DIETARY REGULATION AND ACTION OF APOLIPOPROTEIN C-III: A POTENT MODULATOR OF HYPERTIGLYCERIDEMIA AND CARDIOVASCULAR DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007274
• Project Start Date: Nov 19, 2015
• Project End Date: Jan 13, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269

Performing Department
Nutritional Sciences

Non Technical Summary
Cardiovascular disease is a leading cause death in the United States. Though much is known about how to reduce the risk for developing this disease, there are still a large number of people who die from the disease despite taking advantages of all the suggested dietary and pharmaceutical approaches. Therefore, it is necessary to find new targets and approaches to treating and preventing cardiovascular disease. Apolipoprotein C-III (apoC-III) is one such target. It is a known regulator of cardiovascular disease risk, yet little is known about what it does in the intestine, where it is expressed. We propose to determine how apoC-III functions in the intestine and whether diet can positively impact apoC-III in that tissue. We hypothesize that modulation of apoC-III in the intestine may be preferential to the reduction of cardiovascular disease risk.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
704	3719	1010	100%

Knowledge Area
704 - Nutrition and Hunger in the Population;

Subject Of Investigation
3719 - Other cultured finfish;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

cardiovascular disease

dietary fat absorption

apoc-iii

apolipoprotein

Goals / Objectives
Our long-term goal is to understand how the cardiovascular risk factor apolipoprotein C-III (apoC-III) functions in the intestinal lipoprotein synthesis pathway, its expression in response to changes in diet, and how intestinal apoC-III regulates the metabolism of dietary lipid and subsequent cardiovascular risk factors.This is an important gap in our current knowledge about the treatment and prevention of cardiovascular risk factors, and may be clinically valuable for the development of novel therapies targeting the intestine. The specific goal of this proposal is to examine how dietary components regulate apoC-III expression, and how this regulation, as well as genetic variation in the apoC-III gene, alters lipoprotein secretion and thus cardiovascular disease risk.We propose to accomplish these goals through the following objectives:Specific Aim 1: Role of diet in modulating intestinal lipid absorption and apoC-III expression.Overall Rationale: An increase in apoC-III expression has recently been shown to inhibit intestinal lipid absorption and lipoprotein secretion, though the mechanism is unclear. In the liver, an increase in apoC-III expression is associated with an increase in lipoprotein secretion. This dichotomy has potential clinical implications, since changes in intestinal apoC-III expression could potentially result in lowered plasma triglyceride levels (a major cardiovascular disease risk factor). Little is known about the intestinal regulation of apoC-III, though hepatic apoC-III is inhibited by doxosahexaenoic acid (DHA) and stimulated by insulin. Understanding both the regulation of apoC-III in the intestine and the potential functional implications on lipoprotein synthesis and secretion will be required in order to therapeutically target apoC-III in the intestine. For these studies, dietary fat absorption, intestinal lipoprotein synthesis, secretion, and apoC-III expression will be determined after wild-type mice (age 3-months, n=12/group) are maintained on a fish oil diet (which is rich in DHA) and compared to a control diet (corn oil, unsaturated fat) and a saturated fat diet. Future studies will assess the change in apoC-III regulation in models of metabolic disease (including consumption of a western diet rich in saturated fat and carbohydrate, and in apoE KO mice that are prone to atherosclerosis).Specific Aim 2: Mechanism of action of apoC-III in VLDL synthesis and secretion Overall Rationale: The liver and intestine work cohesively in lipid and lipoprotein metabolism. Multiple transcription factors including Foxo1 and PPARα have been implicated in the regulation of apoC-III in the liver, particularly in cell culture systems. However, the molecular mechanisms of apoC-III action on VLDL synthesis and secretion in the liver have yet to be extensively studied. This aim will determine the molecular step that apoC-III expression alters in the VLDL synthesis and secretion pathway. For these studies, wild-type mice (age 3-months, n=12/group) will be maintained on a chow diet in order to assess lipoprotein synthesis and secretion in both liver and intestine in the normal state.Specific Aim 3. The role of human apoC-III polymorphisms on hepatic and intestinal TAG secretion.Overall rationale: This aim will capitalize on 2 novel cell culture models to determine how 1) decreased expression of intestinal apoC-III, as in the R19X variant, and 2) the Ala23Thr and 1100C>T polymorphisms effect TAG secretion in enteroids. In the first study, we will generate primary intestinal enteroids from mice, which grow from undifferentiated intestinal stem cells into a three dimensional culture containing all the cells lining the small intestine. The use of enteroids to examine fat absorption is novel and will support future studies using this powerful new technique. In addition, we will use differentiated human hepatocyte-like cells to determine the role of these polymorphisms in human liver, and to identify the role of apoC-III polymorphisms in the VLDL synthesis and secretion pathway. These experiments are critical for determining how human apoC-III expression may exacerbate metabolic disease and the potential of apoC-III as a therapeutic target.

