Inhibition of adipogenesis and induction of apoptosis and lipolysis by stem bromelain in 3T3-L1 adipocytes

The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein G(i)α(1), as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.     

Motilin stimulates preadipocyte proliferation and differentiation and adipocyte lipid storage

Motilin is a circulating gastrointestinal peptide secreted primarily by duodenal mucosal M cells and recognized for its prokinetic effects on gastrointestinal tissues. Little information is available regarding effects on insulin/glucose homeostasis or adipocyte function. Our aim was to evaluate the effects of motilin on adipocyte proliferation, differentiation, lipolysis, and macronutrient uptake in adipocytes. 3T3-L1 cells and primary rat adipocytes were treated acutely and chronically with varying motilin concentrations, and effects were compared with vehicle alone (control), set as 100% for all assays. In preadipocytes, motilin stimulated proliferation ([(3)H]thymidine incorporation) and mitochondrial activity (141 ± 10%, P < 0.001 and 158 ± 10%, respectively, P < 0.001), in a concentration-dependent manner. Chronic supplementation with motilin during differentiation further increased lipogenesis (Oil red O staining 191 ± 27%, P < 0.05) and was associated with an upregulation of PPARγ (148 ± 8%, P < 0.01), C/EBPα (142 ± 17%, P < 0.05), and Cav3 (166 ± 20%, P < 0.05) expression. In mature 3T3-L1 adipocytes motilin increased fatty acid uptake/incorporation (≤ 202 ± 12%; P < 0.01) and glucose uptake (146 ± 9% P < 0.05) and decreased net fatty acid release (maximal -31%, P < 0.05) without influencing total lipolysis (glycerol release). Similar effects were obtained in primary rat adipocytes. Motilin acutely increased expression of PPARγ, CEBPβ, DGAT1, and CD36 while decreasing adiponectin mRNA and secretion. In human adipose tissue, motilin receptor GPR38 correlated with HOMA-IR and GHSR1 (r = 0.876, P < 0.0001). Motilin binding and fatty acid incorporation into adipocytes were inhibited by antagonists MB10 and [D-lys3]-GRP6 and PI 3-kinase inhibitor wortmannin. Taken together, these results suggest that motilin may directly influence adipocyte functions by stimulating energy storage.     

Dicer has a crucial role in the early stage of adipocyte differentiation, but not in lipid synthesis, in 3T3-L1 cells

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

Resveratrol inhibits cell differentiation in 3T3-L1 adipocytes via activation of AMPK

Resveratrol (Res) is a natural polyphenolic compound with anti-inflammatory and antioxidant properties. Also, Res can inhibit lipogenesis and adipocyte differentiation. However, the underlying mechanisms of Res's functions remain largely unknown. AMP-activated protein kinase (AMPK) is a key player in adipocyte differentiation. Therefore, the purpose of our study was to determine the role played by AMPK in the Res-mediated regulation of adipocyte differentiation. Incubation of 3T3-L1 cells with Res confirmed that Res inhibited adipocyte differentiation. The phosphorylation of AMPKα was increased by Res in a dose-dependent manner, while total AMPKα levels were unchanged, and peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) levels were decreased. Interestingly, pretreatment with AMPKα siRNA and Res promoted adipocyte differentiation, while the decrease of p-AMPKα increased PPARγ, C/EBPα, and SREBP-1c protein expression. Our study shows that Res is capable of inhibiting lipogenesis and differentiation of 3T3-L1 adipocytes via activation of AMPK, suggesting its potential therapeutic application in the treatment or prevention of obesity.     

Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade

Adult mice abundantly express neudesin, an extracellular heme-binding protein with neurotrophic activity, in white adipose tissues. At the early stage of adipocyte differentiation during adipogenesis, however, the expression of neudesin decreased transiently. Neudesin-hemin significantly suppressed adipogenesis in 3T3-L1 cells. The knockdown of neudesin by RNA interference markedly promoted adipogenesis in 3T3-L1 cells and decreased MAPK activation during adipocyte differentiation. The addition or knockdown of neudesin affected the expression of C/EBPα and PPARγ but not of C/EBPβ. These findings suggest that neudesin plays a critical role in the early stage of adipocyte differentiation in which C/EBPβ induces PPARγ and C/EBPα expressions, by controlling the MAPK pathway.     

Fucoxanthin exerts differing effects on 3T3-L1 cells according to differentiation stage and inhibits glucose uptake in mature adipocytes

Progression of 3T3-L1 preadipocyte differentiation is divided into early (days 0–2, D0–D2), intermediate (days 2–4, D2–D4), and late stages (day 4 onwards, D4-). In this study, we investigated the effects of fucoxanthin, isolated from the edible brown seaweed Petalonia binghamiae, on adipogenesis during the three differentiation stages of 3T3-L1 preadipocytes. When fucoxanthin was applied during the early stage of differentiation (D0–D2), it promoted 3T3-L1 adipocyte differentiation, as evidenced by increased triglyceride accumulation. At the molecular level, fucoxanthin increased protein expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), sterol regulatory element-binding protein 1c (SREBP1c), and aP2, and adiponectin mRNA expression, in a dose-dependent manner. However, it reduced the expression of PPARγ, C/EBPα, and SREBP1c during the intermediate (D2–D4) and late stages (D4–D7) of differentiation. It also inhibited the uptake of glucose in mature 3T3-L1 adipocytes by reducing the phosphorylation of insulin receptor substrate 1 (IRS-1). These results suggest that fucoxanthin exerts differing effects on 3T3-L1 cells of different differentiation stages and inhibits glucose uptake in mature adipocytes.     

Berberine exerts anti-adipogenic activity through up-regulation of C/EBP inhibitors, CHOP and DEC2

Berberine exerts an anti-adipogenic activity that is associated with the down-regulation of C/EBPα and PPARγ. Stimulation of AMP-activated kinase (AMPK) caused by inhibition of mitochondrial respiration has been suggested to underlie such molecular regulation. In the present study, we show that berberine up-regulated the expression of two different sets of C/EBP inhibitors, CHOP and DEC2, while down-modulating C/EBPα, PPARγ and other adipogenic markers and effectors in differentiating 3T3-L1 preadipocytes and mature adipocytes. Data also suggested that the berberine-induced up-regulation of CHOP and DEC2 was attributable to selective activation of an unfolded protein response (UPR) and modified extracellular environment, respectively. As a result, the anti-adipogenic activity of berberine was diminished remarkably by adjusting the differentiation culture media and limitedly but consistently by knockdown of CHOP expression. Together, up-regulation of C/EBP inhibitors appears to underlie the berberine-induced repression of C/EBPα and PPARγ and, so, the inhibition of adipogenesis.     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.