Progress 05/21/99 to 05/20/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Genes ultimately regulate the digestion and absorption processes. This research project focuses on the interactions of nutrients and genes, especially the regulation of genes as they contribute to the development of diet associated with degenerative diseases, such as diabetes and atherosclerosis. The nuclear hormone receptors are a group of proteins that control gene expression. It has been determined that the protein known as farnesoid receptor (FXR) functions as a receptor for bile acids, which are essential for the absorption of fats and fat-soluble vitamins in the intestine, and bile also represents the primary route for the exit of cholesterol from the body. A primary effort of this research is to identify genes regulated by FXR and their role in cholesterol metabolism. Another objective is to determine the mechanisms by which dietary starch interacts with the gene expressing maltase-glucoamylase (MGA). Maltase- glucoamylase is the gate-keeping enzyme that determines small intestinal starch digestion into glucose. The function and regulation of maltase- glucoamylase is under investigation in knockout mice and children with deficient starch digestion. The most recent discovery is the presence of a spliced secreted isoform, which participates in starch digestion in the ileum of knockout mice. The regulation mechanism of both isoforms is under study in vitro in a mouse intestinal cell line producing maltase- glucoamylase and in vivo in mice on various starch diets. Research studies will concentrate on the functional role of Mgamme and Mgamso and to identify nutritional products and other natural products that regulate the activity of specific nuclear hormone receptors. Additional research will focus on the knockout of Mgamso gene expression, for the purpose of confirming its role in starch digestion. The function of the protein domains in the two gene products will be investigated, and researchers will characterize the effects of novel receptor ligands as well as the regulation of expression of target genes in appropriate tissues. Finally, studies will be conducted to analyze the molecular interaction between dietary starch and the expression of Mgamme and Mgamso at the gene regulatory level. Successful completion of these objectives will lead to the identification of new therapeutic approaches to metabolic disorders. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2000): Generate the Floxed HNF4 targeting vector. Identify genes regulated by FXR. Determine polymorphisms that exist in the normal maltase-glucoamylase gene. Year 2 (FY2001): Generation and validation of tissue specific Cre expressing mice. Begin to characterize the role of FXR target genes in cholesterol metabolism. Begin linkage search for the trans-factor that decreases maltase- glucoamylase expression in mouse strains. Characterize the interaction between GCNF and DNMTs. Determine the nutritional consequences of maltase-glucoamylase deficiency in knockout mice and the effectiveness of replacement with a yeast glucoamylase enzyme supplement. Year 3 (FY2002): Generation of a conditional knockout of the HNF4a gene. Characterize the broader metabolic consequences of FXR regulators. Identify the transcriptional regulator that suppresses maltase- glucoamylase expression in some humans and mouse strains. Establish the role of GCNF and DNMT3B in regulating Oct4 gene expression through DNA Methylation. Characterize the role of FXR ligands and FXR target genes in cholesterol metabolism. Continue the search for the trans-factors that decrease maltase- glucoamylase expression in some humans. Year 4 (FY2003): Analyze the role of maternal diet on the function of GCNF during embryonic development. Characterize the broader dietary consequences of FXR regulators. Identify the shared transcriptional factors that regulate maltase- glucoamylase, lactase and sucrase activities, and characterize these genes. Year 5 (FY2004): Complete the characterization of the interaction between GCNF and MBDs. Identify new genes regulated by FXR and new compounds that affect FXR activity. Search for the trans-factors that decrease maltase-glucoamylase expression in strain CBA/CaJ mice. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones listed below were scheduled to be completed under Year 5. All milestones were substantially completed. Complete the characterization of the interaction between GCNF and MBDs. Identify new genes regulated by FXR and new compounds that affect FXR activity. Search for the trans-factors that decrease maltase-glucoamylase expression in strain CBA/CaJ mice. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This project has terminated. However, the research will continue under Project 6250-51000-044-00D, which has completed peer review. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: A primary effort of this research is to identify genes regulated by the protein known as farnesoid receptor (FXR). FXR acts as a receptor for bile acids, which are essential for the absorption of fats and fat- solulble vitamines in the intestine, and represent the primary route for the exit of cholesterol from the body. Studies conducted by scientists at the Children's Nutrition Research Center in Houston, Texas, have shown that one of the major targets of FXR is the orphan nuclear receptor SHP, which functions as a transcriptional repressor. Further studies have identified and defined the functions of FXR and SHP in bile toxicity and triglyceride metabolism. These investigations may provide new approaches to both diet-independent and diet-dependent control of cholesterol levels, and novel treatments of hypercholesterolemia. B. Other significant accomplishments: Research conducted at the Children's Nutrition Research Center in Houston, Texas, on mice in which a portion of the maltase-glycoamylase (MGAM) gene had been removed, or knocked out, revealed an alternate version of the gene in a different location. During sequencing of the mouse maltase- glucoamylase (Mgam) cDNA, a construct was made that knocked out Mgamme at exon 2, the membrane-binding domain. While this provided the expected reduction in proximal glucoamylase activity, other expected responses remained unaffected. Investigation revealed that an alternative form (Mgamso) is expressed when Mgamme is absent. Preliminary evidence from biopsies supports the existence of a soluble MGAM (Mgamso) in the human small intestine. These findings have significant implications in the study of dietary starch digestion, and in the treatment of children with chronic diet related abdominal pain. C. Significant activities that support special target populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project has contributed significantly to the identification of FXR as a target for modulation of lipid metabolism. During this research project, the cDNA and gene for human maltase-glucoamylase (MGAM) have been sequenced and published from this lab. The mouse maltase-glucoamylase (Mgam) cDNA has been sequenced. A construct has been made that knocked out Mgamme at exon 2, the membrane-binding domain. Proof of ablation was provided by loss of the membrane peptide, loss of the second exon in DNA and message, correct villus-specific expression of Lac z from the construct, loss of the enterocyte (Mgamme) isoform on Western blot, absence of exon 2 from Mgamso cDNA, and discovery that membrane and soluble form exons 22-44 transcribe from different domains within the full Mgam gene. This reduced proximal glucoamylase activity by 40% but had no effect on distal small intestinal of Mgamme null mice. Sequencing of null mouse cDNA revealed an alternative splicing where exon 2 is excised. By immunohistology, the soluble form (Mgamso) form is expressed in Paneth cells in the small intestinal crypts of null and WT Mgamme KO mice, and is absent in enterocytes. Real-time RT/PCR of RNA from laser- dissected tissues confirmed presence of Mgamso message in WT Paneth Cells. Mgamso makes up all of the null small intestinal homogenate activity and luminal activity. The luminal Mgamso activity binds to starch granules and maize cellular fragments in vivo. Mgamso binding of starch and maize endosperm was confirmed in vitro. Preliminary evidence from biopsies supports existence of a soluble MGAM (MGAMSO) in the human small intestine. The identification of direct interactions between germ cell nuclear factor (GCNF) and DNA methyltransferases and methylated DNA binding domain factors (MBD2 and 3) is a novel discovery, which links GCNF with developmental regulation. We have isolated GCNF knock out cells, which will allow us to study the mechanisms involved in the GCNF DNA methylation interactions. We also have characterized some GCNF ligands that will help in dissecting this process. MBDs are found in large multiprotein complexes that include histone methyltransferases. Thus, GCNF may potentially recruit both DNA and histone methyltransferases to target gene promoters. The investigation of FXR is providing new approaches to both diet independent and diet dependent control of cholesterol levels. In particular, it is anticipated that potent and specific FXR antagonists may provide a novel means to treat hypercholesterolemia. Human glucoamylase deficiency was discovered in 13% of all children biopsied for clinical evaluation of abdominal symptoms. In 90% of these glucoamylase deficient children, the defect lies in transcriptional regulations that also reduce activity of lactase and sucrase. We have begun to study the genetic regulation of all three carbohydrate digesting enzymes mice who mimic the human combined deficiency, to seek more fundamental insights into the mechanisms of shared regulation. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Baylor College of Medicine scientists at the Children's Nutrition Research Center scientists have reported to the Baylor College of Medicine Office of Technology Administration (BCM-OTA) concerning the development of synthetic modulators of FXR. Such compounds should be entering clinical trials within the next five years. Also, CNRC scientists have shown that supplementing the diet with fungal glucoamylase increases the caloric value of dietary starch. About 95% of all children with sucrose intolerance also have starch intolerance. Presently these children are given a sucrase dietary supplement to reduce sucrose intolerance. The pharmaceutical company that supplies the sucrase supplement has contacted CNRC scientists about adding fungal glucoamylase for those children with starch intolerance. Finally, fungal glucoamylase is used for in vitro assay of glycemic index. This index is a measure of the in vivo digestibility of starch to glucose by glucoamylase. A commercial company will be allowed to use human MGAM expressed in COS-1 cells for an improved in vitro assay of food glycemic index. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Moore, D.D. Nuclear Receptors in Regulation of Drug Metabolism. Hyman Zimmerman Award Lecture. American Association for the Study of Liver Disease Annual Meeting, Boston MA. Oct 27, 2003 Nichols, B.L. Of Maize, Mice and Men, Invited Lecture, Department of Food Science, Purdue University, W. Laffayette, IN. Dec 4, 2003 Nichols, B, Avery, S, Finegold, M, Rudman, D, Luginbuhl, U, Sterchi, E. Developmental Regulation Of Membrane And Soluble Mouse Maltase- Glucoamylases At Weaning American Society for Nutritional Sciences (ASNS) Apr 18, 2004. Washington DC Meeting, Program 39, A103.17 (Poster). Nichols, B., Finegold, M., Sterchi, E. 2004. Molecular Biology of weaning in maltase-glucoamylase Null and WT mice [abstract]. Pediatric Academic Societies' Annual Meeting. Pediatric Research 55(4):187A (Poster) . Scientific Publications for FY 2004 may be viewed on 6250-51000-034-00D