Project Methods
We will use two experimental models (transgenic mice and primary intestinal enterocytes), functional assays (enzyme assays, subcellular localization, and nutrient absorption), and a combination of biochemical and molecular approaches to test our hypothesis. The outcome of these experiments will address the current gap in our knowledge surrounding the expression and function of apoC-III.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Scientists and clinicians studying chronic disease, immunometabolism, and nutrition. Changes/Problems:Project will be terminated after this reporting period. A new Hatch proposal was started October 1, 2018, Access Number 1017482. What opportunities for training and professional development has the project provided?Myself and my graduate students have attended and presented at the following meetings: Kohan, A. (2018, June). Intestinal Organoids for Examining Lipid Absorption and Lipoprotein Secretion. Presentation for the Gordon Research Conferences: Lipoprotein Metabolism in the Brain and Circulation and Its Role in Disease. Kohan, A. (2018, June). Career Panel: Careers in Academia and Industry. Panel for the Gordon Research Series. Kohan, A. (2017, November). ApoC-III and lipoprotein synthesis: intersection of chylomicron synthesis and intestinal immune function. Presentation for the South East Lipid Research Conference: University of Kentucky Saha Cardiovascular Research Center. Kohan, A. (2017, July). The role of apoC-III in dietary lipid absorption and the immune system. Presentation for the FASEB Summer Research Conference: Molecular, Physiological and Therapeutic Studies of Intestinal Lipid Transport and Metabolism. Invited seminars: Kohan, A. (2018, June). ApoC-III and intestinal immunity. Presentation for the University of Arizona School of Medicine, Physiology Seminar Series. Kohan, A. (2018, April). ApoC-III regulates intestinal Tregs and protects the gut against colitis. Presentation for the University of Cincinnati School of Medicine Pathology Seminar Series. Kohan, A. (2018, February). Dietary lipid absorption and the immune system. Presentation for the University of North Carolina Chapel Hill Marsico Lung Institue Seminar Series. Kohan, A. (2017, November). ApoC-III at the intersection of dietary fat absorption and intestinal immunity. Presentation for the Harvard School of Medicine Vascular Biology Seminar Series. Kohan, A. (2017, September). The role of apoC-III in intestinal inflammation. Presentation for the University of Kentucky Cardiovascular Seminar Series. How have the results been disseminated to communities of interest?Through public research seminars and publications. What do you plan to do during the next reporting period to accomplish the goals?This progress report marks the end of this grant, but I have received an additional HATCH grant. My proposal is titled "ApoC-III protects against experimental colitis by stimulating intestinal Tregs: is this a potential treatment for IBD?" IBD is hard to diagnose and very difficult to treat, due in part to lack of understanding about the biology of the gut immune system. IBD is hard to bring into remission, and the standard treatment is immunosuppressive drugs that have significant side-effects, including increasing the susceptibility to infection. Nutrition, high levels of stress, and exposure to infection can exacerbate IBD disease, it is critical to determine new targets for IBD therapy that do not include immunosuppressive drugs. My lab studies a key apolipoprotein involved in whole body lipid metabolism - apolipoprotein C-III (apoC-III). We have found that apoC-III is a critical modulator of lipid metabolism in immune cells in the intestine, and these cells, when stimulated with apoC-III become activated to dampen inflammation in the gut. We have now determined that mice expressing the human form of apoC-III (apoC-III transgenic (apoC-IIITg) mice) are highly resistant to colitis (induced with dextran-sulfate sodium (DSS), **P<0.001), and are resistant to all associated pathologies including weight loss, colon shortening, macroscopic damage, diarrhea, and pro-inflammatory secretion of IL-17. We have determined that this dramatic protection from colitis includes a significant increase in anti-inflammatory cytokine IL-10 and in the number of intestinally resident CD25+FOXP3+Tregs, a unique subset of Tregs that are optimally suppressive in the intestinal system. We have identified in vitro that both WT and apoC-IIITg Tregs proliferate rapidly in response to physiologically high levels of plasma apoC-III. Based on these findings, we hypothesize that apoC-III stimulates Treg proliferation, which protects the gut from inflammation, and that increasing plasma apoC-III levels will protect the gut from DSS-induced colitis. We will test this hypothesis using dietary approaches to raise plasma apoC-III levels acutely in mice, as well as in infusion studies of apoC-III during an acute colitis flare-up in mice. We will also use human intestinal organoids as a model system to determine molecular mechanisms for the anti-inflammatory effect of apoC-III and Tregs on gut homeostasis.

