2006 Annual Report
Project 1: Natural products and nuclear receptors: PPARs Metabolic disorders such as obesity and diabetes have reached epidemic proportions in the United States, and a priority objective of the ARS Human Nutrition National Plan (NP107) is to characterize the mechanism of action of nutrients and other agents with potentially beneficial effects on such disorders.
Important metabolic regulatory functions have been identified for several new members of the nuclear hormone receptor superfamily, including the PPARs, LXRs, FXR, CAR and PXR. All of these receptors are relatively promiscuous, with each recognizing both endogenous and exogenous ligands that often share little or no structural similarity. Thus, we hypothesize that these receptors are potential targets and mediators of the beneficial effects of nutrients and other natural products. Two broad objectives are proposed to test this hypothesis. The first is to identify nutritional products and other natural products that regulate the activity of specific nuclear hormone receptors, and define the active agents that modulate receptor functions. The second is to characterize the effects of such novel receptor ligands at the levels of receptor function and regulation of expression of target genes in appropriate tissues. Successful completion of these objectives will lead to the identification of new therapeutic approaches to metabolic disorders.
Project 2: Maltase-glucoamylase, regulator of starch digestion Metabolic disorders such as obesity and diabetes have reached epidemic proportions in the United States, and a primary objective of the ARS Human Nutrition National Plan (NP107) is to characterize the mechanism of action of nutrients and other agents with potentially beneficial effects on such disorders. Genes ultimately regulate the digestion and absorption processes. Additional research will focus on the interactions of nutrients and genes in these processes, especially the regulation of genes as they contribute to the development of diet-associated degenerative diseases, such as diabetes and atherosclerosis. Our research objective is to determine the mechanisms by which dietary starch interacts with the gene expressing maltase-glucoamylase (Mgam in mice). Mgam is the gate-keeping enzyme that determines mammalian small intestinal starch digestion into glucose. The function and regulation of maltase-glucoamylase is under investigation in those human subjects that are deficient in starch digestion and in mice with congenital Mgam deficiencies.
New discoveries in our lab have shown that both Mgam is differentially spliced in a variable region between exons 22-44. These spliced forms appear to alter substrate binding regions but not catalytic amino acids. This non-allelic variability exists in the gene as paralogous duplications. The messages are differentially spliced from these paralogous regions. We are now preparing expression constructs of these spliced messages to determine the function of the variable regions. We have also begun mouse feeding studies that test whether types of food starches can alter the splicing of Mgam and to identify the foods and other natural products that regulate splicing. The function of the spliced protein domains in the Mgam will be investigated in expression systems. Successful completion of these objectives will lead to the identification of new therapeutic approaches to metabolic disorders.
Project 3: Nutrient Regulation of Cardiac Gene Expression during Diabetes Diabetes and cardiovascular disease are the primary causes of death in the United States. Diabetes has been described as "starvation in the midst of plenty". As such, understanding the molecular mechanisms responsible for inappropriate handling of excess nutrients (i.e., in excess of energetic requirements) will undoubtedly lead to future targets for the treatment and/or prevention of diabetes co-morbidities, such as cardiovascular disease.
Our research interests include the effects of nutrients (primarily fatty acids and glucose) on myocardial gene expression, and how alterations in these nutrients during disease states might contribute to the development of contractile dysfunction. Diabetes mellitus is a major risk factor for the development of cardiovascular disease. Both type I and type II diabetes are characterized by hyperlipidemia and hyperglycemia. The heart adapts to the diabetic environment, thereby allowing maintenance of cardiac function, and one such adaptation is increased fatty acid oxidation in the face of increased fatty acid availability. The heart is exposed to fatty acid species that vary in chain length and degree of saturation, and it is becoming increasingly clear that different fatty acid species can have distinct physiological effects, including stimulation of insulin secretion, induction of arrhythmias and apoptosis (programmed cell death), as well as modulation of gene expression. Fatty acids influence gene expression through a number of differing mechanisms, including activation of the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs). Of these family members, PPAR(alpha) and PPAR(Beta/Delta) are highly expressed within the cardiomyocytes of the heart. Studies in various cell lines suggest that long chain unsaturated fatty acids are better ligands for PPARs, in comparison to shorter, saturated fatty acids. Despite these observations, no previously published studies have characterized the effects of distinct fatty acid species on metabolic gene expression in adult cardiomyocytes. Our researchers intend to 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). If true, then these observations may provide insight as to why oleate is cardioprotective.
