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Biomedical subjects

Nobuhiro Yamada

Publications and source records attributed to Nobuhiro Yamada.

At least 19 recordsLinked to original sources
PubMed2003

Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene.

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/-) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/- embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/- mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/- and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.

Alleles↗
PubMed2003

p53 Activation in adipocytes of obese mice.

The tumor suppressor p53 is a transcription factor that activates or represses its target genes after various genotoxic stresses. We have previously shown that sterol regulatory element-binding protein-1 (SREBP-1), a key transcriptional regulator of triglyceride synthesis, and the lipogenic enzymes under its control are markedly suppressed in adipocytes from genetically obese ob/ob mice. Here we demonstrate that p53 and its target genes are highly induced in adipocytes of ob/ob mice in a fed state, leading to the negative regulation of SREBP-1 and thereby lipogenic genes. In fact, disruption of p53 in ob/ob mice completely suppressed the p53-regulated genes to wild-type levels and partially restored expression of lipogenic enzymes. Consistently, reporter gene analysis showed that p53 overexpression suppressed the promoter activity of the SREBP-1c gene and its downstream genes. Thus, the activation of p53 might constitute a negative feedback loop against excess fat accumulation in adipocytes. In conclusion, we discovered a novel role of p53 in the pathophysiology of obesity.

Adipose Tissue↗
PubMed2003

Cross-talk between peroxisome proliferator-activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. I. PPARs suppress sterol regulatory element binding protein-1c promoter through inhibition of LXR signaling.

Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are members of nuclear receptors that form obligate heterodimers with retinoid X receptors (RXRs). These nuclear receptors play crucial roles in the regulation of fatty acid metabolism: LXRs activate expression of sterol regulatory element-binding protein 1c (SREBP-1c), a dominant lipogenic gene regulator, whereas PPARalpha promotes fatty acid beta-oxidation genes. In the current study, effects of PPARs on the LXR-SREBP-1c pathway were investigated. Luciferase assays in human embryonic kidney 293 cells showed that overexpression of PPARalpha and gamma dose-dependently inhibited SREBP-1c promoter activity induced by LXR. Deletion and mutation studies demonstrated that the two LXR response elements (LXREs) in the SREBP-1c promoter region are responsible for this inhibitory effect of PPARs. Gel shift assays indicated that PPARs reduce binding of LXR/RXR to LXRE. PPARalpha-selective agonist enhanced these inhibitory effects. Supplementation with RXR attenuated these inhibitions by PPARs in luciferase and gel shift assays, implicating receptor interaction among LXR, PPAR, and RXR as a plausible mechanism. Competition of PPARalpha ligand with LXR ligand was observed in LXR/RXR binding to LXRE in gel shift assay, in LXR/RXR formation in nuclear extracts by coimmunoprecipitation, and in gene expression of SREBP-1c by Northern blot analysis of rat primary hepatocytes and mouse liver RNA. These data suggest that PPARalpha activation can suppress LXR-SREBP-1c pathway through reduction of LXR/RXR formation, proposing a novel transcription factor cross-talk between LXR and PPARalpha in hepatic lipid homeostasis.

Animals↗
PubMed2003

Cross-talk between peroxisome proliferator-activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. II. LXRs suppress lipid degradation gene promoters through inhibition of PPAR signaling.

Fatty acid metabolism is transcriptionally regulated by two reciprocal systems: peroxisome proliferator-activated receptor (PPAR) alpha controls fatty acid degradation, whereas sterol regulatory element-binding protein-1c activated by liver X receptor (LXR) regulates fatty acid synthesis. To explore potential interactions between LXR and PPAR, the effect of LXR activation on PPARalpha signaling was investigated. In luciferase reporter gene assays, overexpression of LXRalpha or beta suppressed PPARalpha-induced peroxisome proliferator response element-luciferase activity in a dose-dependent manner. LXR agonists, T0901317 and 22(R)-hydroxycholesterol, dose dependently enhanced the suppressive effects of LXRs. Gel shift assays demonstrated that LXR reduced binding of PPARalpha/retinoid X receptor (RXR) alpha to peroxisome proliferator response element. Addition of increasing amounts of RXRalpha restored these inhibitory effects in both luciferase and gel shift assays, suggesting the presence of RXRalpha competition. In vitro protein binding assays demonstrated that activation of LXR by an LXR agonist promoted formation of LXR/RXRalpha and, more importantly, LXR/PPARalpha heterodimers, leading to a reduction of PPARalpha/RXRalpha formation. Supportively, in vivo administration of the LXR ligand to mice and rat primary hepatocytes substantially decreased hepatic mRNA levels of PPARalpha-targeted genes in both basal and PPARalpha agonist-induced conditions. The amount of nuclear PPARalpha/RXR heterodimers in the mouse livers was induced by treatment with PPARalpha ligand, and was suppressed by superimposed LXR ligand. Taken together with data from the accompanying paper (Yoshikawa, T., T. Ide, H. Shimano, N. Yahagi, M. Amemiya-Kudo, T. Matsuzaka, S. Yatoh, T. Kitamine, H. Okazaki, Y. Tamura, M. Sekiya, A. Takahashi, A. H. Hasty, R. Sato, H. Sone, J. Osuga, S. Ishibashi, and N. Yamada, Endocrinology 144:1240-1254) describing PPARalpha suppression of the LXR-sterol regulatory element-binding protein-1c pathway, we propose the presence of an intricate network of nutritional transcription factors with mutual interactions, resulting in efficient reciprocal regulation of lipid degradation and lipogenesis.

