Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes

Sirt1, a NAD⁺-dependent histone deacetylase, may regulate senescence, metabolism, and apoptosis. In this study, primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1, FoxO1, and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression, but decreased the expression of FoxO1 and adipocyte marker gene PPARγ2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA, but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion, these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1.     

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.     

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.     

CCAAT/enhancer-binding protein-β participates in oxidized LDL-enhanced proliferation in 3T3-L1 cells

Increased circulating oxidized LDL (oxLDL) have been found in obese subjects. Obesity is characterized by an excess of fat mass resulting from an increase in adipocyte number and size. The generation of new adipocytes is a tightly controlled process where multiple factors acting in a signaling cascade follow a precise temporal expression pattern; oxLDL appear to have a role in the impairment of this process. The purpose of this study was to examine the effects of oxLDL on the mechanisms involved in the proliferative stage of the differentiation process in 3T3-L1 cells. After hormonal induction, 3T3-L1 cells undergo approximately two rounds of mitotic clonal expansion (MCE), a process required for adipogenesis. CCAAT/enhancer-binding protein β (C/EBPβ) is immediately expressed after induction, and plays a crucial role in MCE, but its expression must decrease to allow preadipocytes to mature into adipocytes. We found that, in the presence of stimuli to differentiate, oxLDL induced a higher proliferation rate in this cell line, associated with a sustained up-regulation of C/EBPβ, which remained activated inside the nucleus for several days. RNAi-mediated knockdown of C/EBPβ 24 h after oxLDL treatment counteracted the increase in proliferation rate. Both C/EBPβ expression and proliferation processes appear to be influenced by cAMP/protein kinase A (PKA) and extracellular signal-regulated kinases1/2 (ERK1/2) pathways. OxLDL treatment led to increased levels of cAMP, and to a strong, prolonged phosphorylation of ERK1/2 and C/EBPβ. The addition of cAMP and PKA inhibitors, SQ22536 and H-89, respectively, reduced proliferation only in oxLDL-treated cells, whereas the addition of ERK1/2 inhibitor U0126 blocked proliferation in both control and oxLDL-treated cells. C/EBPβ nuclear expression and DNA-binding activity were reduced by U0126, under all tested conditions. These findings show that the altered expression pattern of C/EBPβ is involved in the increase in the number of proliferating cells induced by oxLDL, in hormone-stimulated 3T3-L1 cells.     

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

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.     

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.     

白藜芦醇对猪原代前体脂肪细胞的增殖与分化及Sirt1基因转录表达时序的影响

分别以0μmol/L(对照组)、10μmol/L(低剂量组),20μmol/L(中等剂量组),50μmol/L,100μmol/L(高剂量组)的白藜芦醇(Resveratrol,RES)处理体外培养1~3日龄健康仔猪前体脂肪细胞,采用MTT比色法检测细胞活性及增殖状况;油红O染色化学比色法定量分析细胞内脂肪生成及细胞分化程度;RT-PCR法分析Sirt1(sirtuin1)mRNA表达情况,探讨Sirt1对猪前体脂肪细胞增殖分化的影响及其分子机制。结果表明,脂肪细胞经RES处理后,各组MTT和油红O染色测得的光密度值(OD值)均低于对照组,50μmol/L,100μmol/L组在96~120h作用极显著(P<0.01),与中低剂量组差异显著(P<0.05);以20μmol/L,100μmol/LRES处理细胞后,Sirt1mRNA表达量随细胞分化的进行而逐渐升高,100μmol/L组均显著高于对照组和20μmol/L组(P<0.05)。RES对猪前体脂肪细胞增殖分化均有一定抑制作用,高剂量RES(50μmol/L和100μmol/L)可显著减少细胞内脂肪的合成、抑制脂肪细胞增殖与分化,Sirt1mRNA表达量显著升高可能是RES抑制细胞分化的重要原因之一。     

Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression

Evidence has accumulated that ginseng and its main active constituents, ginsenosides, possess anti-diabetic and insulin-sensitizing properties which may be partly realized by regulating adipocyte development and functions. In the present study, we explored the effect of ginsenoside Rb(1), the most abundant ginsenoside in ginseng root, on adipogenesis of 3T3-L1 cells. We found that with standard differentiation inducers, ginsenoside Rb(1) facilitated adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner; 10 microM Rb(1) increased lipid accumulation by about 56%. Treatment of differentiating adipocytes with 10 microM Rb(1) increased the expression of mRNA and protein of PPARgamma(2) and C/EBPalpha, as well as mRNA of ap2, one of their target genes. After the treatment of differentiating adipocytes with Rb(1), basal and insulin-mediated glucose uptake was significantly augmented, accompanied by the up-regulation of mRNA and protein level of GLUT4, but not of GLUT1. In addition, ginsenoside Rb(1) also inhibited the proliferation of preconfluent 3T3-L1 preadipocytes. Our data indicate that anti-diabetic and insulin-sensitizing activities of ginsenosides, at least in part, are involved in the enhancing effect on PPARgamma2 and C/EBPalpha expression, hence promoting adipogenesis.     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.     

Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响

长链非编码RNA (long non-coding RNA, lncRNA)是一类长度大于200nt、没有长开放阅读框架但往往具有mRNA结构特征的RNA,可以在转录及转录后水平参与基因的表达调控。近年来,有研究证实lncRNA对脂肪生成具有重要作用。Lnc-RAP3位于小鼠(Mus musculus)17号染色体,其表达量在小鼠脂肪细胞分化前后呈现显著差异,但其具体的生物学功能尚不清楚。为探讨lnc-RAP3在小鼠3T3-L1前脂肪细胞成脂分化中的作用,本文首先构建了lnc-RAP3的真核表达载体pcDNA3.1-RAP3,利用脂质体将pcDNA3.1-RAP3和人工合成的lnc-RAP3的siRNAs分别转染3T3-L1前脂肪细胞,并对转染后的细胞进行诱导分化,并通过油红O染色、qRT-PCR检测成脂分化相关基因表达等方法比较过表达和敲降lnc-RAP3对3T3-L1前脂肪细胞成脂分化的影响。结果显示,过表达lnc-RAP3后,细胞内脂滴聚集显著减少(P<0.05),在诱导分化第0 d、2 d和4 d时C/EBPα、Glut4、PPARγ、LPL和FAS的表达水平均呈显著(P<0.05)或极显著(P<0.01)下降;敲降lnc-RAP3后,细胞内脂滴聚集显著增多(P<0.05),同时在诱导分化第0 d、2 d时PPARγ、LPL、C/EBPα、FAS和Glut4的表达水平呈显著(P<0.05)或极显著(P<0.01)升高。本研究结果表明,lnc-RAP3可能通过影响成脂分化相关基因的表达来抑制3T3-L1前脂肪细胞的成脂分化。     

Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

Although fatty acids enhance preadipocyte differentiation in the presence of adequate hormone cocktails, little is known regarding their effects in the absence of these hormones. We have now shown that palmitate, a common long-chain saturated fatty acid, induced apoptosis in both mouse 3T3-L1 and rat primary preadipocytes grown in a normal serum-containing medium. Treatment of preadipocytes with palmitate induced multiple endoplasmic reticulum (ER) stress responses, evidenced by increased protein content of CHOP and GRP78 and splicing of XBP-1 mRNA, as well as altered phosphorylation of eIF2alpha and increased phosphorylation of JNK and Erk1/2. Intriguingly, palmitate induced an early activation of Akt but diminished both Akt activation and its protein mass after prolonged incubation (>6 h). In association with these changes, palmitate reduced expression of beta-catenin and its downstream target, c-Myc and cyclin D1, two key prosurvival proteins. Overexpression of constitutively active Akt did not block the apoptotic effect of palmitate. Cotreatment with unsaturated fatty acids (oleate, linoleate) or with LiCl (a glycogen synthase kinase-3beta inhibitor) attenuated the palmitate-induced apoptosis. Subsequent analysis suggested that the unsaturated fatty acids probably counteracted palmitate by reducing, not eliminating, ER stress, whereas LiCl probably improved viability by activating the Wnt signaling pathway. Cotreatment of palmitate with a standard adipogenic hormone cocktail also abolished the apoptotic effect and promoted adipocyte differentiation. Collectively, our results suggest that palmitate causes multiple cellular stresses that may lead to apoptosis in preadipocytes in the absence of adipogenic stimuli, highlighting the importance of exogenous hormones in directing cell fate in response to increased fatty acid influx.     