Impacts
(N/A)

Publications

• Urizar, N.L., Liverman, A.B., Dodds, D.T., Valentin Silva, F., Ordentlich, P., Yan, Y., Heyman, R.A., Mangelsdorf, D.J., Moore D.D. A natural product that lowers cholesterol as an antagonist ligand for the farnesoid X receptor. Science. 2002. v. 296. p. 1703-1706.
• Hwang, S., Urizar, N.L., Moore, D.D., Henning, S.J. Bile acids regulate the ontogenic expression of ileal bile acid binding protein in the rat via the farnesoid X receptor. Gastroenterology. 2002. v. 122. p. 1483-1492.
• Nichols, B.L., Avery, S.E., Butte, N.F., Opekun, A.R., Hahn, D., Sterchi, E.E. Dietary supplements of yeast amyloglucosidase increase proximal starch digestion in maltase-glucoamylase (MGA) knockout and wild-type mice. Journal of the Federation of American Societies for Experimental Biology. 2002. v. 16. Abstract p. 245.
• Nichols, B.L., Avery, S.E., Butte, N., Opekun, A.R., Hahn, D., Sterchi, E. E. Starch digestion is enhanced by feeding of yeast amyloglucosidase to maltase-glucoamylase knockout and wild-type mice. Gastroenterology. 2002. v. 122. Abstract p. A-26.
• Fuhrmann, G., Chung, A.C-K., Jackson, K.J., Hummelke, G., Baniahmad, A., Sutter, J., Sylvester, I., Scholer, H.R., Cooney, A.J. Mouse germline restriction of "Oct4" expression by germ cell nuclear factor. Developmental Cell. 2001. v. 1. p. 377-387.
• Donovan, P.J. High Oct-ane fuel powers the stem cell. Nature Genetics. 2001. v. 29. p. 246-247.

Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Genes ultimately regulate the digestion and absorption processes. This research project focuses on the interactions of nutrients and genes, especially the regulation of genes as they contribute to the development of diet associated with degenerative diseases, such as diabetes and atherosclerosis. The methylation of genes during development is a process where embryonic events can be transmitted to later stages in life and may explain some of the well-described fetal origins of Adult Disease (Barker Hypothesis). We are studying the regulation of gene methylation by the nuclear receptor, germ cell nuclear factor (GCNF), which is expressed during embryonic development. GCNF regulates gene expression and DNA and histone methylation, by recruiting DNA methyltransferases and Methyl binding domain proteins to target genes. We are analyzing the mechanism of GCNF dependent DNA and histone methylation. The nuclear hormone receptors are a group of proteins that control gene expression. It has recently been determined that a member of this family known as RIP14 or farnesoid receptor (FXR) is a receptor for bile acids. Bile acids are essential for absorption of dietary fats and fat-soluble vitamins and represent the only means for cholesterol to exit the body. Our primary effort is identification of genes regulated by FXR and examination of their role in cholesterol metabolism. Our research objective is to determine the mechanisms by which dietary starch interacts with the gene expressing maltase-glucoamylase (MGA). Maltase-glucoamylase is the gate- keeping enzyme that determines small intestinal starch digestion into glucose. The function and regulation of maltase-glucoamylase is under investigation in knockout mice and children with deficient starch digestion. The most recent discovery is the presence of a spliced secreted isoform, which participates in starch digestion in the lumen of the ileum of KO mice. The regulation mechanism of both isoforms is under study in a mouse intestinal cell line producing maltase-glucoamylase and mice on various starch diets. 2. How serious is the problem? Why does it matter? It is difficult to establish the extent and severity of dysfunctional DNA methylation in development and disease processes but aberrant DNA methylation has been implicated in such human developmental diseases as Rett syndrome, and there is extensive animal evidence that alterations in DNA methylation may promote development of some cancers. The connection between cholesterol levels and arteriosclerosis is well known. Understanding the role of FXR may allow the identification of new agents with beneficial effects on cholesterol metabolism. Epidemiological reports indicate that about 15-30% of US children suffer from chronic abdominal pain. Twenty-six percent of children with chronic abdominal pain have deficiencies of maltase-glucoamylase enzyme activity and poor small intestinal starch digestion. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This work relates to National Program 107 'Human Nutrition' priority E. that is entitled Genetic Variability. These investigations support directly the National Program Component 'Relationship Between Diet, Genetics and Lifestyle and the Risk For Chronic Disease'. Children's Nutrition Research Scientists' are working to understand the role of regulation of DNA methylation by GCNF in embryonic development and potential role of maternal diet on gene expression; to produce new approaches to the control of cholesterol metabolism that could be of central importance for altering expression of genes that contribute to dietary-based cardiovascular disease; and to document the nutritional significance of poor starch digestion and mechanisms for transcriptional down-regulation of dietary starch digestion. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY2003: Starch from grains and tubors contribute 60-80% of calories in the human diet, thus a greater understanding of starch digestion is needed. Scientists at the Children's Nutrition Research Center in Houston, TX, sequenced membrane-bound glucoamylase, the enzyme known to digest starch. A secreted glucoamylase, which binds to dietary starch granules in the small and large intestinal cavity, was discovered in the membrane-bound glucoamylase knockout mouse; the experiment revealed a redundancy in this digestive pathway that explains why the membrane-bound glucoamylase deficient mouse digested starch normally. This accomplishment is a significant scientific discovery that will assist other scientist conducting research starch digestion. B. Other significant accomplishments? Scientific literature suggests that one in ten humans has a reduced ability to digest starch in the small intestine. Scientists at the Children's Nutrition Research Center, Houston, TX, working with a reference clinical laboratory in Buffalo, NY, studied the frequency of glucoamylase deficiency in 981 children who had small intestinal biopsies as part of clinical evaluations. Frequency of glucoamylase deficiency in these children was 13%, with nine-tenths also deficient in sucrase and lactase, which are required for sugar digestion; starches indigestible in the small intestine are salvaged in the large intestine however the small intestine is 17% more efficient. Such findings are significant as we understand digestion and develop optimum diets for this population. C. Significant accomplishments/activities that support special target populations? None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The identification of direct interactions between GCNF and DNA methyltransferases and methylated DNA binding domain factors (MBD2 and 3) is a novel discovery, which links GCNF with developmental regulation. We have isolated GCNF knockout embryonic stem (ES) cells, which will allow us to study the mechanisms involved in the GCNF DNA methylation interactions. We also have characterized some GCNF ligands that will help in dissecting this process. MBDs are found in large multiprotein complexes that include histone methyltransferases. Thus, GCNF may potentially recruit both DNA and histone methyltransferases to target gene promoters. The investigation of farnesoid receptor (FXR) is providing new approaches to both diet-independent and diet-dependent control of cholesterol levels. In particular, it is anticipated that potent and specific FXR antagonists may provide a novel means to treat hypercholesterolemia. Human glucoamylase deficiency was discovered in 13% of all children biopsied for clinical evaluation of abdominal symptoms. In 90% of these glucoamylase-deficient children, the defect lies in transcriptional regulations that also reduce activity of lactase and sucrase. We have begun to study the genetic regulation of all three carbohydratedigesting enzymes in strain CBA/CaJ mice who mimic the human combined deficiency, to seek more fundamental insights into the mechanisms of shared regulation. 6. What do you expect to accomplish, year by year, over the next 3 years? 2004: Complete the characterization of the interaction between GCNF and MBDs. Identify new genes regulated by FXR and new compounds that affect FXR activity. Search for the trans-factors that decrease maltase- glucoamylase expression in strain CBA/CaJ mice. 2005: Analyze the role of maternal diet on the function of GCNF during embryonic development. Characterize the role of FXR ligands and FXR target genes in cholesterol metabolism, particularly the biochemical mechanism of action of guggulsterone. Identify the shared transcriptional factors that regulate maltase-glucoamylase, lactase and sucrase activities and characterize these genes in mice. 2006: Establish the role of GCNF and DNMT3B and MBD 2 and 3 in regulating Oct4 gene expression through DNA and histone Methylation. Characterize the broader dietary consequences of FXR regulators. Reproduce human glucoamylase deficiency by knockout of wildtype mouse transcriptional factors that regulate maltase-glucoamylase, lactase and sucrase activities. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Children's Nutrition Research Center scientists have reported to the Baylor College of Medicine Office of Technology Administration (BCM-OTA) that a fungal dietary enzyme supplement is corrective in MGA null mice and is to be considered for use in management of human and animal maltase- glucoamylase deficiencies. The results of these studies should be available to the public within the next year. Industry is currently identifying synthetic compounds that control GCNF activity, which is available to the Children's Nutrition Research Center scientists for testing. Industry is currently identifying synthetic compounds that control FXR activity, which should be available for our testing in one to two years. Additional work with industry includes testing a fungal amyloglucosidase dietary supplement for use in glucoamylase deficiency. The test results show that the supplement increases proximal starch digestion and increases growth. The constraints include: GCNF ligands and regulators with desirable nutritional and developmental effects can only be adopted as therapeutic agents if they meet FDA approval after clinical trials. FXR regulators with desirable nutritional effects can only be adopted as therapeutic agents if they meet FDA approval after clinical trials. The yeast glucoamylase supplement may need review by the FDA, as was a fungal sucrase supplement used for congenital sucrase deficiency; however, the fungal beta glucosidase supplement used for lactase deficiency was ruled a dietary supplement and not subject to FDA regulation. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Nichols, B.L. The History of Infant Nutrition, and Maize, Mice and Men: The Evolution Of Starch Digestion. South Dakota State University Nutrition Symposium, Brookings, SD, March 25, 2003. Nichols, B.L., Avery, S.E., Butte, N.F., Luginbuehl, U., Sterchi, E.E., Finegold, M. Paneth cells secrete a soluble isoform of maltaseglucoamylase (MGAs) with full starch digesting capacity in membrane MGA (MGAM) null mice. Digestive Disease Week May 17-22, 2003, American Society of Gastroenterology. 2003. Abstract S954. (Distinguished Poster). Nichols, B.L., Avery, S.E., Butte, N.F., Finegold, M., Quezada-Calvillo, R., Luginbuehl, U., Sterchi, E.E. Starch granules and cell structures soluble maltase-glucoamylase of mouse ileum is active in the colon where it binds to maize. European Society of Pediatric Gastroenterlogy, Hepatology and Nutrition (ESPGHAN), (Oral and Poster presentations). Prague, Czechoslavakia. 2003.

