Differentiation Of 3T3-L1 Preadipocyte Cells Research Articles

Adipocyte differentiation is regulated by mitochondrial trifunctional protein α-subunit via sirtuin 1

Mitochondrial trifunctional protein α-subunit (MTPα) is involved in the fatty acid β-oxidation (FAO) pathway. Two MTPα activities, 3-hydroxyacyl-CoA dehydrogenase and long-chain hydratase, have been linked with the occurrence and development of obesity and obesity-related disorders. These activities catalyze two steps in the FAO pathway (the second and third reactions). However, the role of MTPα in the pathogenesis of obesity has not been evaluated, and the functional role of MTPα in adipocyte differentiation has not been determined. Here, we analyzed the functional role of MTPα using in vitro and in vivo models of adipogenesis. MTPα expression was upregulated during the differentiation of 3T3-L1 preadipocyte cells into adipocytes. MTPα gene silencing stimulated peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein alpha(C/EBPα) expression, which promoted adipocyte differentiation. By contrast, MTPα overexpression blocked adipogenesis in 3T3-L1 cells. Further analysis showed that MTPα positively regulated sirtuin 1 (SIRT1). Injection of preadipocytes overexpressing MTPα into athymic mice significantly impaired de novo fat pad formation compared with that of the control, and furthermore MTPα knockdown enhances fat pad formation at a time point earlier than 5-week, such as week-2 and week-3, when the control fat pad is not fully developed. In summary, our data indicate that MTPα is a novel factor that negatively regulates adipocyte differentiation. We propose a pathway in which MTPα inhibits adipogenesis by promoting SIRT1 expression, which represses PPARγ and attenuates adipogenesis.

Glycoproteins isolated from scallop shells inhibit differentiation of 3T3-L1 preadipocyte cells

We previously demonstrated that the organic components isolated from scallop shells (scallop shell extract) inhibit the differentiation of 3T3-L1 preadipocyte cells. In this study, we show that a pronase-treated scallop shell extract inhibited differentiation, but degradation of sugar chains in the scallop shell extract with trifluoromethane sulfonic acid resulted in the loss of the differentiation-inhibiting activity, suggesting that the sugar chain modifications were responsible for the inhibitory activity. To identify the bioactive substance in the scallop shell extract, we isolated glycoproteins from the scallop shell extract via affinity chromatography using concanavalin A (Con A), wheat germ agglutinin, lens culinaris agglutinin (LCA), and ricinus communis agglutinin. LCA and Con A binding fractions inhibited the differentiation of 3T3-L1 preadipocyte cells. In addition, a glycoprotein with a molecular weight of 16 kDa that was purified from the LCA binding fraction reduced lipid accumulation in 3T3-L1 cells during differentiation. The glycoprotein inhibited differentiation-associated mitotic clonal expansion and suppressed the expression of an adipocyte-specific protein, fatty acid binding protein. These results suggest that the sugar chains of glycoproteins in the scallop shell extract inhibit the differentiation of 3T3-L1 preadipocyte cells.

Effect of miR-205 on 3T3-L1 preadipocyte differentiation through targeting to glycogen synthase kinase 3 beta

MiR-205 plays an important role during adipogenesis by modulating the Wnt signaling pathway. Here, we report that miR-205 can regulate the differentiation of 3T3-L1 preadipocyte cells by targeting glycogen synthase kinase 3 beta (GSK-3β), which is a negative regulatory factor of Wnt signaling. When transiently overexpressed in 3T3-L1 cells, miR-205 suppressed the translation of GSK-3β, resulting in increased expression of β-catenin, which can promote cell proliferation by facilitating the transcription of the Wnt target genes cyclin D1 and c-Myc. However, stable overexpression of miR-205 in 3T3-L1 cells did not show any apparent inhibitory effect on adipogenic differentiation. While endogenous miR-205 was inhibited in 3T3-L1 cells, the adipogenesis marker gene, C/EBPα, was significantly activated and more lipid droplets appeared in differentiated adipocytes. However, systemic silencing of miR-205 in mice by using a locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) did not lead to any observable increase in adipose tissue differentiation, implying that, as opposed to the findings from 3T3-L1 cells, miR-205 is dispensable for adipose tissue development in mice.

Scallop shell extract inhibits 3T3-L1 preadipocyte differentiation

In a previous study, we examined the in vitro and in vivo effects of organic components isolated from scallop shells (scallop shell extract) and found that rats fed with powdered scallop shell showed a reduction in white adipose tissue weight compared to rats fed a control diet. To clarify the mechanism of action, we have investigated the effects of scallop shell extract on the differentiation of 3T3-L1 preadipocyte cells. Scallop shell extract reduced lipid accumulation in 3T3-L1 cells during differentiation in a dose-dependent manner. Glycerol-3-phosphate dehydrogenase activity also decreased following treatment with scallop shell extract, suggesting that the extract inhibits adipocyte differentiation. In addition, scallop shell extract suppressed the expression of adipogenic transcription factors (peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein-α) and inhibited differentiation-associated mitotic clonal expansion. These results support the possibility that scallop shell extract may work as an anti-obesity agent via the inhibition of adipocyte differentiation.

