Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis

CCAAT enhancer-binding protein (C/EBP)beta, C/EBPalpha, and peroxisome proliferator activated receptor (PPAR)gamma act in a cascade where C/EBPbeta activates expression of C/EBPalpha and PPARgamma, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C/EBPbeta is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3beta enters the nucleus, which correlates with hyperphosphorylation of C/EBPbeta and acquisition of DNA-binding activity. Concurrently, 3T3-L1 preadipocytes synchronously enter S phase and undergo mitotic clonal expansion, a prerequisite for terminal differentiation. Ex vivo and in vitro experiments with C/EBPbeta show that phosphorylation of Thr-188 by mitogen-activating protein kinase "primes" C/EBPbeta for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3beta, acquisition of DNA-binding function, and transactivation of the C/EBPalpha and PPARgamma genes. The delayed transactivation of the C/EBPalpha and PPARgamma genes by C/EBPbeta appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C/EBPalpha and PPARgamma are antimitotic.     

Knockdown of macrophage migration inhibitory factor disrupts adipogenesis in 3T3-L1 cells

Obesity is a condition in which adipose tissue mass is expanded. Increases in both adipocyte size and number contribute to enlargement of adipose tissue. The increase in cell number is thought to be caused by proliferation and differentiation of preadipocytes. Macrophage migration inhibitory factor (MIF) is expressed in adipocytes, and intracellular MIF content is increased during adipogenesis. Therefore, we hypothesized that MIF is associated with adipocyte biology during adipogenesis and focused on the influence of MIF on adipogenesis. To examine the effects of MIF on adipocytes, MIF expression in 3T3-L1 preadipocytes was inhibited by RNA interference, and cell differentiation was induced by standard procedures. The triglyceride content of MIF small interfering RNA (siRNA)-transfected 3T3-L1 cells was smaller than that of nonspecific siRNA-transfected cells. In addition, MIF knockdown apparently abrogated increases in adiponectin mRNA levels during differentiation. Gene expression of peroxisome proliferator-activated receptor (PPAR)gamma, CCAAT/enhancer binding protein (C/EBP)alpha, and C/EBPdelta decreased with MIF siRNA transfection, but C/EBPbeta expression increased. Cell number and incorporation of 5-bromo-2-deoxyuridine into cells decreased from 1-3 d and from 14-20 h, respectively, after induction of differentiation in MIF siRNA-transfected cells, thus suggesting that MIF siRNA inhibits mitotic clonal expansion. Taken together, these results indicated that MIF regulates differentiation of 3T3-L1 preadipocytes, at least partially, through inhibition of mitotic clonal expansion and/or C/EBPdelta expression.     

Lipopolysaccharides Reduce Adipogenesis in 3T3-L1 Adipocytes Through Activation of NF-κB Pathway and Downregulation of AMPK Expression

Lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria serve as endotoxin to exert potent immune responses. However, the effect of LPS on adipogenesis has not been elucidated. The present study was designed to examine the effect of LPS on adipogenesis in 3T3-L1 preadipocytes and possible mechanism(s) of action involved. Our results revealed that LPS challenge significantly suppressed adipogenesis in 3T3-L1 preadipocytes mainly through downregulated expression of the late adipogenic markers PPARγ and aP2 as well as AMP-activated protein kinase (AMPK) expression and activity. As an inflammatory factor, LPS was found to lead to an overt reduction in IκBα levels compared with the time-matched controls, consolidating its pro-inflammatory property in 3T3-L1 preadipocytes. Our data also revealed that LPS retarded adipogenesis, the effect of which was partially reversed by the selective inhibitor of IKKβ. IκBα was found to be involved in the anti-adipogenic effect of LPS. In conclusion, LPS is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through activation of NF-κB and inhibition of AMPK. With the activation of NF-κB pathway and inhibition of AMPK, LPS suppresses C/EBP α DNA-binding activity and the expression of late adipogenic markers PPARγ and aP2.     

CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program.

