Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody.

Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.

Isolation and characterization of the circulating truncated form of PCSK9.

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purified from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.

Endothelial lipase activity predicts high-density lipoprotein catabolism in hemodialysis: novel phospholipase assay in postheparin human plasma.

OBJECTIVE: A novel phospholipase assay was used to measure for the first time the behavior of endothelial and hepatic phospholipase activities in postheparin human plasma of hemodialyzed patients and its relationship with atherogenic and antiatherogenic lipoprotein levels. METHODS AND RESULTS: Endothelial and hepatic phospholipase activity was assessed in a total SN1-specific phospholipase assay, using (1-decanoylthio-1-deoxy-2-decanoyl-sn-glycero-3-phosphoryl) ethylene glycol as the substrate. Hemodialyzed patients presented lower values of total and hepatic phospholipase activity than controls: 4.4 (1.9-9.0) versus 7.5 (3.6-18.0) and 2.6 (0.7-6.2) versus 6.6 (1.3-15.2) µmol of fatty acid released per milliliter of postheparin plasma per hour, respectively (P<0.001); however, endothelial lipase (EL) phospholipase activity was increased in patients: 1.7 (0.8-3.0) versus 1.1 (0.1-2.7) µmol of fatty acid released per milliliter of postheparin plasma per hour (P=0.008). EL was negatively associated with high-density lipoprotein (HDL)-cholesterol (r=-0.427; P=0.001), and apolipoprotein A-I levels, total phospholipase, and hepatic lipase activity were directly associated with low-density lipoprotein-cholesterol and apolipoprotein B. The association of EL and HDL-cholesterol remained significant when adjusting for waist circumference (ß=-0.26; P=0.05), and the effect of hepatic lipase on low-density lipoprotein-cholesterol continued after adjusting for age (ß=0.46; P= 0.001). CONCLUSIONS: Our results support the hypothesis that EL is the predominant enzyme responsible for lipolytic catabolism of HDLs in hemodialyzed patients and resolve the apparent paradox observed between low hepatic lipase activity and decreased HDL-cholesterol levels observed in these patients. In addition, the ability to assess total hepatic lipase and EL phospholipase activity in plasma will increase our knowledge of the mechanisms involved in controlling HDL levels and cardiovascular risk in hemodialyzed patients, as well as other populations with low levels of HDL-cholesterol.

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.

Quantitative characterization of the mechanism of action and impact of a 'proteolysis-permitting' anti-PCSK9 antibody.

A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY. A quantitative modeling approach is necessary to further understand this novel mechanism of action. We developed a mechanism-based model to characterize the relationship between antibody pharmacokinetics, PCSK9 and LDL cholesterol levels in animals, and used the model to better understand the underlying drivers for the improved efficiency of LY. Simulations suggested that the allowance of cleavage of PCSK9 resulting in a lack of accumulation of intact PCSK9 is the major driver of the improved potency and durability of LY. The modeling reveals that this novel 'proteolysis-permitting' mechanism of LY is a means by which an efficient antibody can be developed with a total antibody dosing rate that is lower than the target production rate. We expect this engineering approach may be applicable to other targets and that the mathematical models presented herein will be useful in evaluating similar approaches.

Hepatic peroxisomal fatty acid beta-oxidation is regulated by liver X receptor alpha.

