Regulated production of a peroxisome proliferator-activated receptor-gamma ligand during an early phase of adipocyte differentiation in 3T3-L1 adipocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear hormone receptor that is critical for adipogenesis and insulin sensitivity. Ligands for PPARgamma include some polyunsaturated fatty acids and prostanoids and the synthetic high affinity antidiabetic agents thiazolidinediones. However, the identity of a biologically relevant endogenous PPARgamma ligand is unknown, and limited insight exists into the factors that may regulate production of endogenous PPARgamma ligands during adipocyte development. To address this question, we created a line of 3T3-L1 preadipocytes that carry a beta-galactosidase-based PPARgamma ligand-sensing vector system. In this system, induction of adipogenesis resulted in elevated beta-galactosidase activity that signifies activation of PPARgamma via its ligand-binding domain (LBD) and suggests generation and/or accumulation of a ligand moiety. The putative endogenous ligand appeared early in adipogenesis in response to increases in cAMP, accumulated in the medium, and dissipated later in adipogenesis. Organically extracted and high pressure liquid chromatography-fractionated conditioned media from differentiating cells, but not from mature adipocytes, were enriched in this activity. One or more components within the organic extract activated PPARgamma through interaction with its LBD, induced lipid accumulation in 3T3-L1 cells as efficiently as the differentiation mixture, and competed for binding of rosiglitazone to the LBD of PPARgamma. The active species appears to be different from other PPARgamma ligands identified previously. Our findings suggest that a novel biologically relevant PPARgamma ligand is transiently produced in 3T3-L1 cells during adipogenesis.     

SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation

The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARgamma, C/EBPalpha, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function.     

Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.     

Molecular determinants of FGF-21 activity-synergy and cross-talk with PPARgamma signaling

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. The precise mechanisms whereby FGF-21 regulates metabolism remain to be determined. Here we describe the early signaling events triggered by FGF-21 treatment of 3T3-L1 adipocytes and reveal a functional interplay between FGF-21 and peroxisome proliferator-activated receptor gamma (PPARgamma) pathways that leads to a marked stimulation of glucose transport. While the early actions of FGF-21 on 3T3-L1 adipocytes involve rapid accumulation of intracellular calcium and phosphorylation of Akt, GSK-3, p70(S6K), SHP-2, MEK1/2, and Stat3, continuous treatment for 72 h induces an increase in PPARgamma protein expression. Moreover, chronic activation of the PPARgamma pathway in 3T3-L1 adipocytes with the PPARgamma agonist and anti-diabetic agent, rosiglitazone (BRL 49653), enhances FGF-21 action to induce tyrosine phosphorylation of FGF receptor-2. Strikingly, treatment of cells with FGF-21 and rosiglitazone in combination leads to a pronounced increase in expression of the GLUT1 glucose transporter and a marked synergy in stimulation of glucose transport. Together these results reveal a novel synergy between two regulators of glucose homeostasis, FGF-21 and PPARgamma, and further define FGF-21 mechanism of action.     

C/EBPalpha-dependent induction of glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI) expression during the differentiation of mouse fibroblasts into adipocytes

Western blot analysis of 3T3-L1 adipocyte proteins using an anti-C/EBPalpha antibody detected a 24kD polypeptide in addition to the expected 42 and 30kD isoforms of C/EBPalpha. Mass spectrometric sequencing of the protein following its purification by HPLC and preparative 2D gel electrophoresis identified it as glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI). Expression of GSTzeta/MAAI mRNA and protein was induced during the terminal phase of adipogenesis in 3T3-L1 preadipocytes. Ectopic expression of PPARgamma2 in NIH-3T3 fibroblasts exposed to insulin and troglitazone-induced perilipin production, but was incapable of activating GSTzeta/MAAI unless C/EBPalpha was also expressed. Similarly, ectopic expression of C/EBPalpha in PPARgamma +/- or PPARgamma -/- MEFs demonstrated that the C/EBPalpha-dependent induction of GSTzeta/MAAI production was dependent on expression of endogenous PPARgamma. These data suggest a role for GSTzeta/MAAI in mature adipocytes that may be responsive to the thiazolidinedione class of insulin sensitizing PPARgamma ligands.     

Functional role of RXRs and PPARgamma in mature adipocytes

The peroxisome proliferator-activated receptor gamma (PPARgamma) is abundantly expressed in adipocytes, and plays an important role in adipocyte differentiation and fat accretion. It is a heterodimeric partner of the retinoid X receptors alpha, beta and gamma, which are also expressed in the adipose tissue. As lethality of PPARgamma(-/-) and RXRalpha(-/-) mouse fetuses precluded the analysis of PPARgamma and RXRalpha functions in mature adipocytes, we generated RXRalpha(ad-/-) and PPARgamma(ad-/-) mice, in which RXRalpha and PPARgamma are selectively ablated in adult adipocytes, respectively. Even though the adiposity of RXRalpha(ad-/-) mice is similar to that of control mice when fed a regular diet, they are resistant to chemically and dietary-induced obesity. However, mature adipocytes lacking either both RXRalpha and RXRgamma or PPARgamma die, and are replaced by newly formed adipocytes. Thus, in adipocytes, RXRalpha is essential for lipogenesis, but RXRgamma can functionally replace RXRalpha for the adipocyte vital functions exerted by PPARgamma/RXR heterodimers.     

A selective peroxisome proliferator-activated receptor-gamma (PPARgamma) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3-L1 adipocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists such as the thiazolidinediones are insulin sensitizers used in the treatment of type 2 diabetes. These compounds induce adipogenesis in cell culture models and increase weight gain in rodents and humans. We have identified a novel PPARgamma ligand, LG100641, that does not activate PPARgamma but selectively and competitively blocks thiazolidinedione-induced PPARgamma activation and adipocyte conversion. It also antagonizes target gene activation as well as repression in agonist-treated 3T3-L1 adipocytes. This novel PPARgamma antagonist does not block adipocyte differentiation induced by a ligand for the retinoid X receptor (RXR), the heterodimeric partner for PPARgamma, or by a differentiation cocktail containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Surprisingly, LG100641, like the PPARgamma agonist rosiglitazone, increases glucose uptake in 3T3-L1 adipocytes. Such selective PPARgamma antagonists may help determine whether insulin sensitization by thiazolidinediones is mediated solely through PPARgamma activation, and whether there are PPARgamma-ligand-independent pathways for adipocyte differentiation. If selective PPARgamma modulators block adipogenesis in vivo, they may prevent obesity, lower insulin resistance, and delay the onset of type 2 diabetes.     

PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.