In the diabetic environment, despite decreased insulin sensitivity, cardiomyocytes exhibit comparable glucose uptake rates to those observed in the normal environment, due to the combined effects of hyperglycemia and hyperinsulinemia. However, increased fatty acid utilization in the diabetic heart results in inhibition of oxidative glucose metabolism. The combination of normal glucose uptake into the cardiomyocyte, concomitant to the uncoupling of glycolysis and pyruvate oxidation, results in an accumulation of glucose metabolites within the cardiomyocyte. Indeed, elevated intracellular levels of numerous glycolytic intermediates have been observed in the diabetic heart. The latter glucose metabolites may activate glucose responsive transcription factors. A comprehensive understanding of the direct effects of fatty acids and glucose on the adult heart is grossly deficient. We intend to investigate whether distinct fatty acid species differentially influence metabolic gene expression. Therefore, we intend to investigate whether glucose attenuates fatty acid-induced changes in gene expression.
Year 2 (2006) Characterize effects of soy and other compounds on other PPAR isoforms and other nuclear receptors. (milestone moved from year 3)
Year 3 (2007) Define role of soy and other compounds as nuclear receptor ligands at the levels of target genes and appropriate mouse knockouts.
Year 4 (2008) Define role of nuclear receptors in mediating effects of additional potential metabolic regulators. (milestone moved from year 5)
Year 5 (2009) Characterize role of nuclear receptors in metabolic regulatory effects at the levels of target genes using gene arrays and appropriate mouse knockouts.
Project 2: Maltase-glucoamylase, regulator of starch digestion Year 1 (2005) Explore starch partitioning and dietary composition, generate the Mgamso construct and define the effect of weaning on transcription factor binding to the Mgam promoter. Conduct a food starch feeding trial.
Year 2 (2006) Define the phyenotype of deficiency in Mgamso KO mice. Determine the fate of fed 13C-starch in the Mgamme KO, and define the effect of diet on transcription factor binding to the Mgam promoter.
Year 3 (2007) Finalize phenotypic analysis of the Mgamso KO mice and characterize the effect of dietary glucose on transcription factor binding to the Mgam promoter.
Year 4 (2008) Determine the fate of dietary 13C-starch in the Mgamso KO and characterize the effect of microflora on transcription factor binding to the Mgam promoter.
Year 5 (2009) Test the effect of a Mgam enzyme inhibitor used in management of type II diabetes, acarbose, on phenotype and on wild-type Mgamme/Mgamso message levels.
Project 3: Nutrient Regulation of Cardiac Gene Expression during Diabetes Year 1 (2006) Characterization of effects of distinct fatty acid species on metabolic gene expression in adult cardiomyocytes.
Year 2 (2007) Determine the role of PPAR as a mediator of the differential effects of distinct fatty acid species on metabolic gene expression in adult cardiomyocytes.
Year 3 (2008) Determine the role of PPAR as a mediator of the differential effects of distinct fatty acid species on metabolic gene expression in adult cardiomyocytes.
Year 4 (2009) Determine the role of Sp1 as a mediator of the effects of glucose on metabolic gene expression in adult cardiomyocytes.
Project 2: Maltase-glucoamylase, regulator of starch digestion Dominant role of Mgam in mouse starch digestion to glucose The digestion of starch to become glucose requires six different enzymatic activities (salivary and pancreatic endo-alpha-glucosidases and 4 mucosal exo-alpha-glucosidases) and such processes have clouded researcher's understanding of starch digestion. Using recombinant pancreatic alpha-amylase, scientists at the Children's Nutrition Research Center, Houston, TX, have shown that only about 10% of all glucose production from starch is due to a luminal enzyme. The remaining 90% of alpha-glucosidase activity was contributed by the two mucosal peptides, Mgam and sucrase-isomaltase (Si). Our studies have shown that Mgam is about 100 times more active than Si in glucose production. Activity of the 4 mucosal exo-alpha-glucosidases are amplified 2-4 times by alpha-amylase pre-digestion of starch. Such results are different from the traditional tests of starch resistance to digestibility using fungal glucoamylase and these studies may revolutionize the classifications of "resistant starches". (ARS Human Nutrition National Plan (NP107) Component 2)
Confirmation of the dominant role of human MGAM in starch digestion to glucose Using specific antibodies to human mucosal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), the observations in the mouse models have been confirmed and extended by Immunoprecipitation of activities from human jejunum (small intestine). MGAM is about 100 times more active than SI in alpha-glucogenesis. Activity of the 4 mucosal alpha-glucosidases are amplified 2-4 fold by human exo-alpha-amylase predigestion of starch. The products of alpha-amylase predigestion of starch (alpha-limit dextrins: LDx) inhibit MGAM but not SI. The presence of low concentrations of LDx slows total alpha-glucogenesis 50 fold by suppressing MGAM activity. The "LDx brake" may be a physiologic barrier to glucose overloading during starch digestion. (ARS Human Nutrition National Plan (NP107) Component 2)
Project 1: Natural products and nuclear receptors: PPARs The potential health benefits of soy isoflavones have been widely publicized, but the molecular basis for such effects are unclear. Researchers hypothesized that, in addition to their modulation of estrogen receptor activity, they would modulate the activity of other nuclear receptors. This hypothesis has been shown to be correct, by our lab as well as others. CNRC scientists have found that both genistein and daidzein activate the anti-diabetic target receptor PPARgamma. We have also found that the isoflavone metabolite equol activates both PPARs and the xenobiotic receptor PXR. These results directly link isoflavones to metabolic regulatory pathways and provide a basis for specific tests of their biological effects and the mechanisms that underlie them. The customers include the very large number of people with metabolic disorders, including syndrome X, as well as the also large number of women who supplement their diet with soy products to ameliorate menopausal symptoms.