ATP Binding Cassette Transporter 1↗
PubMed2003

Effect of thiazolidinediones and metformin on LDL oxidation and aortic endothelium relaxation in diabetic GK rats.

In this study, using GK diabetic rats, we compared the effects of three insulin sensitizers on lipid oxidation and the aortic relaxation response. Eight-week-old rats were treated for 4 wk with either troglitazone or pioglitazone, both of which are thiazolidinediones, or with metformin. Despite the fact that only troglitazone has a similarity in structure to alpha-tocopherol, a potent antioxidant, the level of thiobarbituric acid-reactive substance was lower, and the lag time of the conjugated dienes was longer, in the blood samples from the rats in both troglitazone- and pioglitazone-treated groups. In contrast, another insulin sensitizer, metformin, failed to inhibit the oxidation of blood samples. The aortic vasorelaxation response was increased in both troglitazone- and metformin-treated groups compared with the untreated group. These findings suggest that thiazolidinediones have a beneficial effect on lipid oxidation irrespective of the drug's structural similarity to alpha-tocopherol. It is also suggested that the thiazolidinediones and metformin improve vascular function in diabetes. These effects may play a role in the prevention of atherosclerosis in diabetic patients.

Acetylcholine↗
PubMed2003

Physiological changes in circulating mannose levels in normal, glucose-intolerant, and diabetic subjects.

Mannose is an essential hexose that is required for glycoprotein synthesis. Although circulating mannose levels are known to be influenced by metabolic disorders, how physiological levels of mannose fluctuate in normal and diabetic subjects is largely unknown. We describe a new accurate and sensitive assay for determining circulating mannose levels, which we used to measure plasma mannose levels in 273 normal and diabetic (DM) subjects. Our results revealed a clear correlation (r = 0.754) between fasting plasma mannose (FPM) and fasting plasma glucose (FPG) levels. Our mannose assay showed sensitivity and specificity comparable to that seen for hemoglobin A(1c) (HbA(1c)) assay in subjects with impaired glucose tolerance (IGT) or DM whose FPG levels were normal. Mannose levels were found to increase less than glucose levels in response to an oral glucose tolerance test (OGTT). Furthermore, plasma mannose levels did not significantly change following a meal and more closely correlated with the coefficient of variation (CV) of daily glucose levels than did glucose itself. In conclusion, the close correlation between FPM and FPG levels taken together with the small fluctuations seen in plasma mannose in response to glucose suggests that the measurement of mannose using our assay could potentially play a supplementary role in the diagnosis and screening of patients with mild DM.

Adult↗
PubMed2003

PKClambda in liver mediates insulin-induced SREBP-1c expression and determines both hepatic lipid content and overall insulin sensitivity.

PKClambda is implicated as a downstream effector of PI3K in insulin action. We show here that mice that lack PKClambda specifically in the liver (L-lambdaKO mice), produced with the use of the Cre-loxP system, exhibit increased insulin sensitivity as well as a decreased triglyceride content and reduced expression of the sterol regulatory element-binding protein-1c (SREBP-1c) gene in the liver. Induction of the hepatic expression of Srebp1c and of its target genes involved in fatty acid/triglyceride synthesis by fasting and refeeding or by hepatic expression of an active form of PI3K was inhibited in L-lambdaKO mice compared with that in control animals. Expression of Srebp1c induced by insulin or by active PI3K in primary cultured rat hepatocytes was inhibited by a dominant-negative form of PKClambda and was mimicked by overexpression of WT PKClambda. Restoration of PKClambda expression in the liver of L-lambdaKO mice with the use of adenovirus-mediated gene transfer corrected the metabolic abnormalities of these animals. Hepatic PKClambda is thus a determinant of hepatic lipid content and whole-body insulin sensitivity.

