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.     

Variations in parametrial white adipose tissue mass during the mouse estrous cycle: relationship with the expression of peroxisome proliferator-activated receptor-gamma and retinoic acid receptor-alpha

Estrogen and progestin participate in the regulation of adipose tissue metabolism, and peroxisome proliferator-activated receptor-gamma (PPARgamma) and retinoic acid receptor-alpha (RXRalpha) are absolutely required for adipose tissue development. The present study is to investigate the changes in parametrial fat mass and expression of PPARgamma and RXRalpha during estrous cycle in mice. Parametrial white adipose tissues (WAT), inter-scapula brown adipose tissues, and uteri from female mice were weighed. Blood samples were collected for the measurement of 17 beta-estradiol and progesterone levels. An RNase protection assay and Western blot analysis were used to compare the expression of PPARgamma and RXRalpha in adipose tissue. The mass of parametrial WAT in diestrus was significantly higher compared with estrus. However, there is no significant difference on the mass of brown adipose tissues during estrous cycle. The expression of PPARgamma in WAT in diestrus was significantly higher than that in estrus. The expression of RXRalpha during estrous cycle was unchanged in both white and brown adipose tissues. In conclusion, the variation in parametrial WAT mass during the mouse estrous cycle correlates with changes in the expression of PPARgamma in WAT.     

Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin

Studies have demonstrated cross talk between beta-catenin and peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways. Specifically, activation of PPARgamma induces the proteasomal degradation of beta-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic beta-catenin is resistant to such degradation and inhibits the expression of PPARgamma target genes. In the present studies, we demonstrate a functional interaction between beta-catenin and PPARgamma that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of beta-catenin and a catenin binding domain (CBD) within PPARgamma. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic beta-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARgamma. Furthermore, these mutations render S37A beta-catenin susceptible to proteasomal degradation in response to activation of PPARgamma. Mutation of F372 within the CBD (helices 7 and 8) of PPARgamma disrupts its binding to beta-catenin and significantly reduces the ability of PPARgamma to induce the proteasomal degradation of beta-catenin. We suggest that in normal cells, PPARgamma can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated beta-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic beta-catenin resists proteasomal degradation by inhibiting PPARgamma activity, which requires its TCF/LEF binding domain.     

Oxidized low density lipoprotein activates peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma through MAPK-dependent COX-2 expression in macrophages

It has been reported that oxidized low density lipoprotein (Ox-LDL) can activate both peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma. However, the detailed mechanisms of Ox-LDL-induced PPARalpha and PPARgamma activation are not fully understood. In the present study, we investigated the effect of Ox-LDL on PPARalpha and PPARgamma activation in macrophages. Ox-LDL, but not LDL, induced PPARalpha and PPARgamma activation in a dose-dependent manner. Ox-LDL transiently induced cyclooxygenase-2 (COX-2) mRNA and protein expression, and COX-2 specific inhibition by NS-398 or meloxicam or small interference RNA of COX-2 suppressed Ox-LDL-induced PPARalpha and PPARgamma activation. Ox-LDL induced phosphorylation of ERK1/2 and p38 MAPK, and ERK1/2 specific inhibition abrogated Ox-LDL-induced COX-2 expression and PPARalpha and PPARgamma activation, whereas p38 MAPK-specific inhibition had no effect. Ox-LDL decreased the amounts of intracellular long chain fatty acids, such as arachidonic, linoleic, oleic, and docosahexaenoic acids. On the other hand, Ox-LDL increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) level through ERK1/2-dependent overexpression of COX-2. Moreover, 15d-PGJ(2) induced both PPARalpha and PPARgamma activation. Furthermore, COX-2 and 15d-PGJ(2) expression and PPAR activity were increased in atherosclerotic lesions of apoE-deficient mice. Finally, we investigated the involvement of PPARalpha and PPARgamma on Ox-LDL-induced mRNA expression of ATP-binding cassette transporter A1 and monocyte chemoattractant protein-1. Interestingly, specific inhibition of PPARalpha and PPARgamma suppressed Ox-LDL-induced ATP-binding cassette transporter A1 mRNA expression and enhanced Ox-LDL-induced monocyte chemoattractant protein-1 mRNA expression. In conclusion, Ox-LDL-induced increase in 15d-PGJ(2) level through ERK1/2-dependent COX-2 expression is one of the mechanisms of PPARalpha and PPARgamma activation in macrophages. These effects of Ox-LDL may control excess atherosclerotic progression.     

