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.     

Klotho protein promotes adipocyte differentiation

Mice with homozygous disruption of the klotho exhibit multiple age-related disorders and have barely detectable amounts of white adipose tissue. Although klotho expression in cultured adipocytes has been reported, little is known about its function in adipocytes. In the present study, we investigated the role of klotho on adipocyte differentiation. Adipocyte differentiation was induced by incubation of confluent 3T3-L1 cells with insulin, dexamethasone, and 1-methyl-3-isobutyl-xanthin. Klotho-siRNA and expression vector were produced for klotho suppression and overexpression, respectively. Klotho protein was purified for determination of the hormonal effect of klotho. Klotho mRNA and protein expression increased up to the 3rd d of differentiation. A peroxisome proliferator-activated receptor-gamma agonist increased klotho expression during the early period of adipocyte differentiation. The mRNA expression of adipocyte differentiation markers, such as CCAAT/enhancer-binding protein (C/EBP)alpha, C/EBPbeta, C/EBPdelta, peroxisome proliferator-activated receptor-gamma, and fatty acid binding protein 4, was decreased by klotho suppression, and increased 1.9- to 3.8-fold by klotho overexpression. The results of Oil Red O staining also suggested that klotho overexpression promoted adipocyte differentiation. Klotho protein stimulation resulted in a 2.4- to 4.6-fold increase in mRNA expression of differentiation markers compared with control, and the time course depended on adipocyte induction status. Western blot analysis showed that protein levels of C/EBPalpha and C/EBPdelta were increased by Klotho protein stimulation. These results suggest that klotho works as a hormonal factor to promote adipocyte differentiation in the early days, during the period of transient proliferation in the differentiation process, and that klotho may play an essential role in adipocyte differentiation.