Source: UNIV OF WISCONSIN submitted to
BENEFICIAL AND ADVERSE EFFECTS OF NATURAL CHEMICALS ON HUMAN HEALTH AND FOOD SAFETY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1009931
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
W-3122
Project Start Date
Oct 1, 2016
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Project Director
Eisenstein, RI, S..
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Nutritional Sciences
Non Technical Summary
Iron is an essential nutrient because it is a required component of proteins that perform essential physiological functions such as oxygen transport, the harvesting of energy from food components so that we can use it for necessary processes of life including work output (muscle function), cognitive function and others. Iron deficiency is common in humans and some production animals, particularly in swine. The only way to alter body iron content is by regulating the efficiency of dietary iron absorption by specific cells in the small intestine. In humans, diseases associated with iron overload and iron deficiency are caused by impaired regulation of the dietary iron absorption. In production animals, iron deficiency is a major issue for swine and all neonatal swine in the US are iron supplemented. Little is known about the developmental regulation of the iron transport machinery in the intestine of neonatal swine and if it limits iron harvest from the diet thus enhancing iron deficiency and anemia. While much has been discovered in recent years about the mechanism of dietary iron absorption focusing on how signals external to the intestine (e.g. from the liver) dictate changes in iron absorption it is not apparent that so-called cell intrinsic mechanism (with the cell type of interest) are also important but less well understood. This proposal focuses on understanding this intrinsic mechanism controlling dietary iron absorption using a genetic approach in mice and applying that information to better understand the developmental control of the intestinal iron absorption machinery in a second proposed study using neonatal swine. Finally, we propose to test the hypothesis that dietary additives that activate the intrinsic mechanism for dietary iron absorption may be of use to the swine industry. The ability of these cells to control dietary iron absorption depends on the level of expression of key iron importer, an iron reductase essential because reduced iron is the form absorbed, and an iron exporter that moves iron from the interior of duodenal cells into the blood for transport to other organs. The expression of these key participants in intestinal iron absorption is controlled by signals within the cell, so called intrinsic signal, and signals from outside these cells (extrinsic signals) that provide information on the size of iron stores in the liver or the demand for iron for production of new red blood cells (the main use of iron in the body). Our work proposes to examine a new participant (a regulatory protein called IRP1) in the machinery through which signals intrinsic to the duodenal cells control expression of the duodenal iron transports and the duodenal iron reductase needed for iron absorption. Our first aim is to examine the role of IRP1 in controlling iron absorption in mice since they are more genetically tractable and a genetically altered mouse line is available that we can use to specifically eliminate IRP1 from intestinal cells. This will allow us to directly determine the role of IRP1 in controlling dietary iron absorption and the extent to which its dictates the ability of animals to modulate absorption in response to physiological or pathologic stresses. We will also focus on a second intrinsic regulator of the iron absorption apparatus, a protein called hypoxia inducible factor 2 alpha (HIF-2). HIF-2 activates expression of the iron transporters and reductase while IRP1 represses HIF-2 synthesis. Thus, in the absence of IRP1 we expect increase dietary iron absorption because the iron transport machinery will be at a higher level. Our second aim proposes to determine the changes in expression of iron transporter, the iron reductase and their regulatory's like IRP1 as a function of development in swine. We will focus on the first two weeks of neonatal life when iron deficiency and associated anemia. We will determine the expression level of IRP1, HIF-2 and the iron transporters/reductase in duodenum. We will also test the hypothesis that the simple organic acids fumarate and acetate, which can activate HIF-2, may serve as an inexpensive dietary additive to enhance dietary iron absorption in swine.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023599104050%
7023599101050%
Goals / Objectives
Determine the mechanisms by which dietary bioactive compounds protect against human diseases. Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention.