Impacts
(N/A)

Publications

• Fuhrmann, G., Chung, A.C-K., Jackson, K.J., Hummelke, G., Baniahmad, A., Sutter, J., Sylvester, I., Scholer, H.R., Cooney, A.J. Mouse germline restriction of "Oct4" expression by germ cell nuclear factor. Developmental Cell. 2001. v. 1. p. 377-387.
• Donovan, P.J. High Oct-ane fuel powers the stem cell. Nature Genetics. 2001. v. 29. p. 246-247.
• Urizar, N.L., Liverman, A.B., Dodds, D.T., Valentin Silva, F., Ordentlich, P., Yan, Y., Heyman, R.A., Mangelsdorf, D.J., Moore D.D. A natural product that lowers cholesterol as an antagonist ligand for the farnesoid X receptor. Science. 2002. v. 296. p. 1703-1706.
• Hwang, S., Urizar, N.L., Moore, D.D., Henning, S.J. Bile acids regulate the ontogenic expression of ileal bile acid binding protein in the rat via the farnesoid X receptor. Gastroenterology. 2002. v. 122. p. 1483-1492.
• Nichols, B.L., Avery, S.E., Butte, N.F., Opekun, A.R., Hahn, D., Sterchi, E.E. Dietary supplements of yeast amyloglucosidase increase proximal starch digestion in maltase-glucoamylase (MGA) knockout and wild-type mice. Journal of the Federation of American Societies for Experimental Biology. 2002. v. 16. Abstract p. 245.
• Nichols, B.L., Avery, S.E., Butte, N., Opekun, A.R., Hahn, D., Sterchi, E. E. Starch digestion is enhanced by feeding of yeast amyloglucosidase to maltase-glucoamylase knockout and wild-type mice. Gastroenterology. 2002. v. 122. Abstract p. A-26.

