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We report the in vitro efficacy of recombinant LCAT produced by lcat gene-transduced proliferative adipocytes (ccdPA/lcat), which has been developed for enzyme replacement therapy. ApoA-I-specific immunodetection in combination with 1D and 2D gel electrophoreses showed that the disturbed high-density lipoprotein subpopulation profile was clearly ameliorated by the in vitro incubation with ccdPA/lcat-derived recombinant LCAT. Thus, these results using ccdPA/lcat strongly suggest the cell implantation could contribute the enzyme replacement for the patients with LCAT deficiency., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Low-density lipoprotein receptor (LDLR) relative with 11 binding repeats (LR11; also known as sorLA) is genetically associated with late-onset Alzheimer's disease and is thought to be involved in neurodegenerative processes. LR11 contains a vacuolar protein-sorting 10 protein (Vps10p) domain. As this domain has been implicated in protein-protein interaction in other receptors, its structure and function are of great biological interest. Human LR11 Vps10p domain was expressed in mammalian cells and the purified protein was crystallized using the hanging-drop vapour-diffusion method. Enzymatic deglycosylation of the sample was critical to obtaining diffraction-quality crystals. Deglycosylated LR11 Vps10p-domain crystals belonged to the hexagonal space group P6(1)22. A diffraction data set was collected to 2.4 Å resolution and a clear molecular-replacement solution was obtained.

There is a growing body of evidence that the formation and accumulation of advanced glycation end products (AGE) have been known to progress under diabetic conditions, thereby being involved in diabetic vascular complications. Further, we, along with others, have recently found AGE could disturb insulin actions in cultured adipocytes and skeletal muscles. However, the pathological role of AGE in insulin resistance in vivo is not fully understood. Therefore, in this study, we examined whether pyridoxamine, an inhibitor of AGE formation could ameliorate insulin resistance in KK-A(y) mice, a model animal of obese, type 2 diabetes. Fasting blood glucose, serum levels of insulin and AGE in KK-A(y) mice were elevated as the mice got older (from 5 weeks old to 15 weeks old). Serum levels of AGE were positively correlated with insulin (R(2)=0.3956, P=0.002) in KK-A(y) mice. Administration of pyridoxamine dose-dependently decreased fasting insulin levels and improved insulin sensitivity in KK-A(y) mice of 10 weeks old, although it did not affect fasting blood glucose levels. Our present study suggests the involvement of AGE in insulin resistance in KK-A(y) mice. Inhibition of AGE formation may be a novel therapeutic target for improving insulin resistance in diabetes with obesity.

LR11, an LDL receptor family member, is expressed in intimal smooth muscle cells. It was found that the soluble form of LR11 (sLR11) is detected in serum, and the circulating sLR11 levels are positively correlated with intima-media thickness of carotid arteries in dyslipidemic subjects. To clarify the significance of serum sLR11, the circulating sLR11 levels in patients with organic coronary stenosis and the contributing risk factors for them were studied. The subjects, 150 patients with symptoms of coronary artery disease, underwent coronary angiographic examination, and were divided into sex- and age-matched two groups; one is organic coronary stenosis group (OCS) and the other is normal coronary group (NC). Serum sLR11 levels were significantly higher in OCS than in NC (4.9+/-2.7 U vs 3.6+/-1.8 U, p<0.05). Multivariate regression analysis showed that circulating sLR11 is independent contributing factor for the OCS, as well as diabetes mellitus and dyslipidemia. Among various coronary risk factors for sLR11 level, HbA1c showed the highest correlation coefficient (p<0.01). These results suggest that the circulating sLR11 might reflect coronary organic stenosis, and that hyperglycemic condition might be promoting factor for expression of LR11 in intimal smooth muscle cells., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Background: Recent genetic and pathological studies have suggested that a lipoprotein receptor, LR11, is intricately implicated in the pathogenesis of Alzheimer disease (AD). We have recently established a novel sandwich ELISA, which enabled the sensitive quantification of a soluble LR11 (sLR11). By this ELISA, we attempted to determine the difference in the levels of CSF sLR11 in AD patients., Methods: We examined CSF from 29 AD patients, 20 frontotemporal lobar degeneration patients and 27 age-matched control subjects. The CSF sLR11 level as well as the levels of tau and beta-amyloid42 (Abeta42) were determined by sandwich ELISA., Results: The CSF tau level and tau/Abeta42 ratio were significantly increased (p < 0.01) in the AD patients. The CSF sLR11 level in the AD patients was significantly higher (p < 0.01) than that of the frontotemporal lobar degeneration patients and the controls. The APOE-epsilon4-positive AD patients have higher sLR11 levels than the APOE-epsilon4-negative patients (p < 0.01)., Conclusions: These results suggest that the quantification of CSF sLR11 may serve as a biomarker of AD, although the diagnostic value for individual patients is limited. An elevated CSF sLR11 level in AD patients may be relevant to AD pathogenesis., (Copyright 2010 S. Karger AG, Basel.)

Background: Vascular smooth muscle cells (SMCs) migrate from the arterial media to the intima in the progression of atherosclerosis, and dysfunction of SMCs leads to enhanced atherogenesis. A soluble form of the LDL receptor relative with 11 ligand-binding repeats (sLR11) is produced by the intimal SMCs, and the circulating concentrations of sLR11 likely reflect the pathophysiological condition of intimal SMCs. Furthermore, polymorphism of the LR11 gene has been found to be related to the onset of Alzheimer disease. This study describes the development of a sandwich immunoassay for quantifying sLR11 in human serum and cerebrospinal fluid., Methods: We used synthetic peptides or DNA immunization to produce monoclonal antibodies (MAbs) A2-2-3, M3, and R14 against different epitopes of LR11., Results: sLR11 was immunologically identified as a 250-kDa protein in human serum and cerebrospinal fluid by SDS-PAGE separation, and was purified from serum by use of a receptor-associated protein and MAb M3. An immunoassay for quantification of sLR11 with a working range of 0.25-4.0 microg/L was developed using the combination of MAbs M3 and R14. Treatment of serum with 5.25% n-nonanoyl-N-methyl-d-glucamine reduced the matrix effects of serum on the absorbance detection in the ELISA system. The linear dynamic range of the ELISA spanned the variation of circulating sLR11 concentrations in individuals with atherosclerosis., Conclusions: A sandwich ELISA was established for quantifying sLR11 in serum and cerebrospinal fluid. This technique provides a novel means for assessing the pathophysiology of atherosclerosis, and possibly neurodegenerative diseases.

