Phospholipid Transfer Research Articles

Lipid transfers to HDL are predictors of precocious clinical coronary heart disease

Background High-density-lipoprotein (HDL) has several antiatherogenic properties and, although the concentration of HDL-cholesterol negatively correlates with incidence of coronary artery disease (CAD), this is not sufficient to evaluate the overall HDL protective role. The aim was to investigate whether precocious CAD patients show abnormalities in lipid transfers to HDL, a fundamental step in HDL metabolism and function. Methods Thirty normocholesterolemic CAD patients aged < 50 y and 30 controls paired for sex, age and B.M.I. were studied. Fasting blood samples were collected for the in vitro lipid transfer assay and plasma lipid determination. A donor nanoemulsion labeled with radioactive free-cholesterol, cholesteryl esters, phospholipids and triglycerides was incubated with whole plasma and after chemical precipitation of non-HDL fractions, supernatant was counted for radioactivity in HDL. Results LDL and HDL-cholesterol and triglycerides were equal in both groups. Transfers of free-cholesterol (3.8 ± 1.2%vs 7.0 ± 3.3%,p < 0.0001) and triglycerides (3.7 ± 1.7%vs 4.9 ± 1.9%, p = 0.0125) were diminished in CAD patients whereas cholesteryl ester transfer increased (6.5 ± 1.9%vs 4.8 ± 1.8%, p = 0.0008); phospholipid transfer was equal (17.8 ± 3.5% vs19.5 ± 3.9%). Conclusion Alterations in the transfer of lipids to HDL may constitute a new marker for precocious CAD and relation of this metabolic alteration with HDL antiatherogenic function should be investigated in future studies.

Phospholipid Transfer Protein in the Placental Endothelium Is Affected by Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) causes alterations in fetal high-density lipoproteins (HDL). Because phospholipid transfer protein (PLTP) is important for HDL (re)assembly and is expressed in the human placenta, we hypothesized that circulating fetal and/or placental PLTP expression and activity are altered in GDM. PLTP levels and activity were determined in maternal and fetal sera from GDM and controls. Placental PLTP was immunolocalized, and its expression was measured in placental tissue. PLTP regulation by glucose/insulin was studied in human endothelial cells isolated from placental vessels (HPEC). Placental Pltp expression was up-regulated in GDM (1.8-fold, P < 0.05). PLTP protein (5-fold, P < 0.01) and activity (1.4- to 2.5-fold) were higher in fetal than in maternal serum. The placental endothelium was identified as a major PLTP location. Insulin treatment of HPEC significantly increased secreted PLTP levels and activity. In GDM, fetal cholesterol, HDL-triglycerides and phospholipids were elevated compared with controls. Fetal PLTP activity was higher than maternal but unaltered in GDM. HPEC contribute to the release of active PLTP into the fetal circulation. Pltp expression is increased in GDM with hyperglycemia and/or hyperinsulinemia contributing. High PLTP activity in fetal serum may enhance conversion of HDL into cholesterol-accepting particles, thereby increasing maternal-fetal cholesterol transfer.

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.

Linkage and association of phospholipid transfer protein activity to LASS4

Phospholipid transfer protein activity (PLTPa) is associated with insulin levels and has been implicated in atherosclerotic disease in both mice and humans. Variation at the PLTP structural locus on chromosome 20 explains some, but not all, heritable variation in PLTPa. In order to detect quantitative trait loci (QTLs) elsewhere in the genome that affect PLTPa, we performed both oligogenic and single QTL linkage analysis on four large families (n = 227 with phenotype, n = 330 with genotype, n = 462 total), ascertained for familial combined hyperlipidemia. We detected evidence of linkage between PLTPa and chromosome 19p (lod = 3.2) for a single family and chromosome 2q (lod = 2.8) for all families. Inclusion of additional marker and exome sequence data in the analysis refined the linkage signal on chromosome 19 and implicated coding variation in LASS4, a gene regulated by leptin that is involved in ceramide synthesis. Association between PLTPa and LASS4 variation was replicated in the other three families (P = 0.02), adjusting for pedigree structure. To our knowledge, this is the first example for which exome data was used in families to identify a complex QTL that is not the structural locus.

