The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARg-dependent and PPARg-independent mechanisms

The adipocyte-derived secretory protein adiponectin functions as an insulin-sensitizing agent. In plasma, adiponectin exists as low, medium, and high molecular weight oligomers. Treatment with trans -10, cis -12 conjugated linoleic acid (t -10, c -12 CLA) reduces levels of adiponectin as well as triglyceride (TG) in mice and adipocyte cell culture models. The aim of this study was to determine whether the effects of t -10, c -12 CLA on adiponectin and TG are mediated through modulation of the transcription factor peroxisome proliferator-activated receptor g (PPARg). 3T3-L1 cells were treated either during or after differentiation into adipocytes with 100 mM t -10, c -12 CLA with or without 10 mM troglitazone, a PPARg agonist, or 1 mM GW9662, a PPARg antagonist, and adiponectin and TG levels were analyzed. Treatment with t -10, c -12 CLA reduced TG as well as cellular and secreted adiponectin levels and impaired the assembly of adiponectin oligomers. These changes were accompanied by decreases in PPARgmass. Troglitazone was able to reverse the t -10, c -12 CLA-mediated decrease in TG levels and restore the assembly of adiponectin oligomers but was unable to restore adiponectin synthesis. Conversely, treatment with GW9662 decreased TG mass and impaired adiponectin oligomer assembly but did not decrease total adiponectin mass. In a reporter assay, t-10, c-12 CLA appeared to be a partial PPARg agonist and prevented the stimulation of reporter activity by troglitazone. Therefore, the t -10, c -12 CLA isomer appears to alter adipocyte adiponectin metabolism through PPARg-dependent and PPARgindependent mechanisms.—Miller, J. R., P. Siripurkpong, J. Hawes, A. Majdalawieh, H-S. Ro, and R. S. McLeod. The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARg-dependent and PPARgindependent mechanisms. J. Lipid Res. 2008. 49: 550–562. Supplementary key words mouse & 3T3-L1 & adipocyte & peroxisome proliferator-activated receptor g Conjugated linoleic acid (CLA) refers to a group of fatty acid isomers that are related to the essential fatty acid, linoleic acid (LA; 18:2, cis -9, cis -12), but differ in both the position and the stereochemistry of their double bonds. The two isomers that have been shown to have biological activity are trans -10, cis -12 (t -10, c -12) CLA and cis -9, trans11 (c -9, t-11) CLA (reviewed in Ref. 1). The t -10, c -12 CLA isomer has been shown to reduce obesity in animals (2–8) and triglyceride (TG) accumulation in adipocyte cell culture models (9–12), with its greatest effects, to date, having been shown in the mouse (13). This has led to the promotion of CLA as a weight-loss supplement in humans. In mice, however, the reduction in obesity is often accompanied by insulin resistance and hepatic steatosis (2, 14–16), suggesting that treatment with t -10, c -12 CLA may adversely affect TG metabolism in other tissues. In human studies, neither the large reductions in adipose tissue nor the hepatic steatosis have been observed. Peroxisome proliferator-activated receptor g (PPARg) is an essential transcription factor in adipogenesis that induces the expression of the genes necessary for the acquisition and maintenance of the mature adipocyte phenotype. These include LPL (17), the adipocyte fatty acid binding protein aP2 (18), and the glucose transport protein GLUT4 (19). Both of the CLA isomers commonly found in dietary supplement preparations have affinities for PPARg that are similar to LA, but of the two, t -10, c -12 CLA was shown to be a slightly better ligand (20). Compared with synthetically designed PPARg agonists, the thiazolidinediones (TZDs) (21), however, the affinities of CLA for PPARg are low. The t -10, c -12 CLA isomer, but not the c -9, t -11 CLA isomer, appears to have profound inhibitory effects on the expression of both PPARg itself and PPARg-induced genes. When cultures of human preadipocytes were difManuscript received 13 June 2007 and in revised form 7 November 2007. Published, JLR Papers in Press, December 4, 2007. DOI 10.1194/jlr.M700275-JLR200 1 Present address of A. Majdalawieh: Department of Biology and Chemistry, American University of Sharjah, Sharjah, United Arab Emirates. 2 To whom correspondence should be addressed. e-mail: [email protected] Copyright D 2008 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org 550 Journal of Lipid Research Volume 49, 2008 by gest, on D ecem er 8, 2017 w w w .j.org D ow nladed fom ferentiated in the presence of individual CLA isomers, only treatment with t -10, c -12 CLA caused a marked reduction in the expression of PPARg as well as LPL, aP2, and GLUT4 (22). Adiponectin (23–26) is an adipocyte-derived secretory protein (adipokine). Plasma levels of adiponectin are normally high but are reduced in obesity and correlate negatively with body fat mass (27). Additionally, low levels of adiponectin are associated with cardiovascular disease (28) and type II diabetes (29). Adiponectin monomers are assembled into large, distinct oligomeric forms that canbedetected within adipocytes and in plasma as low molecular weight (LMW) trimers, medium molecular weight (MMW) hexamers, and high molecular weight (HMW) oligomers of 12–18 monomeric units (30, 31). Posttranslational glycosylation of fourhydroxy-lysine residues (32) isnecessary for theassembly of the HMW oligomers (33) and may be necessary for the assembly of the MMW and LMW oligomers as well (34). Adiponectin is an insulin-sensitizing protein, and there are adiponectin receptors in both skeletal muscle and liver. In both tissues, adiponectin binding stimulates the adenosine monophosphate-activated protein kinase pathway (35). In the muscle, this serves to increase b-oxidation as well as the translocation of GLUT4 to the plasma membrane (36). In the liver, activation of adenosine monophosphate-activated protein kinase also increases boxidation and adiponectin binding decreases hepatic glucose output by downregulating gluconeogenesis (35). TZDs are used to improve insulin sensitivity in type II diabetics and to correct hyperglycemia and hyperinsulinemia in animal models of obesity and diabetes, although their precise mechanism of action remains unknown. TZDs increase the conversion of preadipocytes to adipocytes, thereby increasing the number of small, insulinsensitive adipocytes. Recent studies have shown that treatment with TZDs increased plasma levels of adiponectin in normal, obese, and type II diabetic subjects (37) and in obese-diabetic (db/db) mice (38, 39). In cell culture studies, adiponectin secretion from 3T3-L1 adipocytes was also increased by TZDs (39). The aim of our study was to gain insight into the mechanisms of the effects of t -10, c -12 CLA on adiponectin metabolism in both differentiating preadipocytes and fully differentiated adipocytes using the mouse 3T3-L1 model. We also examined whether the t -10, c -12 CLA-mediated reductions in TG and adiponectin mass occur through a PPARg-dependent mechanism by comparison with a known PPARg agonist, the TZD troglitazone, and a known PPARg antagonist, GW9662. Finally, we used a reporter assay to examine the agonist/antagonist properties of the individual CLA isomers. MATERIALS AND METHODS