Deletion of peroxisome proliferator-activated receptor-alpha induces an alteration of cardiac functions

The peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a major role in the control of cardiac energy metabolism. The role of PPARalpha on cardiac functions was evaluated by using PPARalpha knockout (PPARalpha -/-) mice. Hemodynamic parameters by sphygmomanometric measurements show that deletion of PPARalpha did not affect systolic blood pressure and heart rate. Echocardiographic measurements demonstrated reduced systolic performance as shown by the decrease of left ventricular fractional shortening in PPARalpha -/- mice. Telemetric electrocardiography revealed neither atrio- nor intraventricular conduction defects in PPARalpha -/- mice. Also, heart rate, P-wave duration and amplitude, and QT interval were not affected. However, the amplitude of T wave from PPARalpha -/- mice was lower compared with wild-type (PPARalpha +/+) mice. When the myocardial function was measured by ex vivo Langendorff's heart preparation, basal and beta-adrenergic agonist-induced developed forces were significantly reduced in PPARalpha-null mice. In addition, Western blot analysis shows that the protein expression of beta1-adrenergic receptor is reduced in hearts from PPARalpha -/- mice. Histological analysis showed that hearts from PPARalpha -/- but not PPARalpha +/+ mice displayed myocardial fibrosis. These results suggest that PPARalpha-null mice have an alteration of cardiac contractile performance under basal and under stimulation of beta1-adrenergic receptors. These effects are associated with myocardial fibrosis. The data shed light on the role of PPARalpha in maintaining cardiac functions.     

Ligands of peroxisome proliferator-activated receptor-alpha promote glutamate transporter-1 endocytosis in astrocytes

Astrocytes, a stellate-shape glial population in the central nervous system (CNS), maintain glutamate homeostasis in adult CNS by undergoing glutamate uptake at the synapse through their glutamate transporter-1 (GLT-1). Peroxisome proliferator-activated receptor-α (PPARα) can be activated by endogenous saturated fatty acids to regulate astrocytic lipid metabolism and functions. However, it is unclear if PPARα can exert the regulatory action on GLT-1 expression in astrocytes. This study showed that treatment with palmitic acid (PA) and the other two PPARα agonists (GW 7647 and WY 14,643) caused no change in the morphology of astrocytes, whereas membranous GLT-1 protein levels in astrocytes were significantly decreased by PA and PPARα agonists. Through lentivirus-mediated overexpression of GLT-1 tagged with red fluorescent protein (GLT-1-RFP), we also observed that GLT-1-RFP puncta in the processes of astrocytes were inhibited by the PPARα agonists. This reduction was prevented by the addition of the PPARα antagonist, GW6471. GLT-1-RFP was co-localized to the early endosome marker–EEA1 in astrocytes treated with the PPARα agonists. Moreover, PPARα-induced inhibition in membranous GLT-1 expression was abolished by the addition of dynamin inhibitor (dynasore). Furthermore, the co-treatment of astrocytes with PPARα agonists and dynasore, or with PPARα agonists and protein kinase C (PKC) inhibitor bis-indolylmaleimide 1 (BIS1), prevented the endocytosis of GLT-1-RFP. Based on the results, we conclude that the PPARα agonists increased GLT-1 endocytosis in astrocytes possibly through the PKC signaling pathway. In addition, our findings provide important information of PPARα involvement in the downregulation of astrocytic glutamate uptake via the promoted GLT-1 endocytosis.     

Novel pathway of adipogenesis through cross-talk between adipose tissue macrophages, adipose stem cells and adipocytes: evidence of cell plasticity

Introduction

Previous studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes.

Research Design and Methods

Human adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs.

Results

Co-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes.

Conclusions

Cross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes.     

The peroxisome proliferator-activated receptor gamma regulates expression of the perilipin gene in adipocytes

Recent studies have shown that lipid droplets are covered with a proteinaceous coat, although the functions and identities of the component proteins have not yet been well elucidated. The first identified lipid droplet-specific proteins are the perilipins, a family of proteins coating the surfaces of lipid droplets of adipocytes. The generation of perilipin-null mice has revealed that although they consume more food than control mice, they have normal body weight and are resistant to diet-induced obesity. In one study (Martinez-Botas, J., Anderson, J. B., Tessier, D., Lapillonne, A., Chang, B. H. J., Quast, M. J., Gorenstein, D., Chen, K. H., and Chan, L. (2000) Nat. Genet. 26, 474-479) it was reported that in an animal model obesity was reversible by breeding perilipin -/- alleles into Lepr db/db obese mice, ostensibly by increasing the metabolic rate of the mice. To understand the exact mechanisms that drive the exclusive expression of the perilipin gene in adipocytes, we analyzed the 5'-flanking region of the mouse gene. Treatment of differentiating 3T3-L1 adipocytes with an agonist of proliferator-activated receptor (PPAR) gamma, the putative "master regulator" of adipocyte differentiation, significantly augmented perilipin gene expression. Reporter assays using the -2.0-kb promoter revealed that this region contains a functional PPARgamma-responsive element. Gel mobility shift and chromatin immunoprecipitation assays showed that endogenous PPARgamma protein binds to the perilipin promoter. PPARgamma2, an isoform exclusively expressed in adipocytes, was found to be the most potent regulator from among the PPAR family members including PPARalpha and PPARgamma1. These results make evident the fact that perilipin gene expression in differentiating adipocytes is crucially regulated by PPARgamma2, providing new insights into the adipogenic action of PPARgamma2 and adipose-specific gene expression, as well as potential anti-obesity pharmaceutical agents targeted to a reduction of the perilipin gene product.