The Function of the Nuclear Receptor Peroxisome Proliferator activated Receptor Delta in Energy Homeostasis

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1 22. Castellot JJ Jr, Karnovsky MJ, Spiegelman BM. Potent stimulation of vascular endothelial cell growth by differentiated 3T3 adipocytes. Proc Natl Acad Sci U S A.1980;77: Castellot JJ Jr, Karnovsky MJ, Spiegelman BM. Differentiation-dependent stimulation of neovascularization and endothelial cell chemotaxis by 3T3 adipocytes. Proc Natl Acad Sci U S A. 1982;79: Silverman KJ, Lund DP, Zetter BR, et al. Angiogenic activity of adipose tissue. Biochem Biophys Res Commun. 1988;153: Zhang QX, Magovern CJ, Mack CA, Budenbender KT, Ko W, Rosengart TK. Vascular endothelial growth factor is the major angiogenic factor in omentum: mechanism of the omentum-mediated angiogenesis. J Surg Res. 1997;67: Dobson DE, Kambe A, Block E, et al. 1-Butyrylglycerol: a novel angiogenesis factor secreted by differentiating adipocytes. Cell. 1990;61: Sierra-Honigmann MR, Nath AK, Murakami C, et al. Biological action of leptin as an angiogenic factor. Science. 1998;281: Brooks RA, Burrin JM, Kohner EM. Characterization of release of basic fibroblast growth factor from bovine retinal endothelial cells in monolayer cultures. Biochem J. 1991;276: Folkman J. Is tissue mass regulated by vascular endothelial cells? Prostate as the first evidence. Endocrinology. 1998;139: Varzaneh FE, Shillabeer G, Wong KL, Lau DC. Extracellular matrix components secreted by microvascular endothelial cells stimulate preadipocyte differentiation in vitro. Metabolism. 1994;43: Lau DC, Schillabeer G, Li ZH, Wong KL, Varzaneh FE, Tough SC. Paracrine interactions in adipose tissue development and growth. Int J Obes Relat Metab Disord. 1996;20:S16 S Lau DC, Shillabeer G, Wong KL, Tough SC, Russell JC. Influence of paracrine factors on preadipocyte replication and differentiation. Int J Obes. 1990;14: Morimura M, Ishiko O, Sumi T, Yoshida H, Ogita S. Angiogenesis in adipose tissues and skeletal muscles with rebound weight-gain after diet-restriction in rabbits. Int J Mol Med. 2001;8: Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994;372: Considine RV, Caro JF. Leptin and the regulation of body weight. Int J Biochem Cell Biol. 1997;29: Park HY, Kwon HM, Lim HJ, et al. Potential role of leptin in angiogenesis: leptin induces endothelial cell proliferation and expression of matrix metalloproteinases in vivo and in vitro. Exp Mol Med. 2001; 33: Cohen B, Barkan D, Levy Y, et al. Leptin induces angiopoietin-2 expression in adipose tissues. J Biol Chem. 2001;276: Rupnick MA, Panigrahy D, Zhang CY, et al. Adipose tissue mass can be regulated through the vasculature. Proc Natl Acad Sci U S A.2002;99: Friedman JM, Leibel RL, Siegel DS, Walsh J, Bahary N. Molecular mapping of the mouse ob mutation. Genomics. 1991;11: Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA. 1997;94: Qian H, Azain MJ, Compton MM, Hartzell DL, Hausman GJ, Baile CA. Brain administration of leptin causes deletion of adipocytes by apoptosis. Endocrinology. 1998;139: Carr RH, Ipaktchi M, Thenen SW. Effects of prolonged fenfluramine administration in obese and nonobese mice. Proc Soc Exp Biol Med. 1977;154: Scharrer E. Control of food intake by fatty acid oxidation and ketogenesis. Nutrition. 1999;15: Cao Y, Cao R. Angiogenesis inhibited by drinking tea. Nature. 1999;398: Joussen AM, Rohrschneider K, Reichling J, Kirchhof B, Kruse FE. Treatment of corneal neovascularization with dietary isoflavonoids and flavonoids. Exp Eye Res. 2000;71: The Function of the Nuclear Receptor Peroxisome Proliferator activated Receptor Delta in Energy Homeostasis The metabolic function of the nuclear receptor peroxisome proliferator activated receptor This review was prepared by George Wolf, D.Phil., Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720, USA. Please address all reprint requests to the Nutrition Reviews Editorial Office, 711 Washington Street, Boston, MA 02111, USA. (PPAR ) has been established by transfer of the PPAR gene into adipose tissue of mice in vivo and into adipocytes in culture. Investigators found that PPAR activation by such transfer leads to up-regulation of energy expenditure by fatty acid oxidation. PPAR activation also results in lowered serum triglyceride and free fatty acid levels and decreased lipid accumulation. In vivo activation of PPAR in adipose tissue protects Nutrition Reviews, Vol. 61, No

