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.     

黄芩素对猪前体脂肪细胞增殖分化的影响

研究黄芩素(BAI)对猪前体脂肪细胞增殖分化的影响,并探讨其可能的作用机制。原代培养猪前体脂肪细胞,采用油红O染色观察细胞分化的形态学变化;MTT检测细胞增殖状况;油红O染色提取定量分析细胞内脂肪生成及细胞分化程度;分光光度法测定脂肪酸合酶(FAS)的活性;逆转录-聚合酶链反应(RT-PCR)检测分化特异基因过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA表达变化。结果显示,前体脂肪细胞在分化成脂肪细胞的过程中,其形态由梭形变成椭圆形、圆形,细胞内充满大小不一的脂滴;BAI浓度在160～640μmol/L时显著抑制其增殖(P<0.05)、BAI浓度为40～320μmol/L时显著抑制PPARγ2mRNA表达和FAS的活性,并抑制细胞分化(P<0.05)。以上结果说明,BAI对前体脂肪细胞增殖分化均有一定抑制作用,BAI可能通过抑制PPARγ2mRNA表达和降低FAS活性,从而抑制猪前体脂肪细胞分化。     

Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis In 3T3-L1 adipocytes

AMP-activated protein kinase (AMPK) is a phylogenetically conserved intracellular energy sensor that has been implicated as a major regulator of glucose and lipid metabolism in mammals. However, its possible role in mediating or influencing the adrenergic control of lipolysis in adipocytes remains uncertain. In this study, we utilized the murine cultured preadipocyte line 3T3-L1 to examine this question. Treatment of adipocytes with isoproterenol or forskolin promoted the phosphorylation of AMPK at a critical activating Thr-172 residue in a dose- and time-dependent manner. This correlated well with a stimulation of the activity of AMPK, as measured in the immune complex. Analogs of cAMP mimicked the effect of isoproterenol and forskolin on AMPK phosphorylation. Treatment of adipocytes with insulin reduced both basal and forskolin-induced AMPK phosphorylation via a pathway dependent on phosphatidylinositol 3'-kinase. Overexpression of a dominant-inhibitory mutant of AMPK blocked isoproterenol-induced lipolysis by approximately 50%. These data indicate that there exists a novel pathway by which cAMP can lead to the activation of AMPK, and in adipocytes, this is required for maximal activation of lipolysis.     

Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes

Our previous studies have demonstrated that natriuretic peptides (NPs), peptide hormones with natriuretic, diuretic, and vasodilating properties, exert a potent control on the lipolysis in human adipocytes via the activation of the type A guanylyl cyclase receptor (1, 2). In the current study we investigated the intracellular mechanisms involved in the NP-stimulated lipolytic effect in human preadipocytes and adipocytes. We demonstrate that the atrial NP (ANP)-induced lipolysis in human adipocytes was associated with an enhanced serine phosphorylation of the hormone-sensitive lipase (HSL). Both ANP-mediated lipolysis and HSL phosphorylation were inhibited in the presence of increasing concentrations of the guanylyl cyclase inhibitor LY-83583. ANP did not modulate the activity of the cAMP-dependent protein kinase (PKA). Moreover, H-89, a PKA inhibitor, did not affect the ANP-induced lipolysis. On primary cultures of human preadipocytes, the ANP-mediated lipolytic effect was dependent on the differentiation process. On differentiated human preadipocytes, ANP-mediated lipolysis, associated with an increased phosphorylation of HSL and of perilipin A, was strongly decreased by treatment with the inhibitor of the cGMP-dependent protein kinase I (cGKI), Rp-8-pCPT-cGMPS. Thus, ANP-induced lipolysis in human adipocytes is a cGMP-dependent pathway that induces the phosphorylation of HSL and perilipin A via the activation of cGKI. The present study shows that lipolysis in human adipocytes can be controlled by an independent cGKI-mediated signaling as well as by the classical cAMP/PKA pathway.     

