2008 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 Natural products & nuclear receptors: PPARs. We tested the role of the orphan receptor SHP in the effects of a grape seed procyanidin extract (GSPE) on plasma levels of triglycerides (TG) & apolipoprotein B (ApoB). The TG-lowering effects of GSPE were decreased by inhibiting SHP expression in HepG2 via siRNA & in a SHP-null mouse (Project 1). Maltase-glucoamylase, regulator of starch digestion. This year we differentiated 4 separate activities of the mucosal glucosidases that convert the glucose polymers of starch and amylase treated glucose oligomers to free glucose for digestion. We published the structure of the first such domain, the human C-terminal, & have been working to produce recombinant forms of the other 3 domains. In our Mgam null mouse model we demonstrated that the four mucosal activities are not redundant in vivo & that Mgam contains the most activity for normal amylase treated starch digestion to glucose (Project 2). Nutrient regulation of cardiac gene expression during diabetes. Our studies regarding the regulation of gene expression in adult rat cardiomyocytes by distinct fatty acid species were completed, resulting in publication of the findings. Our focus then changed toward understanding the role that the circadian clock within skeletal myocytes plays in regulating the responsiveness of skeletal muscle to fatty acids. These studies have begun with the initial characterization of a novel mouse model that we generated, in which the circadian clock is specifically impaired within skeletal myocytes (Project 3). Requirement & interorgan transactions of urea cycle intermediates. We have generated preliminary data on different approaches to increase citrulline synthesis in control & mutant spfash (which have a deficiency that reduces the production of citrulline) mice. We are breeding the novel ASL conditional ko mouse model & have conducted infusions in 6 ASL F/F control & 2 F/F Lys Cre mice under basal & LPS challenged conditions. We are testing different liquid diets to sustain mice on intragastric infusion of nutrients. The volume limitations together with the high metabolic rate, and thus the high requirement for nutrients, has made this process challenging. We have identified a maltodextrin-hydrolized casein diet that is highly soluble & offers the potential to sustain mice at low rates of volume delivery (Project 4). Role of circadian clock in controlling food intake. We studied food-intake and body weight phenotypes of several circadian gene-mutant mouse models and found that deregulation of food-intake and energy storage contributes to the phenotypes of abnormal weight gain. We also started to investigate the molecular mechanisms underlying the increased body weight phenotype of these mice & found that the loss of circadian control of leptin expression may play a key role leading to increased body weight in mice (Project 5).[NP107, Component 2 & 6] 4.Accomplishments 1. Understanding the Role of Leptin: Children's Nutrition Research Center researchers investigated if and how the circadian clock controls the balance of food-intake. Our lab analyzed the body weight and food-intake of several circadian gene-mutant mouse models, and studied the plasma level of leptin as well as the expression of leptin mRNA and protein in adipose tissues in wild-type and circadian gene-mutant mice. We found that the expression of leptin gene in adipose tissues is controlled by an endogenous mechanism but not by food cues. This finding could provide the first evidence showing that circadian rhythm of plasma leptin is generated by the circadian clock directly in vivo, but not as it is generally regarded as a response to the circadian variation of food cues during a 24-hr period. Given the significant role leptin plays in energy balance, our finding may have a significant impact on the future of obesity prevention and therapy. [NP107, Component 6] (CNRC Project 5) 2. Regulation of Gene Expression in Cardiomyocytes: Fatty acids of varying degrees of saturation and/or carbon chain length differentially influence myocardial function. Therefore researchers have attempted to identify novel transcriptional mechanisms by which distinct fatty acids species potentially mediate cardiotoxic (heart damage) versus cardioprotective (heart protecting) influences. Children's Nutrition Research Center researchers discovered that cardioprotective long chain monounsaturated fatty acids (e.g., oleate) induce expression of genes promoting fatty acid oxidation to a greater extent than cardiotoxic long chain saturated fatty acids (e.g., palmitate). In contrast, cardiotoxic fatty acids markedly induce genes associated with endoplasmic reticulum and oxidative stress, as well as apoptosis (cell death). This was accomplished through microarray and RT-PCR studies, following time- and concentration-dependent challenge of isolated adult rat cardiomyocytes (heart cells) with distinct fatty acid species. These studies provide novel potential mechanisms for cardiotoxicity/cardioprotection. [NP107, Component 6] (CNRC Project 3) 3. Characterization of Skeletal Muscle Specific Circadian Clock Mutant Mice: Understanding the molecular mechanisms by which skeletal muscle responds to fatty acids is important since fatty acid-induced insulin resistance is a founding cause of type 2 diabetes mellitus. Children's Nutrition Research Center researchers have investigated the contribution of the intramyocellular circadian clock on skeletal muscle metabolism and insulin sensitivity. This research has been accomplished through characterization of a novel mouse model in which the circadian clock is specifically impaired within skeletal myocytes (muscle cells). Transgenic mice were generated in which a dominant negative CLOCK (Circadian Locomoter Output Cycles Kaput) mutant protein is specifically expressed within skeletal muscle cells, through the use of a modified creatine kinase promoter. Preliminary studies show that the disruption of the circadian clock within skeletal myocytes alters skeletal muscle gene expression and metabolism. These studies are the first to directly characterize the role of the circadian clock within skeletal muscle cells and this research may ultimately identify a novel