Phospholipid Transfer Protein Gene Research Articles

C3H/HeNSlc mouse with low phospholipid transfer protein expression showed dyslipidemia

High serum levels of triglycerides (TG) and low levels of high-density lipoprotein cholesterol (HDL-C) increase the risk of coronary heart disease in humans. Herein, we first reported that the C3H/HeNSlc (C3H-S) mouse, a C3H/HeN-derived substrain, is a novel model for dyslipidemia. C3H-S showed hypertriglyceridemia and low total cholesterol (TC), HDL-C, and phospholipid (PL) concentrations. To identify the gene locus causing dyslipidemia in C3H-S, we performed genetic analysis. In F2 intercrosses between C3H-S mice and strains with normal serum lipids, the locus associated with serum lipids was identified as 163–168 Mb on chromosome 2. The phospholipid transfer protein (Pltp) gene was a candidate gene within this locus. Pltp expression and serum PLTP activity were markedly lower in C3H-S mice. Pltp expression was negatively correlated with serum TG and positively correlated with serum TC and HDL-C in F2 mice. Genome sequencing analysis revealed that an endogenous retrovirus (ERV) sequence called intracisternal A particle was inserted into intron 12 of Pltp in C3H-S. These results suggest that ERV insertion within Pltp causes aberrant splicing, leading to reduced Pltp expression in C3H-S. This study demonstrated the contribution of C3H-S to our understanding of the relationship between TG, TC, and PL metabolism via PLTP.

Human cholesteryl ester transport protein transgene promotes macrophage reverse cholesterol transport in C57BL/6 mice and phospholipid transfer protein gene knockout mice.

Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) belong to the same gene family. Liver-specific expression of CETP improves reverse cholesterol transport (RCT) and PLTP knockout (KO) decreases RCT in mice. In this study, we investigate the effect of CETP transgene (CETP-tg) on RCT and whether CETP-tg can partially restore RCT efficiency in PLTP KO mice. Several rounds of crossing were carried out to produce colonies of wild type (WT), CETP-tg, PLTP KO, and CETP-tg × PLTP KO mice were obtained after several generations of reproduction. The efficiency of RCT was detected using [3H]-cholesterol-laden macrophages, and the underlying mechanisms were investigated by multiple techniques. Our data demonstrated that CETP-tg significantly increased the transport rate of [3H]-cholesterol from macrophages to plasma and liver, and finally the excretion through feces compared to the WT littermates. The RCT improving effect of CETP-tg was similar in PLTPKO mice. Furthermore, CETP-tg did not affect the expression of RCT-related proteins, such as low-density lipoprotein receptor. The mechanisms of improving RCT may be attributed to the low level of oxidized lipids in CETP-tg mouse and CETP-mediated lipid transport. Collectively, CETP-tg improves RCT in mice, and CETP can not compensate for PLTP deficiency.

Association between the PLTP rs4810479 SNP and Serum Lipid Traits in the Chinese Maonan and Han Populations

The association between the phospholipid transfer protein (PLTP) gene rs4810479 single-nucleotide polymorphism (SNP) and serum lipid levels is largely unknown. This investigation aimed to evaluate the relationship between the PLTP rs4810479 SNP, several environmental risk factors, and serum lipid parameters in the Chinese Maonan and Han nationalities. Polymerase chain reaction-restriction fragment length polymorphism, gel electrophoresis, and direct sequencing were employed to determine the PLTP rs4810479 genotypes in 633 Maonan and 646 Han participants. The frequencies of CC, CT, and TT genotypes and the C allele were different between Maonan and Han groups (29.07%, 53.08%, 17.85%, and 55.61% vs. 35.60%, 49.70%, 14.70%, and 60.45%, respectively, P < 0.05). The C allele carriers in the Maonan group had higher high-density lipoprotein cholesterol levels than the C allele noncarriers, but this finding was only found in Maonan males but not in females. The C allele carriers in Han males had lower total cholesterol and low-density lipoprotein cholesterol levels than the C allele noncarriers. Serum lipid profiles were also affected by several traditional cardiovascular risk factors in both populations. There might be an ethnic- and/or sex-specific association between the PLTP rs4810479 SNP and serum lipid traits.

