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基因表达与营养相互关系的研究进展
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基因表达与营养相互关系的研究进展
官方公共微信&您当前的位置：&>&
营养物质与基因的相互作用时间： 11:34:20&&来源：&&作者：
项目来源 ： 美国&
项目名称 ： 营养物质与基因的相互作用&
承担机构 ： Children's Nutrition Research Center (Houston, Tx)&
项目编号 ： -044-00&
起止时间 ： -&
项目成员 ： DAN R UPCHURCH&
联系方式 ：
Southern Plains Area
Area Director
Phone: (979) 260-9346
Fax: (979) 260-9415
1 HOLLEMAN DRIVE, EAST
COLLEGE STATION, TX, &
资助机构 ： 美国农业部&
项目简介 ：
1.表征核受体超家族内部新近发现的代谢调节的作用，包括PPARs，LXRs, FXR, CAR和PXR,作
为营养物和其他天然产物的主要对象，它们对代谢途径具有直接的调节作用。2.确定顺式和反式
细胞调控基因，这些基因有助于上调膜麦芽糖-糖化酶KO鼠中多余的可溶性麦芽糖-糖化酶。3.
检验以下的假设，即相对于短饱和脂肪酸（如棕榈酸酯）来说，长链不饱和脂肪酸（如油酸）能
够在更大程度上加强脂肪酸应答基因的表达。 4.了解尿素循环障碍在以下两方面的直接影响：
一方面是维持一种酶紊乱存在下的尿素生产，另一方面是维持一氧化氮的生产。
1.查明调节核激素受体活性的营养制品及其他天然产物，具体侧重于PPAR （过氧化物酶体增
殖物激活受体）亚型；确定积极促进因素，这些因素可以调节受体功能;标明这些因素在受体功
能和目标基因层面上的行为特点。2. 伴随着膜麦芽糖-糖化酶的消融，小鼠体内的可溶性麦芽
糖-糖化酶讯息将增加6倍，这个增幅，存在于哺乳以及断奶的老鼠中。这带来了过饮食碳水化
合物产生的基因调控的一个新模型，这一上调中的调控基因正在通过微阵列分析处于调查中，并
会通过mICcl2细胞系体外研究中的机制予以证实并扩大。3.测量具体的PPAR(alpha) and
PPAR(beta/delta)激动剂对分离心肌细胞中的代谢基因表达的影响；并确定PPAR(alpha) and
PPAR(beta/delta) 的流失是否会减衰独特脂肪酸种类对小鼠模型代谢基因表达的影响。4.调查
鸟氨酸内生供应和尿素循环障碍现状的遗传背景。&
研究进展 ：
4 Annual Report
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?
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. Research is being conducted to
characterize the role of newly identified metabolic regulators within the nuclear
receptor superfamily, as targets of nutrients and other natural products that have direct
regulatory effects on metabolic pathways. Increased understanding of these receptors
will provide greater comprehension of metabolic disorders. Additional research will take
place to determine the cell regulatory genes that contribute to the upregulation of the
redundant soluble maltase-glucoamylase in the membrane maltase-glucoamylase.
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 objective is to
determine the mechanisms by which dietary starch interacts with the gene expressing
maltase-glucoamylase (MGA) which is the gate-keeping enzyme that determines small
intestinal starch digestion into glucose. The function and regulation of
maltase-glucoamylase is under investigation in subjects that are deficient in starch
digestion.
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 Define roles of soy compounds as ligands for PPAR isoforms and begin
characterization of effects of soy compounds on wild type mice.
Explore starch partitioning and dietary composition, generate the Mgamso construct, and
define the effect of weaning on transcription factor binding to the Mgam promoter.
Year 2 Characterize effects of soy compounds on other PPAR isoforms and other nuclear
receptors.
Determine the fate of 13C-starch in the Mgamme KO, generate the Mgamso KO mice, and define
the effect of diet on transcription factor binding to the Mgam promoter.
