Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
EGG XANTHOPHYLLS PREVENT CHRONIC INFLAMMATION IN TYPE 2 DIABETES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005630
Grant No.
2015-67018-23176
Cumulative Award Amt.
$149,998.00
Proposal No.
2014-06642
Multistate No.
(N/A)
Project Start Date
Mar 1, 2015
Project End Date
Feb 28, 2018
Grant Year
2015
Program Code
[A1341]- Food Safety, Nutrition, and Health: Function and Efficacy of Nutrients
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Nutritional Sciences
Non Technical Summary
Chronic inflammation is one of the causative factors mediating the pathogenesis of diabetes. Xanthophylls, the oxygenated carotenoids, alleviate inflammation in both humans and rodents. Egg is a common but unique food containing highly bioavailable xanthophylls. In this proposal, we will employ electron microscopy, immunoblotting and immunohistochemistry, ELISA, real-time PCR and Western blot, and HPLC approaches to investigate the molecular targets of egg and its xanthophylls on prevention of chronic inflammation in type 2 diabetes. Our hypothesis is that whole egg dietary intervention activates AMP-activated protein kinase α2 (AMPKα2) and restores mitochondrial turnover, leading to suppression of TNF-α/NF-κB signaling pathway and subsequent amelioration of chronic inflammation in db/db and high fat-induced C57B/6J (B6), but not AMPKα2 knockout diabetic mice. Ablation of β,β-carotene-9′,10'-oxygenase 2 (BCO2) interrupts xanthophyll metabolism in mice. We will use our unique BCO2 knockout mouse model to further confirm the role of xanthophylls as bioactive compounds in egg prevention of diabetic-associated inflammation. We expect that egg consumption results in elevated xanthophyll contents and enhanced anti-inflammation capability in B6 and db/db mice. We also expect disruption of xanthophyll metabolism and/or AMPK function to worsen inflammation status in BCO2 and AMPKα2 knockout mice, which would not be improved by the egg dietary intervention.
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
7013270101010%
7015010101010%
7023270101010%
7025010101010%
7013270103020%
7015010104010%
7023270103020%
7025010104010%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components; 701 - Nutrient Composition of Food;

Subject Of Investigation
3270 - Eggs; 5010 - Food;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1010 - Nutrition and metabolism;
Goals / Objectives
We propose to test the hypothesis that whole eggs and the bioactive xanthophylls prevent chronic inflammation through activation of mitochondrial AMP-activated protein kinase α2 (AMPKα2), which in turn restores mitochondria turnover, and leads to suppression of TNF-α/NF-κB signaling pathway and subsequent amelioration of chronic inflammation in diabetic mice. In the present proposal, we intend to use our unique animal models to determine the egg preventive effect on inflammation at cellular and molecular levels. The specific objectives are: (1) To determine the preventive effect of whole eggs on chronic inflammation in high fat induced diabetic C57B/6J (B6) and in db/db type 2 diabetic mice.(2) To investigate the role of whole eggs and the xanthophylls on activation of AMPKα2, potentiation of mitochondrial turnover, and suppression of TNF-α/NF-κB signaling in diabetic mice.
Project Methods
1 Animals, diets, and systemic inflammation tests: db/db, B6, and KO mice of AMPKα2 and BCO2 will be used in this proposal. Two egg dietary studies will be conducted. Study #1, Egg in high fat-induced diabetic B6 and KO mice of AMPKα2 and BCO2; Study # 2, Egg in db/db diabetic mice. A big batch of eggs will be purchased from a local grocery store, and will be freeze-dried. The egg lutein content will be used as an internal egg quality control as determined by HPLC. We tested the CD and/or HD with 0.5 %, 1 %, and/or 3 % egg diets, and found that the 3% egg diet achieved most significant effects on prevention of inflammation. We will choose 3% egg diets for thisproposal. Thus, four diets will be prepared in the proposal by the Research Diets Inc: CD (10 % kcal from fat, Catalog # D12450B); the low fat egg diet (ED, CD with 3 % (w/w) eggs); the high fat diet (HD, 45 % Kcal from fat, Catalog # D12451); and the high fat egg diet (HE, HD with 3 % (w/w) eggs). At study termination, the concentrations of blood triglycerides, total cholesterol, HDL, LDL, glucose, and TNF-α, IL-1β, IL-8 will be tested using commercial kits. 2 Real-time PCR and Western blot: Liver total RNA will be extracted using the RNeasy RNA isolation kit (Qiagen). cDNA will