PPAR-β/δ activation promotes phospholipid transfer protein expression

The peroxisome proliferator-activated receptor (PPAR)-β/δ has emerged as a promising therapeutic target for treating dyslipidemia, including beneficial effects on HDL cholesterol (HDL-C). In the current study, we determined the effects of the PPAR-β/δ agonist GW0742 on HDL composition and the expression of liver HDL-related genes in mice and cultured human cells. The experiments were carried out in C57BL/6 wild-type, LDL receptor (LDLR)-deficient mice and PPAR-β/δ-deficient mice treated with GW0742 (10mg/kg/day) or a vehicle solution for 14 days. GW0742 upregulated liver phospholipid transfer protein (Pltp) gene expression and increased serum PLTP activity in mice. When given to wild-type mice, GW0742 significantly increased serum HDL-C and HDL phospholipids; GW0742 also raised serum potential to generate preβ-HDL formation. The GW0742-mediated effects on liver Pltp expression and serum enzyme activity were completely abolished in PPAR-β/δ-deficient mice. GW0742 also stimulated PLTP mRNA expression in mouse J774 macrophages, differentiated human THP-1 macrophages and human hepatoma Huh7. Collectively, our findings demonstrate a common transcriptional upregulation by GW0742-activated PPAR-β/δ of Pltp expression in cultured cells and in mouse liver resulting in enhanced serum PLTP activity. Our results also indicate that PPAR-β/δ activation may modulate PLTP-mediated preβ-HDL formation and macrophage cholesterol efflux.

Identification and analysis of phospholipid transfer protein polymorphisms and their association with marbling score in Hanwoo (Korean cattle)

Phospholipid transfer protein (PLTP) regulates high-density lipoprotein metabolism. The gene encoding PLTP is located on bovine chromosome 13. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the Hanwoo (Bos taurus coreanae) PLTP gene to detect novel mutations affecting economically important traits. Five SNPs were identified in the coding region (C7368T, G7453A, C9888T, and C9905T) and intron (A1750G). G7453A changes amino acid 362 of PLTP from alanine to threonine, and C9888T changes amino acid 491 of PLTP from proline to serine. Statistical analyses revealed that the G7453A and C9888T polymorphisms in the PLTP gene were significantly associated with marbling score (P < 0.05). The relationship between haplotype and economic traits was analyzed and found to be significantly associated with marbling score (P < 0.05). The results suggest that PLTP polymorphisms might be an important genetic influence on economic traits in Hanwoo.

Increased Amyloid-β Peptide-Induced Memory Deficits in Phospholipid Transfer Protein (PLTP) Gene Knockout Mice

Oxidative stress is recognized as one of the earliest and most intense pathological processes in Alzheimer's disease (AD), and the antioxidant vitamin E has been shown to efficiently prevent amyloid plaque formation and neurodegeneration. Plasma phospholipid transfer protein (PLTP) has a major role in vitamin E transfers in vivo, and PLTP deficiency in mice is associated with reduced brain vitamin E levels. To determine the impact of PLTP on amyloid pathology in vivo, we analyzed the vulnerability of PLTP-deficient (PLTP-KO) mice to the toxic effects induced by intracerebroventricular injection of oligomeric amyloid-β 25-35 (Aβ 25-35) peptide, a non-transgenic model of AD. Under basal conditions, PLTP-KO mice showed increased cerebral oxidative stress, increased brain Aβ 1-42 levels, and a lower expression of the synaptic function marker synaptophysin, as compared with wild-type mice. This PLTP-KO phenotype was associated with increased memory impairment 1 week after Aβ25-35 peptide injection. Restoration of brain vitamin E levels in PLTP-KO mice through a chronic dietary supplementation prevented Aβ 25-35-induced memory deficits and reduced cerebral oxidative stress and toxicity. We conclude that PLTP, through its ability to deliver vitamin E to the brain, constitutes an endogenous neuroprotective agent. Increasing PLTP activity may offer a new way to develop neuroprotective therapies.

Atherosclerosis and Vascular Biology

Atherosclerosis and Vascular Biology

Type 2 diabetes mellitus interacts with obesity and common variations in PLTP to affect plasma phospholipid transfer protein activity

