Decreased adiponectin levels in familial combined hyperlipidemia patients contribute to the atherogenic lipid profile

Familial combined hyperlipidemia (FCH) is characterized by increased levels of total cholesterol, triglycerides, and/or apolipoprotein B. Other features of FCH are obesity and insulin resistance. Adiponectin is a secretory product of the adipose tissue. Low levels of adiponectin are associated with insulin resistance and accelerated atherosclerosis. The aim of this study was to determine whether decreased adiponectin levels are associated with FCH and its phenotypes. The study population comprised 644 subjects, including 158 patients with FCH. Serum adiponectin levels were determined using a commercially available ELISA. For both males and females, the mean adiponectin level (microg/ml) was significantly lower in FCH patients [2.0 (1.8-2.2) and 2.5 (2.3-2.8), respectively] compared with normolipidemic relatives [2.3 (2.2-2.5) and 3.1 (2.8-3.3), respectively] and spouses [2.4 (2.1-2.7) and 3.2 (2.8-3.6), respectively]. These differences remain significant after adjusting for waist circumference and insulin resistance. Low adiponectin level in FCH patients was a superior independent predictor of the atherogenic lipid profile, including high triglyceride levels, low HDL-cholesterol levels, and the amount of small, dense LDL present, compared with both obesity and insulin resistance. Low adiponectin levels may contribute to the atherogenic lipid profile in FCH, independent of insulin resistance and obesity, as measured by waist circumference. This finding implies a role of adipose tissue metabolism in the pathophysiology of FCH.     

Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome

We have found that hydrogen (dihydrogen; H₂) has beneficial lipid-lowering effects in high-fat diet-fed Syrian golden hamsters. The objective of this study was to characterize the effects of H₂-rich water (0.9–1.0 l/day) on the content, composition, and biological activities of serum lipoproteins on 20 patients with potential metabolic syndrome. Serum analysis showed that consumption of H₂-rich water for 10 weeks resulted in decreased serum total-cholesterol (TC) and LDL-cholesterol (LDL-C) levels. Western blot analysis revealed a marked decrease of apolipoprotein (apo)B100 and apoE in serum. In addition, we found H₂ significantly improved HDL functionality assessed in four independent ways, namely, i) protection against LDL oxidation, ii) inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to endothelial cells, iii) stimulation of cholesterol efflux from macrophage foam cells, and iv) protection of endothelial cells from TNF-α-induced apoptosis. Further, we found consumption of H₂-rich water resulted in an increase in antioxidant enzyme superoxide dismutase and a decrease in thiobarbituric acid-reactive substances in whole serum and LDL. In conclusion, supplementation with H₂-rich water seems to decrease serum LDL-C and apoB levels, improve dyslipidemia-injured HDL functions, and reduce oxidative stress, and it may have a beneficial role in prevention of potential metabolic syndrome.     

Short-term overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not VLDL triglyceride or apoB production

Increased triglyceride synthesis resulting from enhanced flux of fatty acids into liver is frequently associated with VLDL overproduction. This has led to the common belief that hepatic triglyceride synthesis can directly modulate VLDL production. We used adenoviral vectors containing either murine acyl-coenzyme A:diacylglycerol transferase 1 (DGAT1) or DGAT2 cDNA to determine the effect of a short-term increase in hepatic triglyceride synthesis on VLDL triglyceride and apolipoprotein B (apoB) production in female wild-type mice. Hepatic DGAT1 and DGAT2 overexpression resulted in 2.0-fold and 2.4-fold increases in the triglyceride content of liver, respectively. However, the increase in hepatic triglyceride content had no effect on the production rate of VLDL triglyceride or apoB in either case. Liver subfractionation showed that DGAT1 and DGAT2 overexpression significantly increased the content of triglyceride within the cytoplasmic lipid fraction, with no change in the triglyceride content of the microsomal membrane or microsomal VLDL. The increased cytoplasmic triglyceride content was observed in electron micrographs of liver sections from mice overexpressing DGAT1 or DGAT2. Overexpression of DGAT1 or DGAT2 resulted in enhanced [(3)H]glycerol tracer incorporation into triglyceride within cytoplasmic lipids. These results suggest that increasing the cytoplasmic triglyceride pool in hepatocytes does not directly influence VLDL triglyceride or apoB production. In the presence of adequate cytoplasmic lipid stores, factors other than triglyceride synthesis are rate-limiting for VLDL production.     

