A proline-type fullerene derivative inhibits adipogenesis by preventing PPARγ activation

Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPβ, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.     

Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Delta(12,14)-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2.

Recent studies have demonstrated that bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vivo, and in vitro, and that peroxisome proliferator-activated receptor gamma (PPAR gamma) plays a central role in the control of adipocyte differentiation. In the present study, we treated a murine stromal cell line (TMS-14) with a cocktail of dexamethasone, insulin and glucose (DIG cocktail), which caused the cells to convert to fat-laden cells with adipocyte-like morphology. We also exposed TMS-14 cells to DIG cocktail followed by 15-deoxy Delta(12,14)-prostaglandin J2 (15d-PGJ2), a ligand of PPAR gamma, interleukin- 11 (IL-11), 9-cis retinoic acid (9-cis RA) and vitamin K2. 15d-PGJ2 enhanced DIG cocktail-induced adipogenesis, whereas IL-11, 9-cis RA and vitamin K2 each inhibited adipogenesis induced by DIG cocktail. The gene expressions of four adipogenesis markers, PPAR gamma 2, adipocyte P2 (aP2), adipocyte determination and differentiation factor 1 (ADD1), and fatty acid synthase (FAS) were enhanced by DIG cocktail and these expressions were more enhanced by 15d-PGJ2, in contrast they were attenuated by 9-cis RA. IL-11 also attenuated the adipogenesis markers except ADD1. Western blotting showed that 15d-PGJ2 enhanced the levels of PPAR gamma, C/EBP alpha and RXR alpha proteins, while IL-11 and 9-cis RA decreased the level of PPAR gamma protein, but not C/EBP alpha protein and vitamin K2 decreased the level of C/EBP alpha protein. We also tested the effect of 15d-PGJ2 on osteoblastogenesis, using TMS-12 cells, another stromal cell clone from the same mouse, which differentiate into osteoblasts spontaneously. 15d-PGJ2 did not affect osteoblastogenesis, as detected by von Kossa staining and Cbfa-1 gene expression. These data indicate that 15d-PGJ2 enhances the expression of both PPAR gamma and C/EBP alpha and as a result it stimulates adipogenesis in murine bone marrow cells.     

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes.     

Knockdown of KLF9 promotes the differentiation of both intramuscular and subcutaneous preadipocytes in goat

ABSTRACT

KLF9 is reported to promote adipocyte differentiation in 3T3-L1 cells and pigs. However, the roles of KLF9 in adipocytes differentiation of goat remain unknown. In this study, the expression profiles of KLF9 were different between subcutaneous and intramuscular preadipocytes of goat during differentiation process. After silencing KLF9 gene, the lipid droplets were increased in both two types of adipocytes. In subcutaneous preadipocyte with silencing KLF9, the expressions of C/EBPβ, PPARγ, LPL, KLF1-2, KLF5, and KLF17 genes were up-regulated, while KLF12, KLF4, and KLF13 genes were down-regulated in expression level. In intramuscular preadipocyte, aP2, C/EBPα, KLF2-3, KLF5, and KLF7 gene were up-regulated, and Pref-1 gene was down-regulated. In addition, the binding sites of KLF9 existed in the promoters of aP2, C/EBPα, C/EBPβ, LPL and Pref-1. Taken together, KLF9 play a negative role in the differentiation of both intramuscular and subcutaneous preadipocytes in goats, but the functional mechanism may be different.     

Impaired lipid accumulation by trans10, cis12 CLA during adipocyte differentiation is dependent on timing and length of treatment

Conjugated linoleic acids (CLAs) are a group of polyunsaturated fatty acids found in ruminant products, where the predominant isomers are cis9, trans11 (c9,t11) and trans10, cis12 (t10,c12) CLA. We have previously shown that t10,c12 CLA prevents lipid accumulation in mature adipocytes in part by acting as a peroxisome proliferator-activated receptor gamma (PPAR gamma) modulator. The objective of this study was to further establish the molecular mechanisms underlying the attenuating effect on lipid accumulation by t10,c12 CLA, with focus on time point and duration of treatment during adipogenesis. We have shown that t10,c12 CLA treatment has its most attenuating effect early (day (D) 0-6) during differentiation. Treatment during this period is sufficient to prevent lipid accumulation in mature adipocytes. The adipogenic marker genes PPAR gamma and CCAAT/enhancer binding protein alpha (C/EBP alpha) are both down-regulated after treatment within the period from D0-6, while additional treatment also down-regulates the expression of sterol regulatory element binding protein-1c (SREBP-1c), liver X receptor alpha (LXR alpha), fatty acid binding protein (aP2), fatty acid translocase (CD36) and insulin-sensitive glucose transporter 4 (GLUT4). These effects of t10,c12 CLA reflect the subsequent attenuation of lipid accumulation observed in mature adipocytes. Interestingly, the early B-cell factor (O/E-1), which is known to promote adipogenesis and to be involved in control of genes important for terminal adipocyte differentiation, is unaffected by treatment of t10,c12 CLA. Taken together, our data indicate that inhibition of lipid accumulation induced by t10,c12 CLA treatment during adipocyte differentiation is associated with a tight regulatory cross-talk between early (PPAR gamma and C/EBP alpha) and late (LXR alpha, aP2 and CD36) adipogenic marker genes.     

