A general metabolic profile of macrophages was established by measurement of maximum catalytic activities of enzymes in energy-producing pathways and rates of utilisation of glucose, glutamine, fatty acids and ketone bodies under various conditions. It was found that glucose, glutamine and fatty acids can be used to satisfy the energy requirement of the cell. Although a significant proportion of utilised glutamine or fatty acid was converted to C0₂ by the macrophage, most glucose was not oxidised and was converted, almost stoichiometrically, to lactate. Utilised fatty acids were not only oxidised by the macrophage, but were incorporated into cellular lipid (mainly triacylglycerol and phospholipid). The triacylglycerol rich macrophage was shown to be able to release fatty acids into the culture medium. The importance of glutamine in macrophages was indicated from the high activity of phosphate-dependent glutaminase. Glutamine is probably metabolised by the following enzymes in macrophages: phosphate-dependent glutaminase, aspartate aminotransferase (or other amino acid aminotransferases), oxoglutarate dehydrogenase followed by enzymes of the TCA cycle and metabolism of oxaloacetate by phosphoenolpyruvate carboxykinase. Pyruvate derived via this pathway may be metabolised via pyruvate dehydrogenase or pyruvate carboxylase. A study of the sub-cellular distribution of some of these enzymes suggested that phosphate-dependent glutaminase has a cytosolic as well as a mitochondrial localisation. Further characterisation suggested that the non-mitochondrial activity could be associated with the plasma membrane. To the author's knowledge, this is the first report of a non-mitochondrial localisation for phosphate-dependent glutaminase. Glutaminase was shown to be activated by phosphate and inhibited by glutamate and 2-oxoglutarate. Significant inhibition of glutaminase occurred only at high concentrations of these compounds. Glucose and glutamine were utilised at very high rates by the macrophage, but were not fully oxidised even though the cells were incubated in aerobic conditions. The significance of these high rates of utilisation to the macrophage is discussed.

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Phytosterols play an important role in nature and as synthetic supplements for the modification of cholesterol metabolism in humans. The consensus is that the primary mechanism by which phytosterols modify lipid profiles is through altering the absorption of cholesterol. This thesis examined the effects of phytosterol mixtures on (i) de novo cholesterol synthesis rates and appearance of de novo cholesterol into the cholesterol ester pool through the use of deuterium incorporation, as well as, (ii) plasma lipid and sterol concentrations in normolipidemic humans. The initial investigation of this thesis compared the effects of diets based on either corn oil, olive oil, or olive oil plus phytosterol mixture on cholesterol metabolism. Each treatment was administered for 10 days in a cross-over design to 16 normolipidemic humans. In addition to confirming prior conclusions that corn oil was more effective than olive oil at decreasing plasma total and LDL-cholesterol concentration, this study was one of the first to determine that the differential effects on cholesterol metabolism observed in humans consuming corn versus olive oil is due, in part, to the higher concentrations of plant sterols naturally found in corn oil. The second study presented in this thesis investigated the differential effects on sterol metabolism of phytosterol mixtures either enriched with sitostanol or sitostanol-free administered for 10 days in 11 normolipidemic humans. The results of this study demonstrated that only the phytosterol mixture containing low doses of sitostanol decreased plasma total and LDL-cholesterol concentrations and increased the circulating HDL/LDL ratios; thus, the sitostanol-enriched phytosterol mixture was a more effective cholesterol-lowering agent.