Phospholipid transfer protein (PLTP) is an emerging cardiometabolic risk marker that is important in high-density lipoprotein (HDL) and triglyceride metabolism. Plasma PLTP activity is elevated in type 2 diabetes mellitus, whereas glucose may regulate PLTP gene transcription in vitro. Of interest, common PLTP variations that predict cardiovascular disease have been identified recently. We investigated whether the diabetic state is able to amplify relationships between obesity and PLTP gene variations with circulating PLTP levels. Plasma PLTP activity (using a phospholipid vesicles-HDL system), PLTP gene score [number of PLTP activity-decreasing alleles based on two tagging polymorphisms (rs378114 and rs60- 65904)] and waist circumference were determined in two Dutch cohorts comprising 237 patients with type 2 diabetes and 78 control subjects. Patients with diabetes were more obese (P < 0.001 for prevalence of increased waist circumference) and had 13% higher plasma PLTP activity (P < 0.001). PLTP gene score was not different in diabetic and control subjects (P = 0.40). PLTP activity was highest in patients with diabetes with an enlarged waist and lowest in control subjects with a normal waist circumference (P < 0.001). Multiple linear regression analysis revealed a positive interaction between diabetes status and waist circumference on PLTP activity (β = 0.200, P = 0.005). Furthermore, diabetes status (β = -0.485, P = 0.046) or HbA1c (β = -0.240, P = 0.035) interacted with PLTP gene score to affect PLTP activity. Type 2 diabetes and enlarged waist circumference interact to impact on plasma PLTP activity. Diabetes may also amplify the association between plasma PLTP activity and common PLTP gene variations. Our findings support the hypothesis that diabetes-environment and diabetes-gene interactions govern plasma PLTP activity.

Genetic Association between PLTP Gene Polymorphisms and Alzheimer’s Disease in a Japanese Population

Background/Aims: A recent paper reported that the phospholipid transfer protein (PLTP) reduces phosphorylation of tau in human neuronal cells. In addition, patients with Alzheimer’s disease (AD) have significantly higher levels of PLTP in brain tissue and significantly lower PLTP-mediated phospholipid transfer activity in cerebrospinal fluid. PLTP also affects apolipoprotein E (APOE) secretion from glial cells. This study aimed to investigate whether single nucleotide polymorphisms (SNPs) of the PLTP gene are associated with AD. Methods: Five SNPs, genotyped using TaqMan® technology, were analyzed using a case-control study design. Furthermore, we also checked for a synergetic association between the PLTP gene, APOE and AD. Our case-control dataset consisted of 180 AD patients and 130 age-matched controls. Results: None of the SNPs showed a statistically significant association. We could not confirm any synergetic association between the SNPs and APOE in our AD patients. Conclusion: Our results indicate that there is no genetic association between PLTP and AD. Due to the relatively small sample size and the incomplete coverage of the region surrounding the PLTP gene of this study, larger genetic studies covering the entire PLTP gene region are needed.

Innate immune response triggered by triacyl lipid A is dependent on phospholipid transfer protein (PLTP) gene expression

Hexaacyl lipopolysaccharide (LPS) aggregates in aqueous media, but its partially deacylated lipid A moiety forms monomers with weaker toxicity. Because plasma phospholipid transfer protein (PLTP) transfers hexaacyl LPS, its impact on metabolism and biological activity of triacyl lipid A in mice was addressed. Triacyl lipid A bound readily to plasma high-density lipoproteins (HDLs) when active PLTP was expressed [HDL-associated lipid A after 4.5 h: 59.1+/-16.0% of total in wild-type (WT) vs. 32.5+/-10.3% in PLTP-deficient mice, P<0.05]. In the opposite to hexaacyl LPS, plasma residence time of lipid A was extended by PLTP, and proinflammatory cytokines were produced in higher amounts in WT than PLTP(-/-) mice (remaining lipid A after 8 h: 53+/-12 vs. 35+/-7%, and IL6 concentration after 4.5 h: 45.5+/-5.9 vs. 14.6+/-7.8 ng/ml, respectively; P<0.05 in all cases). After 1 wk, onset of B16-induced melanoma was observed in only 30% of lipid A-treated WT mice, whereas >80% of the untreated WT, untreated PLTP-deficient, or lipid A-treated PLTP-deficient animals bore tumors (P<0.05 in all cases). It is concluded that PLTP is essential in mediating the association of triacyl lipid A with lipoproteins, leading to extension of its residence time and to magnification of its proinflammatory and anticancer properties.

Altered phospholipid transfer protein gene expression and serum lipid profile by topotecan