Project 2: Maltase-glucoamylase, regulator of starch digestion While the digestion of sucrose and lactose is accomplished by a single mucosal enzyme activity, the enzymatic digestion of starch is highly variable as a response to the diverse and complex nature of food starches. The future design of food starches for meeting health objectives will need to recognize the spectrum of mechanisms of food starch digestion. We have determined that paralogous regions exist in the human MGAM and mouse Mgam genes and that variable regions between exons 22-44 exist at the message level. We are currently testing the activity of the recombinant spliced mouse proteins. The general hypothesis is that the "committee" of six alpha-glucosidases and alternative splicing of MGA and Mgam are mechanisms of adaptation to the range of naturally occurring and modified food starches. National starch entities have expressed interest in our assays for starch digestion which use recombinant human alpha-glucogenic enzymes. Other groups have also made inquiries.
Project 3: Nutrient Regulation of Cardiac Gene Expression during Diabetes Diabetes mellitus increases individual's risk for cardiovascular disease, and exposes the heart to high plasma fatty acid levels, which induce genes promoting fatty acids, as well as those suppressing carbohydrate. Little is known regarding the time course and magnitude of the effects of distinct fatty acid species on the expression of metabolic genes in the cardiomyocytes (muscular tissue cells) of the heart. As such, the time and concentration dependent effects of octanoate, palmitate, stearate, oleate, and linoleate on metabolic gene expression were investigated by Children's Nutrition Research Center researchers in isolated adult rat cardiomyocytes. Five known fatty acid responsive genes were studied and the order of responsiveness of cardiomyocytes to the fatty acids was investigated. In terms of initial rates of induction of fatty acid-responsive genes, oleate>stearate>palmitate>=linoleate>octanoate. The effects of stearate and palmitate tended to be transient, while oleate and linoleate caused relatively sustained induction of metabolic genes. Gene-specific effects were also observed; palmitate exhibited relatively small effects, as compared to the other genes investigated. These findings may explain why diets high in unsaturated fatty acids are cardioprotective, while diets rich in saturated fatty acids are not.
We have recently characterized the effects of the long chain monounsaturated fatty acid oleate on the expression of long chain acyl-CoA synthetase family members in adult rat cardiomyocytes. These enzymes likely channel fatty acids into beneficial versus detrimental pathways.
Project 2: Maltase-glucoamylase, regulator of starch digestion The in vitro assay of starch digestibility using human recombinant enzymes has been transferred to the Whistler Center for Carbohydrate Research at Purdue University where it is being used to assay slowly digestible ("Resistant") starches for the purpose of producing alpha-glucogenesis resistant food starches.
