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The Antidiabetic Agent LG100754 Sensitizes Cells to Low Concentrations of Peroxisome Proliferator-activated Receptor γ Ligands

2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês

10.1074/jbc.c200004200

1083-351X

Barry M. Forman,

Metabolism, Diabetes, and Cancer

Insulin resistance and non-insulin-dependent diabetes mellitus are major causes of morbidity and mortality in industrialized nations. Despite the alarming rise in the prevalence of this disorder, the initial molecular events that promote insulin resistance remain unclear. The data presented here demonstrate that LG100754, an antidiabetic RXR ligand, defines a novel type of nuclear receptor agonist. Surprisingly, LG100754 has minimal intrinsic transcriptional activity, instead it enhances the potency of proliferator-activated receptor (PPAR) γ-retinoid X receptor heterodimers for PPARγ ligands. The ability of LG100754 to both increase PPARγ sensitivity and relieve insulin resistance implies that a deficiency in endogenous PPARγ ligands may represent an early step in the development of insulin resistance. Insulin resistance and non-insulin-dependent diabetes mellitus are major causes of morbidity and mortality in industrialized nations. Despite the alarming rise in the prevalence of this disorder, the initial molecular events that promote insulin resistance remain unclear. The data presented here demonstrate that LG100754, an antidiabetic RXR ligand, defines a novel type of nuclear receptor agonist. Surprisingly, LG100754 has minimal intrinsic transcriptional activity, instead it enhances the potency of proliferator-activated receptor (PPAR) γ-retinoid X receptor heterodimers for PPARγ ligands. The ability of LG100754 to both increase PPARγ sensitivity and relieve insulin resistance implies that a deficiency in endogenous PPARγ ligands may represent an early step in the development of insulin resistance. non-insulin-dependent diabetes mellitus peroxisome proliferator-activated receptor retinoid X receptor PPAR response element LG10068 LG100754 PPARγ-binding protein glutathione S-transferase coactivator Insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM)1 have reached epidemic status in industrialized societies (1.Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1941) Google Scholar, 2.Saltiel A.R. Cell. 2001; 104: 517-529Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Over 125 million people worldwide suffer from NIDDM, and these individuals face a dramatically increased risk for developing atherosclerotic heart disease, stroke, renal disease, blindness, and limb amputations. It is thus alarming that the number of NIDDM cases have increased 5-fold in the past decade, a trend that is predicted to continue. Equally worrisome is that NIDDM, initially defined as a disease of adult onset, is now appearing in adolescents. Insulin responsiveness can be modulated by a number of processes including transcriptional cascades controlled by nuclear hormone receptors. Nuclear receptors comprise a superfamily of transcription factors that directly regulate gene expression in response to low molecular weight ligands. Upon binding these ligands, receptors undergo a conformational change that promotes an exchange of coregulatory proteins and ultimately a change in the rate of transcription of specific target genes (3.Glass C.K. J. Biol. Chem. 2001; 276: 36865-36868Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Compounds that bind to and activate the PPARγ subunit of the PPARγ-RXR nuclear receptor heterodimer (4.Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1699) Google Scholar, 5.Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 6.Olefsky J.M. J. Clin. Invest. 2000; 106: 467-472Crossref PubMed Scopus (508) Google Scholar) alter transcription of genes involved in glucose and lipid homeostasis. Included among these target genes are lipid transporters (CD36, aquaporin), key metabolic enzymes (lipoprotein lipase, phosphoenolpyruvate carboxykinase, uncoupling protein-1), adipocyte-enriched signaling molecules (leptin, resistin, ACRP30, FIAF/PGAR), lipid-modulated nuclear receptors (LXRα), and an intermediate in the insulin signaling pathway (c-Cbl-associating protein) (2.Saltiel A.R. Cell. 2001; 104: 517-529Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 5.Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 7.Kersten S. Mandard S. Tan N.S. Escher P. Metzger D. Chambon P. Gonzalez F.J. Desvergne B. Wahli W. J. Biol. Chem. 2000; 275: 28488-28493Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 8.Yoon J.C. Chickering T.W. Rosen E.D. Dussault B. Qin Y. Soukas A. Friedman J.M. Holmes W.E. Spiegelman B.M. Mol. Cell. Biol. 2000; 20: 5343-5349Crossref PubMed Scopus (337) Google Scholar, 9.Kishida K. Shimomura I. Nishizawa H. Maeda N. Kuriyama H. Kondo H. Matsuda M. Nagaretani H. Ouchi N. Hotta K. Kihara S. Kadowaki T. Funahashi T. Matsuzawa Y. J. Biol. Chem. 2001; 276: 48572-48579Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 10.Savage D.B. Sewter C.P. Klenk E.S. Segal D.G. Vidal-Puig A. Considine R.V. O'Rahilly S. Diabetes. 2001; 50: 2199-2202Crossref PubMed Scopus (704) Google Scholar, 11.Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. 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A variety of cellular, molecular, and pharmacologic studies have shown that PPARγ activation results in increased adipogenesis, redistribution of fatty acids and triglycerides into fat, and ultimately improved insulin sensitivity (2.Saltiel A.R. Cell. 2001; 104: 517-529Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 6.Olefsky J.M. J. Clin. Invest. 2000; 106: 467-472Crossref PubMed Scopus (508) Google Scholar, 16.Olefsky J.M. Saltiel A.R. Trends Endocrinol. Metab. 2000; 11: 362-368Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Indeed, PPARγ-specific ligands such as rosiglitazone are currently used for the clinical treatment of NIDDM (17.Inzucchi S.E. Maggs D.G. Spollett G.R. Page S.L. Rife F.S. Walton V. Shulman G.I. N. Engl. J. Med. 1998; 338: 867-872Crossref PubMed Scopus (722) Google Scholar). PPARγ functions as part of a heterodimeric complex with a nuclear receptor known as RXR (4.Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1699) Google Scholar, 5.Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 6.Olefsky J.M. J. Clin. Invest. 2000; 106: 467-472Crossref PubMed Scopus (508) Google Scholar). RXR serves as a common heterodimeric partner for several nuclear receptors and is modulated by a class of ligands known as rexinoids. Since PPARγ functions as an obligate heterodimer with RXR, there has been an interest in developing RXR-specific rexinoids as potential treatments for NIDDM. A particularly interesting compound is LG754, which primarily activates PPAR-RXR heterodimers and retains potent antidiabetic properties (18.Cesario R.M. Klausing K. Razzaghi H. Crombie D. Rungta D. Heyman R.A. Lala D.S. Mol. Endocrinol. 2001; 15: 1360-1369Crossref PubMed Scopus (83) Google Scholar). We now show that LG754 defines a novel rexinoid agonist that paradoxically has little intrinsic transcriptional activity. Instead, LG754 functions by enhancing the affinity of PPARγ for its ligands. LG754 thus defines a new class of receptor agonist that can be described as a ligand sensitizer. The fact that this PPARγ sensitizer relieves insulin resistance suggests that a relative deficiency in endogenous PPARγ ligands may play a primary role in the development of insulin resistance. This notion accounts for several critical paradoxes in our understanding of NIDDM. The PPARγ luciferase reporter construct PPRE × 3 TK-Luc contains the herpesvirus thymidine kinase promoter (−105/+51) linked to three copies of the rat acyl-CoA oxidase PPRE (5′-AGGGGACCAGGACAAAGGTCACGTTCGGGA-3′). The GAL4 reporter was as described previously (19.Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (759) Google Scholar). A cytomegalovirus expression vector with a T7 promoter was used to express the following proteins in cells and/or in vitro: PPARγ (mouse PPARγ1, GenBankTM accession number U10374), RXR (human RXRα, GenBankTM accession number X52773), Gal-PBP (human PBP, GenBankTM accession number AF283812, Val574–Ser649), VP-PPARγ (mouse PPARγ ligand binding domain, GenBankTM accession number U10374, Cys163–Tyr475). For two-hybrid studies, an RXR ligand binding domain expression vector was used that contains the SV40 TAg nuclear localization signal (APKKKRKVG) fused upstream of the RXR ligand binding domain (human RXRα, GenBankTM accession number X52773, Glu203–Thr462). Gal4 fusions contained the indicated fragments fused to the C-terminal end of the yeast Gal4 