Metabolism of triglyceride-rich lipoproteins and transfer of lipids to high-density lipoproteins (HDL) in vegan and omnivore subjects

Background and aimsVegan diet excludes all foodstuffs of animal origin and leads to cholesterol lowering and possibly reduction of cardiovascular disease risk. The aim was to investigate whether vegan diet improves the metabolic pathway of triglyceride-rich lipoproteins, consisting in lipoprotein lipolysis and removal from circulation of the resulting remnants and to verify whether the diet alters HDL metabolism by changing lipid transfers to this lipoprotein. Methods and results21 vegan and 29 omnivores eutrophic and normolipidemic subjects were intravenously injected triglyceride-rich emulsions labeled with 14C-cholesterol oleate and 3H-triolein: fractional clearance rates (FCR, in min−1) were calculated from samples collected during 60 min for radioactive counting. Lipid transfer to HDL was assayed by incubating plasma samples with a donor nanoemulsion labeled with radioactive lipids; % lipids transferred to HDL were quantified in supernatant after chemical precipitation of non-HDL fractions and nanoemulsion. Serum LDL cholesterol was lower in vegans than in omnivores (2.1 ± 0.8, 2.7 ± 0.7 mmol/L, respectively, p < 0,05), but HDL cholesterol and triglycerides were equal. Cholesteryl ester FCR was greater in vegans than in omnivores (0.016 ± 0.012, 0.003 ± 0.003, p < 0.01), whereas triglyceride FCR was equal (0.024 ± 0.014, 0.030 ± 0.016, N.S.). Cholesteryl ester transfer to HDL was lower in vegans than in omnivores (2.7 ± 0.6, 3.5 ± 1.5%, p < 0,05). Free-cholesterol, triglyceride and phospholipid transfer were equal, as well as HDL size. ConclusionRemnant removal from circulation, estimated by cholesteryl oleate FCR was faster in vegans, but the lipolysis process, estimated by triglyceride FCR was equal. Increased removal of atherogenic remnants and diminution of cholesteryl ester transfer may favor atherosclerosis prevention by vegan diet.

Simultaneous transfer of cholesterol, triglycerides, and phospholipids to high-density lipoprotein in aging subjects with or without coronary artery disease

OBJECTIVE:To verify whether the capacity of high-density lipoprotein (HDL) to simultaneously receive non-esterified cholesterol, triglycerides, cholesteryl esters, and phospholipids changes with aging and the presence of coronary artery disease.DESIGN:Cross-sectional study with biochemical analyses.SUBJECTS:Eleven elderly patients with coronary artery disease (74±5 years) were compared with the following groups of non-coronary artery disease subjects (referred to as “healthy”): 25 young (25±5 years), 25 middle-aged (42±6 years), and 25 elderly subjects (75±8 years).METHODS:Plasma samples were incubated with a nanoemulsion labeled with radioactive lipids; the transfer of the lipids from the nanoemulsion to the HDL was measured in chemically precipitated HDL. HDL size and paraoxonase-1 activity were also determined.RESULTS:The transfer of cholesteryl esters and phospholipids to high-density lipoprotein was significantly greater (p<0.001) in healthy elderly subjects than in the middle-aged and younger subjects. Non-esterified cholesterol and triglyceride transfer was not different among these three groups. The HDL size was significantly greater (p<0.001) in healthy elderly subjects than in the middle-aged and younger subjects. The paraoxonase-1 activity was similar among the groups. Compared with healthy elderly subjects, coronary artery disease elderly subjects had significantly less (p<0.05) transfer of non-esterified cholesterol, triglycerides, and cholesteryl esters to the HDL and a significantly smaller (p<0.05) HDL size.CONCLUSION:Because lipid transfer is enhanced in healthy elderly subjects but not in those with coronary artery disease, increasing lipid transfer to HDL may be a protective mechanism against the disease.

Chitosan oligosaccharide decreases very-low-density lipoprotein triglyceride and increases high-density lipoprotein cholesterol in high-fat-diet-fed rats