La synthese endogene du cholesterol (CH) a ete etudie chez 81 nouveau-nes, ages de 4 mois (BRAS 1) ou de 11 a 12 mois (BRAS2), afin d'evaluer les effets a long terme d'un supplement de CH sur l'homeostasie du CH. BRAS 1 etait compose de 32 nouveau-nes recevant soit du lait humain (HUM) (6M, 7F), une formule a base de lait de vache (VAC) (6M, 3F) contenant 3.5 mg CH/dl, ou une formule a base de lait de vache modifiee (VACM) (6M, 7F) contenant 13.5 mg CH/dl, afin d'evaluer l'effet du supplement sur les taux de synthese du CH. BRAS2 etait compose de 49 autres enfants recevant soit HUM (11M, 6F), VAC (7M, 12F) ou VACM (6M, 7F) jusqu'a l'age de 6 mois dans le but d'evaluer une hypothese d'impression genetique. Ceci a ete realise en utilisant un design "cross-over" et en montant un defi journalier de 250 g de CH a l'age de 11 mois. Le taux d'incorporation de deuterium, provenant des reserves d'eau corporelle, dans la structure du CH a servi comme indice du taux de synthese fractionnel (TSF) de ce dernier sur une periode de 48 heures. Les niveaux de CH total et LDL etaient considerablement eleves dans HUM en comparaison avec VAC et VACM a l'age de 4 mois. La concentration sanguine du CH etait semblable a 11 et 12 mois. Le TSF etait 4 fois plus eleve dans VAC et VACM relatif a HUM, mais il n'y avait pas de difference entre VAC et VACM a 11 et 12 mois. Cependant, les TSF de 4 a 12 mois ont augmente dans HUM et baisse dans VAC et VACM. Nos resultats indiquent qu'independamment du contenu des dietes, le defi journalier de CH n'as pas eu d'effet considerable ni sur les taux de synthese, ni sur les niveaux de CH sanguin. Ces resultats appuient l'idee que le CH alimentaire n'a que des effets minimes sur le metabolisme a long terme du CH.

Nutrient sensing pathways in both the brain and gut decrease hepatic glucose production. Hypothalamic activation of lactate metabolism decreases glucose production, but it is unknown whether the hypothalamus detects circulating lactate to maintain glucose homeostasis. In the gut, lipids decrease glucose production via a neuronal network but the downstream signaling mechanisms are unknown. We tested whether circulating lactate activates central lactate metabolism to decrease glucose production and postulated that duodenal protein kinase C (PKC) acts downstream of lipids to decrease glucose production through a neuronal network. We report that central lactate metabolism is required for the maintenance of glucose homeostasis in the presence of circulating lactate and that activation of duodenal PKC is required for lipids to decrease glucose production. This shows the importance of the brain and gut in the regulation of glucose production, and could pave the way for restoration of glucose homeostasis in disease.

Over the past decade, the constraint-based approach to metabolic modeling has become an important tool for understanding and controlling biology. Unfortunately, the application of this novel approach to systems biology in biotechnology has been limited by three significant technical issues: existing metabolic modeling methods cannot completely model the overflow metabolism, cannot model the metabolism of microbial communities, and cannot design strains optimized for productivity and titer. Three computational methods – the Flux Balance Analysis with Membrane Economics (FBAME) method, the Dynamic Multi-species Metabolic Modeling (DyMMM) framework, and the Dynamic Strain Scanning Optimization (DySScO) strategy – have been developed to resolve these issues respectively. First, the FBAME method, which adopts the membrane occupancy limitation hypothesis, was used to explain and predict the phenomenon of overflow metabolism, an important metabolic phenomenon in industrial fermentation, in Escherichia coli. Then, the DyMMM framework was used to investigate the community metabolism during uranium bioremediation, and demonstrated that the simultaneous addition of acetate and Fe(III) may be a theoretically viable uranium bioremediation strategy. Lastly, the DySScO strategy, which combines the DyMMM framework with existing strain design algorithms, was used to design commodity-chemical producing E. coli optimized for a balanced product yield, titer, and volumetric productivity. These novel computational methods allow for broader applications of constraint-based metabolic models in biotechnology settings.

Nutrient sensing pathways in both the brain and gut decrease hepatic glucose production. Hypothalamic activation of lactate metabolism decreases glucose production, but it is unknown whether the hypothalamus detects circulating lactate to maintain glucose homeostasis. In the gut, lipids decrease glucose production via a neuronal network but the downstream signaling mechanisms are unknown. We tested whether circulating lactate activates central lactate metabolism to decrease glucose production and postulated that duodenal protein kinase C (PKC) acts downstream of lipids to decrease glucose production through a neuronal network. We report that central lactate metabolism is required for the maintenance of glucose homeostasis in the presence of circulating lactate and that activation of duodenal PKC is required for lipids to decrease glucose production. This shows the importance of the brain and gut in the regulation of glucose production, and could pave the way for restoration of glucose homeostasis in disease.