Impacts
What was accomplished under these goals? 1) We identified that intestinal apoC-III is uniquely regulated from hepatic apoC-III, and that this does not require dietary nutrients nor FOXO1 (published in BBRC) 2) We identified that apoC-III overexpression results in altered cytosolic lipid droplet formation in the intestine, and this is associated with decreased chylomicron secretion (and and increase in cytosolic lipid droplet catalysis and beta oxidation). We have further identified that this mechanism requires basolateral apoC-III on lipoproteins, and LDLr, and influences intestinal mitochondrial bioenergetics. (Manuscript in preparation for January 2019 submission to JOURNAL OF LIPID RESEARCH) 3) we identified a novel mechanism for apoC-III in the immune system; apoC-III overexpression stimulates intestinal regulatory T cells (Tregs) and their IL-10 secretion. This protects the gut from inflammatory bowel disease. We have further identified that this requires LDLr, and also occurs in LDLr-/- mice. This paper was submitted and revised to NATURE COMMS (and is currently in its second review). Our finding suggest that the role of apoC-III in the gut has significant and unique effects on lipid metabolism in this compartment, and further, has impacts on the intestinal immune system. This is a novel finding, and is a brand new avenue for studying the role of intestine, nutrients, and apoC-III on inflammatory bowel diseases.

Publications

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

Outputs
Target Audience:Scientists and clinicians studying chronic disease, immunometabolism, and nutrition. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students have attended and presented at the following meetings: 1) ATVB 2017 2_ FASEB SRC Lipid Droplets and intestinal lipid processing 2017 How have the results been disseminated to communities of interest?Through published and submitted manuscripts, and through seminars at annual meetings. What do you plan to do during the next reporting period to accomplish the goals?In year 3 of this project, we plan to accomplish the following goals: 1) We will determine how tissue-resident macrophage and T cell populations in the intestine are changed in response to apoC-III expression. 2) We plan to further determine how apoC-III is altering T cell speciation, and whether this requires apoC-III, or simply the high plasma triglycerides that accompany high apoC-III levels (through metabolic analyses and through tissue culture studies). Additionally, we will determine whether diets that include high levels of fish oil also cause beneficial changes in T cells populations and how this relates to cardiovascular disease.

Impacts
What was accomplished under these goals? 1) We identified that intestinal apoC-III is not regulated by FOXO-1 and dietary fat and glucose (published in BBRC) 2) We identified that apoC-III overexpression results in altered cytosolic lipid droplet formation in the intestine, and this is associated with decreased chylomicron secretion (and and increase in cytosolic lipid droplet catalysis and beta oxidation) 3) we identified a novel mechanism for apoC-III in the immune system; apoC-III overexpression stimulates intestinal regulatory T cells (Tregs) and their IL-10 secretion. This protects the gut from inflammatory bowel disease.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: The Isolation, Culture, and Propagation of Murine Intestinal Enteroids for the Study of Dietary Lipid Metabolism. Li D, Dong H, Kohan AB. Methods Mol Biol. 2017 Sep 20. doi: 10.1007/7651_2017_69. [Epub ahead of print] PMID: 28929461
• Type: Journal Articles Status: Published Year Published: 2017 Citation: VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells. Botteri G, Montori M, Gum� A, Pizarro J, Ced� L, Escol�-Gil JC, Li D, Barroso E, Palomer X, Kohan AB, V�zquez-Carrera M.Diabetologia. 2017 Nov;60(11):2262-2273. doi: 10.1007/s00125-017-4401-5. Epub 2017 Aug 23. PMID: 28835988
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Key differences between apoC-III regulation and expression in intestine and liver. West G, Rodia C, Li D, Johnson Z, Dong H, Kohan AB. Biochem Biophys Res Commun. 2017 Sep 23;491(3):747-753. doi:10.1016/j.bbrc.2017.07.116. Epub 2017 Jul 21. PMID: 28739253
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Using primary murine intestinal enteroids to study dietary TAG absorption, lipoprotein synthesis, and the role of apoC-III in the intestine. Jattan J, Rodia C, Li D, Diakhate A, Dong H, Bataille A, hroyer NF, Kohan AB. J Lipid Res. 2017 May;58(5):853-865. doi: 10.1194/jlr.M071340. Epub 2017 Feb 3. PMID: 28159868

