Genes In 3T3-L1 Adipocytes Research Articles

Germinated Brown Rice Extract Inhibits Adipogenesis Through the Down-regulation of Adipogenic Genes in 3T3-L1 Adipocytes

The aim of this study was to examine the anti-adipogenic effect of germinated brown rice methanol extract (GBR) in 3T3-L1 adipocytes. The GBR inhibited adipocyte differentiation was measured by Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner without initiating any cytotoxicity. The mRNA levels of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBPα), proliferator-activated receptorγ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c), and adipogenic genes, such as fatty acid synthase (FAS), adipocyte fatty acid-binding protein (aP2), and lipoprotein lipase (LPL), were significantly down-regulated by treatment with GBR when compared to that of untreated control cells. Moreover, tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) mRNA expressions were attenuated by GBR in mature adipocytes. These data suggest that GBR exhibits an anti-adipogenic effect through the suppression of adipogenesis in 3T3-L1 adipocytes.

Identification of meteorin and metrnl as two novel pro-differentiative adipokines: Possible roles in controlling adipogenesis and insulin sensitivity

Meteorin and its homologue meteorin-like (metrnl) are two novel proteins regulating glial cell differentiation in central nervous system. However, the roles of meteorin and metrnl in other organs or tissues have never been studied. In this study, we showed that meteorin and metrnl were expressed abundantly in white adipose, including visceral and subcutaneous adipose tissues, but to a much lesser extent in brown adipose tissue. Meteorin and metrnl were found to be secreted by explanted human white fat pads, 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes. Moreover, meteorin and metrnl were detected in human blood by immunoblotting assay. The mRNA expression of meteorin and metrnl increased along with the 3T3-L1 cells differentiation. Overexpression of meteorin and metrnl using adenovirus promoted the formation of lipid droplets, increased PPARγ protein expression and enhanced mRNA expression of several other adipogenic genes in 3T3-L1 adipocytes. Injection of adipose-specific aP2-promoter driven adenovirus expressing meteorin and metrnl markedly improved global insulin sensitivity and reversed insulin resistance induced by high fat diet in mice. At last, overexpression of meteorin and metrnl further magnified the phosphorylation of Akt upon insulin stimuli in 3T3-L1 cells. Collectively, our data suggests that meteorin and metrnl are two novel pro-differentiative adipokines participating in adipogenesis and regulation of insulin sensitivity.

A gene expression signature for insulin resistance

Insulin resistance is a heterogeneous disorder caused by a range of genetic and environmental factors, and we hypothesize that its etiology varies considerably between individuals. This heterogeneity provides significant challenges to the development of effective therapeutic regimes for long-term management of type 2 diabetes. We describe a novel strategy, using large-scale gene expression profiling, to develop a gene expression signature (GES) that reflects the overall state of insulin resistance in cells and patients. The GES was developed from 3T3-L1 adipocytes that were made "insulin resistant" by treatment with tumor necrosis factor-α (TNF-α) and then reversed with aspirin and troglitazone ("resensitized"). The GES consisted of five genes whose expression levels best discriminated between the insulin-resistant and insulin-resensitized states. We then used this GES to screen a compound library for agents that affected the GES genes in 3T3-L1 adipocytes in a way that most closely resembled the changes seen when insulin resistance was successfully reversed with aspirin and troglitazone. This screen identified both known and new insulin-sensitizing compounds including nonsteroidal anti-inflammatory agents, β-adrenergic antagonists, β-lactams, and sodium channel blockers. We tested the biological relevance of this GES in participants in the San Antonio Family Heart Study (n = 1,240) and showed that patients with the lowest GES scores were more insulin resistant (according to HOMA_IR and fasting plasma insulin levels; P < 0.001). These findings show that GES technology can be used for both the discovery of insulin-sensitizing compounds and the characterization of patients into subtypes of insulin resistance according to GES scores, opening the possibility of developing a personalized medicine approach to type 2 diabetes.