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.     

Regulating the balance between peroxisome proliferator-activated receptor gamma and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylation-defective mutant of beta-catenin inhibits expression of a subset of adipogenic genes

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of beta-catenin to the proteasome. In fact, sustained activation of beta-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. In this report, we investigated the mechanisms regulating the balance between beta-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes. Specifically, we show that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of beta-catenin, which depends on glycogen synthase kinase (GSK) 3beta activity. Mutation of serine 37 (a target of GSK3beta) to an alanine renders beta-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3beta phosphorylation-defective S37A-beta-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-beta-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant beta-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-beta-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKbeta is a key regulator of the balance between beta-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes.     

Phosphorylation of C/EBPbeta at a consensus extracellular signal-regulated kinase/glycogen synthase kinase 3 site is required for the induction of adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes

Stimulation of adipogenesis in mouse preadipocytes requires C/EBPbeta as well as activation of the MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/glycogen synthase kinase 3 (GSK3) site regulates adiponectin gene expression during the C/EBPbeta-facilitated differentiation of mouse fibroblasts into adipocytes. First, we show that exposure of 3T3-L1 preadipocytes to insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) leads to the phosphorylation of C/EBPbeta at threonine 188. Pretreating the cells with a MEK1-specific inhibitor (U0126) significantly attenuates this activity. Similarly, these effectors activate the phosphorylation of T188 within an ectopic C/EBPbeta overexpressed in Swiss mouse fibroblasts, and this event involves both MEK1 and GSK3 activity. We further show that expression of C/EBPbeta (p34kD LAP isoform) in Swiss mouse fibroblasts exposed to DEX, MIX, and insulin induces expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and some adiponectin but that it does not activate expression of FABP4/aP2. In fact, complete conversion of these fibroblasts into lipid-laden adipocytes, which includes activation of FABP4 and adiponectin expression, requires their exposure to a potent PPARgamma ligand such as troglitazone. Expression of a mutant C/EBPbeta in which threonine 188 has been modified to alanine (C/EBPbeta T188A) can induce PPARgamma production in the mouse fibroblasts, but it is incapable of stimulating adiponectin expression in the absence or presence of troglitazone. Interestingly, replacement of T188 with aspartic acid creates a C/EBPbeta molecule (C/EBPbeta T188D) that possesses adipogenic activity similar to that of the wild-type molecule. The absence of adiponectin expression correlates with a reduced amount of C/EBPalpha in the adipocytes expressing the T188A mutant suggesting that C/EBPalpha is required for expression of adiponectin. In fact, ectopic expression of PPARgamma in C/EBPalpha-deficient fibroblasts (NIH 3T3 cells) produces a modest amount of adiponectin, whereas expression of both PPARgamma and C/EBPalpha in NIH 3T3 cells facilitates production of abundant quantities of adiponectin. These data demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/GSK3 site is required for both C/EBPalpha and adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes.     

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.     

Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L alpha

Adiponectin is secreted from adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. In this report, we demonstrate that the endoplasmic reticulum (ER) oxidoreductase Ero1-L alpha and effectors modulating peroxisome proliferator-activated receptor gamma (PPAR gamma) and SIRT1 activities regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and Ero1-L alpha expression are induced during the early phase of adipogenesis but are then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPAR gamma enhances Ero1-L alpha expression and stimulates secretion of high-molecular-weight complexes of adiponectin in mature adipocytes. Suppression of Ero1-L alpha through expression of a corresponding small interfering RNA reduces adiponectin secretion during the differentiation of 3T3-L1 preadipocytes. Moreover, ectopic expression of Ero1-L alpha in Ero1-L alpha-deficient 3T3 fibroblasts stimulates the secretion of adiponectin following their conversion into adipocytes and prevents the suppression of adiponectin secretion in response to activation of SIRT1 by exposure to resveratrol. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to various metabolic states.