Implication of phosphatidylethanolamine N-methyltransferase in adipocyte differentiation

Phosphatidylethanolamine N-methyltransferase (PEMT) is a small integral membrane protein that converts phosphatidylethanolamine (PE) into phosphatidylcholine (PC). It has been previously reported that, unexpectedly, PEMT deficiency protected from high-fat diet (HFD)-induced obesity and insulin resistance, pointing to a possible role of this enzyme in the regulation of adipose cell metabolism. Using mouse 3T3-L1 preadipocytes as a biological system, we demonstrate that PEMT expression is strongly increased during the differentiation of preadipocytes into mature adipose cells. Knockdown of PEMT reduced the expression of early and late adipogenic markers, inhibited lipid droplet formation, reduced triacylglycerol content and decreased the levels of leptin release from the adipocytes, suggesting that PEMT is a novel and relevant regulator of adipogenesis. Investigation into the mechanisms whereby PEMT regulates adipocyte differentiation revealed that extracellularly regulated kinases (ERK1/2) and AKT are essential factors in this process. Specifically, the activities of ERK1/2 and AKT, which are decreased during adipocyte differentiation, were elevated upon Pemt knockdown. Moreover, treatment of cells with exogenous ceramide 1-phosphate (C1P), which we reported to be a negative regulator of adipogenesis, decreased PEMT expression, suggesting that PEMT is also a relevant factor in the anti-adipogenic action of C1P. Altogether, the data presented here identify PEMT as a novel regulator of adipogenesis and a mediator of the anti-adipogenic action of C1P.     

过表达鸡Gata2或Gata3基因抑制Pparγ基因的转录

脂肪细胞分化是脂肪组织发育的一个重要过程. 目前,鸡脂肪细胞分化的分子调控机制还不十分清楚. 哺乳动物的研究结果表明,转录因子GATA结合蛋白2(Gata2)和GATA结合蛋白3(Gata3)具有抑制脂肪细胞分化的功能,它们在白色脂肪组织和棕色脂肪组织中的表达模式不同. 鸡没有棕色脂肪组织,目前还没有关于Gata2 和Gata3作用于鸡脂肪细胞分化的研究报道. 本研究利用半定量RT PCR的方法分析了Gata2和Gata3基因在鸡腹部脂肪组织和前脂肪细胞中的表达规律,发现鸡腹部脂肪组织中高水平表达Gata2 基因,低水平表达Gata3基因|鸡前脂肪细胞中Gata2 基因的表达水平远高于Gata3基因的表达水平,油酸诱导分化后的鸡前脂肪细胞Gata2基因的表达水平明显下调.此外,鸡过氧化物酶体增殖体激活受体γ(Pparγ)启动子（－1985/－89）报告基因荧光素活性分析和半定量RT PCP发现,在DF1细胞中过表达Gata2 或Gata3抑制鸡PPARγ基因的转录. 本研究结果为进一步研究鸡脂肪细胞分化的分子调控机制和Gata2和Gata3基因的生物学功能提供了参考.     

KLF转录因子家族与脂肪细胞分化

Kruppel样转录因子（Kruppel-like factors, KLF）是一类具有锌指结构的转录因子,其典型结构特征是在其羧基端具有3个C2H2锌指结构. KLF广泛参与细胞增殖、凋亡、分化以及胚胎发育等多个生命活动的调控. 近年来脂肪细胞分化研究的结果显示,KLF家族的多个成员参与脂肪细胞分化过程的调控,既有促进脂肪细胞分化的,也有抑制脂肪细胞分化的. 其中KLF4通过与Krox20协同作用,激活C/EBPβ（CCAAT-enhancer-binding protein β）基因表达,促进脂肪细胞分化;KLF5和 KLF15都通过直接结合到氧化物酶增殖体激活受体γ（peroxisome proliferator-activated receptor γ, PPARγ）基因的启动子,激活PPARγ基因表达,促进脂肪细胞分化;而KLF6则通过抑制前脂肪细胞因子（pre-adipocyte factor 1, PREF1）基因表达,促进脂肪细胞分化. 抑制脂肪细胞分化的KLF2通过结合于PPARγ的启动子,抑制PPARγ基因表达,从而抑制脂肪细胞的分化;KLF3通过募集辅助抑制因子C-末端结合蛋白（c-terminal binding protein, CtBP）形成KLF3 CtBP抑制复合体,结合于C/EBPα（CCAAT-enhancer-binding protein α）基因的启动子,抑制C/EBPα表达,进而抑制脂肪细胞的分化;KLF7通过抑制葡萄糖转运蛋白2（glucose transporter2,GLUT2）基因的表达抑制脂肪细胞的成熟. 本文综述这些KLF转录因子在脂肪细胞分化过程的作用及其作用的机制.     

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur.     

Knockdown of KLF9 promotes the differentiation of both intramuscular and subcutaneous preadipocytes in goat

ABSTRACT

KLF9 is reported to promote adipocyte differentiation in 3T3-L1 cells and pigs. However, the roles of KLF9 in adipocytes differentiation of goat remain unknown. In this study, the expression profiles of KLF9 were different between subcutaneous and intramuscular preadipocytes of goat during differentiation process. After silencing KLF9 gene, the lipid droplets were increased in both two types of adipocytes. In subcutaneous preadipocyte with silencing KLF9, the expressions of C/EBPβ, PPARγ, LPL, KLF1-2, KLF5, and KLF17 genes were up-regulated, while KLF12, KLF4, and KLF13 genes were down-regulated in expression level. In intramuscular preadipocyte, aP2, C/EBPα, KLF2-3, KLF5, and KLF7 gene were up-regulated, and Pref-1 gene was down-regulated. In addition, the binding sites of KLF9 existed in the promoters of aP2, C/EBPα, C/EBPβ, LPL and Pref-1. Taken together, KLF9 play a negative role in the differentiation of both intramuscular and subcutaneous preadipocytes in goats, but the functional mechanism may be different.     

Gene expression patterns of bovine perimuscular preadipocytes during adipogenesis

Bovine perimuscular fat (PMF) preadipocytes were induced to undergo adipogenesis in vitro in our recent study to define the expression patterns of genes involved in the differentiation process. Based on the understanding of the interaction among adipogenic genes, a broad overview of gene expression profile in the differentiating PMF preadipocytes was evaluated using bovine specific DNA microarray from day 2 to 8 post-differentiation induction. A total of 100 significantly differentially expressed genes were detected between differentiated and control cells including those involved in several biochemical pathways and cellular/molecular signaling. In addition, quantitative real-time PCR validated that typical adipogenic genes were up-regulated at early differentiation in the preadipocytes. These results suggest that the PMF preadipocyte system is available as a novel in vitro model for molecular adipogenesis studies in the bovine and that a series of genes are switched on/off during early events associated with adipogenesis.     

LMO4 modulates proliferation and differentiation of 3T3-L1 preadipocytes

Previous microarray analyses revealed that LMO4 is expressed in 3T3-L1 preadipocytes, however, its roles in adipogenesis are unknown. In the present study, using RT-PCR sequencing and quantitative real-time RT-PCR, we confirmed that LMO4 gene is expressed in 3T3-L1 preadipocytes and its expression peaks at the early stage of 3T3-L1 preadipocyte differentiation. Further analyses showed that LMO4 knockdown decreased the proliferation of 3T3-L1 preadipocytes, and attenuated the differentiation of 3T3-L1 preadipocytes, as evidenced by reduced lipid accumulation and down-regulation of PPARγ gene expression. Collectively, our findings indicate that LMO4 is a novel modulator of adipogenesis.