Bioinformatic Analysis of Gene Sets Regulated by Ligand-Activated and Dominant-Negative Peroxisome Proliferator–Activated Receptor γ in Mouse Aorta

Drugs that activate peroxisome proliferator-activated receptor (PPAR) gamma improve glucose sensitivity and lower blood pressure, whereas dominant-negative mutations in PPARgamma cause severe insulin resistance and hypertension. We hypothesize that these PPARgamma mutants regulate target genes opposite to those of ligand-mediated activation, and we tested this hypothesis on a genomewide scale. We integrated gene expression data in aorta specimens from mice treated with the PPARgamma ligand rosiglitazone with data from mice containing a globally expressed knockin of the PPARgamma P465L dominant-negative mutation. We also integrated our data with publicly available data sets containing the following: (1) gene expression profiles in many human tissues, (2) PPARgamma target genes in 3T3-L1 adipocytes, and (3) experimentally validated PPARgamma binding sites throughout the genome. Many classic PPARgamma target genes were induced by rosiglitazone and repressed by dominant-negative PPARgamma. A similar pattern was observed for about 90% of the gene sets regulated by both rosiglitazone and dominant-negative PPARgamma. Genes exhibiting this pattern of contrasting regulation were significantly enriched for nearby PPARgamma binding sites. These results provide convincing evidence that the PPARgamma P465L mutation causes transcriptional effects that are opposite to those mediated by PPARgamma ligand, thus validating mice carrying the mutation as a model of PPARgamma interference.

Early Soy Exposure via Maternal Diet Regulates Rat Mammary Epithelial Differentiation by Paracrine Signaling from Stromal Adipocytes1–3

Diet-mediated changes in transcriptional programs that promote the early differentiation of the mammary gland may lead to reduced breast cancer risk. The disparity in adult breast cancer incidence between Asian women and Western counterparts is attributed partly to high soy food intake. Here, we conducted genome-wide profiling of mammary tissues of weanling rats exposed to soy protein isolate (SPI) or control casein (CAS) via maternal diet to evaluate the contribution of early exposure on mammary gene expression. Of the identified 18 up- and 39 downregulated genes with SPI relative to CAS, a subset was associated with lipid metabolic pathways, consistent with reduced mammary adipocyte size and suggesting stromal adipocyte-specific genomic changes. Female offspring of rats fed SPI tended to have fewer terminal end buds (P = 0.06) and had significantly lower body weight and abdominal fat mass. To demonstrate the functional consequence of SPI-mediated adipocyte metabolic changes on neighboring mammary epithelium, the expression of in vivo regulated genes in 3T3-L1 adipocytes treated with soy isoflavone genistein and effects of the resultant conditioned medium (CM) on the differentiation of HC11 mammary epithelial cells were evaluated by quantitative RT-PCR and/or Western immunoblots. In differentiated 3T3-L1, genistein decreased fatty acid synthase and stearoyl-CoA desaturase and increased hydroxysteroid 11-β dehydrogenase 1 expression. CM from genistein-treated adipocytes had higher adiponectin levels and augmented prolactin-induced, glucocorticoid-regulated β-casein levels. These findings suggest that soy-associated components, by targeting mammary adipocytes, alter paracrine signaling to enhance mammary epithelial differentiation, with important implications for the prevention of breast cancer associated with obesity and obesity-related diseases.