Animals↗
PubMed2003

Preventing angiographic progression of coronary atherosclerosis with pravastatin.

We conducted a prospective study to investigate the relationship between the decrease of serum lipid levels during pravastatin therapy and changes of coronary angiography parameters in Japanese patients with coronary atherosclerosis. The patients were predominantly male, aged between 18 and 75 years (mean: 58 years), had at least 25% stenosis of one or more major coronary arteries, and had a serum total cholesterol ( TC) level > or = 200 mg/dl (5.18 mM/l). Treatment with pravastatin (10 mg/day) was continued for 3 years. Coronary angiography was performed before and 3 years after the start of pravastatin therapy to assess the relationship between the mean segment diameter (MSD), the minimal lumen diameter (MLD), and the annual changes of percent stenosis and TC levels. of 265 patients who were initially registered, 129 were followed for an average of 35 months. Consequently, second angiograms were only obtained in 68 patients for various reasons, so this group was used for analysis. During pravastatin therapy, the TC level significantly decreased from 239 mg/dl (6.19 mM/l) to 210 mg/dl (5.44 mM/l) (a 12% reduction; p<0.001). In addition, HDL-cholesterol increased by 5% (p=0.007), but the triglyceride level did not show a significant change. Both MSD and MLD were significantly improved on follow-up angiography, increasing from 2.67 mm to 2.76 mm and from 2.09 mm to 2.13 mm, respectively. However, no change of percent stenosis was observed. The mean TC level during treatment did not show any significant correlation with the changes of angiography parameters. However, a significant correlation was observed between the percent reduction of TC and the annual change of MSD (r=-0.272, p=0.027). A similar relationship was also found between the change of MLD and the percent reduction of TC (r=-0.260, p=0.035). In conclusion, the percent decrease of serum cholesterol may be a better indicator of clinical efficacy than the absolute cholesterol level during pravastatin therapy.

Coronary Angiography↗
PubMed2002

Resistin is regulated by C/EBPs, PPARs, and signal-transducing molecules.

Expression of the adipocyte-derived protein resistin, which is thought to play a key role in the development of insulin resistance in vivo, is regulated by a variety of hormones and mediators, including insulin and TNFalpha. Here we describe our use of adenovirus-mediated gene transfer to determine which transcription factors and signaling pathways affect resistin expression in 3T3-L1 adipocytes. We found that resistin expression was enhanced by overexpression of C/EBPalpha and suppressed by C/EBPzeta, a negative regulator of C/EBPalpha. Additionally, C/EBPalpha induced resistin even in L6 myocytes. Overexpression of PPARgamma markedly reduced resistin expression, whereas PPARalpha had no significant effect. Resistin expression was markedly suppressed by overexpression of the PI3-kinase p110alpha catalytic subunit and by Akt. Finally, overexpression of MEK1, MKK6, or MKK7 suppressed resistin expression. These findings indicate that resistin expression is regulated by C/EBPalpha and PPARgamma, partly via modulation of signal transduction in the PI3-kinase and MAP kinase pathways.

3T3 Cells↗
PubMed2002

HMG-CoA reductase inhibitor decreases small dense low-density lipoprotein and remnant-like particle cholesterol in patients with type-2 diabetes.

Patients with type 2 diabetes are known to have abnormalities in their remnant metabolism and low density lipoprotein (LDL) subfraction pattern, with a preponderance of small dense LDL. The effects of pitavastatin, a newly synthesized 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, on lipoprotein profiles in patients with type 2 diabetes were determined. Thirty-three patients were treated with pitavastatin with a daily dose of 2 mg for 8 weeks. After treatment, triglyceride, total and LDL cholesterol were significantly reduced by 28.7 +/- 36.7%, 25.2 +/- 14.3% and 36.1 +/- 14.3%, respectively. Remnant-like particle cholesterol (RLP-C), an independent risk factor for CAD which is known to be elevated in diabetic patients, was also significantly reduced (-30.9 +/- 30.5%) by the treatment and this decrease correlated well with the decrease in triglyceride level. The proportion of small dense LDL, which is known for its atherogenisity, decreased from 29.9 +/- 26.2% to 19.7 +/- 22.7% and the mean LDL particle size significantly increased from 26.36 +/- 1.13 nm to 27.10 +/- 1.36 nm. Pitavastatin, which is known to improve triglyceride levels and cholesterol levels, also improves RLP-C level and LDL subfraction profiles, and this in turn may reduce the cardiovascular risk in patients with type 2 diabetes and dyslipidemia.

