The ability to adapt fuel usage to nutrient availability is termed metabolic flexibility, and is influenced by activity of the pyruvate dehydrogenase complex (PDC). The Virginia lines of chickens are a unique model of anorexia and obesity that have resulted from 56 generations of artificial selection for high (HWS) or low (LWS) juvenile body weight. We hypothesized that hyperphagia and obesity in juvenile HWS chickens are associated with altered fatty acid oxidation efficiency and metabolic flexibility in tissues associated with energy sensing and storage, and relative cellular hypertrophy in white adipose tissue. Hypothalamus, liver, Pectoralis major, gastrocnemius, abdominal fat, clavicular fat and subcutaneous fat were collected from juvenile (56-65 day-old) HWS and LWS chickens for metabolic, gene expression and histological assays. The HWS chickens had reduced fatty acid oxidation efficiency in abdominal fat (P < 0.0001) and reduced rates of oxidation in abdominal fat and gastrocnemius (P < 0.0001) as compared to LWS. There was reduced citrate synthase activity in white adipose tissue (P < 0.0001) and greater metabolic inflexibility in skeletal muscle (P = 0.006) of HWS compared to LWS. Greater pyruvate dehydrogenase kinase 4 (PDK4) and forkhead box O1 (FoxO1) mRNA were found in skeletal muscle and white adipose tissue of 56-day-old HWS than LWS. Expression of peroxisome proliferator-activated receptor γ (PPARγ) in all adipose tissue depots was greater (P < 0.05) in LWS than in HWS chickens. The HWS chickens had larger (P < 0.0001) and fewer (P < 0.0001) adipocytes per unit area than LWS. These results suggest that the HWS chickens have impaired metabolic flexibility and fatty acid oxidation efficiency due to an up-regulation of pyruvate dehydrogenase activity to accommodate the influx of acetyl CoA from fatty acid oxidation in skeletal muscle and white adipose tissue. These metabolic adaptations can be linked to differences in gene expression regulation and body composition between the lines. Adipocyte cellularity data are consistent with greater oxidative efficiency in the adipose tissue of LWS, because of the greater number of unfilled cells in all depots that were sampled. Results can be extrapolated to agricultural production in the understanding of factors regulating the amount of lipid deposition in chicken carcass fat. Results may also provide insight into eating disorders and the development of obesity. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / The goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.

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Nous avons étudié les mécanismes adaptatifs induits par une surnutrition hyperlipidique (+757 kcal/j) de 56 jours sur le métabolisme énergétique et le muscle squelettique chez des sujets sains. En parallèle, nous avons étudié les mécanismes adaptatifs induits par une surnutrition en fructose de 7 jours sur le métabolisme énergétique et le muscle squelettique chez des sujets apparentés diabétiques de type 2 et nous avons réalisé des études in vitro sur myotubes humains afin d'identifier le médiateur contribuant aux effets du fructose. Ces deux surnutritions contribuent à augmenter le poids corporel. Ces modifications sont accompagnées par l'oxydation préférentielle des glucides au détriment des lipides. La surnutrition hyperlipidique induit, au niveau musculaire, une diminution de l'expression de PDK4 qui pourrait être la conséquence de la diminution de la concentration en NAD+ associée à la baisse de l'activité de SIRT1 comme supportée par l'hyperacétylation de PGC1alpha. Bien que l'activation de la voie SIRT1/PGC1alpha semble réduite, la surnutrition hyperlipidique est associée à une augmentation de l'expression des gènes liés à la mitochondrie. La surnutrition riche en fructose induit quant à elle, au niveau musculaire, une baisse de l'expression de nombreux gènes liés à l'oxydation des lipides et à la mitochondrie comme CPT1 et MLYCD. Les études in vitro suggèrent que le fructose agit de manière indirecte sur le muscle squelettique. Ce travail de thèse met en lumière les conséquences d'une balance énergétique positive induite par la surconsommation de lipides ou de fructose sur le métabolisme énergétique et l'expression génique du muscle squelettique / We studied the effects of a high-fat overfeeding (+757 kcal/d) during 56 days on energy metabolism and skeletal muscle of healthy subjects. ln parallel, we studied the effects of high fructose overfeeding during 7 days on energy metabolism and skeletal muscle of first-degree relatives of type 2 diabetic patients and we performed in vitro studies with human myotubes to identify the mediator contributing to the fructose effects. High-fat and high-fructose overfeeding both contribute to increase body. These changes are associated with a preferential oxidation of carbohydrates instead of lipid. High-fat overfeeding induces in skeletal muscle, a decrease in PDK4 expression that could be the consequences of decreased NAD+ concentration associated with a decreased SlRT1 activity as supported by the hyperacetylation of PGC1alpha. Although this reduction of the SlRT1/PGC-1alpha pathway appears, the high-fat overfeeding is associated with increased mitochondrial gene expression. The high-fructose overfeeding induces in skeletal muscle a decrease in many genes expression related to lipid oxidation and mitochondria as CPT1 and MLYCD. ln vitro experiments suggest an indirect action of fructose in skeletal muscle. This thesis highlights the consequences of a positive energy balance induced by over- consumption of lipid or fructose, which we can find in the general population, on energy metabolism and skeletal muscle gene expression

Indiana University-Purdue University Indianapolis (IUPUI) / Regulation of the pyruvate dehydrogenase complex (PDC) is important for glucose homeostasis and control of fuel selection by tissues. Knocking out pyruvate dehydrogenase kinase 4 (PDK4), one of four kinases responsible for regulation of PDC activity, lowers blood glucose levels by limiting the supply of three carbon compounds for gluconeogenesis. Down regulation of PDK4 expression is also important for control of blood glucose by insulin. The primary goal was to determine whether PDK4 should be considered a target for the treatment of diabetes. A major concern is that inhibition of fatty acid oxidation by PDK4 deficiency may promote fat accumulation in tissues and worsen insulin sensitivity. This was examined by feeding wild type and PDK4 knockout mice a diet rich in saturated fat. Fasting blood glucose levels were lower, glucose tolerance was better, insulin sensitivity was greater, and liver fat was reduced in PDK4 knockout mice. The reduction in liver fat is contradictory to the finding that fibrate drugs increase PDK4 expression but ameliorate hepatic steatosis in rodents. To investigate this phenomenon, wild type and PDK4 knockout mice were fed the high saturated fat diet with and without clofibric acid. The beneficial effect of clofibric acid on hepatic steatosis was greater in the PDK4 knockout mice, indicating up regulation of PDK4 is not necessary and likely opposes the effect of clofibric acid on hepatic steatosis. Clofibric acid dramatically lowered the amount of hepatic CD36, a plasma membrane translocase required for fatty acid import, suggesting a novel mechanism for prevention of hepatic steatosis by fibrates. PDK4 deficiency had no effect on CD36 expression but reduced the enzymatic capacity for fatty acid synthesis, suggesting clofibric acid and PDK4 deficiency ameliorate hepatic steatosis by independent mechanisms. Investigation of the mechanism by which insulin regulates PDK4 expression revealed a novel binding site for hepatic nuclear factor 4α (HNF4α) in the PDK4 promoter. The stimulatory effect of HNF4α was sensitive to inhibition by Akt which is activated by insulin. The findings suggest PDK4 is a viable target for the treatment of hepatic steatosis and type 2 diabetes.