Patrick I. Eacho

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.

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 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 Alzheimers 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.

Hepatic peroxisomal changes induced by a tetrazole-substituted akoxyacetophenone in rats and comparison with other species

Previous work in this laboratory indicated that compound LY171883, a tetrazole-substituted alkoxyacetophenone with leukotriene D4 antagonist activity, caused dose-related hepatomegaly in rodents without other histological evidence of liver toxicity. In the present studies, administration of LY171883 at dietary concentrations of 0.25 or 0.50% to rats for 2 weeks increased peroxisomal beta-oxidation, catalase activity, and peroxisome volume fraction in the liver. The effects were dose-related and corresponded with increases in liver weight. Dietary concentrations of 0.05 and 0.1% LY171883 did not significantly alter peroxisome morphology, enzyme activity, or liver weight. Serum triglycerides were lowered equivalently by all four dietary concentrations of LY171883, indicating that the hypotriglyceridemia was dissociated from induction of peroxisomal beta-oxidation. The hepatic effects in rats reversed within 16 days after discontinuing treatment with LY171883. Liver weight and peroxisomal enzyme activities were increased in mice by LY171883 in a manner comparable to that observed in rats, whereas hamsters were less responsive. In guinea pigs there was a minor increase in beta-oxidation at a toxic dose of LY171883, but no change in catalase or liver weight. Neither hepatomegaly nor induction of peroxisomal enzymes occurred in beagle dogs or rhesus monkeys given LY171883. Since the hepatic effects of LY171883 in rats are not observed in higher species at a significant multiple of the anticipated clinical dose, it is unlikely that such effects will occur in humans.

Effect of ciprofibrate, bezafibrate, and LY171883 on peroxisomal β-oxidation in cultured rat, dog, and rhesus monkey hepatocytes

Cultured rat hepatocytes have been used extensively to study the mechanisms of chemically induced peroxisome proliferation. Hepatocytes from nonrodent species have been used on a limited scale to study interspecies differences in the response. Because of their importance in pharmaceutical safety assessment, we have developed a model to study the response of beagle dog and rhesus monkey hepatocytes to peroxisome proliferators. Treatment of the hepatocytes with peroxisome proliferators was begun after 20 hr in culture and continued for 72 hr. Untreated rat, dog, and monkey hepatocytes retained 62, 42, and 43% of their initial (20 hr) peroxisomal beta-oxidation activity throughout 92 hr of culture. Ciprofibrate, bezafibrate, and LY171883 caused a dose-related increase in beta-oxidation in rat hepatocytes to a maximum of 10-, 8-, and 5-fold, respectively. In dog and monkey hepatocytes the increases in beta-oxidation were less than 2-fold. Peroxisome morphology in dog and monkey hepatocytes appeared to be unchanged by the drugs. Morphometric analysis in monkey hepatocytes showed no increase in peroxisome volume fraction in response to the chemicals. Treatment of dog and monkey hepatocytes with dexamethasone and glucagon during the final 24 hr in culture caused a 4- to 6-fold increase in tyrosine aminotransferase activity. This induction is characteristic of the in vivo response. The small increase in beta-oxidation reflects the relative insensitivity of the dog and monkey liver to peroxisome proliferators in vivo rather than a loss of sensitivity during culture. Cultured hepatocytes from beagle dog and rhesus monkey may provide a model for studying the mechanisms underlying the interspecies differences. Such information would help clarify the relevance of rodent data in human risk assessment.

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.

Induction of peroxisomal β-oxidation in the rat liver in vivo and in vitro by tetrazole-substituted acetophenones: Structure-activity relationships

LY171883, a leukotriene D4 antagonist in the tetrazole-substituted acetophenone structural class, previously was demonstrated to cause peroxisome proliferation in rodents. In the present studies, several analogs were tested to determine if there are structural requirements for the induction of peroxisomal beta-oxidation in the rat liver in vivo and in cultured rat hepatocytes. Liver weight and serum triglycerides also were measured in vivo. The increases in peroxisomal beta-oxidation caused by the tetrazole-substituted acetophenones in vivo ranged from negligible to greater than 17-fold and there was good agreement with the structure-activity relationships found in cultured hepatocytes. N-methylation of the acidic nitrogen of the tetrazole blocked the peroxisomal effects, indicating that the free acid was required for activity. The length of the alkyl chain linked to the tetrazole also influenced the activity of the compounds. However, the more important determinant of peroxisomal activity may be the spatial orientation of the acidic tetrazole with respect to the planar backbone of the molecule. The data indicate there is a target site for peroxisome proliferation in the liver that is able to distinguish between structurally similar analogs. This site appears to be distinct from the leukotriene receptor since both inducers and noninducers of peroxisomal beta-oxidation were shown previously to be potent leukotriene antagonists.