be generated. Gene expression will be quantified by real-time PCR using the Taqman Real-Time assay. Primers and probes will be purchased from Invitrogen. β-actin will be used as an internal control. Changes of mRNA abundance will then be calculated using the ΔΔCt method (Invitrogen). Liver tissues will be homogenized and whole proteins will be extracted. Western blot will be conducted to detect protein expression as described.3 Hepatic mitochondrial fractionation and measurement of lutein and zeaxanthin contents: Mouse hepatic mitochondria will be enriched and fractionated by traditional centrifugation as described. The pellets of mitochondrial enrich fractions will be collected. The content of mitochondrial lutein and zeaxanthin will be determined by HPLC as described in our previous publication.4 Mitochondrial turnover and respiratory activity: Mitochondrial respiratory activity will be determined with a Clark-type oxygen electrode in a closed chamber containing 500 μl of reaction solution (80 mM KCl, 50 mM MOPS, 5.0 mM Pi, 1.0 mM EGTA, and 1 mg of defatted BSA) at 30°C as described previously. Consumption of oxygen will be measured with glutamate (complex I), rotenone and succinate (complex II), rotenone and duroquinol (complex III), and rotenone, N,N,N′,N′-tetramethyl-p-phenylenediamine, and ascorbate (complex IV). To determine maximal ADP-dependent respiration rates,100 mM ADP will be used. Hepatic mitochondrial turnover will be assessedby determining protein expression of PGC-1α (a biogenesis marker) and ULK1 and p62 (mitophagy markers) and by electron microscopy (EM). For EM, liver tissues will be briefly fixed, and postfixed with osmium tetroxide, dehydrated in ethanol, and embedded. Thin sections (0.5 or 1 μm) will be viewed under a transmission electron microscope at OSU Microscopy Core Facility. Immunohistochemistry will also be conducted to characterize differential expression of BCO2 and AMPKα2 in mitochondria (COXIV will be used as a mitochondrial marker). In brief, liver tissues will be fixed in 4% paraformaldehyde. Cryosections will be labeled with primary antibodies against AMPKα2, BCO2, and/or COXIV, and then labeled with secondary antibodies conjugated with Aluxa Fluor488, 594, and/or 647 (nm). Then the slides will be view under a Leica confocal microscope at OSU Microscopy Core facility.5 AMPK activation, pro-inflammatory cytokine, NF-κB, and TNF-α: To measure mitochondrial AMPKα1 and α2 activities, 100 μg hepatic mitochondrial lysate proteins will be pre-cleaned by Protein A/G PLUS-Agarose according to the manufacturer's instructions (Santa Cruz Biotech, Inc). AMPKα1 and/or α2 proteins will then be pulled down using their specific antibodies, prior to the kinase activity assay using a PathScan phospho-AMPKαhr172) sandwich ELISA kit (Cell Signaling, Inc). The activity will be expressed as the absorbance value at 450 nm. Proinflammatory cytokines IL-1β and IL-8, and TNF-α in plasma and liver tissues will be tested by ELISA and Western blot, respectively. Hepatic NF-κB will be measured by Western blot.

Progress 03/01/15 to 02/28/18

Outputs
Target Audience:Scientists and individuals in the relevant fields. It also includes the industrial personnel interested in health promotion and disease prevention. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant partially supported two graduate students (Lei Wu and Xin Guo) to complete their M.S. and/or Ph.D. thesis/dissertation. The PI and graduate students had presented the results in national nutrition conference and local diabetes research symposium. How have the results been disseminated to communities of interest?Informal discussion and communication during the American Society for Nutrition annual conferences, USDA NIFA review panelists, and other small group meetings. What do you plan to do during the next reporting period to accomplish the goals?We have completed the data analysis, and are submitting two more manuscripts for publication in 2018

Impacts
