MOON, HYUN-SEUK, CHUNG-SOO CHUNG, HONG-GU LEE, TAE-GYU KIM, YUN-JAIE CHOI, AND CHONG-SUCHO. Inhibitory effect of (Ϫ)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells. Obesity. 2007;15:2571-2582. Objective: The objective of this study was to investigate the molecular mechanisms underlying the attenuating effect of (Ϫ)-epigallocatechin-3-gallate (EGCG) on proliferation and lipid accumulation of 3T3-L1 cells, with a focus on the duration of EGCG treatment. Research Methods and Procedures: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and diamidino-2-phenylindole staining. The anti-adipogenic effect of EGCG on 3T3-L1 cells was analyzed by glycerol-3-phosphate dehydrogenase activity and Oil red O staining. Western blot analysis was used to detect adenosine monophosphate-activated protein kinase (AMPK) activation and phosphorylation of its substrate, acetyl-CoA carboxylase (ACC), and expression of insulin (INS) receptor, INS receptor substrate-1 (IRS-1), and adipocyte marker proteins. Results: Exposure to EGCG during the early period of adipogenesis (7 days) was sufficient to prevent lipid accumulation. During this period, EGCG greatly decreased expression of the adipocyte marker proteins peroxisome proliferator-activated receptor ␥2 (PPAR␥2) and liver X receptor (LXR)-␣. Furthermore, EGCG significantly induced generation of reactive oxygen species (ROS), which led to AMPK activation, and these effects were eliminated by N-acetylcysteine (NAC) treatment. Also, EGCG increased the tyrosine phosphorylation of INS receptor and INS-1 with increasing incubation time. In contrast, EGCG treatment did not alter glycerol release in the presence or absence of 2Ј,5Ј-dideoxyadenosine (DDA), indicating that EGCG had no effect on lipolysis. Discussion: Our data demonstrate that EGCG decreased cell viability and inhibited differentiation of 3T3-L1 cells in a manner dependent on the duration of treatment. Also, we showed that inhibition of adipocyte differentiation by EGCG was associated with decreased glycerol-3-phosphate dehydrogenase (GPDH) activity accompanied by a strong inhibition of PPAR␥2-induced transcriptional activity. Furthermore, the inhibition of adipocyte differentiation by EGCG involved generation of ROS and activation of AMPK.

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Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of octadecadienoic acid [linoleic acid (LA), 18:2n-6]. Although ruminant milk and meat products represent the largest natural source of CLA and therefore, their concentration in ruminant lipids are of interest to human health, chemical or physical modifications of CLA should be needed as a means to enhance oxidative stability, to improve post-ruminal bioavailability, and to increase the clinical application. In fact, CLA are rapidly decomposed to form furan fatty acids when its are oxidized in air, and the effectiveness of dietary supplements of CLA may be related to the extent that their metabolisms by rumen bacteria are avoided. For these reasons, many scientists have examined the effect of manufacturing and protection on the stability of CLA in ruminants and food products. In this review, physico-chemical modifications of CLA for ruminal protection such as calcium salt (Ca), formaldehyde protection (FP), lipid encapsulation (LE), and amide linkage (AL), and for oxidative stability such as green tea catechin (GTC), cyclodextrin (CD), arginine (Arg), amylase, and PEGylation are proposed.

We previously reported that PEGylated conjugated linoleic acid (PCLA) as a pro-drug treatment of cultures of 3T3-L1 cells containing differentiated adipocytes caused de-differentiation by downregulation of PPARgamma2-induced adipogenesis, and cell apoptosis induced by PCLA was lower than that induced by conjugated linoleic acid (CLA) owing to the biocompatible and hydrophilic properties of poly(ethylene glycol) (PEG). To further investigate our previous observations, the present study is designed to evaluate the lipolytic action of PCLA and its role in biochemical signaling pathways of 3T3-L1 cells when compared to the CLA itself. Although both CLA and PCLA stimulated lipolysis, our results indicated a sensitivity difference between CLA and PCLA treatment: a time-dependent effect on lipolysis and p-extracellular signal-related kinases (ERK) expression was observed for PCLA-treated, but not for CLA-treated cultures. Also, the induction by PCLA of mitogen-activated protein kinase kinase (MEK)/ERK mitogen-activated protein kinase (MAPK) activation was linked to secretion of adipo-cytokines, interleukin-6 (IL-6), and interleukin-8 (IL-8), in time-dependent manners. Interestingly, adenylyl cyclase inhibitor, 2', 5'-dideoxyadenosine (DDA), pre-treatment did not prevent PCLA-stimulated lipolysis. In fact, isoproterenol, but not PCLA, caused a significant increase in cyclic adenosine monophosphate (cAMP) levels, suggesting that the PCLA-induced lipolysis was not mediated in the conventional cAMP-dependent pathway and the cAMP was the intracellular mediator for isoproterenol-induced lipolysis. Overall, our findings provide support for a role for PCLA as a pro-drug in the regulation of metabolism in adipose tissue.