Camptothecin (CPT) and its structural analogues including topotecan and irinotecan, are inhibitors of topoisomerase I. These drugs are clinically active against a broad spectrum of cancers. To understand the genesis of chemotherapeutic resistance to the CPT family of anticancer drugs, we examined by gene expression profiling the pharmacological response to topotecan in the human hepatoma HepG2 cells and found a striking induction of the phospholipid transfer protein (PLTP) gene expression by topotecan. We showed that activation of PLTP gene expression is specific to CPT and its analogues including specific enantiomers that inhibit topoisomerase I. PLTP-mediated lipid transfer to high-density lipoprotein (HDL) is thought to be important for shuttling and redistribution of lipids between lipoproteins, which are normally returned to the liver for metabolism via the reverse cholesterol transport pathway. Hence, we asked whether elevated PLTP levels might increase the transfer of drugs into HDL. We observed that CPT was not accumulated in HDL and other lipoproteins. In addition, topotecan treatment in mice caused a marked reduction in serum HDL that was accompanied by an increase in triglyceride and cholesterol levels. These results showed that PLTP does not mediate the transfer of topoisomerase I inhibitors to serum lipoproteins. However, elevated serum PLTP levels following treatment with topoisomerase I inhibitors in cancer patients may serve as a biomarker for monitoring the development of hypertriglyceridemia and acute pancreatitis.

Reduction of HDL levels lowers plasma PLTP and affects its distribution among lipoproteins in mice

Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1 −/−). Parallel to the strong reduction in PLTP activity in plasma of ABCA1 −/− mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1 −/− mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1 −/− mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1 −/−, apoE −/− and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1 −/−, apoE −/− and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4- , 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.

Ataxia with vitamin E deficiency with a mutation in a phospholipid transfer protein gene

Sirs, Ataxia with isolated vitamin E deficiency (AVED) is an autosomal-recessive spinocerebellar degeneration caused by a mutation of the a-tocopherol transfer protein gene (aTTP) [3, 8]. This report presents a case of juvenile spinocerebellar ataxia caused by mutations in the phospholipid transfer protein (PLTP) gene as well as the aTTP gene. This is the first report to indentify a mutation in the PLTP gene associated with vitamin E-mediated spinocerebellar ataxia. The proband was a 54-year-old female who had developed resting tremors in her right hand and ataxia at 14 years of age. At around 30 years of age, her bilateral visual acuity was decreased by retinitis pigmentosa. Her parents were second-degree cousins. On neurological examination she exhibited ataxia, dysarthria, hyporeflexia, decreased proprioceptive and vibratory sensations, and resting tremors of the extremities. The serum vitamin E concentration was observed to be about half normal level, 4.2 mg/l (normal: 10.8 ± 3.3 mg/l). The results of the following laboratory tests were normal: other fat-soluble vitamins, liverand pancreas-function tests, serum lipids, lipoproteins and apoproteins. The gene analysis revealed a homozygous H101Q mutation in the aTTP gene (Fig. 1a). In AVED, the genotype–phenotype correlation is confirmed, thus indicating that the H101Q mutation is associated with retinitis pigmentosa, late onset during the 3rd decade of life and very low serum vitamin E levels (Table 1) [3, 10, 11]. The clinical findings of the current patient characteristic of juvenile onset and a mild decrease in the serum vitamin E level suggested that other genes associated with vitamin E metabolism might affect the phenotype. Genomic analyses in the PLTP gene revealed the patient and her mother were heterozygous for a H154R mutation (Fig. 1b, c). This mutation was not found in 96 healthy subjects and was different from any previously reported polymorphisms. Following intestinal absorption of vitamin E, aTTP incorporates vitamin E into VLDL resulting in secretion from the liver to the circulation. The plasma PLTP transfers vitamin E from VLDL to HDL. HDL is the predominant source of vitamin E for the brain capillary endothelial cells which facilitates uptake of vitamin E to deliver it to the central nervous system [2, 9]. The PLTP is expressed in the brain, and PLTP knock-out studies showed that PLTP deficiency caused a decrease of vitamin E content in the brain and elevated plasma concentration of vitamin E [1, 6]. The proband and mother heterozygous for the H154R mutation showed a normal plasma PLTP concentration measured by ELISA [7]; however, the plasma PLTP activity was decreased to half the normal level measured by a PLTP assay kit (BioVision, Mountain View, CA) (Table 2). His154 is located in the N-terminal domain with conserved lipid-binding pockets associated with biochemical activity [4], and therefore this H154R mutation may induce the biological dysfunction of the PLTP without causing any impairment of intracellular trafficking. PLTP is an important factor regulating the size and composition of HDL and controlling plasma HDL levels [5]. The identification of the PLTP mutation may thus make it S. Kono (&) A. Otsuji K. Shirakawa H. Suzuki H. Miyajima First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan e-mail: satokono@hama-med.ac.jp

Genetic variation in phospholipid transfer protein modulates lipoprotein profiles in hyperalphalipoproteinemia