Pre beta1-HDL, a minor HDL subfraction consisting of apolipoprotein A-I (apoA-I), phospholipids and unesterified cholesterol, plays an important role in reverse cholesterol transport. Plasma pre beta1-HDL levels have been reported to be increased in patients with coronary artery disease (CAD) and dyslipidemia. To clarify the clinical significance of measuring plasma pre beta1-HDL levels, we examined those levels in 112 patients with CAD, consisting of 76 patients with stable CAD (sCAD) and 36 patients with unstable angina pectoris (uAP), and in 30 patients without CAD as controls. The pre beta1-HDL levels were determined by immunoassay using a specific monoclonal antibody (Mab55201) that we established earlier. The mean pre beta1-HDL level in the CAD patients was significantly higher than the level in the controls (34.8+/-12.9 mg/L vs. 26.6+/-6.9 mg/L, p<0.001). In addition, the mean pre beta1-HDL level was markedly higher in the uAP subgroup than in the sCAD subgroup (43.1+/-11.5mg/L vs. 30.9+/-11.7 mg/L, p<0.0001). These tendencies remained even after excluding dyslipidemic subjects. These results suggest that elevation of the plasma pre beta1-HDL level is associated with the atherosclerotic phase of CAD and may be useful for identifying patients with uAP.

Diet therapy plays an important role in the prevention, management and therapy of obesity and its related disorders. Although the long-term and effective diet programs for the obese patients are expected to manage their body weights and/or the accompanied disorders, there is not enough accumulation of evidence for diet therapy. However, recent intervention studies approach to dissolve the various problems concerning about weight reduction for the obesity.

Aims: To compare the efficacy and safety of pitavastatin and atorvastatin in Japanese patients with hypercholesterolemia., Methods and Results: Japanese patients with total cholesterol (TC) > or = 220 mg/dL were randomized to receive pitavastatin 2 mg (n=126) or atorvastatin 10 mg (n=125) for 12 weeks. The primary endpoint was percent change from baseline in non-HDL-C level after 12 weeks of treatment. Reduction of non-HDL-C by pitavastatin treatment (39.0%, P=0.456 vs. atorvastatin) was non-inferior to that by atorvastatin (40.3%). Both pitavastatin and atorvastatin also significantly reduced LDL-C by 42.6% and 44.1%, TC by 29.7% and 31.1%, and TG by 17.3% and 10.7%, respectively, at 12 weeks without intergroup differences. HDL-C showed a significant increase at 12 weeks with pitavastatin treatment (3.2%, P=0.033 vs. baseline) but not with atorvastatin treatment (1.7%, P=0.221 vs. baseline). Waist circumference, body weight and BMI were significantly correlated with percent reduction of non-HDL-C in the atorvastatin group, whereas pitavastatin showed consistent reduction of non-HDL-C regardless of the body size. In patients with metabolic syndrome, LDL-C was reduced significantly more in patients receiving pitavastatin when compared with those receiving atorvastatin. AST, ALT and gammaGTP increased significantly in patients receiving atorvastatin but not in those receiving pitavastatin. Both treatments were well tolerated., Conclusion: Pitavastatin 2 mg and atorvastatin 10 mg are equally effective in improving the lipid profile and were well tolerated in Japanese patients with hypercholesterolemia.

Aim: The POSITIVE study assessed whether long-term treatment with probucol. a potent anti-oxidant and cholesteryl ester transfer protein (CETP) activator is associated with a lowered risk of cardiovascular events in a very high-risk population: familial hypercholesterolemia (FH)., Methods: The study cohort included 410 patients with heterozygous FH, diagnosed between 1984 and 1999 by cardiovascular and metabolic experts at fifteen centers. Traceable patients were screened using predefined eligibility criteria. The primary outcome measure for comparison between probucol exposure and non-exposure was the time to the first cardiovascular event involving hospitalization., Results: Analysis revealed significant differences in baseline characteristics and follow-up treatment between exposure and non-exposure. An observed indication bias was the use of probucol in more severe FH at diagnosis, both for primary and secondary prevention. When the multivariate Cox regression procedure was used after adjustment for possible confounding factors, probucol lowered the risk (hazard ratio [HR], 0.13; 95% confidence interval [CI], 0.050.34) in secondary prevention (n=74) and was statistically significant (p<0.001), although not significant (HR, 1.5; 95% CI, 0.484.67; p=0.49) in primary prevention (n=233). Safety assessment found no specific difference between exposure and non-exposure., Conclusion: Long-term probucol treatment may prevent secondary attack in a higher cardiovascular risk population of heterozygous FH.

Infiltrated macrophages (Mphi) are believed to cause pathological changes in the surrounding adipocytes through the secretion of active molecules in visceral fat. Matrix metalloproteinase (MMP)-3 is secreted from Mphi, and enhances expression of the inflammatory cytokines through the activation of toll-like receptor (TLR) 2. Visceral adipocytes express high levels of vascular endothelial growth factor (VEGF), and the degree of visceral fat accumulation is associated with the plasma VEGF concentration in obese subjects. The aim of the study is to clarify the role of MMP-3 in the enhancement of the free fatty acids (FFAs)-induced VEGF expression through TLR2 in visceral adipocytes. One mM FFAs induced VEGF mRNA and protein expression in 3T3-L1 adipocytes. The FFAs-induced VEGF expression was mostly mediated by TLR2. A high fat intake increased the VEGF mRNA expression in visceral fat and the VEGF concentration in plasma, accompanied with the increase in the plasma FFAs concentration in mice. These increases were largely inhibited in TLR2-deficient mice. The FFAs-induced VEGF expression was increased in the presence of Mphi-conditioned medium (CM) in adipocytes, and the enhancement was inhibited by a MMP-3 inhibitor or a neutralizing antibody against MMP-3. The active form of MMP-3 induced the VEGF mRNA expression, as well as TLR2, in adipocytes. The increase in the VEGF expression by MMP-3 was inhibited by the treatment with siRNA for TLR2. The enhancement of FFAs-induced TLR2 expression by Mphi-CM was inhibited by blocking of the MMP-3. The increase in the VEGF mRNA expression by Mphi-CM or MMP-3 was partially inhibited by a neutralizing antibody against TNF-alpha. These results indicate that MMP-3 in Mphi-CM enhances the FFAs-induced VEGF expression in adipocytes through the increase in the TLR2 expression. The MMP-3 secreted from the infiltrated Mphi may be a regulator of the VEGF expression in visceral adipocytes.

Medial-to-intimal migration of SMCs is critical to atherosclerotic plaque formation and remodeling of injured arteries. Considerable amounts of the shed soluble form of the LDL receptor relative LR11 (sLR11) produced by intimal SMCs enhance SMC migration in vitro via upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. Here, we show that circulating sLR11 is a novel marker of carotid intima-media thickness (IMT) and that targeted disruption of the LR11 gene greatly reduces intimal thickening of arteries through attenuation of Ang II-induced migration of SMCs. Serum concentrations of sLR11 were positively correlated with IMT in dyslipidemic subjects, and multivariable regression analysis suggested sLR11 levels as an index of IMT, independent of classical atherosclerosis risk factors. In Lr11-/- mice, femoral artery intimal thickness after cuff placement was decreased, and Ang II-stimulated migration and attachment of SMCs from these mice were largely abolished. In isolated murine SMCs, sLR11 caused membrane ruffle formation via activation of focal adhesion kinase/ERK/Rac1 accompanied by complex formation between uPAR and integrin alphavbeta3, a process accelerated by Ang II. Overproduction of sLR11 decreased the sensitivity of Ang II-induced activation pathways to inhibition by an Ang II type 1 receptor blocker in mice. Thus, we demonstrate a requirement for sLR11 in Ang II-induced SMC migration and propose what we believe is a novel role for sLR11 as a biomarker of carotid IMT.