DNA binding domain; VP16 fusions contained the 78 amino acid herpesvirus VP16 transactivation domain. For bacterial expression, p160 coactivator proteins were expressed as fusion proteins containing GST upstream of the 3 receptor interaction domains of SRC-1 (human SRC-1, GenBankTM accession number U59302, Asp617–Asp769), ACTR (human ACTR, GenBankTM accession number AF036892, Gly615–Gln768) or GRIP1 (mouse GRIP1, GenBankTM accession number U39060, Arg625–Lys765). The PBP fusion contained the two receptor interaction domains of PBP (human PBP, GenBankTM accession number AF283812, Val574–Ser649). CV-1 cells were grown and transfected as described previously (20.Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2731) Google Scholar). GST-coactivator fusion proteins were expressed in Escherichia coli and purified on glutathione-Sepharose columns. In vitrotranslated PPARγ (0.6 μl), RXR (0.4 μl), and coactivator proteins (5 μg) were incubated for 30 min at room temperature with 100,000 cpm of Klenow-labeled probes in 10 mm Tris, pH 8, 50 mm KCl, 6% glycerol, 0.05% Nonidet P-40, 1 mmdithiothreitol, 12.5 ng/μl poly(dI·dC) and the indicated ligands. Complexes were electrophoresed through 7% polyacrylamide gels in 0.5× TBE (45 mm Tris base, 45 mm boric acid, 1 mm EDTA) at room temperature. A32P-labeled rat acyl-CoA oxidase PPRE was used as probe (5′-AGGGGACCAGGACAAAGGTCACGTTCGGGA-3′) in Fig. 2. In Fig. 3 the PPRE was not labeled, instead a radiolabeled PPARγ ligand ([125I]SB-236636) (21.Young P.W. Buckle D.R. Cantello B.C. Chapman H. Clapham J.C. Coyle P.J. Haigh D. Hindley R.M. Holder J.C. Kallender H. Latter A.J. Lawrie K.W. Mossakowska D. Murphy G.J. Roxbee Cox L. Smith S.A. J. Pharmacol. Exp. Ther. 1998; 284: 751-759PubMed Google Scholar) was used to visualize the complexes.Figure 3LG754, an antidiabetic RXR ligand, increases the apparent potency of PPARγ ligands.A, LG754 enhances the response to limiting doses of PPARγ ligands. CV-1 cells were transfected with a PPAR reporter construct along with expression vectors for PPARγ and a βgalactosidase internal control. Cells were treated with 60 nmrosiglitazone alone or in the presence of the indicated concentration of LG754. -Fold activation was plotted (n = 6).B, LG754 shifts the dose-response profile of rosiglitazone toward a higher potency. CV-1 cells were transfected with a PPAR reporter construct along with expression vectors for PPARγ and a β-galactosidase internal control. Cells were treated with the indicated concentrations of rosiglitazone in the absence (■) or presence (▪) of 1 μm LG754, and normalized reporter activity was determined (means ± S.E., n = 4).C, LG754 shifts the dose-response profile of 15-deoxy-Δ12,14-prostaglandin J2 toward a higher potency. CV-1 cells were transfected as in B and treated with the indicated concentrations of 15-deoxy-Δ12,14-prostaglandin J2 in the absence (■) or presence (▪) of 1 μm LG754. Normalized reporter activity was determined (means ± S.E., n= 6). D, LG754 increases binding of PPARγ to its ligand. Mobility shift experiments were performed as described in the legend to Fig. 2 but using unlabeled DNA and [125I]SB-236636 (21.Young P.W. Buckle D.R. Cantello B.C. Chapman H. Clapham J.C. Coyle P.J. Haigh D. Hindley R.M. Holder J.C. Kallender H. Latter A.J. Lawrie K.W. Mossakowska D. Murphy G.J. Roxbee Cox L. Smith S.A. J. Pharmacol. Exp. Ther. 1998; 284: 751-759PubMed Google Scholar) as the PPARγ ligand. GST-PBP was included in the reaction and the DNA-bound PPARγ-RXR-PBP complex is shown. E, schematic diagram of the PPARγ ligand deficiency hypothesis. This model suggests that deficiencies in endogenous PPARγ ligands represent an early step in the development of insulin resistance in lipodystrophic diabetes, obesity-related diabetes, and in individuals with mutations in PPARγ.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cesario et al. (18.Cesario R.M. Klausing K. Razzaghi H. Crombie D. Rungta D. Heyman R.A. Lala D.S. Mol. Endocrinol. 2001; 15: 1360-1369Crossref PubMed Scopus (83) Google Scholar) have shown that the RXR ligand LG754 is a selective activator of PPAR-RXR heterodimers. As expected for a PPARγ-RXR agonist, LG754 was also shown to promote adipogenesis in 3T3-L1 cells and to relieve insulin resistance in db/db mice. This prompted us to directly compare the extent of PPARγ activation by LG754 and known PPARγ ligands. To determine the relative efficacy of LG754, we transiently transfected RXR-expressing CV-1 cells with PPARγ reporter and expression vectors and compared the activation of LG754 with that of rosiglitazone, a standard PPARγ agonist (20.Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2731) Google Scholar) (Fig. 1A). Rosiglitazone (1 μm) produced the expected strong activation (28-fold) of PPARγ, but the RXR ligand LG754 (1 μm) yielded only a 5-fold activation. These findings were unexpected in light of the fact that LG754 effectively mimics the biological effects of PPARγ ligands in vivo. Nuclear hormone receptors activate transcription by recruiting transcriptional coactivator proteins. Using a mammalian two-hybrid assay, Cesario et al. (18.Cesario R.M. Klausing K. Razzaghi H. Crombie D. Rungta D. Heyman R.A. Lala D.S. Mol. Endocrinol. 2001; 15: 1360-1369Crossref PubMed Scopus (83) Google Scholar) demonstrated that LG754 specifically recruits the coactivator PBP (also known as TRAP220 and DRIP205) to the PPARγ-RXR heterodimer. Since LG754 had less intrinsic transcriptional activity than PPARγ ligands (Fig. 1A), we compared the relative ability of LG754 and rosiglitazone to recruit PBP using the same two-hybrid assay described above. In this system, reporter expression is activated if the herpesvirus VP16 transactivation domain becomes tethered to the promoter via a PPAR-coactivator interaction. Using this assay, we found that rosiglitazone produced a strong 10-fold increase in the recruitment of PPARγ to PBP, whereas LG754 had only a 2–3-fold effect on this interaction (Fig. 1B). The relative differences in coactivator recruitment by these two ligands closely paralleled the weak effect of LG754 on activation of the PPARγ-RXR heterodimer (Fig. 1A). To further explore the effect of LG754 on coactivator recruitment, we utilized an electrophoretic mobility shift assay. Unlike the two-hybrid assay, this approach examines the effect of ligands on DNA-bound PPARγ-RXR heterodimers, i.e. native receptor complexes. Thus, PPARγ, RXR, and affinity-purified GST-coactivator fusion proteins were incubated with a 32P-labeled response element and separated by electrophoresis through a nondenaturing gel. In this experiment, we compared LG754 to LG268, another RXR-specific rexinoid with antidiabetic activity (22.Mukherjee R. Davies P.J. Cromble D.L. Bischoff E.D. Cesario R.M. Jow L. Hammann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. Paterniti J.R. Heyman R.A. Nature. 1997; 386: 407-410Crossref PubMed Scopus (576) Google Scholar). LG268 effectively recruited the p160 family of coactivators (SRC1, ACTR, GRIP) but had no effect on the GST control (Fig. 2). A quantitatively smaller, but highly reproducible, shift was also seen with PBP (Fig. 2). This demonstrates that RXR ligands can recruit coactivators to the DNA-bound PPARγ-RXR heterodimer. In contrast, LG754 failed to recruit any of these proteins (Fig. 2), confirming that LG754 does not effectively recruit coactivators to the PPARγ-RXR complex. Thus, in contrast to previously described nuclear receptor agonists, LG754 is unique in that does not possess strong transcriptional activating properties. Since LG754 has only weak intrinsic transcriptional activity, its ability to mimic PPARγ ligands in vivoimplies that this compound is functioning by an alternative mechanism. Previous studies have demonstrated that individual subunits of nuclear receptor heterodimers can have dramatic allosteric effects on its partner receptor. For example, the insect ecdysone receptor does not bind its ligand with high affinity, instead ligand binding requires association of the ecdysone receptor with its heterodimeric partner, ultraspiracle (USP) (23.Yao T.P. Forman B.M. Jiang Z. Cherbas L. Chen J.D. McKeown M. Cherbas P. Evans R.M. Nature. 1993; 366: 476-479Crossref PubMed Scopus (779) Google Scholar). The existence of such allosteric interactions among receptor heterodimers prompted us to ask whether LG754 can increase the affinity of the PPARγ-RXR complex for PPARγ ligands. Cell-based transfection assays were used to examine the effect of LG754 on rosiglitazone-mediated activation of PPARγ. CV-1 cells were transfected with PPARγ reporter and expression vectors and treated with suboptimal amounts of rosiglitazone (60 nm) in the presence of increasing amounts of LG754 (Fig. 3A). As expected, rosiglitazone (60 nm) activated PPARγ, but notably this activation was further enhanced by LG754. The optimal effect of LG754 was seen at a concentration of 1 μm (Fig. 3A), which is similar to the optimal doses required for adipogenesis in 3T3-L1 cells (18.Cesario R.M. Klausing K. Razzaghi H. Crombie D. Rungta D. Heyman R.A. Lala D.S. Mol. Endocrinol. 