黄芩素对3T3-L1小鼠前脂肪细胞分化及对脂肪酸合成酶活性的影响

目的 研究黄芩素对3T3-L1小鼠前脂肪细胞分化及对脂肪酸合成酶活性的影响。方法 油红O染色法测定细胞分化速度;分光光度法测定脂肪酸合成酶活性。结果 黄芩素对3T3-L1小鼠前脂肪细胞向脂肪细胞分化以及对脂肪酸合成酶活性有抑制作用。结论 黄芩素通过阻断脂肪合成而抑制小鼠前脂肪细胞分化;黄芩素具有开发成减肥药物的潜力。     

The influence of preadipocyte differentiation capacity on lipolysis in human mature adipocytes.

The ability of catecholamines to maximally stimulate adipocyte lipolysis (lipolytic capacity) is decreased in obesity. It is not known whether the lipolytic capacity is determined by the ability of adipocytes to differentiate. The aim of the study was to investigate if lipolytic capacity is related to preadipocyte differentiation and if the latter can predict lipolysis in mature adipocytes. IN VITRO experiments were performed on differentiating preadipocytes and isolated mature adipocytes from human subcutaneous adipose tissue. In preadipocytes, noradrenaline-induced lipolysis increased significantly until terminal differentiation (day 12). However, changes in the expression of genes involved in lipolysis (hormone sensitive lipase, adipocyte triglyceride lipase, the alpha2-and beta1-adrenoceptors, perilipin, and fatty acid binding protein) reached a plateau much earlier during differentiation (day 8). A significant positive correlation between lipolysis in differentiated preadipocytes and mature adipocytes was observed for noradrenaline (r=0.5, p<0.01). The late differentiation capacity of preadipocytes measured as glycerol-3-phosphate dehydrogenase activity was positively correlated with noradrenaline-induced lipolysis in preadipocytes (r=0.51, p<0.005) and mature fat cells (r=0.35, p<0.05). In conclusion, intrinsic properties related to terminal differentiation determine the ability of catecholamines to maximally stimulate lipolysis in fat cells. The inability to undergo full differentiation might in part explain the low lipolytic capacity of fat cells among the obese.     

慢病毒载体介导shRNA干扰IL-6Rα基因表达削弱IL-6对猪脂肪细胞分化的影响

为研究白细胞介素-6(IL-6)对猪脂肪细胞分化的影响及其分子机制,构建猪IL 6Rα基因RNA干扰（RNA interference, RNAi）慢病毒载体;用IL-6Rα-RNAi重组慢病毒预处理原代培养的猪前体脂肪细胞或不处理, 然后用100 ng/mL IL-6处理分化第6 d的脂肪细胞48 h．通过测定甘油释放量检测脂肪细胞的脂解率;油红O染色提取法测定脂肪细胞的脂质含量;采用RT-PCR 和Western印迹检测脂肪细胞分化相关基因的mRNA 和蛋白表达．结果显示,IL-6显著抑制猪脂肪细胞分化,并下调PPARγ2、Perilipin A和IRS-1的mRNA及蛋白表达,同时增强ERK1/2磷酸化;IL-6Rα-RNAi预处理前体脂肪细胞则显著逆转IL 6的上述作用．总之,IL-6通过多重机制抑制猪脂肪细胞分化;而且本研究构建的IL-6Rα-RNAi重组慢病毒载体可有效阻断IL-6信号,为进一步研究IL-6的功能奠定了基础．     

Acylated and unacylated ghrelin attenuate isoproterenol-induced lipolysis in isolated rat visceral adipocytes through activation of phosphoinositide 3-kinase γ and phosphodiesterase 3B