Progress 10/01/01 to 09/30/02

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Digestion and absorption are ultimately regulated by genes. This research project is focused upon the interactions of nutrients and genes, especially the regulation of genes as they contribute to the development of diet associated degenerative diseases, such as diabetes and atherosclerosis. The methylation of genes during development is a process where embryonic events can be transmitted to later stages in life without a change in the DNA sequence. We are studying the regulation of gene methylation by the nuclear receptor GCNF, which is expressed during embryonic development. GCNF regulates gene expression and DNA methylation by recruiting DNA methyltransferases to target genes. We are analyzing the mechanism of GCNF-dependent DNA methylation. The nuclear hormone receptors are a group of proteins that control expression of genes involved in many important processes, including cholesterol regulation. A member of this family called FXR has been found to be a receptor for bile acids. Bile acids are produced from cholesterol in the liver and are the most important way for cholesterol to exit the body. From the liver, they are released into the gut, where they are essential for absorption of dietary fats and fat-soluble vitamins. Our broad goal is to increase our understanding of bile acid and cholesterol metabolism by identifying genes regulated by FXR and examining their metabolic functions. Maltase- glucoamylase is the gate-keeping enzyme that determines small intestinal starch digestion into glucose or, by default, colonic fermentation into short chain fatty acids. Does the default digestion of starch by the colon provide benefits? What are the nutritional adaptations to this digestive pathway? To answer these questions, the function and regulation of maltase-glucoamylase is under investigation in knockout mice and in children with deficient starch digestion. 2. How serious is the problem? Why does it matter? It is difficult to establish the extent and severity of dysfunctional DNA methylation in development and disease processes, but aberrant DNA methylation has been implicated in such human developmental diseases as Rett syndrome. The connection between cholesterol levels and arteriosclerosis is well known. Understanding the role of FXR may allow the identification of new agents with beneficial effects on cholesterol metabolism. Epidemiological reports indicate that about 15-30% of US children suffer from chronic abdominal pain. Twenty-six percent of children with chronic abdominal pain have deficiencies of maltase-glucoamylase enzyme activity and poor small intestinal starch digestion 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program #107 - Human Nutrition, Priority E, Genetic Variability. These investigations relate directly to the National Program Component "Relationship Between Diet, Genetics and Lifestyle and the Risk For Chronic Disease." We are working to understand the role of regulation of DNA methylation by GCNF in embryonic development and potential role of maternal diet on gene expression; to produce new approaches to the control of cholesterol metabolism that could be of central importance for altering genes that contribute to dietary based cardiovascular disease; and to document the nutritional significance of poor starch digestion and mechanisms for transcriptional down-regulation of dietary starch digestion. 4. What was your most significant accomplishment this past year? A. Single most significant accomplishment during FY2002? A better understanding of the relationships between diet and the risk for chronic disease is needed. Scientists at the USDA, ARS, Children's Nutrition Research Center at Baylor College of Medicine in Houston, TX, conducted studies to define the critical components of diet, genetics, and lifestyle that modulate the risk of chronic desease. These studies resulted in identification of GCNF and DNA Methyltransferase 3B in a single complex that binds to the Oct4 promoter, a novel linkage between nuclear receptors, gene regulation, DNA methylation and development. Since aberrant DNA methylation has been implicated in such human developmental diseases as Rett syndrome, these studies have implication in the future enhancement of human health. B. Other significant accomplishments? Efforts were made to characterize the GCNF ligands. Scientists at the USDA ARS Children's Nutrition Research Center identified direct interactions between GCNF and DNA methyltransferases and methylated DNA binding domain factors, and made a novel discovery, which links GCNF with developmental regulation. We have now isolated GCNF knock out ES cells. This allows significant progress in our studies of the mechanisms involved in the GCNF DNA methylation interactions. We attempted to identify new genes regulated by the FXR hormone and new compounds that affect FXR activity. Studies were conducted at the USDA ARS Children's Nutrition Research Center at Baylor College of Medicine in Houston, TX, to determine potential activity. These studies demonstrated that the natural product guggulsterone is a potent and specific FXR antagonist, and lowers hepatic cholesterol in wild type mice, but not mice lacking the FXR gene. The investigation of FXR is providing new approaches to both diet-independent and diet-dependent control of cholesterol levels, and we anticipate that potent and specific FXR antagonists may provide a novel means to treat hypercholesterolemia. Efforts continued to develop a genetic "knockout" mouse to aid in the study of chronic gastric distress in children. The work was done at the USDA ARS Children's Nutrition Research Center at Baylor College of Medicine in Houston, TX. A mouse maltase-glucoamylase knockout model was produced which replicates the human glucoamylase deficiency phenotype and responds to enzyme supplement treatment. The successful use of dietary glucoamylase enzyme supplements to increase starch digestion in these mice may prove to be a method to improve the quality of dietary grains and cereals in the human deficient population. C. Significant accomplishments/activities that support special target populations? None. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? The identification of direct interactions between GCNF and DNA methyltransferases and methylated DNA binding domain factors is a novel discovery, which links GCNF with developmental regulation. We have isolated GCNF knock out ES cells, which will allow us to study the mechanisms involved in the GCNF DNA methylation interactions. We also have characterized some GCNF ligands that will help in dissecting this process. The investigation of FXR is providing new approaches to both diet- independent and diet-dependent control of cholesterol levels. In particular, it is anticipated that potent and specific FXR antagonists may provide a novel means to treat hypercholesterolemia. Human maltase-glucoamylase deficiency was discovered in 13 children. The defect lies in transcriptional regulations that also reduce activity of lactase and sucrase. We have begun to study the coordinate regulation of all three carbohydrate-digesting enzymes, in a co-expressing cell line, to seek more fundamental insights into this shared regulation. This combination of three enzyme deficiencies makes dietary management very difficult. The use of dietary glucoamylase enzyme supplements to increase starch digestion in null, and WT knockout mice may be a valuable supplement to improve the quality of dietary grains and cereals in this deficient population. 6. What do you expect to accomplish, year by year, over the next 3 years? 2003: Complete the characterization of the interaction between GCNF and DNMTs. Identify new genes regulated by FXR and new compounds that affect FXR activity. Search for the trans-factors that decrease maltase- glucoamylase expression in some humans. 2004: Analyze the role of maternal diet on the function of GCNF during embryonic development. Characterize the role of FXR ligands and FXR target genes in cholesterol metabolism, particularly the biochemical mechanism of action of guggulsterone. Identify the shared transcriptional factors that regulate maltase-glucoamylase, lactase, and sucrase activities and characterize these genes. 2005: Establish the role of GCNF and DNMT3B in regulating Oct4 gene expression through DNA Methylation. Characterize the broader dietary consequences of FXR regulators. Test fungal amyloglucosidase dietary supplements in humans with poor glucoamylase activities 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Technology transfer functions and activities at the Children's Nutrition Research Center are handled through the Baylor College of Medicine Office of Technology Administration (BCM-OTA). Currently, a patent application on GCNF is pending, a biotechnology company has been founded to develop the potential commercial applications of the FXR hormone, and the discovery that a fungal dietary enzyme supplement is corrective in MGA null mice is being considered for possible use in management of human and animal maltase-glucoamylase deficiencies. Industry is currently identifying synthetic compounds that control GCNF activity, and providing them for testing. Results of GCNF studies should be available to the public within the next year. Industry is also working to identify synthetic compounds that control FXR activity. These compounds should be available for testing in one to two years. Researchers are also working with industry to test a fungal amyloglucosidase dietary supplement for use in glucoamylase deficiency. The test results in KO mice show that the supplement increases proximal starch digestion and increases growth. GCNF ligands and regulators and FXR regulators with desirable nutritional and developmental effects can only be adopted as therapeutic agents if they meet FDA approval after clinical trials. The yeast glucoamylase supplement may need review by the FDA; however, it more likely will be ruled a dietary supplement and not subject to FDA regulation. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Nichols, B.L. The History of Infant Feeding: Progress in the 20th Century. Keynote presentation, South Dakota State, University, Brookings, SD, September 27, 2002. Nichols, B.L. Maltase-Glucoamylase Knockout in Mice and Men, Departmental Seminar, Institute of Biochemistry and Molecular Biology, University of Bern, Bern, Switzerland, 28 May, 2002.