2 against obesity and fatty liver in mice fed a highcalorie diet. PPAR also activates the heat-producing uncoupling enzymes in brown adipose tissue (UCP1 and 3) and muscle (UCP2). Key words: nuclear receptor, peroxisome proliferator activated receptor, PPAR, fatty acid oxidation, uncoupling enzymes, adipose tissue, adipocytes, obesity 2003 International Life Sciences Institute doi: /nr.2003.nov Nuclear hormone receptors are proteins activated by binding lipophilic molecules (ligands), such as steroid hormones, retinoids, or prostaglandins. When activated, they function as transcription factors, attaching to gene promoters and thus causing expression of particular genes. An important triad of nuclear hormone receptors comprises the peroxisome proliferator activated receptors,, and (PPAR,, and ), all of which are involved in energy homeostasis. They have in common a transcription function (termed AF-1) at the N-terminal region; a DNAbinding domain, consisting of two zinc fingers; and a dimerization domain and a hydrophilic pocket, which bind to the ligand in the C-terminal region. In order to bind to DNA, PPARs must form dimers with RXR, another nuclear receptor that is itself activated by 9-cis-retinoic acid. The most widely explored member of the PPAR triad is PPAR, which is expressed mainly in adipocytes. PPAR promotes adipocyte differentiation from fibroblasts (embryonic fibroblasts and embryonic stem cells during development) and promotes lipid storage in mature adipocytes by up-regulating genes for fatty acid binding protein and acyl CoA synthetase. Its ligand is the prostaglandin 15-deoxy-D 12,14 -prostaglandin J 2 and the synthetic ligand, the drug thiazolidenedione (TZD). This drug has been used to improve insulin sensitivity in human patients suffering from type 2 diabetes. 1 The molecular mechanism of this effect is still unknown. The receptor PPAR is confined mainly to liver. In liver it exerts its action in the opposite direction from PPAR : it up-regulates genes in liver for lipid combustion, enhancing -oxidation of fatty acids by acyl CoA oxidase and omega oxidation by cytochrome P450. In the food-deprived state, its activation causes a shift from carbohydrate to fat oxidation. When fed a high-fat diet, PPAR -null mice accumulate fat in their livers. PPAR s ligands, apart from synthetic compounds of the fibrate class, include polyunsaturated fatty acids and eicosanoids, which are precursors of prostaglandins. 2 The function of PPAR was recently elucidated in the laboratory of Wang et al. 3 PPAR occurs in many tissues, but is most abundant in muscle, in which fatty acids are the preferred energy source. PPAR -null mice suffer from placental defects such that only few survive to birth 4 ; those that do show a systemic disturbance in lipid metabolism, by contrast to PPAR -null mice, in which the loss of function is confined to adipocytes and PPAR -null mice that have loss of function in liver. The authors 3 studied the effect of gain-of-function with PPAR in vivo, in particular the expression of the PPAR gene in adipose tissue. The 78 amino acid activation domain (VP16) of the herpes simplex virus was fused to the N-terminal end of PPAR. To ensure that the resulting fusion protein had the expected activity, the authors showed this protein to possess ligand-independent transactivation of a synthetic PPAR response element. In order to obtain expression of the VP16-PPAR selectively in adipose tissue, the VP16-PPAR -cdna was placed downstream of the fatty acid binding protein gene promoter (ap2). The purified transgene was then injected into fertilized ova of C57BL6J CBA F1 mice. Transgene-positive offspring were selected and shown to express the VP16-PPAR gene in adipose tissue under control of the ap2 gene. The transgenic mice, which harbored the VP16-PPAR gene in their adipocytes, displayed a reduction in body weight (35% in 12 months) compared with wild-type litter mates; some of the transgenic mice lacked any visible white fat altogether, suggesting increased fatty acid oxidation. There was