N-Acetylcysteine affects obesity-related protein expression in 3T3-L1 adipocytes

Abstract

Objectives

Oxidative stress plays critical roles in the pathogeneses of diabetes, hypertension, and atherosclerosis, but its effect on fat accumulation is still unclear. In this study, we analyzed the role of the well-known antioxidant and a glutathione (GSH) precursor N-acetylcysteine (NAC) in fat accumulation and the expression of obesity-associated proteins.

Methods

We studied the effects of 10 µM NAC on obesity-related protein expression in cultured 3T3-L1 preadipocytes, which are able to differentiate into mature adipocytes and accumulate lipids.

Results

NAC treatment inhibited fat accumulation and reduced the expression of obesity-related proteins, including monoamine oxidase A, heat shock protein 70 (HSP70), aminoacylase -1 (ACY-1), and transketolase.

Discussion

Our results suggest that the effects of NAC on triglycerides (Tgs) and protein expression are correlated. In support of this, we showed that NAC treatment affected both the Tg synthesis pathway and the expression levels of proteins implicated in human obesity.     

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpβ and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.     

Extracellular redox environments regulate adipocyte differentiation

Oxidized extracellular redox states have been associated with many diseases related to obesity, including heart disease and diabetes, but relatively little is known about the relationship between extracellular redox states and obesity. In 3T3-L1 preadipocytes, oxidizing extracellular redox potentials (E_h) increased intracellular and mitochondrial reactive oxygen species (ROS) production. 3T3-L1 adipocytes showed a greater response to extracellular E_h, producing more intracellular ROS, than preadipocytes. 3T3-L1 adipocytes also produced more extracellular ROS and re-regulated the extracellular E_h to a more oxidizing state than preadipocytes. During 3T3-L1 differentiation, cellular glutathione and mitochondrial thioredoxin-2 become oxidized, suggesting that adipogenesis may be enhanced under conditions promoting intracellular and mitochondrial compartment oxidation. Under various extracellular E_h, 3T3-L1 adipogenesis, as determined by lipid accumulation and the expression of early genetic markers of adipogenesis, was sensitive to the extracellular redox environment, where it was enhanced under oxidizing conditions and lower under reducing conditions. Using a diet-induced obesity mouse model, plasma was collected before and after the 8 week diet regimens. Plasma GSH E_h was unchanged as a consequence of weight gain but plasma cystiene (Cys) E_h was significantly oxidized in overweight animals. Data presented here show that adipocytes/excessive adipose preferentially alter extracellular E_h to a more oxidized state in vivo and in vitro and may promote further adipogenesis.     

Increasing cAMP levels of preadipocytes by cyanidin-3-glucoside treatment induces the formation of beige phenotypes in 3T3-L1 adipocytes

Obesity is a serious health problem and a major risk factor for the onset of several diseases such as heart disease, diabetes, stroke and cancer. The conversion of white adipocytes to brown-like adipocytes, also called beige or brite adipocytes, by pharmacological and dietary compounds has gained attention as an effective treatment for obesity. Cyanidin-3-glucoside (Cy3G), a polyphenolic compound contained in black soybean, blueberry and grape, has several antiobesity effects. However, there are no reports on the role of Cy3G in the induction of differentiation of preadipocytes to beige adipocytes and corresponding phenotypes. Here, the formation of beige adipocyte phenotypes following treatment with Cy3G was evaluated using 3T3-L1 adipocytes. Cy3G induced phenotypic changes to white adipocytes, such as increased multilocular lipid droplets and mitochondrial content. Additionally, the expression of mitochondrial genes (TFAM, SOD2, UCP-1 and UCP-2), UCP-1 protein and beige adipocyte markers (CITED1 and TBX1) in 3T3-L1 adipocytes was increased by Cy3G. Furthermore, Cy3G promoted preadipocyte differentiation by up-regulating of C/EBPβ through the elevation of the intracellular cAMP levels. These results indicated that Cy3G elevates the intracellular cAMP levels, which induces beige adipocyte phenotypes. This is the first report on the effect of Cy3G on induction of differentiation of preadipocytes into beige adipocyte phenotypes.     