Adult↗
PubMed2002

Vascular endothelial growth factor gene expression in a retinal pigmented cell is up-regulated by glucose deprivation through 3' UTR.

Diabetic retinopathy is known to be worsened when hypoglycemia occurs, however the pathogenic mechanisms are not defined. Vascular endothelial growth factor (VEGF) is increased in ocular fluid with diabetic retinopathy and linked with development of retinopathy. In this study, we examined whether glucose deficiency could up-regulate VEGF levels in retinal pigmented cells. Exposure to glucose deprived medium induced 30% up-regulation of VEGF mRNA. In hypoxia, as one of the most well-known inducer of VEGF, its up-regulation mechanism is mainly due to increase of transcription of VEGF gene via hypoxia response element in 5'-untranslated region (5'-UTR). We examined the role of 5'-UTR and 3'-UTR of VEGF gene in glucose deprived conditions using luciferase assay system. 5'-UTR containing reporter vector did not show increase of activity in glucose deprived conditions in contrast to the result in oxygen deprived condition. Both 5'-UTR and 3'-UTR containing vector demonstrated significant increase of activity in glucose deficient conditions compared with 5'-UTR containing vector. These findings suggest 3'-UTR of VEGF gene is important for increasing mRNA level in glucose deprived condition.

3' Untranslated Regions↗
PubMed2002

A missense mutation in kynurenine aminotransferase-1 in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) are the most extensively used animal model for genetic hypertension, increased stroke damage, and insulin resistance syndromes; however, the identification of target genes has proved difficult. SHR show elevated sympathetic nerve activity, and stimulation of the central blood pressure control centers with glutamate or nicotine results in exaggerated blood pressure responses, effects that appear to be genetically determined. Kynurenic acid, a competitive glutamate antagonist and a non-competitive nicotinic antagonist, can be synthesized in the brain by the enzyme kynurenine aminotransferase-1 (KAT-1). We have previously shown that KAT-1 activity is significantly reduced in SHR compared with normotensive Wistar Kyoto rats (WKY). Here we show that KAT-1 contains a missense mutation, E61G, in all the strains of SHR examined but not in any of the WKY or outbred strains. Previous studies on F2 rats from a cross of stroke-prone SHR and WKY have shown a suggestive level of linkage between elevated blood pressure and the KAT-1 locus on chromosome 3. In addition, the mutant enzyme expressed in Escherichia coli displays altered kinetics. This mutation may explain the enhanced sensitivity to glutamate and nicotine seen in SHR that may be related to an underlying mechanism of hypertension and increased sensitivity to stroke.

Alleles↗
PubMed2002

PPAR gamma ligands, troglitazone and pioglitazone, up-regulate expression of HMG-CoA synthase and HMG-CoA reductase gene in THP-1 macrophages.

Recently it has been reported that macrophages express a nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma). Using a ligand of PPAR gamma, troglitazone or pioglitazone, we have shown that the expression of two genes involved in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase, were increased by activation of PPAR gamma through a PPAR response element (PPRE) in THP-1 macrophages. In addition, treatment with troglitazone significantly increased the activity of HMG-CoA reductase and the amount of intracellular cholesterol. Thus, we conclude that PPAR gamma and its agonists increase the cholesterol content of macrophages by the increased expression of genes involved in cholesterol biosynthesis. These findings suggest that PPAR gamma may play a role in cholesterol metabolism in macrophages.

Cell Differentiation↗
PubMed2002

Elimination of cholesterol ester from macrophage foam cells by adenovirus-mediated gene transfer of hormone-sensitive lipase.

Cholesterol ester (CE)-laden foam cells are a hallmark of atherosclerosis. To determine whether stimulation of the hydrolysis of cytosolic CE can be used as a novel therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellular cholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells that had been preloaded with CE by incubation with acetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presence of high density lipoproteins, which might be at least partially explained by the increase in the expression of ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low density lipoprotein was significantly reduced probably by decreased expression of scavenger receptor A and CD36. Notably, the cells with stimulated CE hydrolysis did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1 foam cells not only by increasing efflux but also by decreasing influx of cholesterol.

Acetylation↗