No description available.

京都大学 / 0048 / 新制・論文博士 / 博士(工学) / 乙第13016号 / 論工博第4141号 / 新制||工||1650(附属図書館) / 32944 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 梅田 眞郷 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malnoyl-CoA, which in turn controls the rate of fatty acid metabolism. ACC beta or 2 has been shown to be localized on the mitochondria in close proximity to carnintine palmitoyl transferase 1 (CPT-1), the enzyme responsible for the influx of acyl-CoA into the matrix where beta oxidation takes place. CPT-1 is inhibited by malonyl-CoA produced by ACC. It has been well documented that AMP activated kinase (AMPK) when activated phosphorylates and inactivates ACC. ACC is controlled allosterically by citrate, which activates, and by palmitoyl-COA, which inhibits. In this study, we asked the question, "Does phosphorylation by AMPK effect the inhibition of ACC by palmitoyl-CoA?" ACC was isolated and then subjected to phosphorylation and activity was measured in varying concentrations of acetyl-CoA and citrate. Phosphoryation reduced the substrate (acetyl-CoA) saturation activity curves for ACC at all levels of palmitoyl-CoA. The Ki for palmitoyl-CoA inhibition of ACC was reduced from 1.7 ± 0.25 µM to 0.85 ± 0.13 uM (p<0.05) as a consequence of phosphorylation. In addition the citrate activation curves for ACC were greatly reduced in the presence of palmitoyl-CoA. The data show that skeletal muscle ACC or ACC-beta is more potently inhibited by palmitoyl-CoA after phosphorylation by AMPK. During long-term exercise when AMPK is activated and muscle palmitoyl-CoA is elevated this may contribute to the low malonyl-CoA and increased fatty acid oxidation.

No description available.

<p>This thesis describes the structural studies of four different proteins from two organisms. Xyloglucan endotransglycosylases, XETs, are involved in plant cell wall expansion and remodeling by splitting and reconnecting xyloglucan-cellulose crosslinks. The first crystal structure of a XET enzyme has been determined to 1.8 Å. The structure provides insights into how XETs are able to bind a heavily branched xyloglucan sugar, as well as hints about the XET-transglycosylation mechanism.</p><p><i>Mycobacterium tuberculosis</i> (Mtb) is the cause of enormous human mortality each year. Despite the sequencing of the complete Mtb-genome, the biological function of a large fraction of the <i>M. tuberculosis </i>proteins is still unknown. We here report the crystal structures of three such proteins, Rv2740, Rv0216 and Rv0130. Rv2740 forms a Cystatin α+b fold with a deep active site pocket similar to a limonene-1,2-epoxide hydrolase from <i>Rhodococcus erythropolis</i>. However, in contrast to the small limonene-based substrate of the <i>Rhodococcus</i> enzyme, Rv2740 is able to degrade large fatty acid and sterol epoxides, giving suggestions for the physiological substrates of this enzyme.</p><p>The structure of <i>M. tuberculosis</i> Rv0216 exhibits a so-called double hotdog fold. Rv0216 shows similarity to a number of enzymes using thiol esters as substrates, including several <i>R</i>-enoyl hydratases and β-hydroxyacyl dehydratases. However, only parts of the hydratase / dehydratase catalytic site are conserved in Rv0216. Rv0130 in contrast, contains a highly conserved <i>R</i>-hydratase motif, housed in a dimer of two single hotdog folded molecules. This active site is situated in a long tunnel, formed by a sharp kink in the Rv0130 central helix. A number of previously predicted single / double hotdog folded proteins from <i>M. tuberculosis</i> seem to feature a similar substrate-binding tunnel, indicating that Rv0130 as well as some of these proteins, might act on long fatty enoyl chains. </p>

This thesis describes the structural studies of four different proteins from two organisms. Xyloglucan endotransglycosylases, XETs, are involved in plant cell wall expansion and remodeling by splitting and reconnecting xyloglucan-cellulose crosslinks. The first crystal structure of a XET enzyme has been determined to 1.8 Å. The structure provides insights into how XETs are able to bind a heavily branched xyloglucan sugar, as well as hints about the XET-transglycosylation mechanism. Mycobacterium tuberculosis (Mtb) is the cause of enormous human mortality each year. Despite the sequencing of the complete Mtb-genome, the biological function of a large fraction of the M. tuberculosis proteins is still unknown. We here report the crystal structures of three such proteins, Rv2740, Rv0216 and Rv0130. Rv2740 forms a Cystatin α+b fold with a deep active site pocket similar to a limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis. However, in contrast to the small limonene-based substrate of the Rhodococcus enzyme, Rv2740 is able to degrade large fatty acid and sterol epoxides, giving suggestions for the physiological substrates of this enzyme. The structure of M. tuberculosis Rv0216 exhibits a so-called double hotdog fold. Rv0216 shows similarity to a number of enzymes using thiol esters as substrates, including several R-enoyl hydratases and β-hydroxyacyl dehydratases. However, only parts of the hydratase / dehydratase catalytic site are conserved in Rv0216. Rv0130 in contrast, contains a highly conserved R-hydratase motif, housed in a dimer of two single hotdog folded molecules. This active site is situated in a long tunnel, formed by a sharp kink in the Rv0130 central helix. A number of previously predicted single / double hotdog folded proteins from M. tuberculosis seem to feature a similar substrate-binding tunnel, indicating that Rv0130 as well as some of these proteins, might act on long fatty enoyl chains.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24487号 / 医博第4929号 / 新制||医||1063(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 上野 英樹, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Aims/hypothesis Hypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG). Methods To test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios. Results AMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure. Conclusions/interpretation Taken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes. / Diabetes UK (RD Lawrence Fellowship to CB; 13/0004647); the Medical Research Council (MR/N012763/1) to KLJE, ADR and CB; and a Mary Kinross Charitable Trust PhD studentship to CB, ADR and RW to support PGWP. Additional support for this work came from awards from the British Society for Neuroendocrinology (to CB and KLJE), the Society for Endocrinology (CB), Tenovus Scotland (CB) and the University of Exeter Medical School (CB and KLJE). AR was also supported by a Royal Society Industry Fellowship.