Previous studies showed that CCAAT/enhancer binding protein alpha (C/EBP alpha) is required for differentiation of 3T3-L1 preadipocytes induced by exogenous hormonal agents. It was not possible to ascertain, however, whether C/EBP alpha alone is sufficient to induce differentiation because its antimitogenic activity precluded propagating 3T3-L1 cell lines that constitutively express C/EBP alpha at high levels. This problem was circumvented by using 3T3-L1 preadipocytes stably transfected with an isopropyl beta-D-thiogalactoside (IPTG)-inducible p42 C/EBP alpha expression vector system. IPTG-induced expression of the 42-kDa isoform of C/EBP alpha in preadipocytes caused expression of several endogenous adipocyte-specific genes (genes encoding the 422 adipose P2 protein, glucose transporter 4, and C/EBP alpha) and the accumulation of cytoplasmic triglyceride. Thus, C/EBP alpha is not only necessary but also is sufficient to trigger differentiation of growth-arrested 3T3-L1 preadipocytes without use of exogenous hormonal agents.     

Ceramide and adenosine 5'-monophosphate-activated protein kinase are two novel regulators of 11beta-hydroxysteroid dehydrogenase type 1 expression and activity in cultured preadipocytes

Increased activity of intracellular glucocorticoid reactivating enzyme, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in obese adipose tissue contributes to adipose dysfunction. As recent studies have highlighted a potential role of preadipocytes in adipose dysfunction, we tested the hypothesis that a variety of metabolic stress mediated by ceramide or AMP-activated protein kinase (AMPK) would regulate 11beta-HSD1 in preadipocytes. The present study is the first to show that 1) expression of 11beta-HSD1 in 3T3-L1 preadipocytes was robustly induced when cells were treated with cell-permeable ceramide analogue C(2) ceramide, bacterial sphingomyelinase, and sphingosine 1-phosphate, 2) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced activation of AMPK augmented the expression and enzyme activity of 11beta-HSD1, and 3) these results were reproduced in human preadipocytes. We demonstrate for the first time that C(2) ceramide and AICAR markedly induced the expression of CCAAT/enhancer-binding protein (C/EBP) beta and its binding to 11beta-HSD1 promoter. Transient knockdown of C/EBPbeta protein by small interfering RNA markedly attenuated the expression of 11beta-HSD1 induced by C(2) ceramide or AICAR. The present study provides novel evidence that ceramide- and AMPK-mediated signaling pathways augment the expression and activity of 11beta-HSD1 in preadipocytes by way of C/EBPbeta, thereby highlighting a novel, metabolic stress-related regulation of 11beta-HSD1 in a cell-specific manner.     

S100A16基因促进3T3-L1前脂肪细胞分化

目的 研究S100A16基因在3T3-L1前脂肪细胞分化过程中的作用及机制.方法 构建过表达S100A16的慢病毒载体(PLJMI-S100A16-GFP),转染3T3-L1细胞.以Western印迹法检测S100A16正常3T3-L1细胞分化过程中S100A16的表达;采用油红O观察脂滴堆积情况;采用Western印迹和实时定量PCR方法检测前体脂肪细胞分化过程中相关基因的表达变化.免疫共沉淀方法检测S100A16是否与p53相互作用.结果 成功构建S100A16过表达3T3-L1细胞株;随着3T3-L1前脂肪细胞的分化,S100A16蛋白表达水平逐渐升高;高表达S100A16能够促进3T3-L1前脂肪细胞分化,促进甘油三酯在脂肪细胞内聚集(P＜0.01),同时上调脂肪细胞分化标志基因PPARy、CCAAT增强子结合蛋白α(C/EBP-α)、脂蛋白脂酶、脂肪细胞脂肪酸结合蛋白(aP2)及脂肪酸合成酶的表达(P＜0.05或P＜0.01);免疫共沉淀结果提示,S100A16蛋白与p53相互作用.结论 S100A16通过抑制p53活性进而促进3T3-L1前脂肪细胞的分化.