ACAT2 contributes cholesteryl esters to newly secreted VLDL, whereas LCAT adds cholesteryl ester to LDL in mice

The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 +/- 2.1% vs. 3.9 +/- 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 +/- 3.2% vs. 66.7 +/- 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 +/- 4.0% vs. 54.6 +/- 1.9%) and significantly increased the phospholipid percentage (11.2 +/- 0.9% vs. 19.3 +/- 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 +/- 0.8% in controls to 0 +/- 0.7% in ACAT2-deficient mice. In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.     

Cholesteryl ester transfer protein in metabolic syndrome

Objective: Low high‐density lipoprotein cholesterol (HDL‐C), hypertriglyceridemia, and small dense‐low density lipoprotein (LDL) are key components of metabolic syndrome (MS). Cholesteryl ester transfer protein (CETP) mediates the transfer of triglycerides (TGs) from TG‐rich lipoproteins to HDL and LDL particles in exchange for cholesteryl esters, leading to low HDL‐C and small dense‐LDL. The aim of this study was to investigate the role of CETP in subjects with MS. Research Methods and Procedures: In a cross‐sectional cohort of 234 middle‐aged men and 252 women randomly selected from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study, MS was diagnosed according to the National Cholesterol Education Program guidelines. CETP mass was determined by enzyme‐linked immunosorbent assay and LDL size‐by‐gradient polyacrylamide gel electrophoresis. Results: Men and women with MS had lower HDL‐C (45 ± 7 vs. 58 ± 13 and 48 ± 10 vs. 71 ± 14 mg/dL for men and women, respectively; p < 0.001 for all) and higher TG levels (222 ± 71 vs. 98 ± 54 and 167 ± 67 vs. 90 ± 35 mg/dL for men and women, respectively; p < 0.001 for all) than healthy subjects. LDL size was lower in subjects with MS (256 ± 11 Å vs. 267 ± 11 Å and 262 ± 10 Å vs. 273 ± 8 Å for men and women, respectively; p < 0.001 for all). CETP mass was higher in men with MS (1.87 ± 0.78 vs. 1.40 ± 0.65 μg/mL; p < 0.001) but not in women (1.74 ± 0.79 vs. 1.62 ± 0.62 μg/mL). CETP mass correlated inversely with LDL size in both men and women (r = ?0.19, p < 0.01 and r = ?0.13, p < 0.05 in men and women, respectively). Discussion: MS is associated with increased CETP mass in men. Increased CETP mass may be responsible for reduced HDL‐C and reduced LDL particle diameter in MS.     

Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.     

HDL is redundant for adrenal steroidogenesis in LDLR knockout mice with a human-like lipoprotein profile

The contribution of HDL to adrenal steroidogenesis appears to be different between mice and humans. In the current study, we tested the hypothesis that a difference in lipoprotein profile may be the underlying cause. Hereto, we determined the impact of HDL deficiency on the adrenal glucocorticoid output in genetically modified mice with a human-like lipoprotein profile. Genetic deletion of APOA1 in LDL receptor (LDLR) knockout mice was associated with HDL deficiency and a parallel increase in the level of cholesterol associated with nonHDL fractions. Despite a compensatory increase in the adrenal relative mRNA expression levels of the cholesterol synthesis gene, HMG-CoA reductase, adrenals from APOA1/LDLR double knockout mice were severely depleted of neutral lipids, as compared with those of control LDLR knockout mice. However, basal corticosterone levels and the adrenal glucocorticoid response to stress were not different between the two types of mice. In conclusion, we have shown that HDL is not critical for proper adrenal glucocorticoid function when mice are provided with a human-like lipoprotein profile. Our findings provide the first experimental evidence that APOB-containing lipoproteins may facilitate adrenal steroidogenesis, in an LDLR-independent manner, in vivo in mice.     