Impacts
(N/A)

Publications

• Fuhrmann, G., Chung, A.C-K., Jackson, K.J., Hummelke, G., Baniahmad, A., Sutter, J., Sylvester, I., Scholer, H.R., Cooney, A.J. Mouse germline restriction of "Oct4" expression by germ cell nuclear factor. Developmental Cell. 2001. v. 1. p. 377-387.
• Donovan, P.J. High Oct-ane fuel powers the stem cell. Nature Genetics. 2001. v. 29. p. 246-247.
• Urizar, N.L., Liverman, A.B., Dodds, D.T., Valentin Silva, F., Ordentlich, P., Yan, Y., Heyman, R.A., Mangelsdorf, D.J., Moore D.D. A natural product that lowers cholesterol as an antagonist ligand for the farnesoid X receptor. Science. 2002. v. 296. p. 1703-1706.
• Hwang, S., Urizar, N.L., Moore, D.D., Henning, S.J. Bile acids regulate the ontogenic expression of ileal bile acid binding protein in the rat via the farnesoid X receptor. Gastroenterology. 2002. v. 122. p. 1483-1492.
• Nichols, B.L., Avery, S.E., Butte, N.F., Opekun, A.R., Hahn, D., Sterchi, E.E. Dietary supplements of yeast amyloglucosidase increase proximal starch digestion in maltase-glucoamylase (MGA) knockout and wild-type mice. Journal of the Federation of American Societies for Experimental Biology. 2002. v. 16. Abstract p. 245.
• Nichols, B.L., Avery, S.E., Butte, N., Opekun, A.R., Hahn, D., Sterchi, E. E. Starch digestion is enhanced by feeding of yeast amyloglucosidase to maltase-glucoamylase knockout and wild-type mice. Gastroenterology. 2002. v. 122. Abstract p. A-26.

Progress 10/01/00 to 09/30/01

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Digestion and absorption are ultimately regulated by genes. This research project is focused upon the interactions of nutrients and genes, especially the regulation of genes as they contribute to the development of diet associated degenerative diseases, such as diabetes and atherosclerosis. The methylation of genes during development is a process where embryonic events can be transmitted to later stages in life without a change in the DNA sequence. We are studying the regulation of gene methylation by the nuclear receptor GCNF, which is expressed during embryonic development. GCNF regulates gene expression and DNA methylation by recruiting DNA methyltransferases to target genes. We are analyzing the mechanism of GCNF dependent DNA methylation. The nuclear hormone receptors are a group of proteins that control expression of genes involved in many important processes, including cholesterol regulation. A member of this family called FXR has been found to be a receptor for bile acids. Bile acids are produced from cholesterol in the liver and are the most important way for cholesterol to exit the body. From the liver, they are released into the gut, where they are essential for absorption of dietary fats and fat-soluble vitamins. Our broad goal is to increase our understanding of bile acid and cholesterol metabolism by identifying genes regulated by FXR and examining their metabolic functions. Maltase- glucoamylase is the gate-keeping enzyme that determines small intestinal starch digestion into glucose or, by default, colonic fermentation into short chain fatty acids. Does the default digestion of starch by the colon provide benefits? What are the nutritional adaptations to this digestive pathway? To answer these questions, the function and regulation of maltase-glucoamylase is under investigation in knockout mice and in children with deficient starch digestion. 2. How serious is the problem? Why does it matter? It is difficult to establish the full extent and severity of dysfunctional DNA methylation in development and disease processes since only recently has DNA methylation been studies intensively, but already aberrant DNA methylation has been implicated in such human developmental diseases as Rett syndrome. The connection between cholesterol levels and arteriosclerosis is well known. Understanding the role of FXR may allow the identification of new agents with beneficial effects on cholesterol metabolism. Epidemiological reports indicate that about 15-30% of US children suffer from chronic abdominal pain. 26% of children with chronic abdominal pain have deficiencies of maltase-glucoamylase enzyme activity and poor small intestinal starch digestion. 3. How does it relate to the National Program(s) and National Component(s)? This work relates to National Program 107 - "Human Nutrition" Priority E, entitled Genetic Variability. These investigations relate directly to the National Program Component "Relationship Between Diet, Genetics and Lifestyle and the Risk For Chronic Disease." We are working to understand the role of regulation of DNA methylation by GCNF in embryonic development and potential role of maternal diet on gene expression; to produce new approaches to the control of cholesterol metabolism that could be of central importance for altering genes that contribute to dietary based cardiovascular disease; and to document the nutritional significance of poor starch digestion and mechanisms for transcriptional down-regulation of dietary starch digestion. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2001 year: We tested our idea that some of the natural products thought to affect cholesterol levels would work by affecting the activity of the nuclear hormone receptor, FXR. This was done using model systems to measure FXR activation. We found that guggulsterone, a natural plant product that lowers cholesterol in humans, is an inhibitor of FXR activation. This provides a new and unexpected expanation for the effects of this compound and suggests that other FXR inhibitors may also be useful in lowering cholesterol levels. B. Other Significant Accomplishment(s), if any: The enzyme maltase-glucoamylase (MGA) is essential for the digestion of starch to glucose in the small intestine. We have "knocked out" this enzyme in mice with molecular technology. Using studies of oxygen utilization and carbon dioxide production in breath, we have demonstrated that these mice have a significant reduction of digestion of dietary starch to blood glucose with reduced breath carbon dioxide but oxygen utilization remains unchanged. Since these mice replicate the human glucoamylase deficiency phenotype we have a tool that should greatly enhance human digestive system research. C. Significant Accomplishments/Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. The identification of direct interactions between GCNF and DNA methyltransferases and methylated DNA binding domain factors is a novel discovery, which links GCNF with developmental regulation. The investigation of FXR is providing new approaches to both diet independent and diet dependent control of cholesterol levels. Human maltase- glucoamylase deficiency was discovered in 13 children. The defect lies in transcriptional regulations that also reduce activity of lactase and sucrase. We have begun to study the coordinate regulation of all three carbohydrate-digesting enzymes, in a co-expressing cell line, to seek more fundamental insights into this shared regulation. This combination of three enzyme deficiencies makes dietary management very difficult. The use of dietary glucoamylase enzyme supplements, now under study in knockout mice, may be a valuable alternate. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: Characterize the interaction between GCNF and DNMTs; identify new genes regulated by FXR and new compounds that affect FXR activity; and determine the nutritional consequences of maltase-glucoamylase deficiency in knockout mice and the effectiveness of replacement with a yeast glucoamylase enzyme supplement. Year 2: Establish the role of GCNF and DNMT3B in regulating Oct4 gene expression through DNA Methylation; characterize the role of FXR ligands and FXR target genes in cholesterol metabolism; and continue the search for the trans-factors that decrease maltase-glucoamylase expression in some humans. Year 3: Analyze the role of maternal diet on the function of GCNF during embryonic development; characterize the broader dietary consequences of FXR regulators; and identify the shared transcriptional factors that regulate maltase-glucoamylase, lactase and sucrase activities and characterize these genes. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? Researchers are working with industry to identify synthetic compounds that control FXR activity. These compounds should be available for our testing in one to two years. We are also working with industry to test a dietary supplement to use in glucoamylase deficiency. The test results in "Knock- Out" mice should be complete within a year. FXR regulators with desirable nutritional effects can only be adopted as therapeutic agents if they meet FDA approval after clinical trials. The yeast glucoamylase supplement may need review by the FDA, as was a yeast sucrase supplement used for congenital sucrase deficiency; however, the yeast beta glucosidase supplement used for lactase deficiency was ruled a natural dietary component and not subject to FDA regulation. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) None.