Project Methods
We seek to understand the fundamental processes controlling absorption of dietary non-heme iron, how it is dysregulated in disease and how it may be enhanced in humans and production animals. We will conduct studies in genetically altered mice and also in genetically normal swine to further elucidate the mechanisms through which dietary iron absorption is controlled. The mice studies will use standard genetic methods for deleting a gene (for a protein called iron regulatory protein 1, IRP1) from intestinal cells and we will examine the impact of this on the expression of key proteins required for dietary iron absorption. We will also determine if, as we predict, mice lacking intestinal IRP1 are resistant to dietary iron deficiency.Our studies in swine will focus on determining the developmental changes in expression of the iron transport machinery in duodenum during the first 2 weeks of life and the extent to which supplementation of with iron modulates this process. We also will test our hypothesis that cheap organic acids like fumarate or acetate will enhance dietary iron absorption by activating the expression of genes that encode critical components of the iron transport machinery.Results will be analyzed using standard statistical methods, usually t-tests. The campus statistical consulting service will be brought in for assistance in analyses if need be.Efforts:Our target audience of scientists and clinicians will be informed of the new knowledge from our work through presentations at scientific meetings and through publications in scientific journals. In order to inform lay people such as farmers, we will work with UW-Extension and develop a 1 page non-technical description of our work, should our proposed research lead to a significant improvement of swine iron nutriture and reduce the severity or prevalence of anemia in this species.Evaluation: We will evaluate the success of the research as follows. For the work in genetically altered mice, we will determine if the expression of the iron transport machinery in the intestine responds to the absence or presence of IRP1. We will conclude that our approach is successful if the iron transport machinery is increased in mice lacking IRP1 and if the mice are resistant to dietary iron deficiency. For the work in neonatal swine, we will conclude that our work is successful if there are clear developmental changes in IRP1 expression in duodenum in neonatal swine and if this is associated with predicted changes in the iron transporters that IRP1 controls. We will also conclude that our work is successful if the addition of fumarate or acetate to the diet enhances the expression of duodenal iron transporters as we predict.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audiences we interacted with were other research laboratories at UW Madison, other universities where Dr. Eisenstein presented research talks, or at national scientific meetings. The individuals I met with usually included other faculty, graduate students and other scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities included graduate student James Votava working one-on-one with more senior members in the Eisenstein lab and others to learn new techniques. Professional development included James working with his mentor Prof. Eisenstein on an individual development plan, charting out both his immediate professional development goals including improving his seminar presentation skills. How have the results been disseminated to communities of interest?This has occurred through poster sessions during our annual Winter recruiting events for new graduate students. The poster session is open to the public and held in a visible venue on campus. In addition, the results obtained were presented several times in our own lab group meetings. What do you plan to do during the next reporting period to accomplish the goals?Our work continues under Hatch project-1014006. We plan to determine if mice lacking IRP1 or the regulatory element in HIF2 mRNA that IRP1 binds to are resistant to iron deficiency when mice are fed a moderately iron deficient diet (10 to 15 mg Fe/kg diet). Our previous experiments used diets much lower in iron (less than 6 mg Fe/kg diet) which may have been too low. We will determine the growth rate, blood parameters and liver iron stores as a function of the length of time they have been fed the diet. If time permits we will begin to work out methods to quantify mRNA level and possibly protein level for DCytB, DMT1 and Ferroportin (iron transport machinery) in the duodenum of neonatal swine.

Impacts
What was accomplished under these goals? Iron is a required cofactor for proteins essential for oxygen transport (hemoglobin), respiration by mitochondria (electron transport chain proteins), DNA replication and some pathways of intermediary metabolism (lipogenesis). Iron deficiency can cause reduced work output due to anemia and reduced function of muscle mitochondria, impaired cognitive function which can be irreversible and possibly impaired immune function. Iron deficiency due to dietary insufficiency and/or blood loss (i.e. from parasitic infection) results in nearly one-third of the world being iron deficient and a large fraction of these individuals are anemic. Iron deficiency is also a key issue for the swine industry because given the rapid growth rate of neonatal swine they rapidly become iron deficient unless they receive supplements; essentially all neonatal swine in large production facilities in the US are given an iron injection to prevent iron deficiency and promote optimal growth. Because the iron content of the body can only be regulated through changes in the efficiency of dietary absorption it is essential to fully understand the mechanism of absorption, the regulators that control it and the extent to which it changes through development. In recent years, key participants in the process of absorption of dietary non-heme iron have been identified in mammals. This includes an iron reductase (DCytb) which reduces ferric (+3) iron to ferrous )+2) iron, a transporter for the reduced iron (divalent metal transporter 1, DMT1) and an iron exporter (ferroportin, Fpn) that transfers iron from the interior of duodenal mucosal cells of the small intestine to the blood where it is oxidized toFe+3 by a so-called multicopper oxidase, hephaestin. The Fe+3 is then bound by the serum protein transferrin which safely transports iron to cells and tissues. The levels at which these proteins are expressed is determined: a). transcriptionally by the transcription factor hypoxia inducible factor 2alpha (HIF2) in the case of DCytB, DMT1 and Fpn; and b). post-transcriptionally by iron regulatory protein 1 (IRP1) and IRP2 in the case of DMT1 and Fpn. In addition, IRP1 controls the translation of HIF2 through the so-called IRP1-HIF2 axis. We seek to understand the impact of IRP1 control of HIF2 in modulating dietary iron absorption using two models: a). a genetic approach in mice coupled with; b). a developmental and dietary approach in neonatal swine. These findings will have direct impact on the understanding of how dietary iron absorption is controlled in humans. In the past year, we have conducted diet experiments with mice that lack IRP1 or lack the regulatory element in HIF2 mRNA where IRP1 binds and are determining. Both of our animal models should have had an increased expression of HIF2 in multiple tissues including the intestine where we hypothesize there would be higher expression of the iron transport machinery (DCytb etc). We hypothesize that these animals may be resistant to dietary iron deficiency since they may be more efficient at absorbing dietary iron. Our first trials with a very low iron diet showed that the mutant mice became iron deficient at the same rate as wildtype mice. While this result was not anticipated, we are planning a new study using a moderately iron deficient diet to determine if the mutant mice are better able at scavenging dietary iron under these conditions. We hypothesize that in our first experiments the iron level of the test diet was too low.

Publications