Peroxisomes are the exclusive site for the beta-oxidation of very-long-chain fatty acids of more than 20 carbons in length (VLCFAs). Although the bulk of dietary long-chain fatty acids are oxidized in the mitochondria, VLCFAs cannot be catabolized in mitochondria and must be shortened first by peroxisomal beta-oxidation. The regulation of peroxisomal, mitochondrial, and microsomal fatty acid oxidation systems in liver is mediated principally by peroxisome proliferator-activated receptor alpha (PPARalpha). In this study we provide evidence that the liver X receptor (LXR) regulates the expression of the genetic program for peroxisomal beta-oxidation in liver. The genes encoding the three enzymes of the classic peroxisomal beta-oxidation cycle, acyl-coenzyme A (acyl-CoA) oxidase, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, are activated by the LXR ligand, T0901317. Accordingly, administration of T0901317 in mice promoted a dose-dependent and greater than 2-fold increase in the rate of peroxisomal beta-oxidation in the liver. The LXR effect is independent of PPARalpha, because T0901317-induced peroxisomal beta-oxidation in the liver of PPARalpha-null mice. Interestingly, T0901317-induced peroxisomal beta-oxidation is dependent on the LXRalpha isoform, but not the LXRbeta isoform. We propose that induction of peroxisomal beta-oxidation by LXR agonists may serve as a counterregulatory mechanism for responding to the hypertriglyceridemia and liver steatosis that is promoted by potent LXR agonists in vivo; however, additional studies are warranted.

Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism.

Lipoprotein lipase (LPL) is a key regulator of triglyceride clearance. Its coordinated regulation during feeding and fasting is critical for maintaining lipid homeostasis and energy supply. Angiopoietin-like (Angptl)3 and Angptl4 are secreted proteins that have been demonstrated to regulate triglyceride metabolism by inhibiting LPL. We have taken a targeted genetic approach to generate Angptl4- and Angptl3-deficient mice as well as transgenic mice overexpressing human Angptl4 in the liver. The Angptl4 transgenic mice displayed elevated plasma triglycerides and reduced postheparin plasma (PHP) LPL activity. A purified recombinant Angptl4 protein inhibited mouse LPL and recombinant human LPL activity in vitro. In contrast to the transgenic mice, Angptl4-deficient mice displayed hypotriglyceridemia and increased PHP LPL activity, with greater effects in the fasted compared with the fed state. Angptl3-deficient mice also displayed hypotriglyceridemia with elevated PHP LPL activity, but these mice showed a greater effect in the fed state. Mice deficient in both Angptl proteins showed an additive effect on plasma triglycerides and did not survive past 2 months of age. Our results show that Angptl3 and Angptl4 function to regulate circulating triglyceride levels during different nutritional states and therefore play a role in lipid metabolism during feeding/fasting through differential inhibition of LPL.

Liver X receptors (LXRs) regulate apolipoprotein AIV-implications of the antiatherosclerotic effect of LXR agonists.

Liver X receptors (LXRs) regulate target genes that are critical in lipoprotein metabolism and atherosclerosis. Apolipoprotein AIV (ApoAIV) is an apolipoprotein that is associated with chylomicrons and high-density lipoproteins. Plasma ApoAIV level in humans is inversely correlated with coronary artery events and overexpression of ApoAIV in mice results in significant reduction in atherosclerosis. We report here that LXRs directly regulate apoAIV at the transcriptional level. Treatment of C57B6 mice with a synthetic LXR agonist, T0901317, resulted in significant increases in plasma apoAIV that was associated with high-density lipoprotein. Examination of both intestinal and liver apoAIV mRNA revealed specific increases in liver mRNA only. In a human heptoma HepG2 cell model, apoAIV mRNA was up-regulated upon the treatment with either native or synthetic LXR agonists. Nuclear run-on study revealed a significant increase in the ApoAIV transcriptional rate upon LXR activation. Examination of the human apoAIV proximal promoter revealed a potential LXR response element that demonstrated binding with HepG2 nuclear extracts. Cotransfection studies in HepG2 cells indicated that this responsive element was functional in mediating the human ApoAIV gene response to LXR agonists. In addition, we identified a functional LXR-responsive element at 3' end enhancer region of mouse ApoAIV gene. We conclude that ApoAIV is a direct target gene of LXRs that may contribute to the antiatherogenic effect of LXR activation.

Liver X receptors as potential therapeutic targets for multiple diseases.