2001; 15: 1360-1369Crossref PubMed Scopus (83) Google Scholar). We next examined the effect of LG754 on the PPARγ dose-response curve. As expected from Fig. 1A, LG754 had minimal activity by itself. However, it shifted the rosiglitazone dose response curve leading to a 3-fold increase in the apparent potency of rosiglitazone (Fig. 3B). LG754 had a similar effect on the potency of other PPARγ ligands including 15-deoxy-Δ12,14-prostaglandin J2 (20.Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2731) Google Scholar) (Fig. 3C). These findings suggest that LG754 increases the affinity of PPARγ for its ligands. To assess the effect of LG754 on the ligand binding activity of PPARγ, an assay was required that can efficiently measure ligand binding to PPARγ-RXR heterodimers. Standard solution-based binding assays are useful for measuring binding to PPARγ monomers. While such assays can be performed in the presence of RXR, it can be difficult to ensure that all PPARγ molecules in the reaction are complexed with RXR. Therefore, solution-based assays may measure binding to a mixed population of PPARγ monomers and PPARγ-RXR heterodimers. To overcome this limitation, ligand binding was measured in a modified electrophoretic mobility shift assay where heterodimer-containing complexes can be separated and identified within the gel. Complexes were analyzed as in Fig. 2, but instead of using radiolabeled DNA, the complexes were visualized with a 125I-labeled PPARγ ligand (21.Young P.W. Buckle D.R. Cantello B.C. Chapman H. Clapham J.C. Coyle P.J. Haigh D. Hindley R.M. Holder J.C. Kallender H. Latter A.J. Lawrie K.W. Mossakowska D. Murphy G.J. Roxbee Cox L. Smith S.A. J. Pharmacol. Exp. Ther. 1998; 284: 751-759PubMed Google Scholar). Note that LG754 significantly increased the amount of125I-labeled PPARγ ligand in the complex (Fig. 3D) without affecting the total amount of complex formed (Fig. 2 and data not shown). These data suggest that LG754 enhances the affinity of PPARγ for its ligands. A large body evidence of biochemical, structural, and genetic data have firmly demonstrated that nuclear receptors activate transcription by recruiting transcriptional coactivator proteins (3.Glass C.K. J. Biol. Chem. 2001; 276: 36865-36868Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). This has led to the commonly accepted paradigm that nuclear receptor agonists function by inducing a conformation change that favors a more stable receptor-coactivator complex. The data presented here indicate that LG754 defines a new class of nuclear receptor agonist that has minimal coactivator recruitment activity and therefore minimal inherent transcriptional activity. Instead, this compound activates transcription by allosterically enhancing the ligand binding activity of its partner receptor, PPARγ. We refer to this agonist class as a "sensitizer." LG754 therefore represents the first example of a nuclear receptor-sensitizing agent. In addition to being a PPARγ sensitizer, previous studies have demonstrated that LG754 relieves insulin resistance in vivo(18.Cesario R.M. Klausing K. Razzaghi H. Crombie D. Rungta D. Heyman R.A. Lala D.S. Mol. Endocrinol. 2001; 15: 1360-1369Crossref PubMed Scopus (83) Google Scholar). These findings have important implications, since the molecular events that result in insulin resistance remain obscure. It is well known that PPARγ ligands reverse insulin resistance both in humans and in a variety of animal models. PPARγ agonists have the interesting property of lowering glucose in diabetic animals but not in non-diabetic animals (24.Zhang B. Graziano M.P. Doebber T.W. Leibowitz M.D. White-Carrington S. Szalkowski D.M. Hey P.J. Wu M. Cullinan C.A. Bailey P. Lollmann B. Frederich R. Flier J.S. Strader C.D. Smith R.G. J. Biol. Chem. 1996; 271: 9455-9459Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This implies that PPARγ ligands reverse or replace a deficiency that is unique to the diabetic state. It is intriguing to speculate that insulin resistance arises from a relative deficiency in endogenous PPARγ ligands and that PPARγ agonists are effective antidiabetic agents, because they correct this deficiency. In principle, this hypothesis could be directly tested by determining the levels of endogenous PPARγ ligands in normal and diabetic individuals. Several fatty acid derivatives and prostanoids have been shown to bind to PPARγ (4.Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1699) Google Scholar, 20.Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2731) Google Scholar); however, these ligands are not specific for PPARγ, and the precise identity of the endogenous PPARγ ligand is unknown (25.Forman B.M. Trends Mol. Med. 2001; 7: 331-332Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Therefore, a direct quantitation of endogenous PPARγ ligand levels is not currently possible and an alternative approach is required to test this "PPARγ ligand deficiency" model. Since LG754 is a PPARγ sensitizer, this compound reverses the biological effects that result from a deficiency in PPARγ ligands. The previous demonstration that LG754 relieves insulin in db/db mice (18.Cesario R.M. Klausing K. Razzaghi H. Crombie D. Rungta D. Heyman R.A. Lala D.S. Mol. Endocrinol. 2001; 15: 1360-1369Crossref PubMed Scopus (83) Google Scholar) provides support for the hypothesis that insulin resistance is secondary to suboptimal levels of the yet-to-be identified PPARγ ligand. A genetic test of the "ligand deficiency" hypothesis might include the development of animals expressing PPARγ mutants with diminished ligand binding affinity. These animals would not respond to endogenous PPARγ ligands and would be predicted to develop insulin resistance. Such animals have not been described, although PPARγ-null mice have been established (26.Miles P.D. Barak Y. He W. Evans R.M. Olefsky J.M. J. Clin. Invest. 2000; 105: 287-292Crossref PubMed Scopus (374) Google Scholar, 27.Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Kadowaki T. et al.Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1220) Google Scholar). However, as PPARγ integrates both positive (endogenous ligands) and negative signals (MAP kinase) (28.Hu E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; 274: 2100-2103Crossref PubMed Scopus (937) Google Scholar), the effects of chronic PPARγ ablation cannot be equated with those resulting from ligand deficiency. While appropriate animal models do not exist, several patients have been described with point mutations in PPARγ that result in defects in ligand binding and/or transactivation (29.Barroso I. Gurnell M. Crowley V.E. Agostini M. Schwabe J.W. Soos M.A. Maslen G.L. Williams T.D. Lewis H. Schafer A.J. Chatterjee V.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1161) Google Scholar). These individuals provide insights into the pathological consequences associated with a diminished response to endogenous PPARγ ligands. Indeed, these patients develop lipodystrophy and severe insulin resistance as would be predicted by the PPARγ ligand-deficiency hypothesis (Fig. 3E). There are a number of critical gaps in our understanding of the NIDDM-PPARγ connection. For example, PPARγ is required for adipogenesis (27.Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Kadowaki T. et al.Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1220) Google Scholar, 30.Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Koder A. Evans R.M. Mol. Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1651) Google Scholar, 31.Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. Mol. Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar), and its synthetic agonists increase adipose mass in vivo (24.Zhang B. Graziano M.P. Doebber T.W. Leibowitz M.D. White-Carrington S. Szalkowski D.M. Hey P.J. Wu M. Cullinan C.A. Bailey P. Lollmann B. Frederich R. Flier J.S. Strader C.D. Smith R.G. J. Biol. Chem. 1996; 271: 9455-9459Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This is unexpected, since insulin resistance worsens in most patients as fat mass increases. This raises a question as to how an adipogenic agent can also act as an antidiabetic agent (32.Reginato M.J. Lazar M.A. Trends Endocrinol. Metab. 