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 concentrationand 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.-Qian, Y-W., R. J. Schmidt, Y. Zhang, S. Chu, A. Lin, H. Wang, X. Wang, T. P. Beyer, W. R. Bensch, W. Li, M. E. Ehsani, D. Lu, R. J. Konrad, P. I. Eacho, D. E. Moller, S. K. Karathanasis, and G. Cao. Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency-or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.Metabolic syndrome is a collection of abnormalities in metabolism, including obesity, nonalcoholic fatty liver disease, macrophage inflammation, impaired fasting glucose clearance, dyslipidemia, and hypertension. Insulin resistance appears to be a key feature in metabolic syndrome (47). The de novo sphingolipid synthesis pathway is considered a promising target for pharmacological intervention in insulin resistance. It has been shown that inhibition of serine palmitoyltransferase (SPT; the first enzyme for sphingolipid biosynthesis) increases insulin sensitivity (17). However, the mechanism is incompletely understood, since such an inhibition decreases many bioactive sphingolipids, including sphingomyelin (44), ceramide, and glycosphingolipids. Ceramide levels appear to be important in mediating inflammation, obesity, and insulin sensitivity (4, 17, 18). Sphingomyelin (SM) levels also appear to be important in mediating inflammation and atherosclerosis (11,27,34). However, few in vivo studies have been conducted to investigate the functions of these two metabolism-related sphingolipids separately, since animal models are lacking.The biochemical synthesis of SM occurs through the actions of SPT, 3-ketosphinganine reductase, ceramide synthase, dihydroceramide desaturase, and sphingomyelin synthase (SMS) (36). Mammalian SPT contains two subunits, Sptlc1 and Sptlc2, encoding 53-and 63-kDa proteins, respectively (13, 64). These subunits are homologous, sharing roughly 20% sequence identity (13, 64), and form a heterodimer. A third subunit, Sptlc3, has also been reported (19), but its function remains to be elucidated. Recently, the discovery of two proteins, ssSPTa and ssSPTb, was reported. Each substantially enhances the activity of mammalian SPT, expressed in either yeast or mammalian cells, and...

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 agonistmediated HDL cholesterol and size increase in mice.Epidemiological studies have revealed that plasma HDL 1 cholesterol is inversely correlated to coronary artery disease in humans. Several hypotheses have been proposed to explain the benefits of HDL. Among these, reverse cholesterol transport concept has been widely accepted. This notion, proposed more than 30 years ago by Glomset (1), is defined as the process through which nascent HDL particles remove excessive free cholesterol from peripheral tissues and carry it back to the liver for catabolism. The studies on cellular cholesterol efflux pathway were highlighted by the recent breakthrough defining the genetic defects associated with Tangier disease and hypoalphalipoproteinemia (2-5). The mutations of ATP-binding cassette transport protein 1 (ABCA1) were identified as the underlining cause of the rare genetic disorder that leads to almost total absence of plasma ApoAI and HDL cholesterol and to massive accumulation of cholesterol esters in macrophage cells.Plasma phospholipid transfer protein (PLTP) activity is also closely related to HDL levels. PLTP transfers phospholipids from triglyceride-rich lipoproteins to HDL during lipolysis. Moreover, it also participates the phospholipid exchanges between HDL particles (21). Disruption of PLTP in mice dramatically reduces plasma HDL cholesterol and phospholipid levels (6). Although its role in the circulation has been studied extensively, its potential function in the reverse cholesterol transport pathway and HDL biogenesis awaits further elucidation.Liver X receptors (LXRs) belong to the orphan nuclear receptor superfamily and exist in two isoforms, LXR␣ a...

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.

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Although statins have proven to be effective in reducing coronary artery disease through plasma LDL cholesterol reduction, residual risks of developing cardiovascular disease remain. Epidemiological studies suggest that beyond reducing LDL cholesterol, the inverse correlation of plasma HDL cholesterol to coronary artery disease may provide additional opportunities for further intervention. It is estimated that an elevation of 1 mg/dl plasma HDL cholesterol results in 2-3% reduction in cardiovascular risk ( 1, 2 ). Potential mechanisms for HDL cholesterol protection include its involvement in reverse cholesterol transport ( 3 ), anti-infl ammatory ( 4 ), anti-oxidative ( 5 ), and anti-thrombotic processes, and vessel relaxation ( 6 ). The relative quantitative contribution of each mechanism to coronary artery disease protection remains to be fully elucidated.CETP is a 74 kDa glycoprotein that is primarily synthesized in human liver and adipose tissues and is secreted into the circulation, where it becomes associated with HDL particles. It catalyzes the reciprocal neutral lipid exchange (cholesteryl ester and triglyceride) between HDL and apoB-containing lipoprotein particles, and as a result, plasma HDL cholesterol is reduced ( 7 ). Although plasma CETP activity is inversely correlated to HDL cholesterol levels ( 8 ), the role of CETP in coronary artery disease has not been conclusively established. Recent studies in humans suggest that CETP may function as a pro-atherogenic molecule ( 9 ). The atherogenicity of CETP in animal models appears to be dependent on the background of the animal models. In most atherosclerosis models, CETP functions as a pro-atherogenic molecule

Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St. John's wort (pregnane X receptor, NR1I2). In this study, we identified the first nonoxysterol natural product that functions as a ligand for the liver X receptor (LXR␣ and LXR␤; NR1H3, NR1H2), a NHR that acts as the receptor for oxysterols and plays a key role in regulation of cholesterol metabolism and transport as well as glucose metabolism. We show that paxilline, a fungal metabolite, is an efficacious agonist of both LXR␣ and LXR␤ in biochemical and in vitro cell-based assays. Paxilline binds directly to both receptors and is an activator of LXR-dependent transcription in cell-based reporter assays. We also demonstrate that paxilline binding to the receptors results in efficient activation of transcription of two physiological LXR target genes, ABCA1 and SREBP. The discovery of paxilline, the first reported nonoxysterol natural product ligand of the LXRs, may provide insight into the mechanism of ligand recognition by these receptors and reaffirms the utility of examining natural product libraries for identifying novel NHR ligands.

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