Liver X receptor alpha (LXRalpha) and liver X receptor beta(LXRbeta are oxysterol receptors that regulate multiple target genes involved in cholesterol homeostasis. Recent studies also suggest that the pair of receptors may also be involved in glucose metabolism, inflammation and Alzheimer's disease by regulating critical molecules involved in these pathophysiological processes. Although the prototypic LXR agonists induce liver triglyceride accumulation by regulating the hepatic lipogenesis pathway, it is hoped that a subtype-specific agonist or selective modulators would provide the desired cardioprotection and other benefits without the undesirable concomitant induction of lipogenesis. This review intends to summarize the most recent progress in the field and provide an assessment of LXRs as potential therapeutic targets.

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.

A 15-ketosterol is a liver X receptor ligand that suppresses sterol-responsive element binding protein-2 activity.

Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-alpha and -beta (LXRalpha and LXRbeta). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.

Niacin mediates lipolysis in adipose tissue through its G-protein coupled receptor HM74A.

A G-protein coupled receptor to niacin (nicotinic acid) was identified recently but the physiological/pharmacological role of the receptor remains poorly defined. We present our studies to demonstrate that HM74A, but not HM74, binds niacin at high affinities and effectively mediates Gi signaling events in human embryonic kidney HEK293 cells as well as in 3T3L1 adipocytes expressing HM74A. Furthermore, HM74A, but not HM74, expressed in differentiated 3T3L1 adipocytes effectively mediated inhibition of lipolysis by niacin. Our results provided direct evidence indicating that HM74A, but not HM74, was sufficient to mediate anti-lipolytic effect of niacin in adipose tissue.

Genetic disorders associated with ATP binding cassette cholesterol transporters.

Coronary artery disease is the most prevalent form of mortality and morbidity in Western countries. Studies in the last several decades have identified high LDL cholesterol and low HDL cholesterol as major risk factors leading to the disease. Human genetic studies have provided significant insight into the regulation of lipoprotein metabolism. In the last several years, the genes associated with several rare genetic diseases of lipid metabolism have been revealed. These landmark discoveries that identified mutant ABC cholesterol transporters as the underlying causes of these genetic disorders have paved the way for better understanding of the cellular cholesterol transport process and HDL biogenesis. This summary provides an overview and discussion of the most recent progress that includes molecular mechanism and regulation of cholesterol transport mediated by these ABC transporters.

Estrogen receptor-mediated repression of human hepatic lipase gene transcription.

Estrogen replacement therapy in women decreases hepatic lipase (HL) activity, which may account for the associated increase in HDL cholesterol. To investigate whether estrogen decreases HL transcription, transient cotransfection assays with HL promoter and estrogen receptor-alpha (ERalpha) expression constructs were performed in HepG2 cells. 17beta-estradiol (E(2)) decreased transcription driven by the -1557/+41 human HL promoter by up to 50% at 10(-7) M. Mutation of ERalpha by deletion of its transactivation domains or ligand-binding domain eliminated E(2)-induced repression of the promoter, whereas deletion of the DNA-binding domain of ERalpha resulted in a 7-fold activation by E(2). The E(2)-induced repression was maintained after mutation of a potential estrogen-response element in the promoter. The region of estrogen responsiveness was localized to -1557/-1175 of the HL promoter by deletion analysis. Mutation of an AP-1 site at -1493 resulted in a partial loss of E(2)-induced repression, similar to that caused by deletion of nucleotides -1557 to -1366. Gel shift assays with nuclear extracts from E(2)-treated HepG2 cells stably expressing ERalpha demonstrated an increase in binding to an AP-1 consensus oligonucleotide. The AP-1 activator, phorbol 12-myristate 13-acetate, inhibited the HL promoter by greater than 50%. Collectively, the data suggest that estrogen represses the transcription of the HL gene, possibly through an AP-1 pathway.

