Browsing by Subject "Lipid metabolism"
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Item Glycerol-3-phosphate acyltransferase regulates T cell effector function and metabolism(2013-08) Faris, Robert Allen, Jr.; Jolly, Christopher A.The aged T cell is characterized by decreased responsiveness to stimulation. Aging is associated with reduced membrane glycerophospholipid (GPL) to cholesterol ratios so it is interesting that deletion of mitochondrial glycerol-3-phosphate acyltransferase-1 which catalyzes the first step in de novo GPL synthesis induces an aged T cell phenotype in otherwise healthy mice. GPAT-1 could regulate T cell function through three possible mechanisms: maintenance of membrane GPL ratios and membrane based signaling, providing a specific substrate for downstream signaling, or direct regulation of cellular metabolism. Therefore, the goal of this project was to determine whether these mechanisms contribute to the dysfunctional T cell phenotype observed with decreased GPAT-1 activity. T cell stimulation requires significant upregulation of metabolic processes to drive clonal expansion and cytokine production. T cell dysfunction in GPAT-1 knockout mice may be partially explained by altered metabolic function. We found that GPAT-1 KO T cells have significantly reduced basal respiration rates and spare respiratory capacity which is not compensated for by increased glycolytic metabolism suggesting an inherent metabolic defect in GPAT-1 KO T cells. To better understand mechanistically how GPAT-1 regulates T cell function we moved into the Jurkat T cell line and found that shRNA mediated knockdown of the human isoform of GPAT-1 (GPAM) recapitulated key aspects of the dysfunctional T cell phenotype we observed in the mouse including highly significant reductions in IL-2 production and altered membrane GPL to cholesterol ratios. Phosphatidic acid addition was not capable of rescuing these deficiencies suggesting that GPAT-1/GPAM activity is required for proper T cell function. This was the first time that GPAT-1 activity has been shown to be important for T cell function in a non-murine model system and strongly suggests that GPAT-1/GPAM deficiency regulates T cell function at the cellular level. We further demonstrate that phosphorylation of ZAP-70 a proximal effector of T cell activation is significantly reduced in GPAM knock down Jurkat T cells, suggesting that membrane based signaling is dysfunctional. Taken together these data suggest that GPAT-1 is necessary for regulating cellular energy demands in T cells and essential for optimal T cell activation following stimulation.Item Lipid Metabolism, Gene Expression, Substrate Oxidation, and Meat Quality of Growing-finishing Pigs Supplemented with Conjugated Linoleic Acid and Arginine(2012-02-14) Go, Gwang-Woong, 1979-We hypothesized that supplementation of dietary conjugated linoleic acid (CLA) and arginine singly or in combination would increase animal performance and meat quality by decreasing adiposity and increasing lean mass in growing-finishing pigs. Sixteen pigs (80 kg) were assigned to four treatments in a 2 x 2 factorial design, differing in dietary fatty acid and amino acid composition [control: 2.05% alanine (isonitrogenous control) plus 1% canola oil (lipid control); CLA: 2.05% alanine + 1% CLA; arginine: 1% arginine + 1% canola oil; arginine + CLA: 1% arginine + 1 CLA]. Preliminary tests indicated that up to 2% arginine was acceptable without interfering with lysine absorption. Pigs were allowed to feed free choice until reaching 110 kg. There were no significant differences across treatments in feed intake, weight gain, or feed efficiency. CLA tended to decrease carcass length (P = 0.06), whereas backfat thickness tended to be greater in pigs supplemented with arginine (P = 0.08). Arginine decreased muscle pH at 45 min postmortem (P = 0.001) and tended to increase lightness of muscle at 24 h postmortem (P = 0.07). CLA supplementation increased the concentrations of trans-isomers of 18:1 (P = 0.001) and SFA (P = 0.01) in s.c. and r.p. adipose tissue. CLA supplementation increased palmitate incorporation into total lipids in longissimus muscle (P = 0.01). Glucose oxidation to CO? in r.p. and s.c. adipose tissue were greater in pigs supplemented with CLA in the absence or presence of arginine (P = 0.03 and P = 0.04, respectively). The volume of s.c. adipocytes in s.c. and r.p. adipose tissues was greater in pigs supplemented with CLA, arginine, or CLA plus arginine than in control pigs (P = 0.001). Neither CLA nor arginine affected the expression of PGC-1[alpha],AMPK, mTOR, CPT-1A, FAS, or SCD (P > 0.05) in any tissues. We conclude that there was no significant interaction between arginine and CLA. Supplementary CLA or arginine to finishing-growing pigs did not modulate growth performance and did not reduce adiposity. CLA increased intramuscular fat content without deteriorating meat quality traits and increased saturated fatty acids and substrate oxidation in adipose tissues. In the presence of 1% of canola oil or CLA in the diet, arginine has the potential to deteriorate meat quality by reducing early postmortem pH and by increasing carcass fatness.Item Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis(2013-12-09) Liu, ZhenMycobacterium tuberculosis (M. tuberculosis) contains a wide array of genes responsible for the synthesis and secretion of a variety of bioactive lipids. The genes represent attractive drug-targets due to their involvement in essential cell cycles, the implication in pathogenesis, and the interference with therapeutics. In this thesis, I report our efforts to understand the biological functions of, and to develop inhibitors against, multiple genes related to M. tuberculosis lipid metabolism. Firstly, dioctylamine, a substrate mimic of the mycolic acid cyclopropane synthases, is shown to inhibit CmaA2 in vitro. Its inhibition action is explained by the structural characterization. Together with our collaborators, we have found dioctylamine able to intervene multiple mycolic acid cyclopropane synthases in vivo, and hence established the first model study for the single-drug-multiple-target strategy to inhibit the mycolic acid biosynthesis of M. tuberculosis. In addition, dioctylamine can serve as the platform for the design of more potent and selective drugs in the future. Secondly, the action mechanism of isoniazid and ethionamide, both of which are pro-drugs targeting the mycolic acid biosynthesis, is explored via biochemical, X-ray crystallographic or modeling studies. We have determined that the intracellular target of isoniazid is the enoyl reductase InhA; and we have discovered the correlation between mycothiol and ethionamide susceptibility. Thirdly, I have investigated the function and mechanism of FadD10, an enzyme involved in the synthesis of a virulence-related lipopeptide. The results reveal that FadD10 was mis-annotated as a fatty acyl-CoA ligase, but it indeed transfers fatty acids to an acyl carrier protein (Rv0100). Further crystallographic characterization provides the molecular basis for the mechanism of FadD10, leading to the discovery of a new type of adenylate-forming enzyme.