The Japan Atherosclerosis Society (JAS) guidelines for the prevention of atherosclerotic diseases, proposing management for LDL cholesterol as the primary target, have successfully contributed to the prevention of cardiovascular events; however, recently, the impact of hypertriglyceridemia as an additional cardiovascular risk has become understood, especially in light of the rise in obesity, metabolic syndrome, and diabetes in the Japanese population. Rather than waiting to obtain conclusive domestic data confirming that hypertriglyceridemia is a cardiovascular risk factor and that its management is efficacious, we propose guidelines for hypertriglyceridemia using non-HDL cholesterol as a second target.

Aquaporin 1 (AQP1) was first purified from red blood cell membranes and is now known to be an osmolarity-driven water transporter that is widely expressed in many epithelial and endothelial cells outside the brain. Several recent studies have shown strong expression of AQP1 in proliferating tumor microvessels, suggesting that AQP1 may have an important role in tumor angiogenesis. Hypoxia is thought to be a common precursor to neovascularization in many retinal diseases, including diabetic retinopathy, and therefore we analyzed the expression pattern and function of AQP1 in human retinal vascular endothelial cells cultured under hypoxic conditions. The levels of AQP1 mRNA and protein expression significantly increased under hypoxia, and inhibition of VEGF signaling did not affect AQP1 expression. To examine the effect of AQP1 on hypoxia-inducible angiogenesis, a tube formation assay was performed. Reduction of AQP1 expression using siRNA and inhibition of VEGF signaling both significantly inhibited tube formation, and these effects were additive. Therefore, our data suggest that AQP1 is involved in hypoxia-inducible angiogenesis in retinal vascular endothelial cells through a mechanism that is independent of the VEGF signaling pathway.

The increased population of TLR2/TNF-alpha co-expressing adipocytes is associated with the development of insulin resistance. We have herein shown the significance of low-dose growth hormone (GH) supplementation for the regulation of TLR2 and TNF-alpha expressions in visceral fat using different kinds of mouse models fed with a high-fat diet. Low-dose GH supplementation reduced the increased population of TLR2/TNF-alpha co-expressing adipocytes in high-fat fed mice. The neutralization of IGF-1 abolished the effect of GH supplementation on the TLR2 expression using GH-overexpressing mice. IGF-1, but not GH, inhibited the FFA-induced TLR2 and TNF-alpha expression in 3T3-L1 cells. Finally, low-dose GH supplementation reduced the TLR2 expression without an obvious change in the visceral fat volume in ob/ob mice. These results indicate that low-dose GH supplementation possibly inhibits the high-fat induced change of the adipocytes to TLR2/TNF-alpha co-expressing cells through the action of IGF-1.

Aim: Not all genetic factors predisposing phenotypic features of dyslipidemia have been identified. We studied the association between the low density lipoprotein-related protein 2 gene (LRP2) and levels of plasma total cholesterol (T-Cho) and LDL-cholesterol (LDL-C) among 352 adults in Japan., Methods: Subjects were obtained from among participants in a cohort study that was carried out with health-check screening in an area of east-central Japan. We selected 352 individuals whose LDL-C levels were higher than 140 mg/dL from the initially screened 22,228 people. We assessed the relation between plasma cholesterol levels and single-nucleotide polymorphisms (SNPs) in the LRP2 gene., Results: We identified significant correlations between plasma cholesterol levels and two of 19 examined SNPs in LRP2, c.+193826T/C and IVS55 - 147A/G. In particular, the association of c.+193826T/C with the T-Cho level was prominent (p=0.003), showing a co-dominant effect of the minor C-allele on lowering T-Cho and LDL-C levels: for 24 homozygous C-allele carriers, T-Cho=240.7 +/- 24.2 mg/dL and LDL-C=166.1 +/- 21.0 mg/dL); for 130 heterozygous carriers, 248.5 +/- 23.5 mg/dL and 166.6 +/- 19.3 mg/dL; and for 196 homozygous T-allele carriers, 253.9 +/- 23.5 mg/dL and 172.0 +/- 21.0 mg/dL. Linkage disequilibrium (LD) analyses based on 19 selected SNPs showed that c.+193826T/C and IVS55 - 147A/G were in tight LD and that both were located in an LD block covering the genomic sequence from exon 55 to exon 61., Conclusion: We confirm the association between LRP2 and levels of T-Cho and LDL-C in human plasma. The results suggest that genetic variations in LRP2 are important factors affecting lipoprotein phenotypes of patients with hypercholesterolemia.

Advanced glycation end products (AGE) have been observed in various pathological conditions especially in diabetes mellitus. However, it is unclear as to whether AGE are involved in insulin resistance in adipose tissues. In this study, we examined the effects of AGE on insulin sensitivity in adipocytes by examining the effects of AGE and its mechanisms on the glucose uptake in adipocytes and adipocyte differentiation. Glucose-, glyceraldehyde-, or glycolaldehyde-derived AGE inhibited the differentiation of 3T3-L1 cells. These AGE also inhibited the glucose uptake in the absence or presence of insulin, which were completely prevented by antibody against AGE or receptor for AGE (RAGE). The AGE increased the intracellular reactive oxygen species (ROS) generation in 3T3-L1 adipocytes, and the effects of AGE on glucose uptake were completely reversed by the treatment with an anti-oxidant, N-acetylcysteine. The AGE also induced the expression of monocyte chemoattractant protein-1, which has been implicated in the development of obesity-associated glucose intolerance, in 3T3-L1 adipocytes. Our present study suggests that AGE-RAGE interaction inhibits the glucose uptake through the overgeneration of intracellular ROS, thus indicating that it is involved in the development of obesity-related insulin resistance.