Inhibition of hepatic fatty acid oxidation by bezafibrate and bezafibroyl CoA

The acute effect of the hypolipidemic agent bezafibrate on fatty acid oxidation was studied in rat hepatocytes and mitochondria. Bezafibrate caused a concentration-related inhibition of oleate oxidation in liver cells. In mitochondria bezafibrate inhibited the oxidation of palmitoyl CoA but had no effect on palmitoylcarnitine oxidation, suggesting the site of inhibition was the formation of the carnitine derivative. Bezafibrate and bezafibroyl CoA inhibited the overt carnitine palmitoyltransferase (I) in rat liver mitochondria with comparable potency but with distinct kinetics. The inhibition caused by bezafibrate was not prevented by omission of Mg++-ATP from the assay mixture, indicating activation of bezafibrate to bezafibroyl CoA was not required for inhibition. The data demonstrate that bezafibrate, like several other peroxisome proliferating agents, inhibits mitochondrial fatty acid oxidation in rat liver. The inhibition may be relevant to the mechanism of peroxisome proliferation.

Changes in hepatic lipid metabolism associated with lipid accumulation and its reversal in rats given the peroxisome proliferator LY171883

Dietary administration of 0.05, 0.1, and 0.3% LY171883 to rats for 1 day caused a dose-related increase in hepatic triglycerides. When added to rat liver mitochondria in vitro, LY171883 caused competitive inhibition of carnitine palmitoyltransferase 1 (CPT-1), the rate-limiting enzyme for mitochondrial fatty acid oxidation. This effect appears to be involved in the lipid accumulation. The hepatic triglycerides in rats given 0.1% LY171883 increased progressively through 3 months of treatment. In contrast, hepatic triglycerides in high-dose rats returned to control levels by Day 3 and remained there throughout the study. The regression of the lipid corresponded with increases in hepatic peroxisomal beta-oxidation, mitochondrial beta-oxidation, and CPT-1 activity of up to 13-, 7-, and 3.2-fold, respectively. The 0.1% dose increased these parameters modestly compared to those of high-dose rats (2-, 3-, and 1.6-fold, respectively). Addition of LY171883 to mitochondria from rats given dietary treatment for 2 weeks inhibited CPT-I by the same percentage as in control mitochondria. In mid-dose rats, the induction of CPT-I was largely negated by LY171883 in vitro. Even with the inhibition, CPT-I activity in mitochondria from high-dose rats remained 2-fold higher than that in untreated controls. The data suggest that the induction of CPT-I in high-dose rats was sufficient to overcome the inhibitory action of LY171883. The increased oxidative capacity in peroxisomes and mitochondria led to the regression of the lipid in high-dose rats. The more modest increases in fatty acid oxidation in rats given 0.1% LY171883 were not sufficient to reverse the lipid accumulation.

Effects of Chronic Treatment with the Leukotriene D4-Antagonist Compound LY171883 on B6C3F1 Mice

A 2-year toxicity/oncogenicity study was done to evaluate the potential effects of the leukotriene antagonist LY171883 in B6C3F1 mice. Dietary concentrations of LY171883 during the initial 7 months of the study were 0.0, 0.005, 0.015, or 0.05% but were increased to 0.0, 0.0075, 0.0225, or 0.075% during Months 7 through 24. The estimated average daily compound intake was 0.0, 7.3, 22.5, or 80.5 mg/kg for males and 0.0, 9.2, 27.5, or 95.9 mg/kg for females. Survival was not adversely affected by treatment, however, body weight of males and females in the high dose group was significantly lower than that of controls. The chronic toxicity was localized primarily to the liver. Liver weights were increased in males in the high dose group and in females in the mid and high dose groups. Microsomal p-nitroanisole-O-demethylase activity was increased in mid and high dose females. Hepatic peroxisomal beta-oxidation was increased approximately twofold in both sexes in the high dose group only. Centrilobular eosinophilic granular change of hepatocytes was a common histopathologic finding in male and female mice in the high dose group, with the incidence and severity being greater in females. An increased incidence of hepatocellular carcinomas was observed in female mice in the mid and high dose groups. The number of male mice in the high dose group with hepatocellular carcinomas was higher than that of controls but the change was not statistically significant. Hepatocellular adenomas were increased in females in the high dose group but not in males. All groups of treated females had increased nodular hepatocellular hyperplasia.(ABSTRACT TRUNCATED AT 250 WORDS)