2009 Annual Report 1a.Objectives (from AD-416) 1. Characterize the role of newly identified metabolic regulators within the nuclear receptor superfamily, including the PPARs, LXRs, FXR, CAR and PXR, as targets of nutrients and other natural products that have direct regulatory effects on metabolic pathways. 2. Determine the cis- and trans-Paneth cell regulatory genes that contribute to the upregulation of the redundant soluble maltase-glucoamylase in the membrane maltase-glucoamylase KO mouse. 3. Test the hypothesis that long chain, unsaturated fatty acids (e.g. oleate) enhance fatty acid-responsive gene expression to a greater extent than shorter saturated fatty acids (e.g. palmitate). 4. Understand the direct effects of urea cycle intermediates both in sustaining ureagenesis in the presence of an enzymatic disorder and in maintaining nitric oxide production. 1b.Approach (from AD-416) 1. Identify nutritional products and other natural products that regulate the activity of nuclear hormone receptors, with a specific focus on PPAR (Peroxisome Proliferator Activated Receptor) isoforms, define the active agents that modulate receptor functions, and characterize the actions of such agents at the levels of receptor function and target genes. 2. There is a 6-fold increase in the soluble maltase-glucoamylase message in mice with membrane maltase-glucoamylase ablation. This increase is present in suckling as well as weaned null mice. This presents a novel model of gene regulation by dietary carbohydrates. The regulatory genes involved in this upregulation are under investigation by microarray analysis and will be confirmed and extended by mechanistic in vitro studies in the mICcl2 cell line. 3. Measure the effects of specific PPAR(alpha) and PPAR(beta/delta) agonists on metabolic gene expression in isolated cardiomyocytes; and by determining whether loss of PPAR(alpha) and PPAR(beta/delta) attenuates the effects of distinct fatty acid species on metabolic gene expression in the mouse model. 4. Investigate the genetic background on endogenous supply of ornithine and the presentation of urea cycles disorders. 3.Progress Report Limited progress was made due to a lack of resources. (Project. 1)It has been reported that a chaperone is needed for processing of human SI in cell cultures. We have expressed mouse Nt Mgam and Nt Si and discovered problems with processing in the baculovirus expression system; this was resolved with Si but persists with Mgam. This was not found with expression of human SI and MGAM in drosophila cells which leads to the hypothesis that a chaperone is required for Mgam expression in the baculovirus system but not in the drosophila system. In vitro activities of combined human Nt MGAM and Nt SI and mouse Ct Mgam and Ct Si; a synthesis of native mucosal activities. Studied the effect of Mgam ablation on starch digestion and prandial gluconeogenesis. This study measures the exogenous glucose arising from starch digestion and the endogenous glucose produced by the liver. The null mouse has a reduced glucose from digestion and increased glucose from liver. We studied the effects of weaning on starch digestion in null and WT mice. We have reported that one splicoforms of Mgam lacks the membrane binding domain of the normal peptide. (Project. 2)Scientists investigated the interrelationship between glucose signaling and the cardiomyocyte circadian clock. We found that glucose, through O-GlcNAcylation (reversible addition of a glucose moiety to a protein, analogous to phosphorylation) of critical clock proteins, influences the timing of the cardiomyocyte circadian clock. In turn, we also observed that the cardiomyocyte circadian clock regulates O-GlcNAcylation of myocardial proteins. (Project. 3)We have established the precursors used by the gut for the synthesis of citrulline during feeding and fasting. Citrulline in turn is the precursor for the endogenous synthesis of arginine. Demand for arginine increases during endotoxic challenge for the production of nitric oxide, and thus the contribution of endogenous arginine becomes crucial to sustain the nitric oxide response. We have developed and refined mass spectrometry analysis to study these metabolic processes in mouse models. (Project. 4)We have made progress in studying the mechanism of circadian control of food-intake and peripheral adiposity signals. We have also characterized the molecular pathways for the circadian control of bile acid homeostasis. (Project 5) 4.Accomplishments 1. Role of Circadian Clock in Preventing Obesity and Metabolic Syndromes: Children's Nutrition Research Center researchers studied the effect of the disruption of circadian rhythm on food-intake and body weight control using experimental mice that lack a circadian clock due to circadian gene mutation or constant jetlag. Preliminary results from our experiments have clearly shown that disruption of the circadian rhythm increases body weight in experimental mice. Additionally, we have studied whether restricted feeding affects the metabolic balance in experimental mice and found that restricted feeding disrupts the circadian balance of metabolic parameters, especially the control of bile acid synthesis and signaling, which leads to increased liver damage that mimics cholestatic disease. Our findings contribute to a better understanding of how disruption of circadian rhythms increases the risk of metabolic syndromes such as obesity and other associated diseases. (CNRC Project 5: Role of circadian clock in controlling