The acylated peptide ghrelin (AG) and its endogenous non-acylated isoform (UAG) protect cardiomyocytes, pancreatic β-cells, and preadipocytes from apoptosis, and induce preadipocytes differentiation into adipocytes. These events are mediated by AG and UAG binding to a still unidentified receptor, which determines the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) ERK1/2. AG and UAG also possess antilipolytic activity in vitro, but the underlying mechanism remains unknown. Thus, the objective of the current study was to characterize the molecular events involved in AG/UAG receptor signaling cascade. We treated rat primary visceral adipocytes with isoproterenol (ISO) and forskolin (FSK) to stimulate lipolysis, simultaneously incubating them with or without AG or UAG. Both peptides blocked ISO- and FSK-induced lipolysis. By direct measurement of cAMP intracellular content, we demonstrated that AG/UAG effect was associated to a reduction of ISO-induced cAMP accumulation. Moreover, the cAMP analog 8Br-cAMP abolished AG/UAG effect. As AG and UAG were ineffective against lipolysis induced by db-cAMP, another poorly hydrolyzable cAMP analog, phosphodiesterase (PDE) involvement was hypothesized. Indeed, cilostamide, a specific PDE3B inhibitor, blocked AG/UAG effect on ISO-induced lipolysis. Furthermore, the PI3K inhibitor wortmannin and AKT inhibitor 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate also blocked AG/UAG action, suggesting a role in PDE3B activation. In particular, PI3K isoenzyme gamma (PI3Kγ) selective inhibition through the compound AS605240 prevented AG/UAG effect on ISO-stimulated lipolysis, hampering AKT phosphorylation on Ser(473). Taken together, these data demonstrate for the first time that AG/UAG attenuation of ISO-induced lipolysis involves PI3Kγ/AKT and PDE3B.     

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells’ lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

     

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。     

过表达RBP4抑制体外培养的猪前体脂肪细胞分化

为研究视黄醇结合蛋白4(retinol binding protein 4,RBP4)对猪前体脂肪细胞分化的影响,实验构建了RBP4重组腺病毒表达载体,包装并感染猪前体细胞,采用油红O染色和Real-time PCR等方法,检测了过表达RBP4对成脂分化的作用. 研究结果显示,重组腺病毒RBP4载体构建成功,转染猪前体脂肪细胞后,使RBP4的mRNA水平和蛋白水平分别增加了约400倍和20倍. 过表达RBP4能减少脂肪细胞的脂质积累,降低成脂关键基因过氧化物酶体增生物激活受体γ (peroxisome proliferator-activated receptor gamma, PPARγ)和脂肪酸结合蛋白2 (adipocyte protein 2, aP2)的表达. 结果表明,RBP4对猪前体脂肪细胞分化有抑制作用,为进一步研究RBP4对猪前体脂肪细胞分化的作用机制奠定基础.     

Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous

The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.     

Signaling pathway of magnolol-stimulated lipolysis in sterol ester-loaded 3T3-L1 preadipocyes

The aims of the present study were to examine the effect of magnolol on lipolysis in sterol ester (SE)-loaded 3T3-L1 preadipocytes and to determine the signaling mechanism involved. We demonstrate that magnolol treatment resulted in a decreased number and surface area of lipid droplets, accompanied by release of glycerol. The lipolytic effect of magnolol was not mediated by PKA based on the facts that magnolol did not induce an elevation of intracellular cAMP levels, and protein kinase A (PKA) inhibitor KT5720 did not block magnolol-induced lipolysis. Calcium/calmodulin-dependent protein kinase (CaMK) was involved in this signaling pathway, since magnolol-induced a transient rise of intracellular [Ca(2+)] and Ca(2+) influx across the plasma membrane, and CaMK inhibitor significantly abolished magnolol-induced lipolysis. Moreover, magnolol increased the relative levels of phosphorylated extracellular signal-related kinases (ERK1 and ERK2). In support of the involvement ERK, we demonstrated that magnolol-induced lipolysis was inhibited by PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK), and PD98059 reversed magnolol-induced ERK phosphorylation. Further, the relationship between CaMK and ERK was connected by the finding that CaMK inhibitor also blocked magnolol-induced ERK phosphorylation. Taken together, these findings suggest that magnolol-induced lipolysis is both CaMK- and ERK-dependent, and this lipolysis signaling pathway is distinct from the traditional PKA pathway. ERK phosphorylation is reported to enhance lipolysis by direct activation of hormone sensitive lipase (HSL), thus magnolol may likely activate HSL through ERK and increase lipolysis of adipocytes.     