Year 3 Define role of soy compounds as PPAR ligands at the levels of target genes and
appropriate mouse knockouts.
Initiate phenotypic analysis of the Mgamso KO mice and characterize the effect of dietary
glucose on transcription factor binding to the Mgam promoter.
Year 4 Define role of nuclear receptors in mediating effects of additional potential
metabolic regulators.
Determine the fate of 13C-starch in the Mgamso KO and characterize the effect of
microflora on transcription factor binding to the Mgam promoter.
Year 5 Characterize role of nuclear receptors in metabolic regulatory effects at the
levels of target genes using gene arrays and appropriate mouse knockouts.
Test the effect of a Mgam enzyme inhibitor used in management of type II diabetes,
acarbose, on phenotype and on Mgamme and Mgamso message levels.
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.
This research project has recently received peer
therefore no
milestones were reached during the reporting year.
B. List the milestones (from the list in Question #2) that you expect to address over
the next 3 years (FY , & 2007). What do you expect to accomplish, year by year,
over the next 3 years under each milestone?
Year 1 Define roles of soy compounds as ligands for PPAR isoforms and begin
characterization of effects of soy compounds on wild type mice. -Accomplishment will
include defining the role of PPARa and PPARg, and also for the effects of soy compounds
on wild type mice.
Explore starch partitioning and dietary composition, generate the Mgamso construct, and
define the effect of weaning on transcription factor binding to the Mgam promoter.
-Complete research that relates to dietary composition and starch partitioning, and
understand effects of weaning.
Year 2 Characterize effects of soy compounds on other PPAR isoforms and other nuclear
receptors. - The soy compound studies will identify the biological effects in mice and
critically test the roles of PPAR isoforms in such effects.
Determine the fate of 13C-starch in the Mgamme KO, generate the Mgamso KO mice, and define
the effect of diet on transcription factor binding to the Mgam promoter. - These studies
will define primary functional roles of the Mgamme and Mgamso isoforms in the processing
of 13C-starch.
Year 3 Define role of soy compounds as PPAR ligands at the levels of target genes and
appropriate mouse knockouts. -The soy studies will identify the biological effects in
mice and critically test the roles of PPAR isoforms in such effects. These studies will
provide insights into the potential impact of soy products on glucose metabolism and
Initiate phenotypic analysis of the Mgamso KO mice and characterize the effect of dietary
glucose on transcription factor binding to the Mgam promoter. - These studies will define
primary functional roles of the Mgamme and Mgamso isoforms when absorbing dietary
4.What were the most significant accomplishments this past year?
A. Single most significant accomplishment during FY2004: None. Accomplishments for FY
4 may be viewed on -034-00D.
B. Other significant accomplishments, if any: None. Accomplishments for FY 2004 may be
viewed on -034-00D.
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.
To provide a sense of history and continuity that ties this research project with that
of the previous project, please refer to project -034-00D for past
accomplishments.
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?
7.List your most important publications in the popular press and presentations to
organizations and articles written about your work.
5 Annual Report
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?
This research unit addresses nutrient - gene interactions and investigates these
interactions through two individual research projects:.
1)Natural products and nuclear receptors: PPARs; and.
2)Maltase-glucoamylase, regulator of starch digestion. This research addresses ARS
Human Nutrition National Program (NP107), Component 2: Diet, Genetics, Lifestyle, and
the Prevention of Obesity and Disease, Priority Objective B. Mechanism of Action -
Identify and fully characterize mechanisms of action for beneficial effects of known
nutrients and other potentially benefici measure the size of the
effects associated with specific amounts of the chemical or nutrient component in
question. It conforms to ARS Strategic Plan Goal 4 Improve the Nation's Nutrition and
Health, specifically Objective 4.1: Promote Healthier Individual Food Choices and
Lifestyles and Prevent O Improve Human Health by Better Understanding the
Nutrient Requirements of Individuals and the Nutritional Value of F Determine Food
Consumption Patterns of Americans.