Objective: Macrophages play a key role in lipid-rich unstable plaque formation and interact with intimal smooth muscle cells (SMCs) in early and progressive stages of atherosclerosis. LR11 (also called sorLA), a member of low-density lipoprotein receptor family, is highly and specifically expressed in intimal SMCs, and causes urokinase-type plasminogen activator receptor-mediated degradation of extracellular matrices. Here we investigated whether the secreted soluble form of LR11 (solLR11) enhances adhesion, migration, and lipid accumulation in macrophages using animal models and cultured systems., Methods and Results: Immunohistochemistry showed solLR11 expression in thickened intima of balloon-denuded rat artery. Macrophage infiltration into the cuff-injured artery was markedly reduced in LR11-deficient mice. In vitro functional assays using THP-1-derived macrophages showed that solLR11 (1 microg/mL) significantly increased acetylated low-density lipoprotein uptake by THP-1 cells and cell surface levels of scavenger receptor SR-A 1.7- and 2.8-fold, respectively. SolLR11 dose-dependently increased the migration activity of THP-1 macrophages and adhesion to extracellular matrices 2.0- and 2.1-fold, respectively, at 1 microg/mL. These effects of solLR11 were almost completely inhibited by a neutralizing anti-urokinase-type plasminogen activator receptor antibody., Conclusion: SolLR11, secreted from intimal SMCs, regulates adhesion, migration, and lipid accumulation in macrophages through activation of urokinase-type plasminogen activator receptor. The formation of lipid-laden macrophages in atherosclerotic plaques possibly is regulated by SolLR11 of intimal SMCs.

The relationship between adipocytes and infiltrated macrophages in fat tissue is important for the pathogenesis of insulin resistance through the activation of cytokines. Peroxisome proliferator-activated receptors (PPARs) play a role in the regulation of cytokine secretion in these cells. We studied the effect of the PPARalpha activation of macrophages on the modulation of the tumor necrosis factor alpha (TNFalpha) expression in adipocytes using a cell culture system. A conditioned medium of lipopolysaccharide (LPS)-stimulated RAW264.7 cells, a macrophage cell line, induced the level of TNFalpha mRNA in 3T3-L1 adipocytes. This effect was inhibited by the addition of neutralizing antibody against interleukin 6 (IL-6) in the conditioned medium or the preincubation of RAW264.7 cells with a specific PPARalpha agonist, K-111 (2,2-dichloro-12-(4-chlorophenyl)dodecanoic acid). K-111 reduced both the IL-6 production and mRNA expression in RAW264.7 cells, and its effect was stronger than that of rosiglitazone, a PPARgamma agonist. The activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway and nuclear factor kappa B (NF-kappaB) subunits of p65 was significantly inhibited by K-111. The blocking of IL-6 production through the SAPK/JNK pathway or by transfection with siRNA specific for IL-6 abolished the inhibitory effect of K-111 on the TNFalpha expression in the 3T3-L1 adipocytes. As a result, the IL-6 produced by RAW264.7 cells is an inducer of TNFalpha expression in 3T3-L1 adipocytes, and the IL-6 secretion is inhibited by the activation of PPARalpha. The PPARalpha activators may suppress the pathogenetical secretion of TNFalpha in the adipocytes through the functional modulation of the infiltrated macrophages.

Cytokine production in fat tissue plays a key role in insulin resistance. The aim of study is to know the phenotypic changes of adipocytes with high fat-induced insulin resistance. High fat intake induced the expression of tumor necrosis factor alpha (TNFalpha) in visceral fat tissue as well as development of insulin resistance. Analysis of the gene expression profiles in adipocytes showed that high fat intake induced the expression of toll-like receptor 2 (TLR2) in addition to TNFalpha. Flow cytometry analysis revealed the presence of adipocytes co-expressing TLR2 and TNFalpha (TLR2/TNFalpha-adipocytes), and the number of TLR2/TNFalpha-adipocytes in visceral fat tissues was increased by high fat intake compared to that in subcutaneous fat tissues. Free fatty acids increased TNFalpha expression in 3T3-L1 adipocytes through TLR2 signals. These results indicate that TLR2/TNFalpha-adipocytes possibly cause the induction of TNFalpha expression in visceral fat tissues, being associated with the development of high fat-induced insulin resistance.

The recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid beta peptide (Abeta) in Alzheimer disease. We report here that inherited variants in the SORL1 neuronal sorting receptor are associated with late-onset Alzheimer disease. These variants, which occur in at least two different clusters of intronic sequences within the SORL1 gene (also known as LR11 or SORLA) may regulate tissue-specific expression of SORL1. We also show that SORL1 directs trafficking of APP into recycling pathways and that when SORL1 is underexpressed, APP is sorted into Abeta-generating compartments. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing Alzheimer disease.

Using preadipocyte implantation methods, we recently demonstrated that adipocytes in the visceral area change their function, as the expression of tumor necrosis factor-alpha (TNF-alpha) increases, thereby causing insulin resistance. In order to clarify the mechanism for changes in the function of adipocytes in visceral area, we examined the mRNA expression profiles in visceral fat tissue specimens. Four weeks after cell implantation, we performed a microarray analysis using the RNA of fat tissue specimens implanted either with 3T3-L1 cells or PBS alone. Sixty-three genes were thus isolated and the expression of matrix metalloproteinase-3 (MMP-3) mRNA was found to dramatically increase in the fat tissue specimens. The neutralization of MMP-3 protein inhibited adipogenesis and the free fatty acid-induced TNF-alpha secretion in 3T3-L1 adipocytes. These results suggest a potential role of MMP-3, which promotes the TNF-alpha secretion, thus contributing to the disturbance of the functions in the adipocytes which accumulate in the visceral area.

Statins, inhibitors of HMG-CoA reductase, elicit various actions on vascular cells including the modulation of proliferation and migration of smooth muscle cells (SMCs). Here, we have elucidated the mechanism by which statins, in particular pitavastatin, attenuate the migration activity of SMCs. The expression of LR11, a member of the LDL receptor family and an enhancer of cell surface localization of urokinase-type plasminogen activator receptor (uPAR), is increased in cultured SMCs by treatment with PDGF-BB. Pitavastatin attenuates the PDGF-BB -induced surface expression of LR11 and uPAR. The increased migration of SMCs observed both upon overexpression of LR11 and via stimulation of secretion of soluble LR11 is not reversed by pitavastatin. In vivo studies showed that the SMCs expressing LR11 in plaques are almost congruent with intimal cells expressing nonmuscle myosin heavy chain (SMemb). Pitavastatin reduced the expression of LR11 and SMemb, and the levels of LR11, uPAR, and SMemb in cultured intimal SMCs were reduced to those seen in medial SMCs. We propose that this statin reduces PDGF-induced migration through the attenuation of the LR11/uPAR system in SMCs. Modulation of the LR11/uPAR system with statins suggests a novel treatment strategy for atherogenesis based on suppression of intimal SMC migration.

