Masato Ishigami

Identification of Unique Lipoprotein Subclasses for Visceral Obesity by Component Analysis of Cholesterol Profile in High-Performance Liquid Chromatography

Objective—The contribution of visceral fat accumulation to the development of coronary heart disease was previously reported, but the relation between visceral fat accumulation and serum lipoprotein subclasses was unknown. Methods and Results—We examined the relation of lipoprotein subclasses with visceral fat accumulation in 62 male subjects (aged 22 to 67 years) with visceral fat syndrome or obesity. Cholesterol levels in very low–density, low-density, and high-density lipoprotein subclasses (VLDL, LDL, and HDL) were determined by computer-assisted high-performance liquid chromatography. Subcutaneous fat area and visceral fat area were measured by computed tomographic scanning. There was no significant correlation between the subcutaneous fat area and the cholesterol levels in all lipoprotein subclasses. In contrast, the visceral fat area was correlated positively (P<0.002) with VLDL and LDL subclasses, except for large LDL, but negatively (P<0.001) with those in large and medium HDL subclasses. The observed positive correlations of small and very small LDL subclasses remained significant (P<0.005) after adjustment for serum cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol, respectively, but a significant negative correlation (P<0.005) of large LDL was obtained after adjustment for LDL cholesterol. Conclusion—These findings indicate that this simple high-performance liquid chromatography method may be applied for easy detection and evaluation of abnormal distribution of lipoprotein subclasses.

Matrix Metalloproteinases as Novel Disease Markers in Takayasu Arteritis

Background—Takayasu arteritis (TA) is a chronic vasculitis that primarily affects large elastic arteries. Monitoring of disease activity is crucial because the disease tends to progress despite treatment with glucocorticoid and/or immunosuppressive agents. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) have generally been used as disease activity markers, but these are nonspecific inflammatory markers and lack the sensitivity and specificity to accurately monitor the disease status. Given the histological findings characterized by destruction of elastic fibers, we hypothesized that matrix metalloproteinases (MMPs) could be useful as markers of disease activity in TA. Methods and Results—A consecutive series of 25 patients with TA were enrolled in this study. According to the National Institutes of Health criteria of disease activity, 11 were in an active phase and the remaining 14 were in remission. Circulating levels of MMP-2, MMP-3, and MMP-9 were determined by ELISA in all patients with TA and controls. MMP-2 levels were higher in patients with TA than in controls, but no correlation was found between serum MMP-2 and disease activity score. In contrast, MMP-3 and MMP-9 levels in patients with active disease were higher than in patients in remission and controls, and a positive correlation was demonstrated between circulating levels of MMP-3 or MMP-9 and disease activity score. The high levels of MMP-3 and MMP-9 improved when patients underwent remission. Conclusions—The present results indicate that MMP-2 can be helpful in diagnosing TA and that MMP-3 and MMP-9 can be used as activity markers for TA.

Physiological and pathological roles of a multi-ligand receptor CD36 in atherogenesis; insights from CD36-deficient patients

Oxidized low density lipoprotein (LDL) (Ox-LDL) plays an important role in the pathogenesis of atherosclerosis. Oxidized LDL is taken up by macrophages via scavenger receptors. CD36 is an 88 kDa glycoprotein expressed on platelets, monocyte-macrophages, microvascular endothelial cells, adipose tissue, skeletal muscles and heart. We found patients with CD36 deficiency and identified several mutations in the CD36 gene. We also reported that CD36-deficient macrophages showed a 50% reduction in the binding of Ox-LDL, suggesting that CD36 is one of the major receptors for Ox-LDL. CD36 was expressed on macrophages in the atherosclerotic lesions of human aorta and coronary arteries especially on foamed macrophages. The distribution of CD36 expression was slightly different from that of scavenger receptor class A types I and II. The expression of CD36 on macrophages was up-regulated by Ox-LDL and down-regulated by interferon γ. Since CD36 is a transporter of long-chain fatty acids (LCFA), CD36-deficient patients showed a defect in the uptake of an LCFA analog, BMIPP, by the heart. Furthermore, the secretion of IL-1β and TNF-α from monocyte-derived macrophages induced by Ox-LDL was markedly reduced and the activation of NF-κB was attenuated in CD36-deficient subjects compared with controls, suggesting that CD36-mediated signaling is also impaired in CD36 deficiency.To elucidate the roles of CD36 in vivo, we characterized the clinical profile of CD36-deficient patients. Most of them were accompanied by hyperlipidemia (mainly hypertriglyceridemia), increased remnant lipoproteins and mild elevation of fasting plasma glucose level and blood pressure. Glucose clamp technique revealed mean whole body glucose uptake was reduced in CD36-deficient patients, indicating the presence of insulin resistance. The frequency of CD36 deficiency was higher in patients with coronary heart disease (CHD) than in control subjects. Taken together, CD36 deficiency is accompanied by (1) hyperlipidemia and increased remnant lipoproteins, (2) impaired glucose metabolism based upon insulin resistance, and (3) mild hypertension, and comprises one of the genetic backgrounds of the metabolic syndrome, leading to the development of CHD. (Mol Cell Biochem xxx: 1–4, 2004)

Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages.

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRalpha and PPARgamma was increased by APN. In APN-KO mice, the expression of ABCA1, LXRalpha, PPARgamma, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRalpha and PPARgamma.

Adiponectin deficiency suppresses ABCA1 expression and ApoA‐I synthesis in the liver

Plasma high density lipoprotein (HDL)‐cholesterol levels are inversely correlated with the incidence of cardiovascular diseases. HDL is mainly assembled in the liver through the ATP‐binding cassette transporter (ABCA1) pathway. In humans, plasma HDL‐cholesterol levels are positively correlated with plasma adiponectin (APN) concentrations. Recently, we reported that APN enhanced apolipoprotein A‐I (apoA‐I) secretion and ABCA1 expression in HepG2 cells. In the present study, we investigated HDL assembly in APN‐knockout (KO) mice. The apoA‐I protein levels in plasma and liver were reduced in APN‐KO mice compared with wild‐type‐mice. The ABCA1 expression in liver was also decreased in APN‐KO mice. APN deficiency might cause the impaired HDL assembly by decreasing ABCA1 expression and apoA‐I synthesis in the liver.

Ezetimibe improves postprandial hyperlipidaemia in patients with type IIb hyperlipidaemia

Background  Postprandial hyperlipidaemia is known to be a high‐risk factor for atherosclerotic disease because of rapid and lasting accumulations of triglyceride‐rich lipoproteins and remnants. The Niemann‐Pick C1‐Like 1 (NPC1L1) protein acts as an intestinal cholesterol transporter and ezetimibe, which inhibits NPC1L1, has been used in patients with hypercholesterolaemia. We investigated effects of ezetimibe on fasting lipid and lipoprotein profiles and postprandial hyperlipidaemia in patients with type IIb hyperlipidaemia.

Dominant expression of ATP-binding cassette transporter-1 on basolateral surface of Caco-2 cells stimulated by LXR/RXR ligands

ATP-binding cassette transporter-1 (ABCA1) is a cause of Tangier disease, which is a familial deficiency of plasma high density lipoproteins (HDL). This molecule is known to be expressed in the multiple tissues and organs including small intestines, liver, and macrophages in the blood vessels. Recent in vivo studies suggested that ABCA1 plays some roles in the flux of cholesterol in the intestines. One of the major questions to understand the roles of ABCA1 in the intestines is the expression pattern in the intestinal epithelial cells. To address this issue, we have investigated the expression and regulation of ABCA1 in Caco-2 cells cultured on Transwell as a model, especially focusing on possible polarized expression of ABCA1. The expression of ABCA1 was up-regulated during the differentiation and under the stimulation of LXR/RXR by the addition of 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-OH). Apolipoprotein-AI-mediated cholesterol efflux was dominant toward the basolateral side of polarized cells when stimulated by 9-cis-RA and 22-OH. The cell surface biotinylation experiment followed by Western blot analyses demonstrated a markedly dominant expression of ABCA1 on the basolateral surface, which was clearly confirmed by the confocal laser scanning microscopy. In conclusion, the present study demonstrates that ABCA1 is dominantly expressed on the basolateral surface of Caco-2 cells tested, suggesting that this molecule may play a role in the basolateral movement of cholesterol at least when stimulated by LXR/RXR ligands.