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 Program
(NP107) is to characterize the mechanism of action of nutrients and other agents with
potentially beneficial effects on such disorders.
Project 1: Natural products and nuclear receptors: PPARs 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 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 (MGA). Maltase-glucoamylase is the gate-keeping enzyme that
determines human small intestinal starch digestion into glucose. The function and
regulation of maltase-glucoamylase is under investigation in those subjects that are
deficient in starch digestion.
New discoveries in our lab have shown that both Mgamme and Mgamso are differentially
spliced in a variable region. 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. We are preparing expression constructs of these spliced
forms 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 Mgamme and
Mgamso and to identify the foods and other natural products that regulate splicing. The
function of the protein domains in the Mgamme and Mgamso will be investigated in
expression systems. 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.
Project 1: Natural products and nuclear receptors: PPARs Year 1 (FY 2005): Define roles
of soy compounds as ligands for PPAR isoforms and begin characterization of effects of
soy compounds on wild type mice.
Year 3 (FY 2007): Characterize effects of soy compounds on other PPAR isoforms and other
nuclear receptors. Initial characeterization of nuclear receptor effects of biochanin
A, formononetin and additional potential metabolic regulators.
Define role of soy compounds as PPAR ligands at the levels of target genes and appropriate
mouse knockouts.
Year 5 (FY 2009): Define role of nuclear receptors in mediating effects of additional
potential metabolic regulators.
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 (FY 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. Weaning on
footprinting and EMSA TF confirmation. Conduct a food starch feeding trial.
Year 2 (FY 2006): Determine the fate of 13C-starch in the Mgamme KO, generate the Mgamso
KO mice, and define the effect of diet on transcription factor binding to the Mgam
promoter. Diet on footprinting and EMSA TF confirmation.
Year 3 (FY 2007): Initiate phenotypic analysis of the Mgamso KO mice and characterize
the effect of dietary glucose on transcription factor binding to the Mgam promoter.
Glucose on footprinting, EMSA TF confirmation, and mlCcl2 promoter expression.
Year 4 (FY 2008): Determine the fate of 13C-starch in the Mgamso KO and characterize
the effect of microflora on transcription factor binding to the Mgam promoter. Microflora
on footprinting, EMSA TF confirmation, and mlCcl2 promoter expression.
Year 5 (FY 2009): Test the effect of a Mgam enzyme inhibitor used in management of type
II diabetes, acarbose, on phenotype and on Mgamme and Mgamso message levels. Feedback
regulation of footprint, EMSA TF confirmation, TF KO on Mgamme and Mgamso.
4a.What was the single most significant accomplishment this past year?
Project 1: Natural products and nuclear receptors: PPARs UNDERSTANDING THE MOLECULAR
BASIS OF THE HEALTH BENEFITS ASSOCIATED WITH ISOFALVONES The potential health benefits
of soy isoflavones have been widely publicized, but the molecular basis for such effects
is unclear. Researchers hypothesized that, in addition to their modulation of estrogen
receptor activity, they would also adjust the activity of other nuclear receptors. This
hypothesis has now been shown to be correct, by our lab as well as simultaneously in
the labs of others. Specifically, Children's Nutrition Research Center scientists have
found that both genistein and daidzein activate PPAR, the anti-diabetic target receptor
PPARgamma. These results directly link nutritional isoflavones to metabolic regulatory
pathways and provide a basis for specific tests of their biological effects and the
mechanisms that underlie them.
4b.List other significant accomplishments, if any.
Project 2: Maltase-glucoamylase, regulator of starch digestion GENE REPLICATION IN
MAMMALIAN SPECIES A greater understanding of gene replication in mammalian species is
needed. Researchers at the Children's Nutrition Research Center in Houston, TX, have
discovered that the gene for Mgam is replicated in all mammalian species. The replicated
regions are expressed as a variable region and contain a fixed catalytic site but variable
substrate binding domains. Food starches vary greatly between plant species, and we
hypothesize that the ability to differentially splice Mgam is a response to the
variability of food starch. It is recognized that starch digestion can be just as complex
as the spectrum of food starches. Our strategy is to express the spliced messages as
recombinant proteins so that the substrate binding can be analyzed and recognizing that
Mgam is replicated in all mammalian species is a considerable finding that will enhance
research in this area.