The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) is a risk factor of cardiovascular disease. Plasma Lp-PLA₂ is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA₂. We determined the plasma levels of Lp-PLA₂-bound apoB (apoB/Lp-PLA₂) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA₂ concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA₂ [apoB/Lp-PLA₂(−)] was calculated by subtracting the apoB/Lp-PLA₂ from total apoB. The apoB/Lp-PLA₂ levels were 3.6-fold higher, while apoB/Lp-PLA₂(−) were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA₂ and apoB/Lp-PLA₂(−) levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA₂, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA₂(−). Considering that Lp-PLA₂ is proatherogenic, the predominance of apoB/Lp-PLA₂ particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

Levels of atherogenic lipoproteins are unexpectedly reduced in interstitial fluid from type 2 diabetes patients

At a given level of serum cholesterol, patients with T2D have an increased risk of developing atherosclerosis compared with nondiabetic subjects. We hypothesized that T2D patients have an increased interstitial fluid (IF)-to-serum gradient ratio for LDL, due to leakage over the vascular wall. Therefore, lipoprotein profiles in serum and IF from 35 T2D patients and 35 healthy controls were assayed using fast performance liquid chromatography. The IF-to-serum gradients for VLDL and LDL cholesterol, as well as for apoB, were clearly reduced in T2D patients compared with healthy controls. No such differences were observed for HDL cholesterol. Contrary to our hypothesis, the atherogenic VLDL and LDL particles were not increased in IF from diabetic patients. Instead, they were relatively sparser than in healthy controls. The most probable explanation to our unexpected finding is that these lipoproteins are more susceptible to retainment in the extravascular space of these patients, reflecting a more active uptake by, or adhesion to, tissue cells, including macrophages in the vascular wall. Further studies are warranted to further characterize the mechanisms underlying these observations, which may be highly relevant for the understanding of why the propensity to develop atherosclerosis is increased in T2D.     

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.     

Autoantibody titers against OxLDL are correlated with Achilles tendon thickness in patients with familial hypercholesterolemia

Achilles tendon xanthomas are associated with increased cardiovascular risk in patients with familial hypercholesterolemia (FH). Oxidized low density lipoprotein (OxLDL), the antibodies against OxLDL, and the LDL-associated phospholipase A(2) (Lp-PLA(2)) may play important roles in atherogenesis. We investigated the possible association between plasma levels of OxLDL, Lp-PLA(2) activity, and autoantibody titers against various types of mildly OxLDL with Achilles tendon thickness (ATT). ATT was determined by sonography in 80 unrelated heterozygous FH patients. Three different types of mildly OxLDL were prepared: OxLDL(L), OxLDL(P), and OxLDL(D), at the end of the lag, propagation, and decomposition phases of oxidation, respectively. Similar types of OxLDL were also prepared after inactivation of the LDL-associated Lp-PLA(2). These types were denoted OxLDL(-)(L), OxLDL(-)(P), and OxLDL(-)(D). FH patients exhibited significantly higher plasma OxLDL levels and serum IgG titers against OxLDL(P) and OxLDL(D) compared with 40 normolipidemic apparently healthy controls. ATT values were positively correlated with autoantibody titers against OxLDL(P) and OxLDL(D); however, in multiple regression analysis, ATT was independently associated only with the autoantibody titers against OxLDL(D). We conclude that the IgG autoantibody titers against OxLDL(D) but not OxLDL or Lp-PLA(2) may play an important role in the pathogenesis of Achilles tendon xanthomas in FH patients.     

Persistence of an atherogenic lipid profile after treatment of acute infection with brucella

Serum lipid changes during infection may be associated with atherogenesis. No data are available on the effect of Brucellosis on lipids. Lipid parameters were determined in 28 patients with Brucellosis on admission and 4 months following treatment and were compared with 24 matched controls. Fasting levels of total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides, apolipoproteins (Apo) A, B, E CII, and CIII, and oxidized LDL (oxLDL) were measured. Activities of serum cholesterol ester transfer protein (CETP), paraoxonase 1 (PON1), and lipoprotein-associated phospholipase A₂ (Lp-PLA₂) and levels of cytokines [interleukins (IL)-1β, IL-6, and tumor necrosis factor (TNFa)] were also determined. On admission, patients compared with controls had 1) lower levels of TC, HDL-C, LDL-cholesterol (LDL-C), ApoB, ApoAI, and ApoCIII and higher LDL-C/HDL-C and ApoB/ApoAI ratios; 2) higher levels of IL-1b, IL-6, and TNFa; 3) similar ApoCII and oxLDL levels and Lp-PLA₂ activity, lower PON1, and higher CETP activity; and 4) higher small dense LDL-C concentration. Four months later, increases in TC, HDL-C, LDL-C, ApoB, ApoAI, and ApoCIII levels, ApoB/ApoAI ratio, and PON1 activity were noticed compared with baseline, whereas CETP activity decreased. LDL-C/HDL-C ratio, ApoCII, and oxLDL levels, Lp-PLA₂ activity, and small dense LDL-C concentration were not altered. Brucella infection is associated with an atherogenic lipid profile that is not fully restored 4 months following treatment.     