Decreased microsomal triglyceride transfer protein activity contributes to initiation of alcoholic liver steatosis in rats

BACKGROUND/AIMS To elucidate the role of microsomal triglyceride transfer protein (MTP) in the pathogenesis of alcoholic fatty liver, the effects of ethanol on MTP activity and gene expression were investigated. METHODS AND RESULTS Male Sprague-Dawley rats fed an ethanol-containing liquid diet for 37 days, respectively, showed 2.9- and 4.9-fold increases in hepatic cholesterol and triglyceride content in comparison with rats fed an isocaloric ethanol-free diet (P<0.01). Furthermore, a significant decrease in MTP activity and mRNA expression (by 27 and 58%, respectively) was observed after ethanol administration. Intravenous injection of human recombinant hepatocyte growth factor (hrHGF) on each of the last 7 days markedly suppressed ethanol-induced lipid accumulation in the liver. This inhibition of fatty change by hrHGF was accompanied by recovery of MTP activity and gene expression. No inhibitory effect of hrHGF on ethanol-induced acyl-CoA synthetase activation was observed. Experiments using human hepatoma-derived HepG2 cells indicated a direct positive effect of hrHGF on MTP gene expression as well as apolipoprotein B secretion. CONCLUSIONS These results suggest that reduced MTP activity is crucial to development of alcoholic fatty liver, while promotion of MTP activity by HGF might serve as a therapeutic measure against alcoholic liver steatosis.

Prevalence and phenotypic spectrum of cholesteryl ester transfer protein gene mutations in Japanese hyperalphalipoproteinemia.

A patient with cholesteryl ester transfer protein (CETP) deficiency presents with marked hyperalphalipoproteinemia (HALP). To investigate the contribution of CETP deficiency to the cause of HALP (HDL-C> or =1.94 mmol/l, 75 mg/dl), we investigated the CETP activities and the prevalence of genetic CETP mutations among 624 Japanese HALP subjects. The subjects were screened for four known genetic CETP mutations (intron 14 splicing defect (In14), exon 15 missense mutation (Ex15), intron 10 splicing defect (In10) and exon 6 nonsense mutation (Ex6)). We found the frequency of the patients with reduced CETP activity (<75% of normal controls) to be 55.5 and 64.1% in a high HDL group (1.94< or =HDL-C<2.59 mmol/l) and a marked HALP group (HDL-C> or =2.59 mmol/l, 100 mg/dl), respectively. At least one of the four mutations was identified in 65.7% of subjects with reduced CETP activities and 57.5% of subjects with marked HALP. The In14 and Ex15 mutations were very common in HALP subjects and the frequency of In10 mutation and Ex6 mutation was quite low. To investigate the impact of genetic CETP mutation on the phenotypes, we compared the plasma lipid levels and CETP activities between the subjects with two common mutations. All In14 homozygotes showed marked HALP, while marked HALP is less frequent (64.3%) in Ex15 homozygotes. HDL-C levels in Ex15 heterozygotes were significantly higher than those of In14 heterozygotes, suggesting the mutation has dominant negative effects on CETP activity in vivo. Some cases with In14 (5.7%) or Ex15 (7.2%) mutation showed low HDL-C levels. We conclude that CETP deficiency is a major cause of HALP; nevertheless CETP deficiency is not necessarily HALP.

Decreased affinity of low density lipoprotein (LDL) particles for LDL receptors in patients with cholesteryl ester transfer protein deficiency

Abstract. We have reported that the disorder of lipoprotein metabolism in hyperalphalipoproteinae‐mic patients with a deficiency of cholesteryl ester transfer protein (CETP) is characterized by the poly‐disperse low density lipoprotein (LDL) particles and the accumulation of cholesteryl ester (CE) in high density lipoprotein (HDL) particles, forming cholesterol‐induced HDL (HDLc)‐like particles. In the present study we have investigated the interaction of these abnormal LDL with LDL receptors of normal human fibroblasts. Since the ultracentrifugally separated LDL fraction (1.019 < d < 1.063 gmL‐1) from the CETP‐deficient patients contained HDLc‐like particles, these particles were removed by anti‐apolipoprotein (apo) A‐I immunoaffinity column chromatography. The lipoproteins eluted in the unbound fraction of this column did not contain apo A‐I, so this fraction was considered to be authentic LDL. The authentic LDL of the patients were deficient in CE and rich in triglycerides and apo B. The authentic LDL itself showed polydispersity, ranging in size from 23 nm to 30 nm. The affinity of these abnormal LDL particles for LDL receptors was analysed by a competitive assay in which cold LDL from the patients or control compete with 125I‐labelled LDL for fibroblast LDL receptors. The concentration of LDL particles at which 50% of 125I‐labelled normal LDL was replaced was two to three times higher for the patients than for the normal control. Therefore, the affinity of patient LDL was thought to be reduced compared to that of control LDL. These results demonstrate that CETP may play an important role in making LDL particles homogeneous and rich in CE. This modulation of LDL by CETP may enhance the affinity of LDL for LDL receptors to deliver cholesterol to peripheral tissues.