It is well known that chitosan has beneficial lipid-regulating effects, but it remains unknown whether chitosan oligosaccharide (COS), the chitosan degradation product, has the same lipid benefits. High-fat-diet-fed Wistar rats were administrated with COS by gastric gavage for three weeks. The effects of COS on lipids, lipoprotein components and lipid metabolism related protein activities were investigated. Plasma lipids level assays by an enzyme method showed that COS decreased triglyceride (TG) by 29-31%, and increased high-density lipoprotein (HDL) cholesterol by 8-11%, but did not affect low-density lipoprotein (LDL) cholesterol. Lipid distribution analysis through fast protein liquid chromatography indicated that COS significantly decreased TG content distributed in very-low-density lipoprotein (VLDL)/LDL fractions but increased cholesterol content in HDL fractions. Apolipoprotein analysis through plasma ultracentrifugation and sodium dodecyl sulfate polyacrylamide gel electrophoresis displayed that COS decreased apolipoprotein B-100 of LDL and increased apolipoprotein E of LDL and apolipoprotein B-100 of VLDL, but did not change apoA-I content of HDL particles. Lipoprotein formation associated protein determination showed that COS also increased plasma activity of lecithin cholesterol acyl transferase but not phospholipid transfer protein. The present study suggests that COS may play a beneficial role in plasma lipid regulation of rats with dyslipidemia induced by high-fat diet. The COS-decreased VLDL/LDL TG and -enhanced HDL cholesterol may be related to the upregulated activity of lecithin cholesterol acyl transferase.

Post-menopausal hormone therapy reduces autoantibodies to oxidized apolipoprotein B100

The aim of the study was to verify whether post-menopausal hormone replacement therapy (HRT) modifies autoantibody titers against oxidized low-density lipoprotein (LDL) (anti-LDLoxi), against epitopes of oxidized apolipoprotein B100 and common carotid intima-media thickness (IMT) in these women. Sixty-eight women in pre-menopause (PMW) and 216 in post-menopause (POMW) were recruited; eighty-three had undergone HRT for at least 12 months, where 48 received conjugated estrogens alone (EHRT) and 35 received conjugated estrogen and medroxyprogesterone acetate (CHRT). ELISA was used to determine autoantibodies. Lipoprotein lipase (LPL), hepatic lipase (HL), cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activities were assayed by radiometric methods. IMT was measured using Doppler ultrasound. Anti-oxidized LDL and anti-D antibodies increased by 40% (p ≤ 0.003) and 42% (p ≤ 0.006), respectively, with menopause. There was a surprising and significant 7% reduction in anti-D2 antibody titers with HRT (p ≤ 0.050), indicating a positive effect of treatment on the immune response to oxidized LDL. Combined HRT decreased activities of HL and LPL. HRT did not change common carotid IMT, which was increased by 32% as expected after menopause (p ≤ 0.030). This study describes, for the first time, the protective effect of HRT on decreasing autoantibody titers against oxidized apolipoprotein B in LDL.

Novel aspects of HDL level and function in a clinical setting

HDL is composed of heterogeneous particles ranging between 7 and 14 nm. ApoA1, the main HDL apolipoprotein, is synthesized in hepatic and intestinal cells and is a determinant of the formation and stability of HDL and the two main HDL functions: cholesterol esterification and reverse cholesterol transportation [1]. HDL is secreted by the liver as disk-shaped particles constituted by ApoA1, phospholipids and unesterified cholesterol. Nascent HDL becomes progressively spherical by cholesterol esterification. HDL also originates in the plasma compartment, by accretion of ApoA1 to phospholipids and unesterified cholesterol derived from lipolysis of triglyceride-rich lipoproteins [1]. Continuous exchange of lipids and proteins between HDL and the other lipoproteins and cholesterol esterification keeps HDL particles in constant remodeling. Lipid exchange is mediated by cholesteryl ester transfer protein (CETP), mainly for cholesteryl esters and triglycerides and phospholipid transfer protein mainly for phospholipids [2]. Reverse cholesterol transportation begins with the binding of HDL ApoA1 to the ABCA1 protein complex and the transfer of unesterified cholesterol from the cells to HDL. The transfer of cholesterol to nascent HDL is facilitated by ABCA1, whereas ABCAG1 shuttles cholesterol to mature HDL particles. Subsequently, reverse cholesterol transportation is driven by the cholesterol esterification process in HDL particles that are taken up by the liver by SR-BI receptors or transferred to other lipoproteins that are also taken up by the liver for excretion in bile [2].