Liver X receptor and retinoic X receptor mediated ABCA1 regulation and cholesterol efflux in macrophage cells-messenger RNA measured by branched DNA technology.

ABCA1 is an ATP binding cassette transporter that plays an essential role in cholesterol and phospholipid efflux and HDL biogenesis. ABCA1 expression in macrophage cells is subject to regulation by cAMP, cholesterol loading, and ligands of the nuclear receptors liver X receptor (LXR) and retinoid X receptor (RXR). We report here the development of a rapid and high volume branched DNA (bDNA) method to measure ABCA1 mRNA. By using the bDNA method, we show that both LXR and RXR ligands effectively regulate ABCA1 expression in three macrophage cell types: mouse RAW264.7 cell line, mouse peritoneal macrophage cells, and human macrophage THP-1 cells and their regulation is additive. Furthermore, by using a radiolabeled cholesterol efflux assay, we show that both LXR and RXR ligands are sufficient to mediate cholesterol efflux in macrophage cells and their efficacy correlates with ABCA1 regulation. These studies strengthen further the notion that LXR and RXR mediate ABCA1 expression and cholesterol efflux in macrophage cells as a permissive heterodimer and development of small molecule ligands of these nuclear receptors may represent a promising approach to modulating cholesterol efflux and plasma HDL cholesterol level in humans.

Enlargement of high density lipoprotein in mice via liver X receptor activation requires apolipoprotein E and is abolished by cholesteryl ester transfer protein expression.

The factors involved in the generation of larger high density lipoprotein (HDL) particles, HDL1 and HDLc, are still not well understood. Administration of a specific synthetic liver X receptor (LXR) agonist, T0901317, in mice resulted in an increase of not only HDL cholesterol but also HDL particle size (Cao, G., Beyer, T. P., Yang, X. P., Schmidt, R. J., Zhang, Y., Bensch, W. R., Kauffman, R. F., Gao, H., Ryan, T. P., Liang, Y., Eacho, P. I., and Jiang, X. C. (2002) J. Biol. Chem. 277, 39561-39565). We have investigated the roles that apoE and CETP may play in this process. We treated apoE-deficient, cholesterol ester transport protein (CETP) transgenic, and wild type mice with various doses of the LXR agonist and monitored their HDL levels. Fast protein liquid chromatography and apolipoprotein analysis revealed that in apoE knockout mouse plasma, there was neither induction of larger HDL formation nor increase of HDL cholesterol, suggesting that apoE is essential for the LXR agonist effects on HDL metabolism. In CETP transgenic mice, CETP expression completely abolished LXR agonist-mediated HDL enlargement and greatly attenuated HDL cholesterol levels. Analysis of HDL particles by electron microscope and nondenaturing gel electrophoresis revealed similar findings. In apoE-deficient mice, LXR agonist also produced a significant increase in very low density lipoprotein/low density lipoprotein cholesterol and apolipoprotein B content. Our studies provide direct evidence that apoE and CETP are intimately involved in the accumulation of the enlarged HDL (HDL1 or HDLc) particles in mice.

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes.

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.

Coadministration of a liver X receptor agonist and a peroxisome proliferator activator receptor-alpha agonist in Mice: effects of nuclear receptor interplay on high-density lipoprotein and triglyceride metabolism in vivo.