no reduction in number of adipocytes, but rather a reduction in the size and triglyceride content of adipocytes. Serum triglyceride levels were much reduced, although cholesterol levels remained unchanged. To further explore their hypothesis that PPAR in adipose tissue enhances fatty acid oxidation, the authors 3 fed a 35% (by weight) fat diet to 8-week-old transgenic mice and wild-type litter mates for 32 days (Figure 1). The transgenic mice gained only g (mean SD) compared with the wild-type mice, which gained g. The low weight gain in the transgenic mice was mainly due to inguinal, reproductive, and peritoneal fat of the transgenic mice, which weighed only 1/3 of that of the wild-type mice. Serum triglycerides and free fatty acids in the transgenic mice fed the high-fat diet remained at a low level; this result was similar to controls fed a chow diet, whereas the high-fat diet approximately doubled these parameters in the wild-type mice. The authors 3 concluded that PPAR expressed in adipose tissue promoted fat oxidation and prevented diet-induced obesity. To determine whether the leptin gene was involved in this effect, the authors 3 used db/db mice. This strain of mice becomes obese as a result of a mutation in the leptin receptor gene. 5 The VP16-PPAR gene was transferred into adipocytes of db/db mice. Sixteen-week-old db/db mice were grossly obese, weighing g, whereas the db/db-vp16-ppar mice weighed Nutrition Reviews, Vol. 61, No. 11

3 Figure 1. Effects of activation of peroxisome proliferator activated receptor (PPAR ) in adipose tissue. (A) Eight-week-old male mice (n 6 7) were fed a high-fat diet. The increases in body weights were calculated based on the initial body weights at day 0 of high-fat feeding. (B) Photographs of representative control littermate and transgenic mice after 5 weeks of high-fat feeding. (C) Weights of white fat depots normalized by body weight (n 7 9). (D) Steady-state serum levels of triglycerides (n 4) and free fatty acids (n 4). WT wild-type, TG transgenic, IWAT inguinal, RDWAT reproductive, RETROWAT retroperitoneal white adipose tissue. Adapted from reference 3, with permission g, similar to wild-type littermates; this result demonstrated that the PPAR activation in adipose tissue is independent of leptin signaling. Acting as ligand for PPAR, an agonist named GW was developed by Oliver et al. 6 by means of structure-based drug design. Administering this drug to obese db/db mice for only 7 days prevented the huge fat accumulation both in brown fat and liver in the mutant mice. Even without gene transfer into the adipocytes, therefore, a synthetic agonist can activate the PPAR receptor of adipocytes. To study the molecular mechanism of the greatly increased fatty acid oxidation promoted by PPAR in adipocytes, the authors 3 determined expression of genes coding for fatty acid oxidation enzymes by Northern blot analysis of the enzymes mrna. They found that mrnas for acyl CoA synthetases, acyl CoA oxidase, acyl CoA dehydrogenase, and carnitine palmitoyltransferase all had doubled in the adipose tissue of the transgenic mice. Most interesting was the finding of the effect of PPAR activation on brown fat: mrna for the uncoupling enzymes UCP1 and UCP3 was increased threefold. Therefore, not only does PPAR in adipose tissue increase expression of enzymes for fatty acid oxidation, but it also causes uncoupling of oxidation from ATP formation in brown fat, leading to heat production at the expense of ATP. The authors 3 therefore designated PPAR a thermogenic transcription factor. The conclusions arrived at from the in vivo experiments discussed above were confirmed in vitro. The authors 3 used 3T3L1 pre-adipocytes and allowed them to differentiate into mature adipocytes filled with triglycerides. They then infected the cells with adenoviruses harboring either the PPAR or the VP16-PPAR genes (or the VP16 gene, for control), and measured triglyceride metabolism by assay of glycerol released into the media. They found that overexpression of PPAR in the adipocytes by infection with the PPAR containing virus doubled the release of glycerol compared to control cells with the VP16 gene. Infection with the VP16-PPAR gene containing virus tripled the release. When the adipocytes were incubated with the specific synthetic PPAR ligand, GW501516, the release of glycerol was stimulated even further, all within 6 hours. Intracellular triglycerides declined simultaneously with the increase in glycerol in the media. No free fatty acids were detected in the media along with the glycerol, Nutrition Reviews, Vol. 61, No