Morus alba L. root bark stimulates adipocyte differentiation in 3T3-L1 cells

Mori Cortex (MC), the root bark of the mulberry tree (Morus alba L.), has been used traditionally in several Asian countries for medicinal purposes due to its anti-inflammatory, hypoglycemic, and antibacterial activities, as well as its antiviral effects. In the present study, we found that ethanolic extract of Morus alba L. root bark (MC) accelerated differentiation of 3T3-L1 preadipocyte cells. Treatment with MC resulted in enhanced triglyceride (TG) accumulation and increased mRNA levels of C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome proliferator-activated receptors γ); however, their upstream regulator, C/EBPδ, showed a dose-dependent decrease during adipocyte differentiation. Immunoblot analysis indicated that MC induced an increase in expression of adipogenic maker proteins, such as PPARγ, adipocyte-specific fatty acid binding protein 4 (aP2), and GLUT4 (glucose transporter 4). Results of the reporter gene assay revealed that MC did not show PPARγ ligand activity even at a high dose (50 μg/mL), suggesting that enhanced expression of PPARγ resulted not from PPARγ ligand activity but by other mechanisms that might be related to the PPARγ signal pathway. In conclusion, our findings suggest the potential for use of MC as a possible therapeutic material for treatment of dyslipidemia and type 2 diabetes by increasing PPARγ transcriptional activities.

Fraxinus excelsior seed extract FraxiPure™ limits weight gains and hyperglycemia in high-fat diet-induced obese mice

The aim of this study was to determine whether a Fraxinus excelsior L. seed extract, FraxiPure™ (0.5% in the diet), limits weight gain and hyperglycemia in mice. In a previous report, we identified several secoiridoids in FraxiPure™, some of which activated peroxisome proliferator-activated receptor alpha (PPARα) in vitro and inhibited the differentiation of 3T3-L1 preadipocyte cells. In a separate study, FraxiPure™ reduced glycemia in healthy volunteers, following an oral glucose tolerance test. These findings suggest that FraxiPure™ has antiobesity and antihyperglycemia effects. FraxiPure™ was tested in mice that were fed a high-fat diet over 16 weeks and compared with low-fat and high-fat diet controls. Weight gain, omental and retroperitoneal fat, fasting blood glucose, and fasting blood insulin were measured. FraxiPure™ reduced gains in body weight by 32.30% (p < 0.05), omental fat by 17.92%, and retroperitoneal fat by 17.78%. FraxiPure™ also lowered fasting blood glucose levels by 76.52% (p < 0.001) and plasma insulin levels by 53.43% (p < 0.05) after 16 weeks. Moreover, FraxiPure™ lowered liver weight gains by 63.62% (p < 0.05) and the incidence of fatty livers by 66.67%. Our novel results demonstrate the antiobesity effects of chronic administration of an F. excelsior seed extract and confirm its ability to regulate glycemia and insulinemia. In addition, this extract, which is rich in secoiridoid glucosides, protects against obesity-related liver steatosis.

Cloning and Characterization of Inducible Genes at the Beginning of Adipocyte Differentiation

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Cloning and characterization of inducible genes at the beginning of adipocyte differentiation

Adipocyte differentiation takes place via a complex series of steps. While PPAR gamma and C/EBP alpha are known to be master regulators, the events at the earliest stage of adipocyte differentiation are not yet known. In this study, we cloned the genes that are induced at the beginning of the differentiation of 3T3-L1 preadipocyte cells. Of 102 clones obtained, only several clones were already reported as genes that are expressed differentially during adipocyte development. The expression of TCL/TC10 beta L (TC10-like/TC10 beta Long) and RGS2 (regulators of G protein signaling 2) genes isolated here rapidly increased after the addition of inducers (insulin, dexemethasone, 3-isobutyl-1-methylxanthine, fetal bovine serum [FBS]). Further, the antisense TCL/TC10 beta L inhibited the adipogenesis of mouse 3T3-L1 preadipocyte cells, prevented cytoplasmic triglyceride accumulation, and decreased the expression of PPAR gamma and C/EBP alpha. Moreover, the constitutive overexpression of TCL/TC10 beta L or RGS2 in the mouse fibroblast cell line NIH-3T3 results in efficient adipocyte conversion when stimulated with 10% FBS, insulin, 3-isobutyl-1-methylxanthine, dexamethasone, and PPAR gamma ligand BRL49653. These results strongly suggest that TCL/TC10 beta L and RGS2 have crucial roles in the program of adipocyte differentiation, probably linked to the PPAR gamma pathway. Using a subtraction protocol, the genes specifically regulated by TCL/TC10 beta L were also isolated. The expression pattern of some was similar to TCL/TC10 beta L expression in adipogenesis, suggesting that these genes are regulated by TCL/TC10 beta L.

Identification of Inducible Genes at the Early Stage of Adipocyte Differentiation of 3T3-L1 Cells

Adipocyte differentiation takes place via a complex series of steps. While PPARγ2 and C/EBPα are known to be master regulators, the events at the earliest stage of adipocyte differentiation are not yet known. In this study, we cloned the genes which are induced at the beginning of differentiation of 3T3-L1 preadipocyte cells. Of 58 clones obtained, only a few were already reported as the genes that are expressed differentially during adipocyte development. More than 30 clones are known but have been newly identified here as differentially expressed genes. Nineteen clones seemed to be unknown genes. The expression of RGS2, HSP105, Rho (TC10), VDR, and HIF-1α genes isolated here rapidly increased after the addition of inducers, and after 3–12 h the levels of expression decreased. The expression patterns of these mRNAs were different among growth-arrested and proliferating 3T3-L1 cells and NIH-3T3 cells, strongly indicating that some of the proteins identified here have crucial roles in the program of adipocyte differentiation.