Liver X receptors are nuclear receptors that regulate metabolism of cholesterol. They are activated by oxysterols resulting in increased transcription of the ABCA1 gene, promoting cholesterol efflux and HDL formation. We have identified podocarpic acid anhydride as a 1nM agonist of LXRalpha and beta receptors. Functionally this agonist was over 8-10-fold better activator of LXR receptors compared to one of the natural ligands, 22-(R)-hydroxy cholesterol, in HEK-293 cells. An imide analog increased the level of HDL by 26%, decreased LDL by 10.6%, and increased triglyceride by 51% in hamsters. Discovery, synthesis, SAR and details of the activities of dimers have been described.

Patients with type 2 diabetes mellitus exhibit hyperglycemia and dyslipidemia as well as a markedly increased incidence of atherosclerotic cardiovascular disease. Here we report the characterization of a novel arylthiazolidinedione capable of lowering both glucose and lipid levels in animal models. This compound, designated TZD18, is a potent agonist with dual human peroxisome proliferator-activated receptor (PPAR)alpha/gamma activities. In keeping with its PPARgamma activity, TZD18 caused complete normalization of the elevated glucose in db/db mice and Zucker diabetic fatty rats. TZD18 lowered both cholesterol and triglycerides in hamsters and dogs. TZD18 inhibited cholesterol biosynthesis at steps before mevalonate and reduced hepatic levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Moreover, TZD18 significantly suppressed gene expression of fatty acid synthesis and induced expression of genes for fatty acid degradation and triglyceride clearance. Studies on 17 additional PPARalpha or PPARalpha/gamma agonists showed that lipid lowering in hamsters correlated with the magnitude of hepatic gene expression changes. Importantly, the presence of PPARgamma agonism did not affect the relationship between hepatic gene expression and lipid lowering. Taken together, these data suggest that PPARalpha/gamma agonists, such as TZD18, affect lipid homeostasis, leading to an antiatherogenic plasma lipid profile. Agents with these properties may provide favorable means for treatment of type 2 diabetes and dyslipidemia and the prevention of atherosclerotic cardiovascular disease.

The LXR nuclear receptors are intracellular sensors of cholesterol excess and are activated by various oxysterols. LXRs have been shown to regulate multiple genes of lipid metabolism, including ABCA1 (formerly known asABC1). ABCA1 is a lipid pump that effluxes cholesterol and phospholipid out of cells. ABCA1 deficiency causes extremely low high density lipoprotein (HDL) levels, demonstrating the importance of ABCA1 in the formation of HDL. The present work shows that the acetyl-podocarpic dimer (APD) is a potent, selective agonist for both LXRα (NR1H3) and LXRβ (NR1H2). In transient transactivation assays, APD was ∼1000-fold more potent, and yielded ∼6-fold greater maximal stimulation, than the widely used LXR agonist 22-(R)-hydroxycholesterol. APD induced ABCA1mRNA levels, and increased efflux of both cholesterol and phospholipid, from multiple cell types. Gas chromatography-mass spectrometry measurements demonstrated that APD stimulated efflux of endogenous cholesterol, eliminating any possible artifacts of cholesterol labeling. For both mRNA induction and stimulation of cholesterol efflux, APD was found to be more effective than was cholesterol loading. Taken together, these data show that APD is a more effective LXR agonist than endogenous oxysterols. LXR agonists may therefore be useful for the prevention and treatment of atherosclerosis, especially in the context of low HDL levels.

The liver X receptors, LXRalpha and LXRbeta, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRalpha knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F(3)MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F(3)MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F(3)MethylAA induced cyp7a mRNA, confirming conclusions from the knockout mouse studies. Furthermore, in rat in vivo studies, F(3)MethylAA induced liver cyp7a mRNA and enzyme activity. A critical species difference is also reported in that neither F(3)MethylAA nor 22(R)-hydroxycholesterol induced cyp7a in human primary hepatocytes. However, other LXR target genes, ABCA1, ABCG1, and SREBP1, were regulated.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARalpha and PPARgamma, relatively little is known about the most widely expressed PPAR subtype, PPARdelta. Here we show that treatment of insulin resistant db/db mice with the PPARdelta agonist L-165041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L-165041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARdelta ligand, but was increased by a PPARgamma agonist. These data suggest both that PPARdelta is involved in the regulation of cholesterol metabolism in db/db mice and that PPARdelta ligands could potentially have therapeutic value.

The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15-30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [(3)H] cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [(3)H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [(3)H]cholesterol. Caco-2 cells took up 2- to 5-fold more [(3)H]cholesterol than fluoresterol from bile acid micelles, and esterified 21-24% of the fluoresterol but only 1-4% of the [(3)H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.

Monocytes can be activated by incubation with opsonized zymosan (Zop), and under these conditions can oxidize low density lipoprotein (LDL). We have characterized the biochemical changes in the lipoprotein after this oxidation. We found that monocyte-oxidized LDL has increased mobility on agarose gels, increased absorbance at 234 nm, increased content of lysophosphatidylcholine, and fluorescence at 430 nm when excited at 350 nm. All these features were somewhat less pronounced in monocyte-oxidized LDL than in LDL oxidized by 5 μm CuSO4. Under appropriate conditions, Zop-stimulated monocytes oxidized LDL to a form recognized by macrophage scavenger receptors. Monocytes stimulated by Zop produced superoxide and also oxidized LDL, whereas monocytes stimulated by phorbol ester produced slightly more superoxide but did not oxidize LDL. We found that the chelators EDTA and diethylenetriaminepentaacetic acid inhibited LDL oxidation by Zop-stimulated monocytes, implying a requirement for transition metal ions. We found that Zop contained approximately 5 nmol iron per mg, probably as Fe3+. Zop stripped of its iron supported superoxide production by monocytes, but did not support LDL oxidation. Furthermore, Fe2+ appeared in the medium when monocytes were incubated with Zop, but not with iron-stripped Zop. Taken together, these results imply that monocytes stimulated by Zop are able to oxidize LDL only because of contaminating iron in the commercial zymosan preparations. —Xing, X., J. Baffic, and C. P. Sparrow. LDL oxidation by activated monocytes: characterization of the oxidized LDL and requirement for transition metal ions. J. Lipid. Res. 1998. 39: 2201–2208.