Quezada, Roberto, Claudia Robayo, Bridget Adams, Stephen E Avery, Robert D. Baker, Bruce Hamaker, Erwin Sterchi, Buford L. Nichols, Susan S. Baker. Characterizations of Substrate and Enzyme Specificity of Maltase Assays of Mucosal Starch Digestion With Determinations of Group and Single Biopsy Reference Values From 980 Unselected Pediatric Endoscopic Clinical Duodenal Biopsies. NASPGHAN 2005, Salt Lake City, Utah. Oct. 20-23, 2005: Poster
Quezada-Calvillo, Roberto, Claudia Robayo, Stephen E Avery, Buford L. Nichols, Bridget Adams, Robert D. Baker, Susan S. Baker, Ursi Lugenbuhl, Erwin Sterchi. Kinetic Analysis of Terminal Starch Digestion Reveals Major Role of Sucrase-Isomaltase and Inhibition of Maltase-Glucoamylase by Maltotriose. NASPGHAN 2005, Salt Lake City, Utah. Oct. 20-23, 2005: Poster
Quezada-Calvillo, Roberto, Claudia C Robayo-Torres, Susan S Baker, Bruce Hamaker, Erwin E Sterchi, Buford L Nichols. Maltotriose, product of alpha-Amylase Starch Hydrolysis, Suppresses Maltase-Glucoamylase Activity and Slows Terminal Starch Digestion 44.5 Fold. (T2060) APS/SP, SF, CA. April 29-May 2, 2006: Poster Robayo, Claudia, Antone R Opekun, Roberto Quezada-Calvillo, Susan S Baker, Buford L Nichols. 13C-Sucrose Breath Test to differentiate Congenital Sucrase Isomaltase Deficiency from Pandisaccharidase Deficiency. (M1132) Digestive Disease Week, LA, CA. May 21-25, 2006: Poster
Robayo-Torres, Claudia C, Roberto Quezada-Calvillo, Susan S Baker, Erwin E Sterchi, Buford L Nichols. Maltotriose Brake: Alpha-Amylase Hydrolysis Product Maltotriose Regulates Maltase-Glucoamylase Activity and Controls Total Rates of Starch Digestion to Glucose. (T2060) Digestive Disease Week, LA, CA. May 21-25, 2006: Presentation
Invited Speaker: Nichols, BL. "Let them eat cake: The digestion of sugar and starch". Gulf Coast DDC Seminar, Houston, TX. March 23, 2006.
Nichols, BL. "Let them eat cake, digestion of starch and sugar". Institute Fur Biochemie & Molekular Medizen, University of Bern, Bern, Switzerland June 8, 2006.
Nichols, BL "Let them eat cake: digestion of sugar and starch". Institut fur Physiologische Chemie, Medizinischen Hochschule Hannover, Hannover, Germany. June 12, 2006.
Project 3: Nutrient Regulation of Cardiac Gene Expression during Diabetes Young, M. XX Annual Meeting of the Brazilian Federation of the Societies for Experimental Biology (FeSBE), Sao Paulo, Brazil. "Potential Role for the Circadian Clock in Metabolic Adaptation of the Heart" Young, M. Albert Einstein College of Medicine, Bronx, NY "Linking the Circadian Clock within the Cardiomyocyte to Myocardial Metabolism"
Young, M. University of Alberta, Edmonton, Canada "Linking the Circadian Clock within the Cardiomyocyte to Myocardial Metabolism"
Young, M. International Society for Heart Research, Toronto, Canada "Intrinsic Circadian Rhythms in the Cardiomyocyte"
Young, M. American Heart Association Scientific Sessions, Dallas, TX "The Intrinsic Circadian Clock within the Cardiomyocyte"
Young, M. American Diabetes Association Scientific Sessions, Washington D.C. "Differential Influence of Distinct Fatty Acids on Cardiomyocyte Metabolic Gene Expression"
Lockbridge, J.B., Durgan, D.J., Egbejimi, O., Stanley, W.C., Young, M.E. 2006. Differential influence of distinct fatty acids on cardiomyocyte metabolic gene expression. Diabetes. 55(Suppl.1):A442.
Young, M.E. 2006. Intrinsic circadian rhythms in the cardiomyocyte [abstract]. 28th Annual International Society for Heart Research. p. 2.
Okere, I.C., Chandler, M.P., McElfresh, T.A., Rennison, J.H., Sharov, V., Sabbah, H.N., Tserng, K.Y., Hoit, B.D., Ernsberger, P., Young, M.E., Stanley, W.C. 2006. Differential effects of saturated and unsaturated fatty acid diets on cardiomyocyte apoptosis, adipose distribution, and serum leptin. American Journal of Physiology - Heart and Circulatory Physiology. 291(1):H38-H44.
Wilson, C.R., Tran, M.K., Guthrie, P.H., King, T.M., Young, M.E., Taegtmeyer, H. 2006. Maladaptive skeletal muscle metabolism precedes maladaptive changes in cardiac metabolism and function with "western" diet in the Wistar rat [abstract]. Diabetes. 55(Suppl.1):A393.
Burgmaier, M., Philip, F., Guthrie, P., Young, M.E., Wilson, C.R., Taegtmeyer, H. 2006. Loss of cardiac metabolic adaptation and dysfunction of the heart with western diet in the obese Zucker rat [abstract]. Diabetes. 55(Suppl.1):A388.
Rossi, E.J., Sim, L., Kuntz, D.A., Hahn, D., Johnston, B.D., Ghavami, A., Szczepina, M.G., Kumar, N.S., Sterchi, E.E., Nichols, B.L., Pinto, B.M., Rose, D.R. 2006. Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives. FEBS Journal. 273:2673-2683.