Progress 11/19/15 to 09/30/16

Outputs
Target Audience:The proposed studies will add to our knowledge regarding cardiovascular disease risk factors, dietary fat absorption, and dietary strategies for cardiovascular disease risk reduction. Therefore the target audience includes basic/clinical researchers interested in lipoprotein metabolism, apolipoproteins, and the intestine, as well as physicians and individuals who are interested in determining nutritional approaches to metabolic and cardiovascular disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?A publication is currently under review at Journal of Lipid Research. What do you plan to do during the next reporting period to accomplish the goals?In year 2 of this project, we plan to accomplish the following goals: 1) We will complete the 12-week diet studies, and complete our comparison of apoC-III regulation between the liver and intestine in response to diet. These studies will thoroughly determine how intestinal apoC-III is regulated, and allow us to compare tissue-resident macrophage and T cell populations in the intestine when apoC-III expression is altered. 2) We plan to further determine how apoC-III is altering T cell speciation, and whether this requires apoC-III, or simply the high plasma triglycerides that accompany high apoC-III levels. Additionally, whether diets that include high levels of fish oil are protected from these T cell speciation changes is a major goal of the 2nd year of funding 3) We will also continue our studies in apoC-III KO mice, to assess VLDL synthesis and secretion defects, and how diet impacts apoC-III expression.

Impacts
What was accomplished under these goals? In this year of this project, we made major strides towards our stated goals. Specifically: 1) We hypothesized that intestinal apoC-III is down-regulated by dietary intake of DHA via inhibition of FoxO1. We found that duodenal apoC-III is particularly responsive to chronic low fat, high carbohydrate feeding. ChREBP appears to be implicated in the increased expression of apoC-III in response to dietary carbohydrate. These results also confirm that fish oil feeding reduces hepatic apoC-III gene expression. However, the mechanism by which intestinal apoC-III expression is blunted by fish oil remains to be elucidated. In the course of these studies, we have found it necessary to culture primary mouse hepatocytes, both from wild-type and human apoC-III transgenic mice. This additional model enables us to dissect out the differences in regulation between the liver and intestine, and how diet plays into this. We have also taken on a 12-week dietary challenge, in order to see whether raising plasma lipids via western diet may change the regulation of apoC-III in wild-type mice. We are preparing this data for publication. 2) We have undertaken LPS-treatment of wild-type mice, as well as mice overexpressing apoC-III. Interestingly, we have not found any differences in macrophage activation in response to changes in apoC-III levels. Since atherogenesis has been associated with increased activated T-cells and differentiation of T-cells in to pro-inflammatory subsets, we have also discovered a potential role for apoC-III, or the resulting hypertriglyceridemia, in the speciation of circulating T cells. This has involved a novel technique for our lab, flow cytometry, and we are currently preparing this manuscript. 3) We uncovered a relationship between apoC3 overexpression and CD36 regulation and cell localization, and have developed completely new techniques (cell fractionation in intestine, lipid droplet quantification, and enzyme assays). 4) We also uncovered a completely novel immune cell phenotype in my colony of apoC-III transgenic mice, in which T cell populations are skewed to the Treg subset, and with increases in Treg cytokine secretion (IL-10). We have recently found that when these mice are challenged with an atherogenic diet, these Treg differences persist, and seem to involve changes in the concentration of intestinal dendritic cells. This has significant implications for the known roles of apoC-III in cardiovascular disease, diabetes, and other inflammatory diseases. 5) We have obtained and bred a colony of apoC-III KO mice, critical to studies of VLDL synthesis and secretion.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Nothing to report