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.     

Interleukin-4 mediates STAT6 activation in 3T3-L1 preadipocytes but not adipocytes

STAT6 is abundantly expressed in 3T3-L1 preadipocytes and adipocytes but activating ligands are not well defined. In this report, we provide evidence that interleukin 4 (IL-4) induced JAK2-mediated STAT6 tyrosine phosphorylation and DNA binding in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. Loss of IL-4-mediated STAT6 tyrosine phosphorylation occurred 2 days after preadipocytes were induced to differentiate into adipocytes but when cells remained phenotypically preadipocytes. 3T3-L1 adipocytes were still responsive to IL-4 through tyrosine phosphorylation of other cellular proteins. We conclude that IL-4 signals through STAT6 in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. This differentiation-dependent loss of STAT6 activation may be critical for distinct biological effects of IL-4 in 3T3-L1 preadipocytes and adipocytes.     

Efficient adenoviral transduction of 3T3-F442A preadipocytes without affecting adipocyte differentiation

The preadipocyte cell lines 3T3-L1 and 3T3-F442A are widely used to study the cellular mechanisms of preadipocyte differentiation and mature adipocyte functions. However, transfection with naked DNA is inefficient in these cell lines. Adenoviral gene transfer is a powerful technique to induce high levels of transgene expression. After failing to obtain 3T3-F442A stable transfectants, we studied different techniques designed to enhance the efficiency of adenoviral transduction in fat cells. First, we compared the effects of two agents known to significantly enhance adenoviral transgene transduction, namely the cationic lipid lipofectamine and the cationic polymer polylysine. We show here that lipofectamine-assisted adenoviral transduction was more efficient in 3T3-F442A than in 3T3-L1 preadipocytes at all tested multiplicity of infection. Lipofectamine, and more efficiently polylysine, yielded high and sustained levels of adenoviral transgene expression in 3T3-F442A preadipocytes. Adenoviral transgene expression was maintained throughout the differentiation process. Furthermore, the two agents also efficiently enhanced adenoviral transduction in mature 3T3-F442A adipocytes. Interestingly, neither protocol affected the differentiation process, morphological features or protein expression of mature adipocytes. These approaches could be of interest to study fat cell differentiation and the functions of mature adipocytes.     

Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway

Thyroid-stimulating hormone (TSH) has been shown to play an important role in the regulation of triglyceride (TG) metabolism in adipose tissue. Adipose triglyceride lipase (ATGL) is a rate-limiting enzyme controlling the hydrolysis of TG. Thus far, it is unclear whether TSH has a direct effect on the expression of ATGL. Because TSH function is mediated through the TSH receptor (TSHR), TSHR knockout mice (Tshr-/- mice) (supplemented with thyroxine) were used in this study to determine the effects of TSHR deletion on ATGL expression. These effects were verified in 3T3-L1 adipocytes and potential underlying mechanisms were explored. In the Tshr-/- mice, ATGL expression in epididymal adipose tissue was significantly increased compared with that in Tshr+/+ mice. ATGL expression was observed to increase with the differentiation process of 3T3-L1 preadipocytes. In mature 3T3-L1 adipocytes, TSH significantly suppressed ATGL expression at both the protein and mRNA levels in a dose-dependent manner. Forskolin, which is an activator of adenylate cyclase, suppressed the expression of ATGL in 3T3-L1 adipocytes. The inhibitory effects of TSH on ATGL expression were abolished by H89, which is a protein kinase A (PKA) inhibitor. These results indicate that TSH has an inhibitory effect on ATGL expression in mature adipocytes. The associated mechanism is related to PKA activation.     