mechanism regulating skeletal muscle metabolism and insulin sensitivity. [NP107 – Component 6 Prevention of Obesity and Disease] (CNRC Project 3) 4. Understanding Citrulline Precursors: Children's Nutrition Research Center researchers have determined the contribution of different endogenous precursors for citrulline, a non-standard amino acid that is not normally present in protein, to citrulline and arginine production. Our data show that plasma ornithine and proline are the main precursors, while plasma glutamate is a minor player in the synthesis of citrulline. The determination of the correct precursor(s) for citrulline synthesis might be useful in devising strategies to increase citrulline production, endogenous arginine synthesis, and nitric oxide status. This is relevant because in some physiological (pregnancy) or pathophysiological (sepsis, urea cycle disorders) citrulline production is reduced, and arginine and nitric oxide production might be compromised. [NP107, Component 4] (CNRC Project 4) 5. Roles of "Resistant" and "Slowly Digested" Starch: The roles of "resistant" and "slowly digested" starch in human health are still unclear. Children's Nutrition Research Center researchers successfully demonstrated that endogenous and exogenous glucose are co-regulated together in the normal mouse and that normal starch digestion is necessary for insulin response to feeding. This investigation in null mice addresses the benefits of reduced food starch digestibility since the null mouse has a 40% reduction in digestion of starch to glucose in the small intestine, which is due to the loss of Mgam activity with maintenance of the 10x slower sucrase-isomaltase activity. This is important since the Mgam null mouse allows the mechanistic study of mucosal glucogenic enzymes with and without Mgam. This research is important because starches are the chief contributors of food glucose and play a role in the disorders of glucose metabolism, including diabetes and obesity. More details of the mechanisms of mucosal starch digestion are needed before researchers can understand the resistance of some food starches to glucose production and their modulating effects on insulin. [NP107, Component 2] (CNRC Project 2) 6. The Role of the Nuclear Receptor SHP in Hypolipidemic Effects of Grape Seed Extract: High blood levels of triglycerides, referred to as hypertriglyceridemia, is an independent risk factor in the development of cardiovascular diseases, and it has previously been reported that ingesting of a grape seed procyanidin extract decreases the plasma levels of triglycerides and apolipoprotein B (protein that is involved in the formation of bad cholesterol or LDL) in normal rats. Children's Nutrition Research Center researchers examined whether the nuclear receptor small heterodimer partner is required for the reduction of triglyceride-rich apolipoprotein Bcontaining lipoproteins triggered by grape seed procyanidin extract. Grape seed procyanidin extract inhibited triglycerides and apolipoprotein B secretion in human cancerous liver cells and had a hypotriglyceridemic effect in wild-type mouse. The triglycerides -lowering action of grape seed procyanidin extract was abolished in HepG2 cells transfected with a small heterodimer partner-specific siRNA and in a small heterodimer partner-null mouse. Moreover, in mouse liver, GSPE downregulated several lipogenic genes, including steroid response element binding protein 1c (SREBP-1c), and upregulated carnitine palmitoyltransferase-1A (CPT-1A) and apolipoprotein A5 (ApoA5), in a small heterodimer partner-dependent manner. In HepG2 cells GSPE also inhibited apolipoprotein B secretion, but in a SHP-independent manner. Our lab concluded that small heterodimer partner is a key mediator of the hypotriglyceridemic response triggered by grape seed procyanidin extract. This novel signaling pathway of procyanidins through small heterodimer partner may be relevant for the health effects ascribed to the regular consumption of dietary flavonoids. [NP107 – Component 6 Prevention of Obesity and Disease] (CNRC Project 1) 6.Technology Transfer None Review Publications Durgan, D.J., Moore, M.W.S., Ha, N.P., Egbejimi, O., Fields, A., Mbawuike, U., Egbejimi, A., Shaw, C.A., Bray, M.S., Nannegari, V., Hickson-Bick, D.L., Heird, W.C., Dyck, J.R.B., Chandler, M.P., Young, M.E. 2007. Circadian rhythms in myocardial metabolism and contractile function; influence of workload and oleate. American Journal of Physiology - Heart and Circulatory Physiology. 293(4):H2385-H2393. Razeghi, P., Baskin, K.K., Sharma, S., Young, M.E., Stepkowski, S., Essop, M.F., Taegtmeyer, H. 2006. Atrophy, hypertrophy, and hypoxemia induce transcriptional regulators of the ubiquitin proteasome system in the rat heart. Biochemical and Biophysical Research Communications. 342(2):361-364. Buroker, N.E., Young, M.E., Wei, C., Serikawa, K., Ge, M., Ning, X., Portman, M.A. 2007. The dominant negative thyroid hormone receptor beta-mutant delta337T alters PPAR-alpha signaling in heart. American Journal of Physiology - Endocrinology and Metabolism. 292:E453-E460. Tong, Q., Hotamisligil, G.S. 2007. Developmental biology: Cell fate in the mammary gland. Nature. 445(7129):724-726. Ao, Z., Quezada-Calvillo, R., Sim, L., Nichols, B.L., Rose, D.R., Sterchi, E.E., Hamaker, B.R. 2007. Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant). FEBS Letters. 581:2381-2388. Quezada-Calvillo, R., Robayo-Torres, C.C., Opekun, A.R., Sen, P., Ao, Z., Hamaker, B.R., Quaroni, A., Brayer, G.D., Wattler, S., Nehls, M.C., Sterchi, E.E., Nichols, B.L. 2007. Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal starch alpha-glucogenesis. Journal of Nutrition. 137(7):1725-1733. Fu, L. 2007. Understanding the mechanism of circadian modulation to improve the quality of life for cancer patients. Journal of Supportive Oncology. 5(4):176-177. Marini, J.C., Fox, D.G., Murphy, M.R. 2008. Nitrogen transactions along the gastrointestinal tract of cattle: A meta-analytical approach. Journal of Animal Science. 86(3):660-679. Marini, J.C., Erez, A., Castillo, L., Lee, B. 2007. Interaction between murine spf-ash mutation and genetic background yields different metabolic phenotypes. American Journal of Physiology - Endocrinology and Metabolism. 293(6):E1764-E1771.