The global epidemic of obesity is rapidly becoming a major public health problem in many parts of the world. Unhealthy diets and physical inactivity are two modifiable risk factors for prevention of obesity and its associated chronic diseases. Their influence on muscle energy metabolism and fat mass is not completely elucidated. A decreased capacity for fatty acid oxidation (FAO) may be a metabolic risk factor for weight gain and is found to be depressed in obese individuals; and exercise training may promote an increased capacity for FAO. In addition to the interest in whole-body FAO, the role of site specific lipid accumulation including visceral adipose tissue (VAT), intrahepatic lipids (IHL) and intramyocellular lipids (IMCL) has become a focus of interest because of their reported association with insulin resistance (IR), a key metabolic defect associated with obesity and type 2 diabetes mellitus (T2DM). However, ambiguity persists regarding the importance of IMCL as a metabolic substrate for energy production in obesity. A better understanding of the factors regulating FAO, body fat distribution and IMCL mobilisation is important for the development of interventions allowing effective treatment of conditions in which these are disturbed. The study of individuals with metabolic myopathies can give more information about the energy metabolism of muscle. McArdle disease (MD) affects glucose availability to muscle for energy production. Investigations into IMCL storage and mobilisation in MD have not been reported. The aims of this thesis are to investigate 1) the effects of weight-loss via dietary restriction plus modest but clinically-relevant exercise training on FAO, body fat distribution and mobilisation of IMCL during exercise in obese non-diabetic adults; 2) the effect of an exercise training intervention on IMCL storage and mobilisation in a subject with MD. All obese subjects underwent a 4 month lifestyle intervention with weekly meetings with a dietitian and an exercise physiologist. Of the 92 subjects, 73 completed the intervention. They showed significant decreases in body weight (8%), fat mass (14%) and total cholesterol (5%). The exercise prescription of 1500 kcal.week-1 resulted in variable compliance with the prescription (1224 ± 1085 kcal.week-1) measured by heart rate monitor. Those who did most exercise and also those who had less weekly variability in their exercise, had greater reductions in body weight and fat mass. The total activity energy expenditure measured by accelerometry did not change post-intervention but there was a reduction in low intensity activity and an increase in moderate and high intensity activity. A submaximal treadmill test and resting metabolic rate (RMR) using indirect calorimetry was measured before and after the intervention to investigate factors regulating FAO and energy expenditure. Subjects showed increases in FAO without change in energy expenditure for the same walking speed post intervention, but the volume of exercise completed during the intervention was not associated with these changes. To investigate body fat distribution in obesity, VAT, IHL and soleus muscle IMCL was measured in a sub-group of 18 males by magnetic resonance imaging (MRI) and spectroscopy (MRS) along with measurement of maximal aerobic capacity. Fitness increased significantly with significant decreases in VAT (29%) and IHL (54%), without significant change in IMCL. Subjects who had the greatest decrease in VAT were those who exercised for longer durations during the intervention. IHL was the only measure of excess lipid that correlated with IR. The measurement of IMCL before and after 1-hour of cycle ergometer exercise showed no significant mobilisation of IMCL either at baseline or after the lifestyle intervention. The intensity of the acute exercise was adjusted to correspond to each individual’s maximal fatty acid oxidation (MFAO) which increased by over 60% post intervention. In the subject with MD, an 8 week exercise training intervention without dietary intervention increased IMCL stores by 27%, but there was no marked change in IMCL with acute exercise at both time points. The findings of this thesis demonstrate that a clinically relevant and achievable lifestyle intervention incorporating weight loss through diet and objectively measured exercise can achieve improvements in blood lipid profile, body composition and FAO. The differential effects of the intervention on the various fat depots and their associations to metabolic markers suggest that individualised strategies may be required dependent upon body fat distribution. The non detection of mobilisation of IMCL by MRS suggests that these lipids may not be present as a substrate source in this population but rather an ectopic lipid depot related to increased energy consumption in diet. The relatively low capacity for FAO in both the obese and MD subjects may have affected the results. This thesis discusses implications for clinical practice, discusses novel findings related to the energy metabolism in obesity and MD and informs clinical and basic science about important future directions.