up-regulating the expression of ATGL and HSL to maintain the increasing energy demand in adipocytes. Meanwhile, the lipolysis rate is decreased.
|
PDF Full Text Request