4c.List any significant activities that support special target populations.
5.Describe the major accomplishments over the life of the project, including their
predicted or actual impact.
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
is unclear. Researchers hypothesized that, in addition to their modulation of estrogen
receptor activity, they would adjust 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 PPAR the anti-diabetic target
receptor PPARgamma. These results directly link nutritional 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 women who supplement
their diet with soy products.
Project 2: Maltase-glucoamylase, regulator of starch digestion While the digestion of
sucrose and lactose is accomplished by a single message, the digestion of starch is either
redundant or highly variable in the response to complex nature of food starches. The
future design of food starches for meeting health objectives will need to recognize the
spectrum of Mgam splicing and starch digestion. We have determined that the paralogous
regions also exist in the human MGAM gene and that two variable regions between exons
22-44 exist at the message level. The design of food starches will need to consider these
findings for considering appropriate consumer digestion.
This research addresses ARS Human Nutrition National Program (NP107), Component 2: Diet,
Genetics, Lifestyle, and the Prevention of Obesity and Disease, Priority Objective B.
Mechanism of Action - Identify and fully characterize mechanisms of action for beneficial
effects of known nutrients and other potentially benefici measure
the size of the effects associated with specific amounts of the chemical or nutrient
component in question. It conforms to ARS Strategic Plan Goal 4 Improve the Nation's
Nutrition and Health, specifically Objective 4.1: Promote Healthier Individual Food
Choices and Lifestyles and Prevent O Improve Human Health by Better Understanding
the Nutrient Requirements of Individuals and the Nutritional Value of F Determine
Food Consumption Patterns of Americans.
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?
7.List your most important publications in the popular press and presentations to
organizations and articles written about your work. (NOTE: List your peer reviewed
publications below).
Project 1: Natural products and nuclear receptors: PPARs Moore, D. 2005. Nuclear hormone
receptors are targets for new metabolic regulators. Invited presentation, October 15,
5, Beijing, China.
Moore, D. 2005. New targets for old treatments - An interface between traditional and
modern medicine. Invited presentation, University of Houston, March 30, 2005.
Moore, D. 2004. The coffee diterpene cafestol as an FXR and PXR ligand. FASEB 2004, San
Diego, California.
6 Annual Report
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? Why does it matter?
This CRIS research unit addresses nutrient - gene interactions and investigates these
interactions through three individual research projects:.
1)natural products and nuclear receptors: PPARs;.
2)maltase-glucoamylase, regulator and 3)nutrient regulation of
cardiac gene expression during diabetes. This research addresses ARS Human Nutrition
National Plan (NP107), Component 2: Diet, Genetics, Lifestyle, and the Prevention of
Obesity and Disease, Priority Objective B. Mechanism of Action - Identify and fully
characterize mechanisms of action for beneficial effects of known nutrients and other
potentially benefici measure the size of the effects associated
with specific amounts of the chemical or nutrient component in question, as well as
Strategy 3.1.1 - Human Nutrition Requirements: Determine requirements for nutrients and
other food components of children, pregnant and lactating women, adults, and elderly
of diverse racial and ethnic backgrounds. It conforms to ARS strategic plan Goal #4
Improve the Nation's Nutrition and Health, specifically Objective 4.1: Promote Healthier
Individual Food Choices and Lifestyles and Prevent O Improve Human Health by
Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value
of F Determine Food Consumption Patterns of Americans.
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.
2.List by year the currently approved milestones (indicators of research progress)
Project 1: Natural products and nuclear receptors: PPARs Year 1 (2005) Define roles of
soy compounds as ligands for PPAR isoforms and begin characterization of effects of soy
compounds on wild type mice.
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.
4a.List the single most significant research accomplishment during FY 2006.