4 Table 1. Simplified Summary of Peroxisome Proliferator Activated Receptors Effects on Lipid Metabolism PPAR * PPAR * PPAR * Liver TG storage 1 TG storage 2 TG storage 2 TG uptake 1 FA oxidation 1 FA oxidation 2 Serum unclear TG Level 2 TG Level 2 Muscle unclear FA oxidation 1 FA oxidation 1 Uncoupling enzyme 1 White Fat TG storage 1 No change TG storage 2 TG uptake 1 FA oxidation 1 Adipocyte differentiation 1 Brown Fat TG uptake 1 No change TG storage 2 Uncoupling enzymes 1 *Activation by ligand. PPAR peroxisome proliferator-activated receptor, TG triglyceride, FA fatty acid. showing that they were all oxidized. To clinch their conclusion that PPAR stimulated fatty acid oxidation, the authors 3 infected adipocytes with viruses harboring PPAR or VP16-PPAR genes or treated them with the synthetic ligand GW and then incubated them with [ 3 H]palmitic acid. In all cases, the treatment resulted in doubled [ 3 H]H 2 O production compared with control. An added synthetic PPAR ligand had no effect. Next, the investigators 3 determined the action of PPAR on muscle cells, for muscle is the tissue in which this receptor is most abundant. Skeletal muscle cells C2C12 were allowed to differentiate into myotubules. When treated with the PPAR agonist, GW501516, these muscle cells showed stimulated fatty acid oxidation and a much greater stimulation when infected with the VP16-PPAR containing virus. Upon treatment of the cultured muscle cells with agonist, the mrna of the heat-producing uncoupling enzyme, UCP2, increased significantly. The authors 3 concluded that PPAR enhances -oxidation and energy uncoupling (i.e., heat production) in muscle as well as in fat. In summary, the in vivo activation of PPAR in adipose tissue leads to up-regulation of energy expenditure by fatty acid oxidation. It results in lowered serum triglyceride and free fatty acid levels and decreased lipid accumulation. In vivo activation of PPAR in adipose tissue protects against obesity and fatty liver in mice fed a high-calorie diet by activating the genes for -oxidation of fatty acids. At the same time, PPAR activates the heat-producing uncoupling enzymes in brown adipose tissue (UCP1 and 3) and muscle (UCP2). By accompanying increased uncoupling (i.e., decline in ATP production) with increased fatty acid oxidation (i.e., leading to ATP production), the organism can maintain its normal ATP/ADP ratio. 3 Wang et al. 3 have called PPAR a sensor for energy homeostasis, protecting against dietary and genetic obesity. A comparison with other members of the PPAR family of receptors (Table 1) shows that PPAR and act in parallel, enhancing fatty acid combustion; however, PPAR functions in adipose tissue and muscle and PPAR in liver, with polyunsaturated fatty acids acting as ligands. PPAR and function in opposition to each other, however, with PPAR promoting fatty acid oxidation and PPAR enhancing adiposity by stimulating fatty acid accumulation in adipose tissue. 1. Lee C-H, Olson P, Evans RM. Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator activated receptors. Endocrinology. 2003; 144: Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids and eicosanoids are ligands for peroxisome proliferator activated receptors and. Proc Nat Acad Sci USA.1997;94: Wang Y-X, Lee C-H, Tiep S, et al. Peroxisome-proliferator activates fat metabolism to prevent obesity. Cell. 2003;113: Barak Y, Liao D, He W, et al. Effects of peroxisome proliferator activated receptor on placentation, adiposity and colorectal cancer. Proc Nat Acad Sci USA.2002;99: Chen H, Charlat O, Tartaglia LA, et al. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 1996;84: Oliver WR Jr, Shenk JL, Snaith MR, et al. A selective peroxisome proliferator activated receptor delta agonist promotes reverse cholesterol transport. Proc Nat Acad Sci U S A.2001;98: Nutrition Reviews, Vol. 61, No. 11

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