Impacts
(N/A)

Publications

• Nichols, B.L., Avery, S., Swallow, D.M., Hahn, D., Sterchi, E.E. Sequence of maltase-glucoamylase gene: common ancestry with sucrase-isomaltase with complementary starch digestion activity. Journal of Pediatric Gastroenterology and Nutrition. 2001. v. 31(2). p. S101.
• Karnsakul, W., Lifschitz, C., Kitagawa, S., Olive, A., Villa, X., Avery, S., Sterchi, E., Hahn D., Luginbuehl, U., Swallow, D., Nichols, B. Pandisaccharidase deficiencies in chronic abdominal pain. Gastroenterology. 2001. v. 120(1). p. A210.
• Nichols, B.L., Avery, S.E., Karnsakul, W., Hahn, D., Sterchi, E.E. Genomic organization and granulocyte expression of small intestinal maltase-glucoamylase. FASEB Journal. 2001. v. 15(5). p. A736.
• Cooney, A.J., Lee, C.T., Lin, S-C., Tsai, S.Y., Tsai, M-J. The use of gene targeting to elucidate orphan nuclear receptor function: Knock-outs of GCNF and COUP-TFs. Trends in Endocrinology and Metabolism. 2001. v. 12(6). p. 247-251.
• Chung, A.C.K., Katz, D., Pereira, F.A., Jackson, K.J., DeMayo, F.J., Cooney, A.J., O'Malley, B.W. Loss of orphan receptor Germ Cell Nuclear Factor (GCNF) function results in ectopic development of the tail bud and a novel posterior truncation. Molecular and Cellular Biology. 2001. v. 21(2). p. 663-677.
• Urizar, N.L., Dowhan, D.H., Moore, D.D. The farnesoid X-activated receptor mediates bile acid activation of phospholipid transfer protein gene expression. Journal of Biological Chemistry. 2000. v. 275(50). p. 39313-39317.
• Laffitte, B.A., Kast, H.R., Nguyen, C.M., Zavacki, A.M., Moore, D.D., Edwards,P.A. Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor. Journal of Biological Chemistry. 2000. v. 275(14). p. 10638-10647.
• Nichols, B.L., Karnsakul, W., Avery, S.E., Swallow, D., Hahn, D., Sterchi, E.E. Trans-suppression of maltase-glucoamylase activity in an infant with congenital lactase and sucrase deficiencies. FASEB Journal. 2000. v. 14. p. A290.