Relation of Increased Prebeta-1 High-Density Lipoprotein Levels to Risk of Coronary Heart Disease

Preβ-1 high-density lipoprotein (HDL) plays a key role in reverse cholesterol transport by promoting cholesterol efflux. Our aims were (1) to test previous associations between preβ-1 HDL and coronary heart disease (CHD) and (2) to investigate whether preβ-1 HDL levels also are associated with risk of myocardial infarction (MI). Plasma preβ-1 HDL was measured by an ultrafiltration-isotope dilution technique in 1,255 subjects recruited from the University of California-San Francisco Lipid and Cardiovascular Clinics and collaborating cardiologists. Preβ-1 HDL was significantly and positively associated with CHD and MI even after adjustment for established risk factors. Inclusion of preβ-1 HDL in a multivariable model for CHD led to a modest improvement in reclassification of subjects (net reclassification index 0.15, p = 0.01; integrated discrimination improvement 0.003, p = 0.2). In contrast, incorporation of preβ-1 HDL into a risk model of MI alone significantly improved reclassification of subjects (net reclassification index 0.21, p = 0.008; integrated discrimination improvement 0.01, p = 0.02), suggesting that preβ-1 HDL has more discriminatory power for MI than for CHD in our study population. In conclusion, these results confirm previous associations between preβ-1 HDL and CHD in a large well-characterized clinical cohort. Also, this is the first study in which preβ-1 HDL was identified as a novel and independent predictor of MI above and beyond traditional CHD risk factors.

PLTP regulates STAT3 and NFκB in differentiated THP1 cells and human monocyte-derived macrophages

Phospholipid transfer protein (PLTP) plays an important role in regulation of inflammation. Previously published studies have shown that PLTP binds, transfers and neutralizes bacterial lipopolysaccharides. In the current study we tested the hypothesis that PLTP can also regulate anti-inflammatory pathways in macrophages. Incubation of macrophage-like differentiated THP1 cells and human monocyte-derived macrophages with wild-type PLTP in the presence or absence of tumor necrosis factor alpha (TNFα) or interferon gamma (IFNγ) significantly increased nuclear levels of active signal transducer and activator of transcription 3, pSTAT3Tyr705 (p<0.01). Similar results were obtained in the presence of a PLTP mutant without lipid transfer activity (PLTPM159E), suggesting that PLTP-mediated lipid transfer is not required for activation of the STAT3 pathway. Inhibition of ABCA1 by chemical inhibitor, glyburide, as well as ABCA1 RNA inhibition, reversed the observed PLTP-mediated activation of STAT3. In addition, PLTP reduced nuclear levels of active nuclear factor kappa-B (NFκB) p65 and secretion of pro-inflammatory cytokines in conditioned media of differentiated THP1 cells and human monocyte-derived macrophages. Our data suggest that PLTP has anti-inflammatory capabilities in macrophages.

C-Terminus of Apolipoprotein A-I Removes Phospholipids from a Triolein/Phospholipids/Water Interface, but the N-Terminus Does Not: A Possible Mechanism for Nascent HDL Assembly

Apolipoprotein A-I (ApoA-I) is the principle protein component of HDL, also known as “good cholesterol,” which is an inverse marker for cardiovascular disease. The N-terminal 44 amino acids of ApoA-I (N44) are predicted to be responsible for stabilization of soluble ApoA-I, whereas the C-terminal 46 amino acids (C46) are predicted to initiate lipid binding and oligomerization. In this work, we apply what we believe to be a novel application of drop tensiometry to study the adsorption and desorption of N44 and C46 at a triolein/POPC/water (TO/POPC/W) interface. The amount of peptide that adsorbed to the surface was dependent on the surface concentration of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and pressure (Π) before adsorption. At a TO/POPC/W interface, the exclusion pressure (ΠEX) of C46 was 25.8 mN/m, and was 19.3 mN/m for N44. Once adsorbed, both peptides formed a homogeneous surface with POPC but were progressively ejected from the surface by compression. During a compression, C46 removed POPC from the surface whereas N44 did not. Repeated compressions caused C46 to deplete entirely the surface of phospholipid. If full-length ApoA-I could also remove phospholipid, this could provide a mechanism for the transfer of surface components of chylomicrons and very low density lipoprotein to high density lipoprotein with the assistance of phospholipid transfer protein.