长链非编码RNAIFNG-AS1靶向miR-19b-1-5p调控oxLDL诱导的血管内皮细胞增殖、凋亡的机制研究

为了探讨长链非编码RNA干扰素活化基因的反义核糖核酸(lncRNA IFNG-AS1)对氧化型低密度脂蛋白(oxLDL)诱导的人脐静脉血管内皮细胞EVC-304增殖、凋亡的影响和调控机制,该研究采用100 μg/mL的oxLDL分别处理转染si-IFNG-AS1、miR-19b-1-5p mimics或共转染si-IF...     

SiRNA against Fabp5 induces 3T3-L1 cells apoptosis during adipocytic induction

Fatty acid-binding protein 5 (Fabp5), exhibits an important role in binding free fatty acids, as well as regulating lipid metabolism and transport. The purpose of this study was to evaluate the role of Fabp5 during adipogenesis. 3T3-L1 preadipocytes were selected as cell differentiation model and short interfering RNAs (siRNA) against Fabp5 (siFabp5) were prepared. Our results showed that two potent siFabp5 specifically inhibited endogenous expression of Fabp5 at both mRNA and protein level. SiFabp5 had little effect on undifferentiated 3T3-L1 fibroblasts. However, during adipocytic induction, 3T3-L1 preadipocytes transfected with siFabp5 significantly reduced cell viability, as well as increased both caspase-3 activity and procaspase-3 cleavage. Furthermore, we illustrated that knockdown Fabp5 inhibited the expression of PPARγ and C/EBPα during adipocytic induction. In conclusion, our data suggests that Fabp5 is crucial in maintaining the viability of preadipocytes during adipogenesis via the activation of Akt cascade, and decreased Fabp5 expression induce differentiating preadipocytes apoptosis via caspase-3 activation.     

alpha2- to beta3-Adrenoceptor switch in 3T3-L1 preadipocytes and adipocytes: modulation by testosterone, 17beta-estradiol, and progesterone

Sex steroid hormones are important factors in the determination of fat distribution and accumulation. The aim of this study was to investigate the effect of testosterone (T), 17beta-estradiol (17betaE), and progesterone (P) on adrenergic receptor (AR) gene expression in 3T3-L1 preadipocytes and adipocytes and their relation to the proliferation and differentiation processes. Our data clearly show that alpha(2A)-AR was the highest AR subtype expressed in preadipocytes, whereas in mature adipocytes was by far beta(3)-AR. In the differentiation process to adipocytes, alpha(2A)-AR expression was decreased to 0.3-fold (P < 0.01), whereas beta(3)-AR was upregulated 578-fold (P < 0.001) compared with preadipocytes. In addition, the expression of alpha(2A)-AR in preadipocytes was increased upon incubation with T, 17betaE, and P, and a stimulation of proliferation was also observed in 17betaE- and P-treated cells. In mature adipocytes, 17betaE and P enhanced both alpha(2A)- and beta(3)-AR gene expression (although the effects on beta(3)-AR mRNA levels could be more relevant, since beta(3)-AR was the most highly expressed), whereas T only increased alpha(2A)-AR mRNA levels. Leptin and adipocyte fatty acid-binding protein mRNA levels were higher after 17betaE and P treatment, possibly indicating a proadipogenic effect of these hormones. In conclusion, this study indicates that AR gene expression is affected by these hormones in both preadipocytes and adipocytes, which could have potential importance when considering the role of ARs in the mechanisms underlying the sex-related differences in adipose tissue regional distribution.