Regulation of adipocytokine secretion and adipocyte hypertrophy by polymethoxyflavonoids, nobiletin and tangeretin

AimsThe polymethoxyflavonoids nobiletin and tangeretin possess several important biological properties such as neuroprotective, antimetastatic, anticancer, and anti-inflammatory properties. The present study was undertaken to examine whether nobiletin and tangeretin could modulate adipocytokine secretion and to evaluate the effects of these flavonoids on the hypertrophy of mature adipocytes.Main methodsAll experiments were performed on the murine preadipocyte cell line 3T3-L1. We studied the formation of intracellular lipid droplets in adipocytes and the apoptosis-inducing activity to evaluate the effects of polymethoxyflavonoids on adipocyte differentiation and hypertrophy, respectively. The secretion of adipocytokines was measured using ELISA.Key findingsWe demonstrated that the combined treatment of differentiation reagents with nobiletin or tangeretin differentiated 3T3-L1 preadipocytes into adipocytes possessing less intracellular triglyceride as compared to vehicle-treated differentiated 3T3-L1 adipocytes. Both flavonoids increased the secretion of an insulin-sensitizing factor, adiponectin, but concomitantly decreased the secretion of an insulin-resistance factor, MCP-1, in 3T3-L1 adipocytes. Furthermore, nobiletin was found to decrease the secretion of resistin, which serves as an insulin-resistance factor. In mature 3T3-L1 adipocytes, nobiletin induced apoptosis; tangeretin, in contrast, did not induce apoptosis, but suppressed further triglyceride accumulation.SignificanceOur results suggest that nobiletin and tangeretin are promising therapeutic candidates for the prevention and treatment of insulin resistance by modulating the adipocytokine secretion balance. We also demonstrated the different effects of nobiletin and tangeretin on mature adipocytes.     

Inhibitory effect of p53 on mitochondrial content and function during adipogenesis

The p53 protein is known as a guardian of the genome and is involved in energy metabolism. Since the metabolic system is uniquely regulated in each tissue, we have anticipated that p53 also would play differential roles in each tissue. In this study, we focused on the functions of p53 in white adipose tissue (adipocytes) and skeletal muscle (myotubes), which are important peripheral tissues involved in energy metabolism. We found that in 3T3-L1 preadipocytes, but not in C2C12 myoblasts, p53 stabilization or overexpression downregulates the expression of Ppargc1a, a master regulator of mitochondrial biogenesis. Next, by using p53-knockdown C2C12 myotubes or 3T3-L1 preadipocytes, we further examined the relationship between p53 and mitochondrial regulation. In C2C12 myoblasts, p53 knockdown did not significantly affect Ppargc1a expression and mtDNA, but did suppress differentiation to myotubes, as previously reported. However, in 3T3-L1 preadipocytes and mouse embryonic fibroblasts, p53 downregulation enhanced both differentiation into adipocytes and mitochondrial DNA content. Furthermore, p53-depleted 3T3-L1 cells showed increase in mitochondrial proteins and enhancement of both Citrate Synthase and Complex IV activities during adipogenesis. These results show that p53 differentially regulates cell differentiation and mitochondrial biogenesis between adipocytes and myotubes, and provide evidence that p53 is an inhibitory factor of mitochondrial regulation in adipocyte lineage.     

Phenols from Potentilla anserina L. Improve Insulin Sensitivity and Inhibit Differentiation in 3T3-L1 Adipocytes in Vitro

Potentilla anserina L., a well-known perennial herb, is widely used in traditional Tibetan medicine and used as a delicious food in humans. The present investigation reports on the activity of P. anserina phenols (PAP) in regulating glycolipid metabolism in 3T3-L1 adipocytes. Insulin sensitivity tests showed that PAP improved insulin-stimulated glucose uptake by promoting the phosphorylation of serine/threonine kinase Akt. Moreover, an assay involving the differentiation of 3T3-L1 preadipocytes demonstrated that PAP also decreased the accumulation of lipid droplets by suppressing the expression of adipokines during the differentiation process. In addition, the underlying mechanism from the aspects of energy metabolism and oxidative stress is also discussed. The improvement in energy metabolism was supported by an increase in mitochondrial membrane potential (MMP) and intracellular ATP. Amelioration of oxidative stress was supported by decreased levels of intracellular reactive oxygen species (ROS). In summary, our findings suggest that PAP can ameliorate the disorder of glycolipid metabolism in insulin resistant 3T3-L1 adipocytes by improving energy metabolism and oxidative stress and might be an attractive candidate for the treatment of diabetes.     