food intake process during cancer treatment) 2. Influences on the Circadian Clock: Circadian clocks regulate energy metabolism. This has lead many investigators to suggest that impairment of circadian clocks plays a role in the fundamental beginnings of various metabolic diseases, such as obesity and diabetes mellitus. Children's Nutrition Research Center scientists investigated whether glucose influenced the timing of the circadian clock using the heart as a model system. Our research team has revealed a direct interrelationship between O-GlcNAcylation and the cardiac muscle circadian clock. We found that O-GlcNAcylation regulates the cardiac muscle circadian clock, and that the cardiac muscle circadian clock in turn regulates O-GlcNAcylation. As such, O-GlcNAcylation can be considered as a novel integral post-translational component of the mammalian circadian clock. Our studies reveal that the nutrient glucose has profound effects on the timing of the circadian clock. These observations may aid our understanding of how nutrition influences metabolism under physiological and pathological conditions. (CNRC Project 3: Nutrient regulation of cardiac gene expression during diabetes) 3. Importance of Sugar Digestion to Starch Digestion: Proper starch digestion is of importance for children for energy uptake. In order to determine the role of the sugar digesting protein, Children's Nutrition Research Center researchers studied starch digestion in children with mutant sucrase-isomaltase (a protein that participates in the digestion of starch) and sucrose malabsorption. In these children with a sugar digesting defect, we determined that 30% have very deficient starch digestion. This experiment confirms the significant role of sucrase-isomaltase in human starch digestion. In the future our research team will test enzyme supplements and botanical starch variants for improving the digestion of starches in these deficient children, which will be significant for health, nutrition, food, and agriculture sciences. (CNRC Project 2: Maltase-glucoamylase, regulator of starch digestion) 4. Impact of Insulin Deficiency on Starch Digestion to Glucose: Diabetes is a major disorder of glucose metabolism and has an increasing national prevalence. Conventional wisdom is focused on altered fasting glucose production by the liver. In order to determine if there are alterations in glucose digestion from fed starch, Children's Nutrition Research Center scientists developed a model of insulin deficiency in mice using an inhibitor of insulin production. These experiments will become validations for test-tube studies of starch digestion inhibitors. This will be significant for public health, nutrition, and clinical sciences. (CNRC Project 2: Maltase-glucoamylase, regulator of starch digestion) Review Publications Makhosazane, Z., Alcolea, M.P., Garcia-Palmer, F.J., Young, M.E., Essop, M.F. 2007. Genomic modulation of mitochondrial respiratory genes in the hypertrophied heart reflects adaptive changes in mitochondrial and contractile function. American Journal of Physiology - Heart and Circulatory Physiology. 293(5):H2819-H2825. Makhosazane, Z., Young, M.E., Stanley, W.C., Essop, M.F. 2008. Expression of mitochondrial regulatory genes parallels respiratory capacity and contractile function in a rat model of hypoxia-induced right ventricular hypertrophy. Molecular and Cellular Biochemistry. 318(1-2):175-181. Sim, L., Quezada-Calvillo, R., Sterchi, E.E., Nichols, B.L., Rose, D.R. 2008. Human intestinal maltase-glucoamylase: Crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity. Journal of Molecular Biology. 375:782-792. Quezada-Calvillo, R., Simm, L., Ao, Z., Hamaker, B.R., Quaroni, A., Brayer, G.D., Sterchi, E.E., Robayo-Torres, C.C., Rose, D.R., Nichols, B.L. 2008. Luminal starch substrate "Brake" on maltase-glucoamylase activity is located within the glucoamylase subunit. Journal of Nutrition. 138:685-692. Delgado, A.F., Okay, T.S., Leone, C., Nichols, B., Del Negro, G.M., Costa Vaz, F.A. 2008. Hospital malnutrition and inflammatory response in critically ill children and adolescents admitted to a tertiary intensive care unit. Clinics. 63:357-362. Khairallah, R.J., Khairallah, M., Gelinas, R., Bouchard, B., Young, M.E., Allen, B.G., Lopaschuk, G.D., Deschepper, C.F., Des Rosiers, C. 2008. Cyclic GMP signaling in cardiomyocytes modulates fatty acid trafficking and prevents triglyceride accumulation. Journal of Molecular and Cellular Cardiology. 45(2):230-239. Durgan, D.J., Young, M.E. 2008. Linking the cardiomyocyte circadian clock to myocardial metabolism. Cardiovascular Drugs and Therapy. 22(2):115-124. Rennison, J.H., McElfresh, T.A., Okere, I.C., Patel, H.V., Foster, A.B., Patel, K.K., Stoll, M.S., Minkler, P.E., Fujioka, H., Hoit, B.D., Young, M.E., Hoppel, C.L., Chandler, M.P. 2008. Enhanced acyl-CoA dehydrogenase activity is associated with improved mitochondrial and contractile function in heart failure. Cardiovascular Research. 79(2):331-340. Garcia, R.A., Afeche, S.C., Scialfa, J.H., Amaral, F.G., Santos, S.H., Lima, F.B., Young, M.E., Cipolla-Neto, J. 2008. Insulin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland. Life Sciences. 82(1-2):108-114. Robayo-Torres, C.C., Opekun, A.R., Quezada-Calvillo, R., Villa, X., Smith, E.0., Navarette, M., Baker, S.S., Nichols, B.L. 2008. 13C-breath tests for sucrose digestion in congenital sucrase isomaltase-deficient and sacrosidase-supplemented patients. Journal of Pediatric Gastroenterology and Nutrition. 48:412-418.