Insulin Increases Tristetraprolin and Decreases VEGF Gene Expression in Mouse 3T3–L1 Adipocytes

Tristetraprolin (TTP) family proteins (TTP/ZFP36; ZFP36L1, ZFP36L2, ZFP36L3) destabilize adenylate uridylate-rich element-containing mRNAs encoding cytokines, such as tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Little is known about the expression and insulin regulation of TTP and related genes in adipocytes. We analyzed the relative abundance of TTP family mRNAs in 3T3-L1 adipocytes compared to RAW264.7 macrophages and investigated insulin effects on the expression of 43 genes in 3T3-L1 adipocytes. Insulin was added to mouse 3T3-L1 adipocytes. Relative abundance of mRNA levels was determined by quantitative real-time PCR. TTP and ZFP36L1 proteins were detected by immunoblotting. Zfp36l1 and Zfp36l2 genes were expressed at eight- to tenfold higher than Ttp in adipocytes. Zfp36l3 mRNA was detected at approximately 1% of Ttp mRNA levels in adipocytes and its low level expression was confirmed in RAW cells. Insulin at 10 and 100 nmol/l increased Ttp mRNA levels by five- to sevenfold, but decreased those of Zfp36l3 by 40% in adipocytes after a 30-min treatment. Immunoblotting showed that insulin induced TTP but did not affect ZFP36L1 protein levels in adipocytes. Insulin decreased mRNA levels of Vegf and a number of other genes in adipocytes. Insulin induced Ttp mRNA and protein expression and decreased Vegf mRNA levels in adipocytes. Zfp36l3 mRNA was detected, for the first time, in cells other than mouse placenta and extraembryonic tissues. This study established a basis for the investigation of TTP and VEGF genes in the regulation of obesity and suggested that Vegf mRNA may be a target of TTP in fat cells.

Effects of Statins on Adipose Tissue Inflammation

Macrophage-mediated chronic inflammation of adipose tissue is causally linked to insulin resistance in obesity. The beneficial effects of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors (statins) on glucose metabolism have been suggested, but the effects of these agents on adipose tissue inflammation are unclear. The aim of the present study is to define the effects of statins on adipose tissue inflammation and macrophages. Pravastatin or pitavastatin treatment of obese mice attenuated an increase in mRNA expressions of proinflammatory genes, including MCP1 and IL6, in adipose tissue. The supernatant of TLR4-stimulated RAW264 macrophages strongly induced the expression of these genes in 3T3-L1 adipocytes, which was inhibited by pretreatment of macrophages with either statin. Statins inhibited TLR4-mediated activation of interferon (IFN) regulatory factor (IRF)3 by either lipopolysaccharide (LPS) or palmitic acid, resulting in suppression of IFN-beta expression, but not that of NF-kappaB or JNK. Moreover, statins strongly downregulated TLR3-mediated gene expressions by poly(I:C), but not TLR2-stimulation by zymosan A. Neutralization of IFN-beta attenuated proinflammatory activities of the macrophage supernatant. Statins partially attenuated the development of adipose tissue inflammation in obese mice, which might be associated with an inhibitory effect of statins on TLR4-triggered expression of IFN-beta via MyD88-independent signaling pathway in macrophages.

Glucocorticoids reciprocally regulate expression of the CCAAT/enhancer-binding protein alpha and delta genes in 3T3-L1 adipocytes and white adipose tissue.

Glucocorticoid agonists, i.e. dexamethasone or triamcinolone acetonide, rapidly induce expression of CCAAT/enhancer-binding protein (C/EBP) delta and repress expression of C/EBP alpha in fully differentiated 3T3-L1 adipocytes. Within 30 min of glucocorticoid treatment, the cellular level of C/EBP delta rises dramatically, increasing > 100-fold within 6 h. Concurrently, the level of C/EBP alpha decreases, reaching a minimum within 4 h. The dexamethasone concentration dependence and steroid specificity of these responses suggest that both processes are mediated by the glucocorticoid receptor. The reciprocal effects of dexamethasone on the steady-state levels of C/EBP alpha and C/EBP delta can be accounted for kinetically and quantitatively by changes in their mRNA levels and by the transcription rates of their respective genes. The glucocorticoid-induced changes in expression of the C/EBP isoforms are correlated with the transcriptional activation of the SCD1 gene, an adipocyte gene known to be transactivated by C/EBP isoforms. Glucocorticoids also regulate expression of the C/EBP isoforms in vivo. Within 4 h of administration of dexamethasone or triamcinolone acetonide to adult rats, expression of C/EBP delta is induced in white adipose tissue while expression of C/EBP alpha is repressed. Like the response in 3T3-L1 adipocytes, the effects of dexamethasone on C/EBP alpha in white adipose tissue are rapid and transient.