1999; 10: 9-13Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar)? Another gap is highlighted by the fact that certain rare forms of NIDDM are paradoxically associated with diminished fat mass (lipodystrophy) (33.Arioglu E. Duncan-Morin J. Sebring N. Rother K.I. Gottlieb N. Lieberman J. Herion D. Kleiner D.E. Reynolds J. Premkumar A. Sumner A.E. Hoofnagle J. Reitman M.L. Taylor S.I. Ann. Intern. Med. 2000; 133: 263-274Crossref PubMed Scopus (262) Google Scholar), and perhaps even more surprising is the observation that PPARγ activators can effectively treat both obesity-dependent and lipodystrophic diabetes (6.Olefsky J.M. J. Clin. Invest. 2000; 106: 467-472Crossref PubMed Scopus (508) Google Scholar, 33.Arioglu E. Duncan-Morin J. Sebring N. Rother K.I. Gottlieb N. Lieberman J. Herion D. Kleiner D.E. Reynolds J. Premkumar A. Sumner A.E. Hoofnagle J. Reitman M.L. Taylor S.I. Ann. Intern. Med. 2000; 133: 263-274Crossref PubMed Scopus (262) Google Scholar). The PPARγ ligand deficiency hypothesis (Fig. 3E) is intriguing as it provides a rationale to close these gaps. Given the dual role of PPARγ ligands in enhancing adipose mass and insulin responsiveness, I suggest that a primary defect in the synthesis or accumulation of an endogenous PPARγ ligand might be the molecular event that underlies lipodystrophic diabetes. Although the identity of the endogenous PPARγ ligand is unknown, it has been suggested that the transcription factor SREBP-1c (ADD1) is required to produce an endogenous ligand in adipocytes (34.Kim J.B. Wright H.M. Wright M. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4333-4337Crossref PubMed Scopus (558) Google Scholar). The PPARγ ligand deficiency hypothesis would predict that treatments which lower SREBP-1c levels should result in lipodystrophy, insulin resistance, and decreased adipogenesis. Indeed, two human immunodeficiency virus protease inhibitors (indinavir and nelfinavir) that promote lipodystrophic diabetes in vivo (35.Carr A. Samaras K. Burton S. Law M. Freund J. Chisholm D.J. Cooper D.A. AIDS. 1998; 12: F51-F58Crossref PubMed Scopus (2191) Google Scholar) have been shown to inhibit adipogenesis and to reduce SREBP-1 activity (36.Dowell P. Flexner C. Kwiterovich P.O. Lane M.D. J. Biol. Chem. 2000; 275: 41325-41332Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 37.Caron M. Auclair M. Vigouroux C. Glorian M. Forest C. Capeau J. Diabetes. 2001; 50: 1378-1388Crossref PubMed Scopus (280) Google Scholar). These observations provide further support for the PPARγ ligand deficiency hypothesis. In the opposing state of obesity, it is reasonable to imagine that feedback mechanisms are triggered in an attempt to limit further lipid storage. In principle, this could be accomplished by decreasing lipogenic signals such as those represented by endogenous PPARγ ligands. Indeed, several groups have shown that SREBP-1c levels are lower in obesity (38.Kolehmainen M. Vidal H. Alhava E. Uusitupa M.I. Obes. Res. 2001; 9: 706-712Crossref PubMed Scopus (73) Google Scholar, 39.Ducluzeau P.H. Perretti N. Laville M. Andreelli F. Vega N. Riou J.P. Vidal H. Diabetes. 2001; 50: 1134-1142Crossref PubMed Scopus (226) Google Scholar, 40.Nadler S.T. Stoehr J.P. Schueler K.L. Tanimoto G. Yandell B.S. Attie A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11371-11376Crossref PubMed Scopus (322) Google Scholar), suggesting that the obese state may be associated with a corresponding decrease in endogenous PPARγ ligands. While this response may provide short term benefits, a chronic deficiency in PPARγ ligands could eventually lead to the development of insulin resistance. In effect, lipodystrophy may represent a primary PPARγ ligand deficiency, whereas in obesity-related diabetes the deficiency would be secondary to increasing fat mass. Thus, the ligand deficiency hypothesis accounts for the paradoxical association of NIDDM with both obesity and lipodystrophy. It also explains how PPARγ ligands can treat both disorders. Given the rising toll of NIDDM, these findings provide further impetus to identify and quantitate the endogenous PPARγ ligand. I thank Kevin Hollister, Eric Wang, and Karol Rostamiani for technical assistance and Richard Bergman and Gregg Van Citters for comments. [125I]SB-236636 was provided by Dr. Stephen A. Smith. I am grateful to Leslie and Susan Gonda for support of research facilities and infrastructure at the City of Hope.