The distribution of fat in obese persons is related to the risk of developing various metabolic disorders, such as glucose intolerance, dyslipidemia and hypertension, and the combination of these conditions is known as the metabolic syndrome. The aim of this study was to investigate the role of subcutaneous fat in regulating insulin resistance and its influence on TNF-alpha expression in visceral fat, by using mice that were subjected to subcutaneous lipectomy with or without subsequent fat transplantation. After partial subcutaneous lipectomy, mice showed significantly greater accumulation of visceral fat compared with sham-operated control mice. Lipectomy led to higher plasma insulin and lower plasma glucose levels after loading with glucose and insulin, respectively, compared with the levels in control mice. Insulin-induced phosphorylation of IRS-1 was decreased in the skeletal muscles of lipectomized mice. Subcutaneous transplantation of fat pads into lipectomized mice reversed the above-mentioned changes indicating insulin resistance in these animals. The fat storage area of adipocytes and TNF- alpha expression by adipocytes in visceral fat were significantly higher in the lipectomized mice than in controls, while subcutaneous transplantation of fat reduced both the fat storage area and TNF-alpha expression. The insulin resistance of lipectomized mice was also ameliorated by systemic neutralization of TNF-alpha activity using a specific antibody. These findings obtained in mice subjected to subcutaneous lipectomy with/without subsequent fat transplantation indicate that subcutaneous fat regulates systemic insulin sensitivity, possibly through altering fat storage and the expression of TNF-alpha by adipocytes in visceral fat. The balance between accumulation of subcutaneous fat and visceral fat may be important with respect to the occurrence of systemic insulin resistance in the metabolic syndrome.

The tumor necrosis factor-alpha (TNF-alpha) expression has been reported to be largely dependent on the size of adipocytes. We herein investigated the gene regulation of diacylglycerol O-acyltransferase (DGAT) in order to clarify the mechanism of TNF-alpha expression induced in large adipocytes. 3T3-L1 cells were cultured in the presence of 5 mM or 25 mM glucose to generate adipocytes from which the triglyceride content differs. The expression of TNF-alpha, DGAT1, and DGAT2 were upregulated in adipocytes cultured with 25 mM glucose. Furthermore, knockdown of DGAT1 gene significantly inhibited the TNF-alpha expression. Finally, the DGAT1 expression levels were closely related to the TNF-alpha level in 3T3-L1 adipocytes.

Low-density lipoprotein receptor family members (LRs) play a key role in the catabolism of many membrane-associated proteins, such as complexes between proteinases and their receptors, in addition to being involved in lipoprotein metabolism as suspected by the hitherto well-established functions of low-density lipoprotein receptor, in a variety of tissues. Recent studies using receptor-deficient or -overexpressing animals and cells have suggested that certain LRs are important regulators of the migration (and proliferation) of vascular smooth muscle cells (SMCs). LR expression is markedly induced in intimal or medial SMCs during the formation of atherosclerotic lesions. Because LRs can modulate the activity of the urokinase-type plasminogen activator (uPA) receptor and possibly of the platelet-derived growth factor (PDGF) receptor, LRs may influence the migration of SMCs through functional modulation of these membrane receptors. Therefore, SMC migration may be regulated by time-restricted expression of LRs. In agreement with the concept of functional interaction between LRs and membrane signaling receptors, a negative regulator of uPA receptor protein catabolism, LR11, has been identified. Statins modulate the PDGF-induced migration of intimal SMCs via the LR11/uPA receptor cascade. Selective modification of the LRs/uPA receptor/PDGF receptor systems in SMCs may be important for suppression of atherosclerotic plaque formation as well as for preventing intimal thickening after angioplasty.

Low-density lipoprotein (LDL) apheresis has been applied to patients with familial hypercholesterolemia (FH) with coronary artery disease (CAD). To examine the efficacy and safety of a new type of LDL adsorption column (KLD01, Kaneka, Osaka, Japan), which deals with whole blood without separating plasma, the new system was evaluated in a multicenter trial. The present study included 33 FH patients with CAD (24 males, 9 females, 57 +/- 13 years) who were treated five times with a mean interval of 2.12 +/- 0.60 weeks between treatments. We studied the removal efficacies for serum LDL cholesterol, Lipoprotein(a) (Lp(a)) and triglyceride, the times for the preparation of the system and for treatment, symptoms, and the biochemical data. The scheduled treatments were completed by 31 patients. Serum levels of LDL cholesterol, Lp(a) and triglycerides were all significantly reduced with KLD01; 61.5 +/- 6.2%, 72.4 +/- 5.9% and 69.5 +/- 9.7%, respectively. The times for both setting up the column system (26 +/- 7 min) and treatment (138 +/- 20 min) were shorter with KLD01 than conventional methods. Adverse reactions occurred in eight cases (17 episodes), but the patients fully recovered immediately after each apheresis therapy session. We conclude that the new type of LDL adsorption column, one that deals with whole blood, is a promising apheresis therapy for FH patients in view of its efficacy, reduced time for treatment, and safety.

Autologous fat transplantation is a popular and useful technique in plastic and reconstructive surgery. The efficiency and survival of such grafts is predictable in many cases, but there are still issues to be resolved, such as how to improve graft volume retention. To address the issue of volume retention, we studied the effect of revascularization from the recipient on the size and function of adipocytes in fat grafts. Treatment of mice with TNP-470, an angiogenesis inhibitor, reduced blood flow from the recipient into the graft after subcutaneous transplantation of epididymal fat. The weight of transplanted tissues and the size of adipocytes in the grafts were significantly lower in mice treated with TNP-470 (TNP mice) than in control mice. Expression of genes for enzymes related to lipid accumulation was decreased in the grafts of TNP mice compared with control mice. Moreover, the expression of adipocyte-derived angiogenic peptides, VEGF and leptin, was significantly lower in the grafts of TNP mice than in grafts from control animals. The expression of VEGF and leptin by cultured human adipocytes was increased in the presence of conditioned medium from cultured vascular endothelial cells. These results show that the inhibition of the revascularization of fat grafts after transplantation reduces graft volume retention and cellular function. Early and adequate revascularization may be important for both the supply of nutrients and vasoactive interactions between vascular endothelial cells and adipocytes in graft.

The migration of cultured cultured smooth muscle cells (SMCs) is regulated by the time-specific expression of members of the LDL receptor family (LRs). LRP1B, a member of LRs, modulates the catabolism of PDGF beta-receptor, affecting the migration of SMCs. An involvement of PDGF beta-receptor in atherosclerosis is focused because of its abundant expression in intimal SMCs. Here, in order to know a functional significance of LRP1B in the increased migration of intimal SMCs, the functions of three groups of cultured SMCs with different origins in atherosclerotic arteries were studied. Each group of SMCs (central, marginal or medial SMCs) was isolated from explanted pieces of central or marginal area of thickened intima, or media prepared from rabbit aortic plaques. The LRP1B expression levels were significantly decreased in intimal SMCs, particularly in marginal SMCs, compared to medial SMCs. The expression levels of LRP1B in SMCs were negatively correlated with those of PDGF beta-receptor. The level of PDGF beta-receptor-mediated phosphorylation of ERK 1/2 in central SMCs was increased to 5.2-fold with the functional inhibition of LRP1B using anti-LRP1B IgY. The antibody increased the PDGF-BB-stimulated migration and invasion activities in SMCs. The increase in the PDGF beta-receptor-mediated outgrowth activity of SMCs from the explants was also inhibited by the functional inhibition of LRP1B. These results indicate that LRP1B regulated the migration activity of SMCs through the modulation of PDGF beta-receptor-mediated pathway. The regulation of LRP1B expression is possibly involved in the activated migration of intimal SMCs in the course of atherosclerosis.