Project 3: Nutrient Regulation of Cardiac Gene Expression during Diabetes Understanding
Fatty Acid impact on Heart Cells Diabetes mellitus increases risk for cardiovascular
disease, and exposes the heart to high plasma fatty acid levels, which induce genes
promoting fatty acids. 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
muscular tissue cells (cardiomyocytes) of the heart. 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 investigated, in
terms of initial rates of induction of fatty acid-responsive genes, was
oleate&stearate&palmitate&=linoleate&octanoate. Oleate and linoleate caused
relatively sustained induction of metabolic genes, and gene-specific effects were also
palmitate exhibited relatively small effects, as compared to the other fatty
acids investigated. These findings may explain why diets high in unsaturated fatty acids
are cardioprotective, while diets rich in saturated fatty acids are not. (ARS Human
Nutrition National Plan (NP107) Component 2)
4b.List other significant research accomplishment(s), if any.
Project 1: Natural products and nuclear receptors: PPARs Equol Activation of Xenobiotic
Receptors Vegetables and fruits (especially soybean) contain substances termed
phytochemicals that alter certain body functions, and isoflavones are just one type of
phytochemical found in plant foods. Increasing our knowledge base of these isoflavones
is important as scientists understand their biological impact on consumers. Equol
(7-Hydroxy-3-(4'-hydroxyphenyl)-chroman) is an end product metabolite of the soy
isoflavone daidzein that accumulates in some individuals exposed to soy products.
Additionally, the presence or absence of equol is thought to alter the biological effects
of isoflavones, yet environmental and/or genetic factors also affect equol production.
Through their studies of this metabolite, Children's Nutrition Research Center
researchers have discovered that equol activates the xenobiotic receptor,
pregnane-x-receptor (PXR). Such activation could increase overall drug metabolism,
indicating the existence of potential food-drug interactions, and also alter the
metabolism of soy isoflavones themselves. (ARS Human Nutrition National Plan (NP107)
Component 2)
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
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)
4c.List significant activities that support special target populations.
5.Describe the major accomplishments to date and their predicted or actual impact.
This research addresses ARS Human Nutrition National Plan (NP107), Component 2: Diet,
Genetics, Lifestyle, and the Prevention of Obesity and Disease, Priority Objective B.
Mechanism of Action - Identify and fully characterize mechanisms of action for beneficial
effects of known nutrients and other potentially benefici measure
the size of the effects associated with specific amounts of the chemical or nutrient
component in question, as well as Strategy 3.1.1 - Human Nutrition Requirements:
Determine requirements for nutrients and other food components of children, pregnant
and lactating women, adults, and elderly of diverse racial and ethnic backgrounds. It
conforms to ARS strategic plan Goal #4 Improve the Nation's Nutrition and Health,
specifically Objective 4.1: Promote Healthier Individual Food Choices and Lifestyles
and Prevent O Improve Human Health by Better Understanding the Nutrient
Requirements of Individuals and the Nutritional Value of F Determine Food
Consumption Patterns of Americans.
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
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.
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?
Project 1: Natural products and nuclear receptors: PPARs Results of studies with soy
isoflavones have been summarized in recent review articles. More detailed and
mechanistic peer-reviewed studies will also be published. The impact of soy isoflavones,
particularly equol, on drug metabolism may have practical effects on application of
dietary and nutriceutical strategies employing such compounds.
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.
7.List your most important publications in the popular press and presentations to
organizations and articles written about your work. (NOTE: List your peer reviewed
publications below).
Project 2: Maltase-glucoamylase, regulator of starch digestion Nichols, BL,
Quezada-Calvillo R, Robayo, CC, Sterchi EE, Baker SS. Sucrase-Isomaltase Damps Rate Of
Mealtime Small Intestinal Mucosal Starch Oligomer Digestion To Glucose While
Maltase-Glucoamylase Amplifies During Snacking. ASN/EB, SF, CA. April 3, 2006: Poster
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,
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,
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,
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&&
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