Fenofibrate, homocysteine, cholesterol efflux and primum non nocere

The FIELD study investigated the effect of long-term treatment ith fenofibrate on cardiovascular events in diabetic patients [1]. he primary outcomes were somewhat disappointing, with no eduction in coronary events and less than the expected reducion in total cardiovascular events. However, the long duration and onsiderable size of the study, as well as thoroughness of analyis, has generated many exciting opportunities to investigate the nterrelationship between variousmetabolic parameters. One such pportunitywas explored in the study byMaranghi et al. published n this issue ofAtherosclerosis [2]. The authors took advantage of the nverse correlation between changes in plasma levels of homocyseine and HDL found in the FIELD study to investigate the possible echanismsof suchassociation. Thehypothesiswas thathomocyseinemay affect cholesterol efflux, and cholesterol efflux is amajor eterminant of plasma HDL concentration and, possibly, functionlity. The effect of homocysteine on cholesterol efflux could be irect or indirect; therefore several possibilities of indirect effect formationof pre -HDLandactivities of Phospholipid Transfer Proein (PLTP) and PON-1) were also investigated. If functionality of DL was impaired by fenofibrate, through homocysteine or somehing else, that may have negated the beneficial effects of higher DL concentration and in part explain the outcomes of the FIELD tudy. However, no effect of fenofibrate, or higher plasma homoysteine level, on cholesterol efflux was found. Cholesterol efflux is the first, and often the rate limiting, process n two pathways, HDL biogenesis and reverse cholesterol transort. The rate of cholesterol efflux depends on the ability of cells o release cholesterol and on the ability of plasma lipoproteins

Macrophage cholesterol efflux to plasma and HDL in subjects with low and high homocysteine levels: A FIELD substudy

ObjectivesIncreases of homocysteine (Hcy) by fenofibrate correlated inversely to changes in HDL-C and apoA-I in the FIELD study. This finding raised the question whether high Hcy may influence HDL function and counteract benefits of fenofibrate on cardiovascular outcomes.In a subset of the FIELD study we investigated whether fenofibrate therapy or high Hcy, separately or in concert, modulate: (1) ability of plasma or HDL to facilitate cholesterol efflux from THP-1 foam cells; (2) plasma potential to generate preβ-HDL; (3) plasma phospholipid transfer protein (PLTP) activity, serum PON-1 mass and activity, HDL particle size and distribution. MethodsWe selected 33 subjects in the FIELD fenofibrate arm according to quartiles of Hcy at 5th year: 17 subjects were in the lowest (Low Hcy group) and 16 subjects were in the highest quartile (High Hcy group). In addition, 14 subjects allocated to placebo were matched by close-out Hcy levels to Low Hcy group. This design allowed us to examine the effects of both fenofibrate (comparison between placebo vs Low Hcy groups) and Hcy (comparison between close-out Low and High Hcy groups) on plasma and HDL ability to facilitate cellular cholesterol removal in the efflux assay in vitro using THP-1 foam cells. ResultsHcy levels were 13.3±0.7μmol/L (placebo), 13.2±2μmol/L (Low Hcy) and 27.4±6.5μmol/L (High Hcy). Cholesterol efflux values to HDL and plasma, percentage of plasma preβ-HDL, PLTP activity, serum PON-1 mass and HDL particle size and distribution were similar in both fenofibrate groups and comparable to those of the placebo group. ConclusionsIn the present study cohort fenofibrate and high Hcy levels did not modulate HDL and plasma functions in the first step of reverse cholesterol transport, cholesterol efflux from foam cells.

A reduction of CETP activity, not an increase, is associated with modestly impaired postprandial lipemia and increased HDL-Cholesterol in adult asymptomatic women

BackgroundThe relationship between CETP and postprandial hyperlipemia is still unclear. We verified the effects of varying activities of plasma CETP on postprandial lipemia and precocious atherosclerosis in asymptomatic adult women.MethodsTwenty-eight women, selected from a healthy population sample (n = 148) were classified according to three CETP levels, all statistically different: CETP deficiency (CETPd ≤ 4.5%, n = 8), high activity (CETPi ≥ 23.8, n = 6) and controls (CTL, CETP ≥ 4.6% and ≤ 23.7%, n = 14). After a 12 h fast they underwent an oral fat tolerance test (40 g of fat/m2 of body surface area) for 8 hours. TG, TG-rich-lipoproteins (TRL), cholesterol and TRL-TG measurements (AUC, AUIC, AR, RR and late peaks) and comparisons were performed on all time points. Lipases and phospholipids transfer protein (PLTP) were determined. Correlation between carotid atherosclerosis (c-IMT) and postprandial parameters was determined. CETP TaqIB and I405V and ApoE-ε3/ε2/ε4 polymorphisms were examined. To elucidate the regulation of increased lipemia in CETPd a multiple linear regression analysis was performed.ResultsIn the CETPi and CTL groups, CETP activity was respectively 9 and 5.3 higher compared to the CETPd group. Concentrations of all HDL fractions and ApoA-I were higher in the CETPd group and clearance was delayed, as demonstrated by modified lipemia parameters (AUC, AUIC, RR, AR and late peaks and meal response patterns). LPL or HL deficiencies were not observed. No genetic determinants of CETP deficiency or of postprandial lipemia were found. Correlations with c-IMT in the CETPd group indicated postprandial pro-atherogenic associations. In CETPd the regression multivariate analysis (model A) showed that CETP was largely and negatively predicted by VLDL-C lipemia (R2 = 92%) and much less by TG, LDL-C, ApoAI, phospholipids and non-HDL-C. CETP (model B) influenced mainly the increment in ApoB-100 containing lipoproteins (R2 = 85% negatively) and phospholipids (R2 = 13%), at the 6thh point.ConclusionThe moderate CETP deficiency phenotype included a paradoxically high HDL-C and its sub fractions (as earlier described), positive associations with c-IMT, a postprandial VLDL-C increment predicting negatively CETP activity and CETP activity regulating inversely the increment in ApoB100-containing lipoproteins. We hypothesize that the enrichment of TG content in triglyceride-rich ApoB-containing lipoproteins and in TG rich remnants increases lipoproteins' competition to active lipolysis sites,reducing their catabolism and resulting on postprandial lipemia with atherogenic consequences.