Vascular endothelial growth factor (VEGF) is an important angiogenic factor and is expressed in wide variety of cell types. In this study, we investigated the mechanism of VEGF production in adipocytes in three sets of experiments. First, to clarify the relation between plasma VEGF concentrations and their expressions in adipose tissues, we investigated the genetically obese db/db and KK-Ay mice. Plasma VEGF concentrations in obese mice were significantly higher than in control and were related to adiposity. VEGF expressions in visceral fat were enhanced during growth and were related to fat deposition. Next, to demonstrate the relation between VEGF production and lipid accumulation in adipocytes, we analyzed VEGF mRNA expression and its protein secretion in 3T3-L1 cells. VEGF production was enhanced during lipid accumulation in 3T3-L1 cells after adipocyte conversion. Next, to clarify the role of anatomic localization on VEGF expression in adipocytes, we implanted 3T3-L1 cells into visceral or subcutaneous fat in athymic mice. 3T3-L1 cells implanted into the mesenteric area expressed more VEGF mRNA than that into the subcutaneous area. Plasma VEGF concentration in the mice implanted in visceral fat was higher than in controls. These results suggest that both the anatomic localization and the lipid accumulation are important for the VEGF production in adipocytes.

Factors predisposing to the phenotypic features of high total cholesterol (T-Cho) in human plasma have not been clearly defined. Here we report an association between two variations in the apolipoprotein B48 receptor gene (APOB48R) and plasma T-Cho levels among 352 adult individuals in Japan. By analyzing phenotypic associations between age- and gender-adjusted levels of plasma T-Cho, low-density lipoprotein (LDL) cholesterol (LDL-C), and high-density lipoprotein (HDL) cholesterol (HDL-C), we detected a significant correlation between genotypes of the A419P variation and adjusted T-Cho levels. Among homozygous G-allele carriers (n=265), heterozygous carriers (n=78), and homozygous minor C-allele carriers (n=9), T-Cho levels were 2.43+/-0.21 mg/cm(3), 2.48+/-0.24 mg/cm(3), and 2.63+/-0.21 mg/cm(3), respectively, indicating a codominant T-Cho-elevating effect of the minor C-allele (r=0.15, P=0.007). A similar effect was detected for c.934-960/del (r=0.13, P=0.015). Linkage disequilibrium (LD) analysis detected significant LD among eight variant sites that included neighboring loci. Our results indicate that variations in APOB48R and nearby genes are among the many factors involved in hypercholesterolemia. The etiological studies should now include consideration of this novel aspect of the mechanism(s) leading to hypercholesterolemic disease.

Peroxisome proliferator-activated receptors (PPARs) regulate the vascular cell functions as well as systemic lipid and glucose metabolism. Here, we studied the effect of TAK-559, a newly developed potent activator both for PPARalpha and gamma, on the vascular cell recruitment. TNF-alpha- or interleukin-1beta (IL-1beta)-induced THP-1 cell attachment to cultured endothelial cells was significantly reduced in the presence of 10 microM TAK-559 (P < 0.05). The secretion of monocyte chemoattractant protein-1 (MCP-1) from endothelial cells is reduced by 36% in the presence of 10 microM TAK-559, accompanied with the decreased mRNA expression in the cells. The proliferation and migration of cultured smooth muscle cells (SMCs) were significantly decreased in the presence of TAK-559 (P < 0.05). TAK-559-treated hypercholesterolemic rabbits showed the significant reduction of intimal thickning after balloon catheterization by 51% compared with control (P < 0.05), although the plasma lipid and glucose level was not changed between them. The numbers of macrophage and SMCs were decreased to 34% and 49% in the hyperplastic intima of arteries from TAK-559-treated rabbits compared to those from control, respectively. These results suggest that the PPARalpha and gamma activator inhibits the recruitment of macrophages and SMCs in intima, possibly leading to the reduction of intimal hyperplasia in hypercholesterolemia.

We investigated effects of weight loss from diet and exercise regimen in obese subjects with normal fasting plasma glucose or impaired glucose tolerance (IGT) on insulin release capacity and insulin sensitivity. Eight subjects were recruited among visceral obesity patients (4 men, 4 women; age range, 24 to 57 years; body mass index [BMI], 32.8 to 60.3 kg/m(2)). All were admitted to Chiba University Hospital for 2 weeks, were treated with a tapering 5,023 to 2,930 kJ diet, and were given exercise equivalent to 628 kJ/d. For assessments, we used a combination of C-peptide secretion rate determination and minimal model analysis as previously reported. BMI and visceral fat area (V) significantly decreased (BMI on initiation v after intervention, 43.0 +/- 3.2 v 40.3 +/- 3.1 kg/m(2), P <.05; V, 224 +/- 22 v 188 +/- 22 cm(2); P <.05). Fasting immunoreactive insulin (F-IRI) and leptin concentrations decreased significantly. Capacity for insulin release in response to glucose increased in all subjects (first-phase insulin secretion [CS1], 4.66 +/- 4.05 v 6.81 +/- 4.57 ng/mL/5 min, P <.05), but the insulin sensitivity index (S(i)) did not change significantly. These data suggest that weight reduction early in development of type 2 diabetes can oppose progression of diabetes by improving capacity for insulin release.

Background: Genetic, epidemiologic, and biochemical evidence suggests that apolipoprotein E, low-density lipoprotein receptors, and lipid metabolism play important roles in sporadic Alzheimer disease (AD)., Objective: To identify novel candidate genes associated with sporadic AD., Design: We performed an unbiased microarray screen for genes differentially expressed in lymphoblasts of patients with sporadic AD and prioritized 1 gene product for further characterization in AD brain., Setting: Emory University, Atlanta, Ga., Subjects: Cell lines were used from 14 patients with AD and 9 normal human control subjects., Results: Six genes were differentially expressed in lymphoblasts of 2 independent groups of patients with probable AD and autopsy-proven AD. We hypothesized that 1 of the genes, termed low-density lipoprotein receptor relative with 11 binding repeats (LR11) (reduced 1.8- and 2.5-fold in AD lymphoblasts vs controls), might be associated with sporadic AD on the basis of its function as neuronal apolipoprotein E receptor. We found dramatic and consistent loss of immunocytochemical staining for LR11 in histologically normal-appearing neurons in AD brains. This reduction of LR11 protein was confirmed by quantitative Western blotting (P =.01)., Conclusions: There is loss of the microarray-derived candidate, LR11, in neurons of AD brains. This study shows that microarray analysis of widely available lymphoblasts derived from patients with AD holds promise as a primary screen for candidate genes associated with AD.