Effect of fenofibrate and atorvastatin on VLDL apoE metabolism in men with the metabolic syndrome

We examined the effects of fenofibrate and atorvastatin on very low density lipoprotein (VLDL) apolipoprotein (apo)E metabolism in the metabolic syndrome (MetS). We studied 11 MetS men in a randomized, double-blind, crossover trial. VLDL-apoE kinetics were examined using stable isotope methods and compartmental modeling. Compared with placebo, fenofibrate (200 mg/day) and atorvastatin (40 mg/day) decreased plasma apoE concentrations (P < 0.05). Fenofibrate decreased VLDL-apoE concentration and production rate (PR) and increased VLDL-apoE fractional catabolic rate (FCR) compared with placebo (P < 0.05). Compared with placebo, atorvastatin decreased VLDL-apoE concentration and increased VLDL-apoE FCR (P < 0.05). Fenofibrate and atorvastatin had comparable effects on VLDL-apoE concentration. The increase in VLDL-apoE FCR with fenofibrate was 22% less than that with atorvastatin (P < 0.01). With fenofibrate, the change in VLDL-apoE concentration was positively correlated with change in VLDL-apoB concentration, and negatively correlated with change in VLDL-apoB FCR. In MetS, fenofibrate and atorvastatin decreased plasma apoE concentrations. Fenofibrate decreased VLDL-apoE concentration by lowering VLDL-apoE production and increasing VLDL-apoE catabolism. By contrast, atorvastatin decreased VLDL-apoE concentration chiefly by increasing VLDL-apoE catabolism. Our study provides new insights into the mechanisms of action of two different lipid-lowering therapies on VLDL-apoE metabolism in MetS.     

ACAT2 stimulates cholesteryl ester secretion in apoB-containing lipoproteins

Previous studies in nonhuman primates revealed a striking positive correlation between liver cholesteryl ester (CE) secretion rate and the development of coronary artery atherosclerosis. CE incorporated into hepatic VLDL is necessarily synthesized by ACAT2, the cholesterol-esterifying enzyme in hepatocytes. We tested the hypothesis that the level of ACAT2 expression, in concert with cellular cholesterol availability, affects the CE content of apolipoprotein B (apoB)-containing lipoproteins. In a model system of lipoprotein secretion using COS cells cotransfected with microsomal triglyceride transfer protein and truncated forms of apoB, ACAT2 expression resulted in a 3-fold increase in microsomal ACAT activity and a 4-fold increase in the radiolabeled CE content of apoB-lipoproteins. After cholesterol-cyclodextrin (Chol-CD) treatment, CE secretion was increased by 27-fold in ACAT2-transfected cells but by only 7-fold in control cells. Chol-CD treatment also caused the percentage of CE in the apoB-lipoproteins to increase from 3% to 33% in control cells and from 16% to 54% in ACAT2-transfected cells. In addition, ACAT2-transfected cells secreted 3-fold more apoB than control cells. These results indicate that under all conditions of cellular cholesterol availability tested, the relative level of ACAT2 expression affects the CE content and, hence, the potential atherogenicity, of nascent apoB-containing lipoproteins.     

Critical role of cholesterol ester transfer protein in nicotinic acid-mediated HDL elevation in mice

Nicotinic acid is a commonly used anti-dyslipidemic agent that increases plasma levels of HDL-cholesterol and decrease triglycerides (TG), and VLDL- and LDL-cholesterol. The most well-studied effect of nicotinic acid is its ability to lower plasma free fatty acids, which has been observed in humans and many animal models. However, its ability to raise HDL in humans has not been replicated in animal models, which precludes studying the mechanism of HDL elevation. Here we studied lipid-modulating effects of nicotinic acid in mice carrying genomic DNA fragments that drive expression of various human genes in the mouse liver. Treatment with nicotinic acid reduced serum levels of HDL cholesterol in wild-type and human apolipoprotein B100 (apoB100)-transgenic mice. In contrast, nicotinic acid treatment of mice that express human cholesteryl ester transfer protein (CETP), with or without concomitant apoB100 expression, resulted in a significant increase of HDL cholesterol and reduction of TG, VLDL- and LDL-cholesterol. These data demonstrate a critical role of CETP in nicotinic acid-mediated HDL elevation, and suggest that mice carrying the human CETP gene may be useful animal models for studying the HDL-elevating effect of nicotinic acid.