Liver X receptors (LXRs) are master transcription factors regulating cholesterol and fatty acid metabolism. Treatment of C57B6 mice with a specific synthetic LXR agonist, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317), resulted in elevated high-density lipoprotein (HDL) cholesterol as well as plasma and liver triglycerides. Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists are known to induce peroxisomal fatty acid beta-oxidation and also mediate HDL cholesterol metabolism. We have explored the hypothesis that simultaneous activation of PPARalpha and LXR may lead to additive effects on HDL cholesterol elevation as well as attenuation of triglyceride accumulation. Coadministration of T0901317 and the specific PPARalpha agonist [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy14643)] in mice led to synergistic elevation of HDL cholesterol that was primarily associated with enlarged HDL particles enriched with apoE and apoAI. Liver phospholipid transfer protein (PLTP) mRNA and plasma PLTP activity were additively elevated, suggesting a role of PLTP in the observed HDL cholesterol elevation. Moderate increases in plasma triglyceride levels induced by LXR activation was reduced, whereas the accumulation of triglyceride in the liver was not altered upon coadministration of the PPARalpha agonist. Peroxisomal fatty acid beta-oxidation in the liver was dramatically elevated upon PPARalpha activation as expected. Interestingly, activation of LXRs via T0901317 also led to a significant increase in peroxisomal fatty acid beta-oxidation. Sterol regulatory element binding protein 1c expression was dramatically up-regulated by the LXR agonist but was not changed with PPARalpha agonist treatment. Liver lipoprotein lipase expression was additively increased upon LXR agonist and PPARalpha agonist coadministration. Our studies mark the first exploration of nuclear receptor interplay on lipid homeostasis in vivo.

A chemical switch regulates fibrate specificity for peroxisome proliferator-activated receptor alpha (PPARalpha ) versus liver X receptor.

Fenofibrate is clinically successful in treating hypertriglyceridemia and mixed hyperlipidemia presumably through peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent induction of genes that control fatty acid beta-oxidation. Lipid homeostasis and cholesterol metabolism also are regulated by the nuclear oxysterol receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta). Here we show that fenofibrate ester, but not fenofibric acid, functions as an LXR antagonist by directly binding to LXRs. Likewise, ester forms, but not carboxylic acid forms, of other members of the fibrate class of molecules antagonize the LXRs. The fibrate esters display greater affinity for LXRs than the corresponding fibric acids have for PPARalpha. Thus, these two nuclear receptors display a degree of conservation in their recognition of ligands; yet, the acid/ester moiety acts as a chemical switch that determines PPARalpha versus LXR specificity. Consistent with its LXR antagonistic activity, fenofibrate potently represses LXR agonist-induced transcription of hepatic lipogenic genes. Surprisingly, fenofibrate does not repress LXR-induced transcription of various ATP-binding cassette transporters either in liver or in macrophages, suggesting that fenofibrate manifests variable biocharacter in the context of differing gene promoters. These findings provide not only an unexpected mechanism by which fenofibrate inhibits lipogenesis but also the basis for examination of the pharmacology of an LXR ligand in humans.

Phospholipid transfer protein is regulated by liver X receptors in vivo.

Liver X receptors (LXR) belong to the nuclear receptor superfamily that can regulate important lipid metabolic pathways. The plasma phospholipid transfer protein (PLTP) is known to mediate transfer of phospholipids from triglyceride-rich lipoproteins to high density lipoprotein (HDL) and plays a critical role in HDL metabolism. We report here that a specific LXR agonist, T0901317, elevated HDL cholesterol and phospholipid in C57/BL6 mice and generated enlarged HDL particles that were enriched in cholesterol, ApoAI, ApoE, and phospholipid. The appearance of these HDL particles upon oral dosing of T0901317 in C57/BL6 mice was closely correlated with the increased plasma PLTP activity and liver PLTP mRNA levels. Nuclear run-on assay indicated that the effect of LXR agonist on PLTP expression was at the transcriptional level. In mouse peritoneal macrophage cells, PLTP expression was also up-regulated by the LXR/RXR (retinoid X receptor) heterodimer. However, cholesterol efflux in mouse peritoneal macrophage cells from PLTP-deficient mice (PLTP0) was not significantly different from wild type animals. Although in PLTP-deficient mice, the induction of HDL cholesterol as well as HDL particle size increase persisted, the extent of the induction was greatly attenuated. We conclude that PLTP is a direct target gene of LXRs in vivo and plays an important role in LXR agonist-mediated HDL cholesterol and size increase in mice.