Objective: Studies on the involvement of low-density lipoprotein receptor relatives (LRs) in atherosclerosis have recently gained new focus because of the specific expression of certain of these receptors in the thickened intima. Here, we show that LRP1B, a member of LRs, modulates the migration of smooth muscle cells (SMCs) by increasing the degradation of membrane receptors, urokinase-type plasminogen activator receptor (uPAR), and platelet-derived growth factor receptor (PDGFR) beta., Methods and Results: Immunohistochemistry showed that LRP1B expression in human coronary arteries is localized to the intimal SMCs near the plaque surface as well as to medial SMCs. LRP1B expression levels in cultured SMCs increase at the late phase of proliferation. Cell surface and internalization assays, in combination with coimmunoprecipitation experiments, showed that LRP1B binds and internalizes uPAR. Metabolic labeling analysis demonstrated that anti-LRP1B IgY decreased the catabolism of uPAR. In addition, the anti-LRP1B antibody raised PDGFRbeta protein and PDGFR-mediated phosphorylation levels of ERK1/2. Finally, the anti-LRP1B IgY enhanced the migration and invasion of SMCs in the presence of PDGF-BB., Conclusions: LRP1B modulates the catabolism of uPAR and PDGFR, affecting the migration of SMCs. This functional characterization of LRP1B opens novel avenues for elucidating the (patho)physiological significance of SMC migration in atheromatous plaques.

It becomes now evident that the localization of accumulated adipose mass is more important than the quantity of those in humans for the development of insulin resistance, which is based on the metabolic syndrome. The fat accumulation in visceral or subcutaneous area is closely related with the occurrence and severity of abnormality of glucose or lipid metabolism. The functional disturbance of adipose cells depending on the accumulated areas might cause the formation of metabolic disorders in the metabolic syndrome.

LR11, a member of the LDL receptor family, is highly expressed in vascular smooth muscle cells (SMCs) of the hyperplastic intima, and induces enhanced migration of SMCs in vitro via its upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. In this study, we have delineated the mechanism by which LR11 elevates the expression levels of uPAR in SMCs. Secretion of soluble LR11 is induced in SMCs during the rapidly proliferating phase, and the secreted LR11 induces the migration activities of SMCs. Both the cell-anchored and secreted forms of LR11 have the capacity to bind to and form complexes with uPAR. LR11-overexpressing cells show significantly enhanced uPAR binding, but decreased uPAR internalization. LR11 colocalizes with uPAR on the cell surface and inhibits the LDL receptor-related protein (LRP)-mediated binding and internalization of uPAR. Thus, LR11 mediates the uPAR localization to the plasma membrane. LR11 is highly expressed in the atheromatous plaque areas of apoE knockout mice, particularly in the intimal SMCs at the border between intima and media. The neutralization of LR11 function with anti-LR11 antibody reduced cuff-induced intimal thickness in mice. The novel mechanism of regulation of uPAR localization in SMCs accompanied with enhanced migration activity possibly constitutes an important factor in the process of atherosclerosis and arterial remodeling.

Rosuvastatin is a new statin that has been shown to produce substantial dose-dependent reductions in low-density lipoprotein cholesterol (LDL-C) in Western and Japanese hypercholesterolemic patients. Rosuvastatin efficacy and safety were assessed in an open-label, dose-titration trial of 37 Japanese patients with heterozygous familial hypercholesterolemia. After an 8-week dietary lead-in period, patients received rosuvastatin on the following schedule: 10 mg/day during weeks 0-6; 20 mg/day during weeks 6-12, and 40 mg/day for weeks 12-18. Mean percentage reductions from baseline in LDL-C (49.2-56.7%), total cholesterol (39.4-45.4%), and non-high-density lipoprotein cholesterol (non-HDL-C) (46.7-54.3%) were highly significant at each dose (p < 0.0001). Similar significant reductions in triglycerides (18.2-25.0%; p < 0.006) and increases in HDL-C (9.6-13.6%; p < 0.005) were observed. Rosuvastatin was well tolerated. Two patients withdrew from the study because of adverse events unrelated to the study treatment. No patients had clinically significant elevations in liver transaminases. Two patients exhibited a single increase in creatine kinase (one unrelated to study treatment, the other possibly related) with no muscle symptoms. Rosuvastatin produced significant beneficial changes in all lipid parameters in Japanese patients with heterozygous familial hypercholesterolemia and was well tolerated.

Familial hypercholesterolemia (FH) is one of the most common primary hyperlipidemias, characterized by a heterozygous or homozygous phenotype for a severe serum low-density lipoprotein (LDL)-cholesterol level and advanced atherosclerosis, leading to coronary artery diseases (CAD). Various kinds of mutations in the LDL receptor gene responsible for the genetic disease have been identified since the human LDL receptor gene has been identified. In this study, the clinical features of FH were investigated using a database based on nationwide surveillance for primary hyperlipidemia and related disorders by the Research Committee on Primary Hyperlipidemia. The clinical features and the frequencies of accompanying vascular diseases in 660 cases of FH homozygotes and heterozygotes showed that the incidence of CAD was negatively associated with plasma HDL-cholesterol levels, but not with plasma LDL-cholesterol levels, in 641 FH heterozygotes. Risk factor analyses revealed that hypertension, male, smoking, low HDL-cholesterol levels, age > 50 y, diabetes mellitus, and hypertriglyceridemia were positive risk factors for CAD. The summarized gene analysis in FH heterozygotes showed at least 4 mutations in the LDL receptor gene as common mutations in Japan. The average serum lipids and frequency of CAD based on each common mutation suggested that their clinical features are in part determined by responsive mutations in the LDL receptor gene.

Factors predisposing to the phenotypic features of higher total cholesterol (T-Cho) have not been clearly defined. Here we report an association between a C/T single nucleotide polymorphism at IVS17+8 in the inter-alpha-trypsin inhibitor heavy chain 4 gene (ITIH4) and plasma total cholesterol levels in 351 adult individuals from an east-central area of Japan. Age and gender-adjusted levels of plasma T-Cho, LDL-cholesterol, triglyceride, and HDL-cholesterol were analyzed. When we separate the subjects into two genotypic groups regarding this single nucleotide polymorphism (SNP), those who lack the T-allele had significantly higher plasma T-Cho levels than the others who bear T-allele (mean 252.3 mg/dl versus 241.7 mg/dl; p=0.009). Of the 309 individuals without the T-allele, approximately 90% presented with hypercholesterolemia, whereas only 10% were hypercholesterolemic among 42 individuals with the T-allele (p <0.0001). These data suggest that genetic variation at ITIH4 locus is one of the likely candidate determinants for plasma cholesterol metabolisms.