Transcriptional repression of the C/EBP-alpha and GLUT4 genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha. Regulations is coordinate and independent of protein synthesis.

We have previously demonstrated the ability of tumor necrosis factor-alpha (TNF) to down-regulate the expression of GLUT4 (insulin-responsive glucose transporter) and C/EBP-alpha (CCAAT/enhancer-binding protein) (Stephens J. M., and Pekala, P. H. (1991) J. Biol. Chem. 266, 21839-21845). As C/EBP-alpha has been suggested to control GLUT4 expression, we have examined the time course for attenuation of transcription of these genes. Run-on transcription assays indicate a coordinate transcriptional repression of both GLUT4 and C/EBP-alpha genes (as well as the 422/aP2 gene, the adipocyte lipid-binding protein, whose expression has also been proposed to be controlled by C/EBP-alpha). Inhibition of transcription was observed within 1 h of TNF addition, with maximal suppression observed after 4 h. The inhibition was not blocked by cycloheximide. Okadaic acid treatment (1 h, 0.5 microM) also resulted in the coordinate transcriptional repression of the C/EBP-alpha, GLUT4, and 422/aP2 genes, consistent with involvement of a kinase-phosphatase system in the regulation of these genes. The decrease in C/EBP-alpha protein content was detectable 4 h after TNF addition and declined to 25% of controls within 24 h. A minor decrease in the protein content of GLUT4 was observed during the first 24 h of exposure to TNF; however, after 72 h of exposure GLUT4 protein was not detectable. The rapid coordinate transcriptional regulation of C/EBP-alpha, GLUT4, and 422/aP2 by TNF in the presence of cycloheximide suggests that the TNF-induced loss of GLUT4 protein may be mediated by a post-translational modification of an existing transcription factor. However, the rapid loss of C/EBP-alpha protein may be a contributing factor to further transcriptional suppression of the GLUT4 gene at the later time points. In addition to the transcriptional effect, we report that TNF-induced destabilization of these mRNAs contributes to decreased expression of all three genes.

Transcriptional repression of the GLUT4 and C/EBP genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha.

Fully differentiated 3T3-L1 adipocytes were chronically exposed to 5 nM tumor necrosis factor-alpha (TNF). This resulted in the development of an insulin resistance based on the inability of insulin to stimulate hexose uptake. Western blot analysis for glucose transporter protein in isolated membrane fractions indicated a total depletion of GLUT4 protein (insulin-responsive glucose transporter) in cells chronically treated with TNF. Plasma membrane content of GLUT1 protein (growth-related glucose transporter) was similar in both control and TNF-treated cells; however, the GLUT1 content of the intracellular membrane compartment had decreased markedly after TNF treatment. Continuous exposure to TNF resulted in an 85-90% decrease in the mRNA content for both GLUT4 and 422 (aP2, a lipid binding protein) genes relative to age matched controls, whereas insulin receptor mRNA levels declined by at least 50%. This was preceded by a marked decrease in mRNA accumulation for C/EBP, a transcription factor proposed to control expression of both GLUT4 and 422. The specificity of these observations was demonstrated by the lack of an effect of the chronic TNF treatment on either beta-actin or lipoprotein lipase mRNA content. The decreased content of GLUT4 and C/EBP mRNA was judged to be regulated at least in part at the level of transcription, based on the results of transcription run-on assays. Thus, the lack of response to insulin appeared due to a suppression of GLUT4 expression as well as a decreased intracellular content of GLUT1.