This study was performed to determine the rates of in vitro cholesterol synthesis, as measured by ³H incorporation into cholesterol, in gluteal white adipose tissue and the intestine of infant rats during the early postnatal period. The reason for studying these parameters was to hopefully further elucidate the cause of the differences in blood cholesterol levels between rats raised in different litter sizes. Rats were raised in small (4/dam), medium (8/dam) and large (14/dam) litters. The rate of cholesterol synthesis in the proximal and distal region of the small intestine decreased from birth to a low 14 days later and then increased again by day 21. The rate of the decreases in cholesterol synthesis, from birth to the low on day 14, varied amongst the three litter sizes; the rate of synthesis in both regions of the intestine on day 7 was lowest in the larger litter, and was not significantly different from the values seen on day 14. Previous work has shown that plasma cholesterol levels were low in rats from large litters on day 7, thus in the early postnatal period low rates of intestinal cholesterol synthesis correlate with low plasma cholesterol values. After weaning, on day 21, the rate of synthesis in distal intestine was higher than that of proximal intestine in the medium and small litters, whereas the opposite was found in the larger litter. Cholesterol synthesis in gluteal white adipose tissue remained at a very low rate up to 21 days in the small and medium sized litters. In contrast, the rate of synthesis increased continuously in the large litter to nearly threefold the rate of that in the other two litter sizes on day 21. This study has shown that the rates of cholesterol synthesis in intestinal and adipose tissue can be altered during the early postnatal period by raising the rats in different litter sizes. Also, the pattern of development in the rates of cholesterol synthesis as measured by ³H incorporation into cholesterol are in general agreement with results reported for the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase for this same period in development. / Land and Food Systems, Faculty of / Graduate

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The purpose of this study was to investigate whether increased endogenous cholesterol synthesis contributes to the elevated plasma cholesterol levels observed in type III hyperlipoproteinemia (type III HLP). Eight apolipoprotein (apo) E2 subjects with type III HLP and 8 apo E2 non-hyperlipidemic control subjects (controls) were given a priming bolus dose of deuterium oxide (D₂O) (0.7 g D₂O/kg body H2O). Daily M1 (central) pool free cholesterol fractional synthetic rate (FSR) was calculated as the incorporation rate of deuterium from body water into plasma free cholesterol. Blood samples were collected one half hour prior to, and at 12 hour intervals over 48 hours following, the bolus D₂O dose. Drinking water labelled at 1.4 and 0.7 g D₂O/liter H₂O was given on the fed and fasted days, respectively. Over 0-24 hours, subjects consumed a diet of three isocaloric meals which, in composition, approximated average North American intakes. Subjects fasted over 24-48 hours. The deuterium enrichment of plasma free cholesterol and plasma water was determined by isotope ratio mass spectrometry. When all subjects were included, mean (±SEM) free cholesterol overall FSR in type III HLPs (0.031 ± 0.006 per day) was not significantly different from controls (0.037 ± 0.004 per day). Estimated Ml total cholesterol pool size in type III HLPs (26.1 ± 1.9 g) and controls (24.9 ± 0.6 g) was not significantly different. When free cholesterol net synthesis was calculated as the absolute amount of cholesterol synthesized per day, based on Ml total cholesterol pool size, overall free cholesterol net synthesis in type III HLPs (0.304 ± 0.034 g/day) was not significantly different from controls (0.364 ± 0.035 g/day). When all subjects were included, overall free cholesterol FSR and overall free cholesterol net synthesis were significantly greater (p<0.001) in the fed (0.066 ± 0.006 day⁻¹ and 0.655 + 0.048 g/day, respectively) as compared to the fasted state (0.001 ± 0.004 day⁻¹ and 0.010 ± 0.037 g/day, respectively). In the fed state, type III HLPs tended to synthesize cholesterol at a lower rate and in a lower absolute amount as compared to controls, while the reverse was observed in the fasted state. These results suggest that: (1) the elevated plasma cholesterol levels observed in type III HLPs are not due to excess de novo cholesterol synthesis; (2) fasting significantly reduces cholesterol synthesis from the fed state. / Land and Food Systems, Faculty of / Graduate

Hyperhomocysteinemia, an elevation of blood homocysteine levels, is a metabolic disorder associated with dysfunction of multiple organs. Previous studies have shown that hyperhomocysteinemia is related to fatty liver. However, the underlying mechanism remains speculative. The objective of the present study is to investigate the regulatory mechanism of hepatic inflammatory response and cholesterol metabolism during metabolic disorders. In the present study, hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet. The mRNA and protein expression of cyclooxygenase-2 (COX-2), a pro-inflammatory factor, were significantly elevated in the liver of hyperhomocysteinemic rats. An activation of NF-B and a stimulation of oxidative stress were observed in the same liver tissue in which COX-2 was induced. Inhibition of NF-B or oxidative stress effectively abolished hepatic COX-2 expression, inhibited the formation of inflammatory foci, and improved liver function. Activity of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis, was markedly elevated in the liver of hyperhomocysteinemic rats, which may contribute to the hepatic lipid accumulation induced by hyperhomocysteinemia. Administration of Berberine (5mg/ kg body weight/ day for 5 days) inhibited HMG-CoA reductase activity via upregulating AMP-activated protein kinase (AMPK)-mediated phosphorylation of HMG-CoA reductase. Berberine treatment reduced hepatic cholesterol content and ameliorated liver function. In addition, the regulatory mechanism of HMG-CoA reductase activation was investigated in C57BL/6 mice fed a high-fat diet. There was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 (a transcription factor of HMG-CoA reductase) and Sp1 (a transcription factor of SREBP-2) were both increased in the liver of mice fed a high-fat diet. The in vitro study in palmitic acid-treated HepG2 cells further confirmed that inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. In conclusion, the present study have demonstrated that (1) Hepatic COX-2 expression is induced via oxidative stress mediated NF-B activation during hyperhomocysteinemia; (2) Dietary berberine reduces cholesterol biosynthesis by elevating AMPK-mediated HMG-CoA reductase phosphorylation; (3) HMG-CoA reductase is upregulated by Sp1-mediated SREBP-2 activation in the liver during high-fat diet feeding.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21810号 / 農博第2323号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5182(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 橋本 渉, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24551号 / 薬科博第168号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 金子 周司, 教授 竹島 浩, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