Primary hyperlipidemia is caused by various molecular defects in lipid metabolism. The Research Committee on Primary Hyperlipidemia organized by the Ministry of Health and Welfare of Japan (present: the Ministry of Health, Labour and Welfare) has investigated reported mutations in Japanese patients with primary hyperlipidemia and related disorders (including hypolipidemia), and has created a database based on the questionnaire sent to the members of council board of the Japan Atherosclerosis Society. Mutations in the following genes were investigated: low density lipoprotein receptor, lecithin: cholesteryl acyltransferase, lipoprotein lipase (LPL), hepatic lipase, apolipoproteins A-I, A-II, A-IV, B, C-II, C-III and E, microsomal triglyceride transfer protein, and cholesterol ester transfer protein (CETP). Until 1998, 922 patients with primary hyperlipidemia and related disorders has been registered with the Research Committee, and 190 mutations in 15 genes had been reported, showing a marked variation in Japanese patients with primary hyperlipidemia and related disorders. So-called "common mutations" have been described in Japanese patients with familial hypercholesterolemia, LPL deficiency and CETP deficiency. The genetic defect of familial combined hyperlipidemia (FCHL) is still unknown although FCHL is speculated to be the most prevalent genetic hyperlipidemia, and further investigations should be performed to elucidate the molecular mechanisms of FCHL

Familial combined hyperlipidemia (FCHL) is characterized by elevated levels of serum total cholesterol (TC), triglyceride (TG), or both. The increased incidence of coronary artery diseases (CAD) in the patients with FCHL is believed to be caused by circulating atherogenic lipoproteins associated with the complex phenotype. Recent establishment of sensitive detection system for malondialdehyde-modified (MDA)-LDL, which is one of oxidized lipoproteins, showed its increased circulating level in the patients with CAD. In order to know the atherogenic lipoproteins resulted from the dyslipidemia observed in FCHL, we measured the serum MDA-LDL level in the patients. The circulating MDA-LDL level and the ratio of MDA-LDL and LDL-C in FCHL were significantly higher (P<0.05) than those in control, which are adjusted about the age, serum TC, LDL-C and HDL-C levels, respectively. Furthermore, the circulating MDA-LDL level and the ratio of MDA-LDL and LDL-C were negatively correlated (R=-0.635, P<0.01 and R=-0.702, P<0.01, respectively) with hepatic lipase (HL) activity in FCHL. The serum MDA-LDL level and the ratio of MDA-LDL and LDL-C were in the subjects with T/T genotypes in the HL C-514T polymorphism were significantly increased compared to those with C/C genotype, respectively. The subjects with T/T genotype showed the activities to 65 and 79% of those in the subjects with C/C genotype in male and female, respectively. The intima-media thickness (IMT) of carotid artery was significantly higher (P<0.05) in the subjects with T/T genotype than those with C/C genotype in male. These findings indicate that the circulating MDA-LDL level is possibly contributing the atherogenic process in FCHL, and the common HL polymorphism might be a determinant of the serum level of oxidized LDL in the patients with FCHL.

Aims/hypothesis: Adipose tissue expresses some bioactive molecules, which may be involved in the development of obesity-associated metabolic disorders and cardiovascular diseases. Vascular endothelial growth factor (VEGF) an important angiogenic factor is implicated in normal and pathological vessel formation. The aim of this study is to investigate clinically the association between blood serum VEGF concentrations and body fat accumulation as well as distribution. The study also aims to show the effect of serum VEGF protein on gene expression of transcriptional factor E26 transformation-specific-1 (Ets-1) and matrix metalloproteinase (MMP)-3., Methods: Serum VEGF concentrations were measured in 38 overweight or obese subjects. Fat distribution in the abdominal subcutaneous as well as visceral fat areas was assessed by computed tomography scans at umbilical level. Furthermore, the changes of serum VEGF concentrations following body weight reduction therapy were analyzed in eight subjects recruited from the original pool of subjects. Semi-purified circulating VEGF proteins were obtained by heparin-sepharose and its biological activities were shown to alter gene expressions in human aortic endothelial cells., Results: Serum VEGF concentrations were positively correlated with BMI (r=0.433, p=0.007) and visceral fat area (r=0.488, p=0.002). Stepwise regression analysis showed the visceral fat area as the most important determinant factor for VEGF circulating levels. Following body weight reduction therapy, VEGF concentrations as well as visceral fat area were decreased. The serum semi-purified VEGF protein enhanced expressions of Ets-1 and MMP-3 in human aortic endothelial cells., Conclusion/interpretation: Increased serum VEGF concentrations associated with visceral fat accumulation could influence vascular endothelial function.

Visceral fat accumulation is accompanied by several metabolic disorders. Here, we investigate the improvement of visceral fat accumulation in the early phase of diet. Hyperlipidemic obese patients received a low-calorie diet (1000 kcal/day) for 14 days. Visceral and subcutaneous fat accumulation was analyzed using ultrasonography. After 14 days of the diet, the average visceral fat of obese patients obviously decreased (P < 0.05), as well as the visceral fat-related secreted proteins, whereas subcutaneous fat did not decrease in these patients. These results show that visceral fat is reduced significantly in the early phase of diet therapy in humans. Therefore, to clarify its mechanism, we analyzed the expression of lipid metabolism-related genes in visceral and subcutaneous fat using obese rats. The Long-Evans Tokushima Otsuka (LETO) rats, as an obese model, were divided into two groups: fasting and non-fasting. The gene expressions in visceral and subcutaneous fat were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of beta(3)-adrenergic receptor (AR), hormone sensitive lipase (HSL), peroxisome proliferator-activated receptor (PPAR)-gamma, and uncoupling protein (UCP)-2 genes increased by 3.2-, 2.3-, 2.2-, and 2-fold in visceral fat (P < 0.01), but remained almost unchanged in subcutaneous fat. Taken together, the responsiveness of lipid metabolism-related genes to fasting is more sensitive in visceral fat than in subcutaneous fat in rats, suggesting that the different responsiveness to calorie restriction in fat tissues is due to the different induction of metabolism-related gene expression.

Thiazolidinediones (TZDs), agonists for PPARs, have been shown to block the inhibitory effects of TNF-alpha on insulin action using cultured cells. In order to clarify the in vivo effects of TZDs on the inhibition of insulin sensitivity by TNF-alpha, insulin action in muscles and adipose tissues was assessed in the TNF-alpha-overexpression mice model using transplantation of cells secreting the TNF-alpha protein. After the pioglitazone treatment for 4 weeks, glucose uptake, insulin-induced IRS-1 phosphorylation, and lipoprotein lipase mRNA levels were analyzed. Pioglitazone did not ameliorate TNF-alpha-induced hyperinsulinemia in this model, as assessed by the OGTT. Glucose uptake and lipoprotein lipase mRNA levels were decreased by TNF-alpha in adipose tissues from the TNF-alpha-overexpressing mice, and pioglitazone blocked these inhibitions by TNF-alpha. On the other hand, in muscles, pioglitazone did not reverse the effects of TNF-alpha on insulin-induced phosphorylation of IRS-1, glucose uptake, and lipoprotein lipase mRNA levels. Present study revealed the different sensitivities of pioglitazone for the recovery of decreased insulin action in a TNF-alpha-overexpressing model using cell transplantation. These results suggest that the effect of TZDs is dependent on the fat distribution and accumulation in humans.