High-density lipoprotein metabolism in human apolipoprotein B100transgenic/brown adipose tissue deficient mice: a model of obesity-induced hyperinsulinemia

Obese and diabetic humans display decreased plasma high-density lipoprotein cholesterol (HDL-C) concentrations and an increased risk for coronary heart disease. However, investigation on HDL metabolism in obesity with a particular emphasis on hepatic ATP-binding cassette transporter A1 (ABCA1), the primary factor for HDL formation, has not been well studied. Human apolipoprotein B(100) transgenic (hApoB(tg)) and brown adipose tissue deficient (BATless) mice were crossed to generate hApoB(tg)/BATless mice. Male and female hApoB(tg) and hApoB(tg)/BATless mice were maintained on either a regular rodent chow diet or a diet high in fat and cholesterol until 24weeks of age. The hApoB(tg)/BATless mice that were fed a HF/HC diet became obese, developed hepatic steatosis, and had significantly elevated plasma insulin levels compared with their hApoB(tg) counterparts, but plasma concentrations of total cholesterol, HDL-C, triglycerides, and free fatty acids and lipoprotein distribution between genotypes were not significantly different. Hepatic expression of genes encoding HDL-modifying factors (e.g., scavenger receptor, class B, type I, hepatic lipase, lecithin:cholesterol acyltransferase, and phospholipid transfer protein) was either altered significantly or showed a trend of difference between 2 genotypes of mice. Importantly, hepatic protein levels of ABCA1 were significantly lowered by ∼35% in male obese hApoB(tg)/BATless mice with no difference in mRNA levels compared with hApoB(tg) counterparts. Despite reduced hepatic ABCA1 protein levels, plasma HDL-C concentrations were not altered in male obese hApoB(tg)/BATless mice. The result suggests that hepatic ABCA1 may not be a primary contributing factor for perturbations in HDL metabolism in obesity-induced hyperinsulinemia.

Sterol Regulatory Element–Binding Protein-1 Determines Plasma Remnant Lipoproteins and Accelerates Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice

Sterol regulatory element-binding protein-1 (SREBP-1) is nutritionally regulated and is known to be a key transcription factor regulating lipogenic enzymes. The goal of this study was to evaluate the roles of SREBP-1 in dyslipidemia and atherosclerosis. Transgenic mice that overexpress SREBP-1c in the liver and SREBP-1-deficient mice were crossed with low-density lipoprotein receptor (LDLR)-deficient mice, and the plasma lipids and atherosclerosis were analyzed. Hepatic SREBP-1c overexpression in LDLR-deficient mice caused postprandial hypertriglyceridemia, increased very-low-density lipoprotein (VLDL) cholesterol, and decreased high-density lipoprotein cholesterol in plasma, which resulted in accelerated aortic atheroma formation. Conversely, absence of SREBP-1 suppressed Western diet-induced hyperlipidemia in LDLR-deficient mice and ameliorated atherosclerosis. In contrast, bone marrow-specific SREBP-1 deficiency did not alter the development of atherosclerosis. The size of nascent VLDL particles secreted from the liver was increased in SREBP-1c transgenic mice and reduced in SREBP-1-deficient mice, accompanied by upregulation and downregulation of phospholipid transfer protein expression, respectively. Hepatic SREBP-1c determines plasma triglycerides and remnant cholesterol and contributes to atherosclerosis in hyperlipidemic states. Hepatic SREBP-1c also regulates the size of nascent VLDL particles.