Progress 10/01/99 to 09/30/00

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Digestion and absorption are regulated by genes. The regulation of these genes as they contribute to the development of diet associated degenerative diseases, such as diabetes and atherosclerosis, is under investigation. The following three approaches are underway. A. (Dr. Cooney) Understanding the regulation of intestinal, liver and pancreatic functions by the factor HNF4, a transcription factor. We are generating mouse models in which the HNF4 gene has been knocked out in each of these tissues. The objective of this CRIS is 1) generation of a floxed HNF4a allele; 2) generation and validation of tissue specific Cre expressing mice; 3) generation of a conditional knockout of the HNF4a gene. B. (Dr. Moore) The nuclear hormone receptors are a group of proteins that control gene expression. It has recently been determined that a member of this family known as RIP14 or FXR is a receptor for bile acids. Bile acids are essential for absorption of dietary fats and fat-soluble vitamins and represent the only means for cholesterol to exit the body. We hypothesize that FXR mediates important functions of bile acids in metabolic regulation. Our primary aim is to identify the specific genes regulated by FXR and examine their role in cholesterol metabolism. C. (Dr. Nichols) The dietary starches make up two thirds of the human caloric intake. Maltase-glucoamylase is the gate-keeping enzyme that determines small intestinal starch digestion into glucose or, by default, colonic fermentation into short chain fatty acids. The proof of this key role of maltase-glucoamylase is under investigation in mice. The objective of this CRIS is: 1) determination the fate of dietary starch when maltase-glucoamylase is decreased or absent, 2) determination of the nutritional significance of reduced maltase- glucoamylase activity on small and large intestinal starch digestion. 2. How serious is the problem? Why does it matter? The diet plays a vital role in the development of common degenerative disease3s such as type II diabetes and atherosclerosis. It is therefore essential to study the interaction between the diet and the regulation of the genes underlying these degenerative disorders. Non-insulin dependant diabetes mellitus (NIDDM) is a serious disease within the US population. Mutations in the HNF4 gene have been implicated in the cause of maturity onset diabetes of the young (MODY1). The most common cause of morbidity and mortality in Western societies is atherosclerotic cardiovascular disease. The connection between cholesterol levels and arteriosclerosis is well known. Understanding the role of FXR may allow the identification of new agents with beneficial effects on cholesterol metabolism. Preliminary data suggest that one in ten normal humans is a carrier of a maltase- glucoamylase mutation. Does the default digestion of starch by the colon provide benefits to these citizens? What risks are associated with increased production of fatty acids from starch digestion in the colon? 3. How does it relate to the National Program(s) and National Component(s)? National Program Number 107 Human Nutrition. We are working 1) to identify the function of HNF4 in the intestine, liver and pancreas and how it influences nutrient requirements through effects on absorption, transport, storage, etc., 2) to identify specific maltase- glucoamylase genetic mutations that influence nutrient requirements through effects on starch digestion, absorption, transport, storage, etc., and to identify MGA mutant populations and subgroups with genetic predispositions to chronic diseases and disorders, and 3) to produce new insights into the control of cholesterol metabolism that would be of central importance for understanding of the many factors that contribute to cardiovascular disease. These three investigations relate directly to the National Program Component "Relationship Between Diet, Genetics, and Lifestyle and the Risk for Chronic Disease." 4. What were the most significant accomplishments this past year? A.) Single Most Significant Accomplishment during FY 2000 year: Cloning of the murine SvEv129 gene to make the targeting construct for the inactivation of the HNF4 gene. B.)Other Significant Accomplishment(s), if any: Identified the phospholipid transport (PLTP) gene as a target for FXR mediated bile acid regulation. PLTP is involved in the formation of High Density Lipoprotein (HDL) particles that carry serum cholesterol. Thus, this finding provides direct support for the hypothesized role of FXR in cholesterol metabolism. C.) Significant Accomplishments/Activities that Support Special Target Populations: Documented that an infant with starch malabsorption has a defect in a trans-acting factor that reduces messenger RNA for maltase-glucoamylase and other carbohydrolases. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. The 1995 national expense for care of atherosclerotic heart disease was $58 billion and diabetes was $26 Billion for citizens less than 65 years of age. These citizens made up 13% (33 million) of the national population (Health, United States, 1999, CDC A 10% reduction in frequency of these two major degenerative diseases would result in a present day savings of greater than $8 billion each year. The number of citizens greater thaan 65 will increase to 68 million in 2030. The study of HNF4 function has just been initiated, thus cloning and characterization of the murine 129 gene and designing the targeting vector are the achievements to date. The investigation of FXR as a receptor for bile acids is a relatively new project. We anticipate that the study will provide new insights into lipid metabolism, particularly the control of cholesterol levels. The study of starch digestion has opened the way for future studies of genetic variation in nutrient requirement. It is believed that variations in efficiency of starch digestion may play a role in genetic risk for development of type II diabetes. A non-invasive screening test of human in vivo starch digestion was developed to determine phenotype and the human maltase-glucoamylase gene was sequenced to permit non-invasive determination of genotype. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1 - Generate the Floxed HNF4 targeting vector; identify both new genes regulated by FXR; and determine polymorphisms that exist in the normal maltase-glucoamylase gene. Year 2 - Generation and validation of tissue specific Cre expressing mice; begin to characterize the role of FXR target genes in cholesterol metabolism; and begin linkage search for the trans-factor that decreases maltase-glucoamylase expression in mouse strains. Year 3 - Generation of a conditional knockout of the HNF4a gene; characterize the broader metabolic consequences of FXR regulators; and identify the transcriptional regulator that suppresses maltase-glucoamylase expression in some humans and mouse strains. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? The completion of the HNF4 CreLox knockout in mice in 2 years should provide various mouse models to use in determining the function of HNF4 in the intestine, liver and pancreas. Industry is currently identifying synthetic compounds that control FXR activity. The completion of a maltase-glucoamylase knockout in mice next year should provide a model for others to use in determining whether loss of glucoamylase will protect against type II diabetes in genetically susceptible mice. Potential constraints are that FXR regulators with desirable metabolic effects will only be adopted as therapeutic agents if they do not have additional, undesirable side effects. It will be necessary to confirm all investigations in the human. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)

Impacts
(N/A)

Publications

• Laffitte, B.A., Kast, H.R., Nguyen, C.M., Zavacki, A.M., Moore, D.D., Edwards, P.A. Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor. Journal of Biological Chemistry. 2000. v. 275(14). p. 10638-10647.
• Nichols, B.L., Nichols, V.N., Putman, M., Avery, S.E., Fraley, L.K., Quaroni, A., Shiner, M., Sterchi, E.E., Carrazza, F.R. Contribution of villus atrophy to reduced maltase expression and activity in infants with malnutrition. Journal of Pediatric Gastroenterology and Nutrition. 2000. v. 30. p. 494-502.