Dicer has a crucial role in the early stage of adipocyte differentiation, but not in lipid synthesis, in 3T3-L1 cells

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis.     

The novel gene fad158, having a transmembrane domain and leucine-rich repeat, stimulates adipocyte differentiation

Adipocyte differentiation is known to be regulated by a complex array of genes known as master regulators. Using a subtraction method, we previously isolated 102 genes that are expressed in the early stage of adipocyte differentiation. One of these genes named fad158 (factor for adipocyte differentiation 158) seems to be a novel gene, since there is no significantly similar gene listed in databases. Both mouse and human fad158 encode 803 amino acids and contain 4 transmembrane regions and 8 leucine-rich repeat motifs. Expression of fad158 was induced at an early stage in differentiating 3T3-L1 cells and was observed in the skeletal muscle. When the expression was knocked down with an antisense method in 3T3-L1 cells, the accumulation of oil droplets was reduced. Moreover, on overexpression of fad158 in NIH-3T3 cells, which are fibroblasts and do not usually differentiate into adipocytes, stable transformants accumulated oil droplets and showed an elevated expression of adipocyte marker genes, indicating that these cells had differentiated into mature adipocytes. fad158 has the ability to regulate adipocyte differentiation positively, especially at an early stage.     

peg10, an imprinted gene, plays a crucial role in adipocyte differentiation

An imprinted gene, paternally expressed gene (peg) 10, was isolated as one of the genes expressed early in adipogenesis. The expression of peg10 was elevated after the addition of inducers, and was detected in adipocyte differentiable 3T3-L1 cells, but not observed in the non-adipogenic cell line NIH-3T3. Moreover, the knockdown of peg10 by RNA interference (RNAi) inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Interestingly, peg10 RNAi-treatment reduced the expressions of C/EBPbeta and C/EBPdelta, and inhibited mitotic clonal expansion. These findings strongly indicate that peg10 plays a crucial role at the immediate early stage of adipocyte differentiation.     

Vanillin induces adipocyte differentiation in 3T3-L1 cells by activating extracellular signal regulated kinase 42/44

AimsTo investigate the effect of vanillin, a dietary component, on adipocyte differentiation and the mechanism involved in the process using 3T3-L1 murine preadipocytes.Main methodsThe effect of vanillin on adipocyte differentiation was detected by Oil Red O analysis. The activation of extracellular signal regulated kinase 42/44 (ERK 42/44), Akt, expression of the key regulator of adipocyte differentiation peroxisome proliferators-activated receptor (PPARγ) and its target gene glucose transporter 4 (GLUT4) were detected by western blotting. Glucose uptake assay was used to determine the insulin sensitivity of adipocytes differentiated by vanillin treatment. To confirm the role of ERK 42/44 and Akt, Oil Red O analysis was performed with cells differentiated in the presence or absence of ERK inhibitor U0126 or Akt kinase 1/2 inhibitor.Key findingsVanillin induced adipocyte differentiation in 3T3-L1 cells in a dose dependent manner and also increased the expression levels of PPARγ and its target gene GLUT4. The adipocytes differentiated by vanillin exhibited insulin sensitivity as demonstrated by a significant increase in glucose uptake. Vanillin treatment activated the phosphorylation of ERK 42/44 during the initial phase of adipocyte differentiation but there was no significant change in the Akt phosphorylation status.SignificanceThe data show that vanillin induces adipocyte differentiation in 3T3-L1 cells by activating ERK42/44 and these adipocytes are insulin sensitive in nature.     

Reducing selenoprotein P expression suppresses adipocyte differentiation as a result of increased preadipocyte inflammation