Dry Eye Symptoms Are Increased in Mice Deficient in Phospholipid Transfer Protein (PLTP)

In the tear fluid the outermost part facing the tear-air interface is composed of lipids preventing evaporation of the tears. Phospholipid transfer protein (PLTP) mediates phospholipid transfer processes between serum lipoproteins and is also a normal component of human tears. To study whether PLTP plays any functional role in tear fluid we investigated PLTP-deficient mice, applying functional and morphologic analyses under normal housing and experimentally induced dry eye conditions. Aqueous tear fluid production, corneal epithelial morphology, barrier function, and occludin expression were assessed. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression were shown. These pathologic signs were worsened by experimentally induced dry eye both in wild-type and PLTP knock-out mice. Deficiency in the production of tear PLTP in mice is accompanied by corneal epithelial damage, a feature that is typical in human dry eye syndrome (DES). To complement animal experiments we collected tear fluid from human dry eye patients as well as healthy control subjects. Increased tear fluid PLTP activity was observed among DES patients. In conclusion, the presence of PLTP in tear fluid appears to be essential for maintaining a healthy and functional ocular surface. Increased PLTP activity in human tear fluid in DES patients suggests an ocular surface protective role for this lipid transfer protein.

Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein

The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed mutagenesis and expressed in Chinese hamster ovary Flp-In cells. The apparent size of each of the six PLTP mutants was slightly less than that of wild type by Western blot, indicating that all six sites are glycosylated or utilized. The size of the carbohydrate at each N-glycosylation site ranged from 3.14 to 4.2 kDa. The effect of site-specific N-glycosylation removal on PLTP secretion varied from a modest enhancement (15% and 60%), or essentially no effect, to a reduction in secretion (8%, 14% and 32%). Removal of N-glycosylation at any one of the six glycosylation sites resulted in a significant 35-78% decrease in PLTP activity, and a significant 29-80% decrease in PLTP specific activity compared to wild type. These data indicate that although no single N-linked carbohydrate chain is a requirement for secretion or activity, the removal of the carbohydrate chains had a quantitative impact on cellular secretion of PLTP and its phospholipid transfer activity.

Plasma phospholipid transfer protein activity is independently determined by obesity and insulin resistance in non-diabetic subjects

BackgroundPhospholipid transfer protein (PLTP) is an emerging cardio-metabolic risk factor which is intricately involved in lipoprotein metabolism. Elevated plasma PLTP activity levels are reported in obesity and diabetes mellitus, but the relative contributions of obesity and insulin resistance to plasma PLTP activity remain unclear. We tested whether plasma PLTP activity is independently related to (central) obesity and insulin resistance in non-diabetic subjects. MethodsThe relationships of plasma PLTP activity levels (phospholipid vesicles–HDL system) with waist circumference, waist/hip ratio, body mass index (BMI) and insulin resistance (homeostasis model assessment (HOMAir)) were determined in 313 non-diabetic subjects (273 men). ResultsPLTP activity was higher in 67 subjects with enlarged waist circumference (NCEP-ATP-III criteria; 102±11AU) compared to 246 subjects with normal waist (98±11AU, P=0.027). In univariate analysis PLTP activity correlated positively with waist (r=0.188), waist/hip ratio (r=0.143), BMI (r=0.125) and HOMAir (r=0.192) (P<0.05 to P<0.001). The relationship of PLTP with HOMAir was confined to subjects with the highest waist circumference and waist/hip ratio. In age- and sex-adjusted multiple linear regression models, waist circumference (β=0.158, P=0.025), but not BMI, predicted PLTP activity independently of HOMAir (β=0.126, P=0.047). Furthermore, both waist and waist/hip ratio interacted positively with HOMAir on PLTP activity (β=0.109, P=0.056 and β=0.156, P=0.034, respectively). ConclusionsIn non-diabetic subjects, both obesity and insulin resistance influence plasma PLTP activity, resulting in elevated plasma PLTP activity particularly with combined increases in obesity and insulin resistance. Higher PLTP activity could contribute to elevated cardiovascular risk in the presence of obesity and insulin resistance.