We previously demonstrated the role of a phospholipid transfer protein (PLTP) gene variation (rs2294213) in determining levels of high-density lipoprotein cholesterol (HDL-C) in hypoalphalipoproteinemia (HypoA). We have now explored the role of PLTP in hyperalphalipoproteinemia (HyperA). The human PLTP gene was screened for sequence anomalies by DNA melting in 107 subjects with HyperA. The association with plasma lipoprotein levels was evaluated. We detected 7 sequence variations: 1 previously reported variation (rs2294213) and 5 novel mutations including 1 missense mutation (L106F). The PLTP activity was unchanged in the p.L106F mutation. The frequency of the rs2294213 minor allele was markedly increased in the HyperA group (7.0%) in comparison with a control group (4.3%) and the hypoalphalipoproteinemia group (2.2%). Moreover, rs2294213 was strongly associated with HDL-C levels. Linear regression models predict that possession of the rs2294213 minor allele increases HDL-C independent of triglycerides. These findings extend the association of rs2294213 with HDL-C levels into the extremes of the HDL distribution.

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.

Phospholipid Transfer Protein–Deficient Mice Absorb Less Cholesterol

Phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism and atherosclerosis. PLTP gene knockout (KO) mice show significant reduction of plasma cholesterol levels. Because small intestine is one of the major tissue expressing PLTP, we hypothesize that PLTP deficient small intestine absorbs less cholesterol, thus contributing to the diminishing of cholesterol levels in the plasma. We used dual-labeled cholesterol/sitostanol feeding approach to study cholesterol absorption in PLTP KO and WT mice. We found that PLTP KO mice absorb significant less cholesterol than WT mice. Primary enterocytes isolated from PLTP KO enterocytes took up significant less cholesterol. Moreover, we observed that Niemann-Pick C1-like 1 (NPC1L1) mRNA levels were significantly decreased in the small intestine of PLTP KO mice. Next, we studied the secretion of cholesterol by enterocytes. The amounts of cholesterol transported to plasma and liver were significantly reduced in PLTP KO mice, compared with WT animals. Studies with isolated PLTP KO enterocytes revealed that the secretion of cholesterol via chylomicron and intestinal-HDL was significantly reduced. Furthermore, ATP-binding cassette transporters (ABC) A1 mRNA and microsomal triglyceride transfer protein (MTP) activity levels were significantly decreased in PLTP KO small intestine. These results indicate that PLTP deficiency results in reduced cholesterol uptake as well as secretion by the intestine. We suggest that PLTP could be a useful target to lower plasma cholesterol levels, thus reducing atherosclerosis.

Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

Farnesoid X Receptor Agonist Reduces Serum Asymmetric Dimethylarginine Levels through Hepatic Dimethylarginine Dimethylaminohydrolase-1 Gene Regulation

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

Genistein Affects Adipose Tissue Deposition in a Dose-Dependent and Gender-Specific Manner

The soy isoflavone genistein targets adipose tissue and elicits physiological effects that may vary based on dietary intake. We hypothesized that the adipose effects of genistein are dose and gender dependent. Four-week-old C57BL/6 male and female mice received daily oral doses of genistein (50-200,000 microg/kg.d) or 17beta-estradiol (E2) (5 microg/kg.d) for 15 d or a diet containing 800 ppm genistein. Genistein increased epididymal and renal fat pad and adipocyte size at doses up to 50,000 microg/kg.d or at 800 ppm in the diet in males but not in females. The alteration in adipocity correlated with changes in peripheral insulin resistance. These treatments increased genistein serum concentrations from 35+/-6 to 103+/-26 nM 12 h after treatment and lowered plasma triglycerides and cholesterol levels. The 200,000 microg/kg.d genistein dose decreased adipose tissue weight similarly to E2. This genistein dose down-regulated estrogen receptor (beta more than alpha) and progesterone receptor expression and induced estrogen-dependent adipose differentiation factors; it did not change expression of the minimal consensus estrogen-responsive element in ERE-tK-LUC mice, which was positively modulated in other tissues (e.g. the lung). E2 down-regulated almost all examined adipogenic factors. Gene microarray analysis identified factors in fat metabolism and obesity-related phenotypes differentially regulated by low and high doses of genistein, uncovering its adipogenic and antiadipogenic actions. The lower dose induced the phospholipase A2 group 7 and the phospholipid transfer protein genes; the 200,000 microg/kg.d dose inhibited them. The antiadipogenic action of genistein and down-regulation of adipogenic genes required the expression of ERbeta. In conclusion, nutritional doses of genistein are adipogenic in a gender-specific manner, whereas pharmacological doses inhibited adipose deposition.

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.