Oxidative stress and low-grade inflammation have been implicated in obesity and insulin resistance. As a selenium transporter, ubiquitously expressed selenoprotein P (SeP) is known to play a role in the regulation of antioxidant enzyme activity. However, SeP expression and regulation in adipose tissue in obesity and its role in inflammation and adipocyte biology remain unexplored. In this study, we examined Sepp1 gene expression and regulation in adipose tissue of obese rodents and characterized the role of Sepp1 in adipose inflammation and adipogenesis in 3T3-L1 adipocytes. We found that Sepp1 gene expression was significantly reduced in adipose tissue of ob/ob and high-fat diet-induced obese mice as well as in primary adipose cells isolated from Zucker obese rats. Rosiglitazone administration increased SeP protein expression in adipose tissue of obese mice. Treatment of either TNFα or H(2)O(2) significantly reduced Sepp1 gene expression in a time- and dose-dependent manner in 3T3-L1 adipocytes. Interestingly, Sepp1 gene silencing resulted in the reduction in glutathione peroxidase activity and the upregulation of inflammatory cytokines MCP-1 and IL-6 in preadipocytes, leading to the inhibition of adipogenesis and adipokine and lipogenic gene expression. Most strikingly, coculturing Sepp1 KD cells resulted in a marked inhibition of normal 3T3-L1 adipocyte differentiation. We conclude that SeP has an important role in adipocyte differentiation via modulating oxidative stress and inflammatory response.     

Identification and characterization of a transcript for a novel Rac GTPase-activating protein in terminally differentiating 3T3-L1 adipocytes

Using differential display, we sought to identify novel genes expressed in the early stages of 3T3-L1 adipocyte differentiation. A gene which we have named "band25" was identified, and a full-length cDNA sequence was assembled. Sequence analysis revealed that the 2842-bp cDNA encodes a putative 628-amino acid protein product, which is a member of the GTPase-activating protein (GAP) family. This gene may be the murine homolog of the human MgcRacGAP protein, which was identified in male germ cells. Other closely related proteins include the Drosophila protein Rotund, several chimerins, and the human breakpoint cluster region (Bcr) protein. These GAP proteins all specifically inactivate Rac, a member of the Ras-like family of proteins. A consensus sequence for a diacyl glycerol/phorbol ester-binding domain was also found in the Band25 sequence. The expression of band25 mRNA is regulated during the differentiation of both adipocytes and myoblasts. Its mRNA was shown to be expressed at a low level in confluent 3T3-L1 preadipocytes and in differentiated 3T3-L1 adipocytes. Expression of band25 was increased 15.5 fold by 24 h after the induction of differentiation, when 3T3-L1 cells undergo several rounds of postconfluent cell division. Expression was also high in growing 3T3-L1 and C2C12 cells but decreased progressively as C2C12 cells underwent differentiation. These observations suggest that the expression of band25 is growth regulated and that the protein could play a role in the regulation of growth-related processes.     

The novel gene fad104, containing a fibronectin type III domain, has a significant role in adipogenesis

A novel gene named fad104 (factor for adipocyte differentiation-104), whose expression level quickly increased in the early stage of adipogenesis, was isolated and characterized. The deduced amino acid sequence of fad104 revealed the possible presence of a fibronectin type III domain and transmembrane domain. The expression of fad104 was detected in adipocyte differentiable 3T3-L1 cells but not observed in the non-adipogenic cell line NIH-3T3. Moreover, the ability of 3T3-L1 cells to differentiate declined with the knockdown of fad104 by RNA interference, strongly indicating that fad104 functions as a positive regulator of adipogenesis.     

A novel gene, fad49, plays a crucial role in the immediate early stage of adipocyte differentiation via involvement in mitotic clonal expansion

Adipogenesis is accomplished via a complex series of steps, and the events at the earliest stage remain to be elucidated. To clarify the molecular mechanisms of adipocyte differentiation, we previously isolated 102 genes expressed early in mouse 3T3-L1 preadipocyte cells using a PCR subtraction system. About half of the genes isolated appeared to be unknown. After isolating full-length cDNAs of the unknown genes, one of them, named factor for adipocyte differentiation 49 (fad49), appeared to be a novel gene, as the sequence of this clone showed no identity to known genes. FAD49 contains a phox homology (PX) domain and four Src homology 3 (SH3) domains, suggesting that it may be a novel scaffold protein. We found that the PX domain of FAD49 not only has affinity for phosphoinositides, but also binds to its third SH3 domain. Expression of fad49 was transiently elevated 3 h after differentiation was induced, and diminished 24 h after induction. Induction of the fad49 gene was observed in adipocyte differentiable 3T3-L1 cells, but not in non-adipogenic NIH-3T3 cells. RNAi-mediated knockdown of fad49 significantly impaired adipocyte differentiation. Moreover, the knockdown of fad49 by RNAi inhibited mitotic clonal expansion, and reduced the expression of CCAAT/enhancer-binding protein beta (C/EBPbeta) and C/EBPdelta at the immediate early phase. Taken together, these results show that fad49, a novel gene, plays a crucial role in the immediate early stage of adipogenesis.     

Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade

Adult mice abundantly express neudesin, an extracellular heme-binding protein with neurotrophic activity, in white adipose tissues. At the early stage of adipocyte differentiation during adipogenesis, however, the expression of neudesin decreased transiently. Neudesin-hemin significantly suppressed adipogenesis in 3T3-L1 cells. The knockdown of neudesin by RNA interference markedly promoted adipogenesis in 3T3-L1 cells and decreased MAPK activation during adipocyte differentiation. The addition or knockdown of neudesin affected the expression of C/EBPα and PPARγ but not of C/EBPβ. These findings suggest that neudesin plays a critical role in the early stage of adipocyte differentiation in which C/EBPβ induces PPARγ and C/EBPα expressions, by controlling the MAPK pathway.     

Insulin-like growth factor-1/insulin bypasses Pref-1/FA1-mediated inhibition of adipocyte differentiation

Pref-1 is a highly glycosylated Delta-like transmembrane protein containing six epidermal growth factor-like repeats in the extracellular domain. Pref-1 is abundantly expressed in preadipocytes, but expression is down-regulated during adipocyte differentiation. Forced expression of Pref-1 in 3T3-L1 cells was reported to inhibit adipocyte differentiation. Here we show that efficient and regulated processing of Pref-1 occurs in 3T3-L1 preadipocytes releasing most of the extracellular domain as a 50-kDa heterogeneous protein, previously isolated and characterized as FA1. Unexpectedly, we found that forced expression of the soluble form, FA1, or full-length Pref-1 did not inhibit adipocyte differentiation of 3T3-L1 cells when differentiation was induced by standard treatment with methylisobutylxanthine, dexamethasone, and high concentrations of insulin. However, forced expression of either form of Pref-1/FA1 in 3T3-L1 or 3T3-F442A cells inhibited adipocyte differentiation when insulin or insulin-like growth factor-1 (IGF-1) was omitted from the differentiation mixture. We demonstrate that the level of the mature form of the IGF-1 receptor is reduced and that IGF-1-dependent activation of p42/p44 mitogen-activated protein kinases (MAPKs) is compromised in preadipocytes with forced expression of Pref-1. This is accompanied by suppression of clonal expansion and terminal differentiation. Accordingly, supplementation with insulin or IGF-1 rescued p42/p44 MAPK activation, clonal expansion, and adipocyte differentiation in a dose-dependent manner.     

Activation of early phase of adipogenesis through Krüppel-like factor KLF9-mediated,enhanced expression of CCAAT/enhancer-binding protein β in 3T3-L1 cells

In this study, we found that Krüppel-like factor (KLF) 9 activate the progression of the early phase of adipocyte differentiation in mouse adipocytic 3T3-L1 cells. KLF9 mRNA was detected in preadipocytes; and its level increased after the initiation of adipocyte differentiation, reached its maximum at 1 h, and gradually decreased thereafter. Functional suppression of KLF9 mRNA by its siRNAs repressed the accumulation of the intracellular lipids with a reduction in the expression of CCAAT/enhancer-binding protein (C/EBP) β, but not in that of C/EBPδ. In contrast, C/EBPβ and C/EBPδ did not affect the expression of KLF9 in 3T3-L1 cells. A chromatin immunoprecipitation assay revealed that KLF9 bound the KLF binding element at position − 874 of the mouse C/EBPβ promoter. Moreover, the ability of KLF9 to bind to this element was enhanced, with a peak at 1–2 h after the initiation of adipogenesis, whose profile well resembled that of the expression of the C/EBPβ gene in 3T3-L1 cells. These results indicate that KLF9 activated the early phase of adipogenesis by enhancing the expression of the C/EBPβ gene in 3T3-L1 cells.