While the role of cholesterol in liver carcinogenesis remains controversial, hepatocellular carcinoma generally prevails in males. Herein, we uncover pathways of female-prevalent progression to hepatocellular carcinoma due to chronic repression of cholesterogenic lanosterol 14α-demethylase (CYP51) in hepatocytes. Tumors develop in knock-out mice after year one, with 2:1 prevalence in females. Metabolic and transcription factor networks were deduced from the liver transcriptome data, combined by sterol metabolite and blood parameter analyses, and interpreted with relevance to humans. Female knock-outs show increased plasma cholesterol and HDL, dampened lipid-related transcription factors FXR, LXRα:RXRα, and importantly, crosstalk between reduced LXRα and activated TGF-β signalling, indicating a higher susceptibility to HCC in aging females. PI3K/Akt signalling and ECM-receptor interaction are common pathways that are disturbed by sex-specific altered genes. Additionally, transcription factors (SOX9)2 and PPARα were recognized as important for female hepatocarcinogenesis, while overexpressed Cd36, a target of nuclear receptor RORC, is a new male-related regulator of ECM-receptor signalling in hepatocarcinogenesis. In conclusion, we uncover the sex-dependent metabolic reprogramming of cholesterol-related pathways that predispose for hepatocarcinogenesis in aging females. This is important in light of increased incidence of liver cancers in post-menopausal women.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25356号 / 農博第2622号 / 新制||農||1109(附属図書館) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 佐々木 努, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Made available in DSpace on 2016-08-10T10:39:08Z (GMT). No. of bitstreams: 1 STANLEY SILVANO SOUSA.pdf: 1573160 bytes, checksum: 779804c156f10ab50a1b61b85240a0ab (MD5) Previous issue date: 2015-08-27 / The main regulatory enzyme of cholesterol biosynthesis is hydroxyl-methylglutaryl-CoA reductase (HMGCR) and several polymorphisms are described in the gene encoding this enzyme. Currently, associations between genetic polymorphisms and cardiovascular disease are investigated in order to better understand the genetic factors associated with such diseases. The objective of this study was to evaluate the frequency of -911 polymorphism (rs3761740) in the promoter region of HMGCR gene in patients with coronary artery disease (CAD), as well as the possible associations between the resultant genotypes and clinical features of patients with CAD. Genomic DNA isolated from patients blood samples were analyzed for the detection of genetic polymorphism, by using polymerase chain reaction (PCR) analysis and restriction fragment length polymorphism (RFLP). Allele frequencies obtained for the -911 polymorphism (rs3761740) in the promoter region of the HMGCR gene were: A (51.2%) and C (48.8%). The genotype frequencies obtained were: AA (11.9%), AC (78.6%) and CC (9.5%). Significant associations between the diferente genotypes and clinical features of the patients with CAD were not detected in this study. Our results show that -911 polymorphism (rs3761740) in the promoter region of the HMGCR gene was not associated with clinical and laboratory characteristics in patients with coronary artery disease. / A principal enzima regulatória da biossíntese do colesterol é a hidrox-imetilglutaril-CoA redutase (HMGCR) e vários polimorfismos são descritos no gene que codifica esta enzima. Atualmente, associações entre tais polimorfismos genéticos e as doenças cardiovasculares são investigadas no sentido de compreender melhor os fatores genéticos associados a essas doenças. O objetivo deste estudo foi avaliar a frequência do polimorfismo -911(rs3761740) na região promotora do gene da HMGCR em pacientes com doença arterial coronariana (DAC), bem como as possíveis associações entre os genótipos encontrados e os aspectos clínicos dos pacientes com DAC. O DNA genômico isolado das amostras de sangue dos pacientes foi analisado para detecção do polimorfismo genético, por meio da reação em cadeia da polimerase (PCR) e análise de polimorfismos de comprimento de fragmentos de restrição (RFLP). As frequências alélicas obtidas para o polimorfismo -911 (rs3761740) na região promotora do gene HMGCR foram: A (51,2%) e C (48,8%). As frequências genotípicas obtidas foram: AA (11,9%), AC (78,6%) e CC (9,5%). Associações significativas entre os genótipos encontrados e os aspectos clínicos dos pacientes com DAC não foram detectadas neste estudo. Nossos resultados permitem concluir que polimorfismo -911(rs3761740) na região promotora do gene da HMGCR não esteve associado aos aspectos clínicos e laboratoriais em pacientes com doença arterial coronariana.