胰岛素对3T3-L1脂肪细胞中极低密度脂蛋白受体基因表达的影响

体外培养3T3-L1细胞分化模型,研究不同浓度胰岛素及慢性胰岛素刺激对3T3-L1脂肪细胞中极低密度脂蛋白受体（VLDLR）基因表达的影响.在不同浓度胰岛素及胰岛素慢性刺激的干预下,用半定量RT-PCR检测细胞VLDLR mRNA水平的变化.微量化GOD-PAP法检测培养基中残存的葡萄糖.在细胞诱导分化过程中,胰岛素浓度的增高促进VLDLR的表达;胰岛素慢性刺激下,VLDLR表达因浓度差异呈现不同变化.研究结果表明,胰岛素的浓度及慢性刺激对3T3-L1脂肪细胞的成熟和VLDLR基因的表达有显著作用,而胰岛素抵抗明显减低成熟脂肪细胞VLDLR的表达.     

Distinct hypoxic regulation of preadipocyte factor-1 (Pref-1) in preadipocytes and mature adipocytes

Preadipocyte factor-1 (Pref-1) is a secretory soluble protein, which exerts pleiotropic effects on maintenance of cancer stem cell characteristics and commitment of mesenchymal stem cell lineages by inhibiting adipogenesis. Observations that obesity renders the microenvironment of adipose tissues hypoxic and that hypoxia inhibits adipogenesis lead us to investigate whether hypoxia increases the expression of anti-adipogenic Pref-1 in preadipocytes, mature adipocytes, and adipose tissues from obese mouse. In 3T3-L1 preadipocytes, hypoxia induces Pref-1 by a hypoxia-inducible factor 1 (HIF-1)-dependent mechanism accompanied by increase in the levels of the active histone mark, acetylated H3K9/14 (H3K9/14Ac). Adipogenesis increased the levels of the heterochromatin histone mark, trimethylated H3K27 (H3K27me3), whereas it decreased the levels of H3K4me3 and H3K9/14Ac euchromatin marks of the mouse Pref-1 promoter. However, differently from preadipocytes, in mature adipocytes hypoxia failed to reverse the repressive epigenetic changes of Pref-1 promoter and to increase its expression. Short term (8 weeks) high fat diet (HFD) increased HIF-1α protein in subcutaneous and epididymal adipose tissues, but did not increase Pref-1 expression. Unlike in 3T3-L1 preadipocytes, HIF-1α did not increase Pref-1 expression in adipose tissues in which mature adipocytes constitute the main population. Interestingly, long term (35 weeks) HFD increased Pref-1 in serum but not in obese adipose tissues. This study suggests that Pref-1 is an endocrine factor which is synergistically increased by obesity and age.     

Rehmannia inhibits adipocyte differentiation and adipogenesis

Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPβ, as well as C/EBPα and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPβ to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.     

Changes in IGF-I receptor and IGF-I mRNA during differentiation of 3T3-L1 preadipocytes

Insulin-like growth factor-1 (IGF-I) is an essential factor for the differentiation of preadipocytes into adipocytes. We investigated the expression of IGF-I receptor and IGF-I RNA messenger during 3T3-L1 preadipocyte differentiation. Levels of IGF-I receptor decreased in the mature adipocytes compared to cells before the initiation of differentiation. In addition, cultures not induced to differentiate showed a decrease on the receptor levels after 4 days in the presence of insulin compared to cultures without treatment. The levels of the IGF-I RNA messenger were shown to be higher in mature adipocytes compared to preadipocytes. We propose an autocrine and/or paracrine action of IGF-I in this adipocyte differentiation model, where IGF-I produced by the differentiating preadipocytes acts over their adjacent cells and, in this way, diminishes the expression of IGF-I receptor.