二十二碳六烯酸对大鼠脂肪细胞增殖分化的影响

体外培养大鼠脂肪细胞,分别以0 μmol/L（对照组）、40 μmol/L（低剂量组）和160 μmol/L（高剂量组）的二十二碳六烯酸（DHA）处理细胞。采用台盼蓝排斥试验和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;逆转录聚合酶链反应 (RT-PCR) 分析过氧化物酶增殖物激活受体γ2（PPARγ2）mRNA表达情况,探讨DHA对前体脂肪细胞增殖分化的影响及其可能机制。结果显示,各组细胞活力及MTT测得的光密度值(OD值)均低于对照组,160μmol/L组在60～72h作用显著（P<0.05）;脂肪细胞经DHA处理后, 160μmol/L组细胞油红O染色的OD值及PPARγ2 mRNA表达量均显著下降(P<0.01)。以上结果说明,DHA对脂肪细胞增殖分化均有一定抑制作用,高剂量DHA（160μmol/L）可显著减少细胞内脂肪的合成、抑制脂肪细胞分化,PPARγ2 mRNA表达量的下降可能是DHA抑制细胞分化的部分原因。     

Late expression of alpha 2-adrenergic-mediated antilipolysis during differentiation of hamster preadipocytes.

In order to study the ontogenesis of the beta- and alpha 2-adrenergic control of lipolysis during the adipose conversion process, a model based on preadipocytes isolated from the stromal-vascular fraction of hamster adipose tissue was developed. When cultured in an ITT (insulin, transferrin, triiodothyronine) medium supplemented with 2% fetal calf serum, adipose precursors differentiated into adipose-like cells. On 8-day-post-confluent differentiating preadipocytes, the rank order of potency of activation of lipolysis by various beta-adrenergic agonists (BRL37344 greater than norepinephrine = isoproterenol greater than epinephrine greater than fenoterol) was equivalent to that determined in mature adipocytes isolated from adult hamster adipose tissue. On 8-day-post-confluent differentiating preadipocytes, phenylisopropyladenosine (A1-adenosine agonist) and prostaglandin E1 evoked a strong antilipolytic response whereas that evoked by UK 14304 and clonidine (alpha 2-adrenergic agonists) remained undetectable at this step of differentiation. The activity of UK 14304 and clonidine only appeared on 20- to 25-day-post-confluent differentiating preadipocytes. They induced dose-dependent antilipolysis with a maximal effect reaching 80-85% inhibition of adenosine deaminase-stimulated lipolysis. Their action was blocked by increased concentrations of different alpha 2-adrenergic antagonists with the following order of potency, RX 821002 greater than phentolamine much greater than yohimbine. This order of potency was similar to that determined on mature adipocytes isolated from adult hamsters. Both the density of the alpha 2-adrenoceptors, identified with the selective alpha 2-adrenergic radioligand [3H]RX-821002 (19 +/- 1 vs. 30 +/- 1 fmol/mg protein: P less than 0.01) and the amount of Gi proteins identified by pertussis toxin-catalyzed ADP-ribosylation (31 +/- 4 vs. 43 +/- 4% of the amount defined in mature fat cells from adult hamsters: P less than 0.05) were significantly increased between 8 days and 20-25 days after confluence, explaining the late emergence of the alpha 2-adrenergic control of lipolysis during preadipocyte differentiation. In conclusion, the late emergence of the alpha 2-adrenergic control of lipolysis, which is also supported by previous data obtained in vivo that demonstrated the age and/or the fat cell size dependence of alpha 2-adrenoreceptor expression in mature adipocytes, allows the alpha 2-adrenoceptor to be considered as a marker of adipocyte hypertrophy.