What was accomplished under these goals? We successfully completed the egg dietary interventions in two separate animal studies as proposed, including the long-term, high-fat diet induced obese/diabetic C57BL/6J (B6) and BCO2/B6 BCO2 knockout mice, and the db/db type 2 diabetic mice. In both studies, consumption of egg decreased plasma pro-inflammatory cytokines levels, and increased anti-inflammatory cytokine IL10 level, especially in BCO2 knockout mice, suggesting that intact egg-derived carotenoids, might be superior in prevention of inflammation compared to their metabolites by cleavage of BCO2. Most significantly, egg consumption for 8 weeks significantly improved the diabetic parameters in db/db type 2 diabetic mice, e.g., lowered blood glucose, insulin, ghrelin, but increased glucagon-like peptide 1 (GLP-1). Biochemical and molecular assessments were performed in the final year of the funding period. The results demonstrated that egg dietary intervention significantly elevated the protein expression of BCO2, but not BCO1 in the liver, suggesting that egg xanthophylls might play a critical roles in regulation of systemic inflammation in mice. Therefore, we further investigated molecular markers, for oxidative stress, and glucose metabolism. Egg consumption decreased protein expression of mTOR and HSP60, though GLP1R and DNMT1 were consistent. Intriguingly, GLUT4 protein expression was largely enhanced with no changes in hepatic gluconeogenesis. Biochemical data suggested thatdietary egg intervention attenuated inflammation and lowered blood glucose through regulation of cellular oxidative stress and the host immune responses. However, we did find unexpected results in cholesterol metabolism in those experimental mouse models. Egg dietary intervention caused the elevation of total blood cholesterol, HDL, LDL, and plasminogen activator inhibitor (PAI-1), but decreased the ratio of HDL/LDL. That might indicate that those mice might have increased risks of thrombosis and atherosclerosis. Also, in the long term egg intervention study, we observed that the incidence of high fat diet-induced liver cancer was significantly lower in the high fat with egg group, compared to the high fat diet ((as a control group). Extra biochemical and histophathophysilogical study was also completed.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Y Lyu, L Wu, X Guo, A Davis, S Clarke, E Lucas, B Smith, W Wang, Denis M Medeiros, Xinchun Shen, D Lin. Targeted metabolomics Reveals Abnormal Hepatic Energy Metabolism in B, B-Carotene-9B, 10B-Oxygenase 2 Knockout Mice. FASEB Journal 31 (1 Supplement), 2017, 655.5-655.5 (poster presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: X Shen, Y Lyu, L Wu, D Lin. Astaxanthin mediates mitochondrial dynamics in mice. FASEB Journal 31 (1 Supplement), 2017, lb422-lb422 (poster presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: L Wu, X Guo, Y Lyu, SL Clarke, EA Lucas, BJ Smith, W Wang, D M Medeiros, X Shen, D Lin. Deficiency of B, B-Carotene-9B, 10B-Oxygenase 2 Links to Systemic Inflammation, Dyslipidemia and Glucose Intolerance. FASEB Journal 31 (1 Supplement), 2017, 797.16-797.16 (oral presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: D Lin, L Wu, X Guo, Y Lyu, S Clarke, E Lucas, B J Smith. Deficiency of B,B-carotene-9,10-oxygenase 2 links to dyslipidemina and glucose intolerance. Harold Hamm Diabetes Center Research Symposium, Oct. 28, 2016
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: L Wu, X Guo, A Davis, T P Soh, S Clarke, E Lucas, B Smith, W Wang, D Medeiros, D Lin. Ablation of BCO2 leads to increased susceptibility to high fat diet-induced metabolic disorders in mice. Experimental Biology 2016, San Diego (oral presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: X Guo, L Wu, T Soh, W Chowanadisai, S Clarke, E Lucas, B Smith, W Wang, D Medeiros, D Lin. Altered fatty acid metabolism remodels the hypothalamic metabolome to stimulate feeding behavior in BCO2 knockout mice. Experimental Biology 2016, San Diego (oral presentation)
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Yi Lyu, Lei Wu, Fan Wang, Xinchun Shen, Dingbo Lin. 2018. Carotenoid supplementation and retinoic acid in regulation of immunoglobulin A production and gut microbiome dysbiosis. Exp Biol Med. 2018, 243:613-620
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Lei Wu, Xin Guo, Yi Lyu, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Deana Hildebrand, Weiqun Wang, Denis M. Medeiros, Xinchun Shen, Dingbo Lin. 2017. Targeted metabolomics reveals abnormal hepatic energy metabolism by depletion of Bcarotene oxygenase 2 in mice. Scientific Reports 2017 Nov 7;7(1):14624. doi: 10.1038/s41598-017-15222-x.
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Lei Wu, Xin Guo, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Deana Hildebrand, Xinchun Shen, Dingbo Lin. 2018. Dietary egg intervention attenuates chronic inflammation and lowers fasting blood glucose in db/db diabetic mice. Mol Nutr Food Res. (In submission)
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Lei Wu, Xin Guo, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Deana Hildebrand, Xinchun Shen, Dingbo Lin. 2018. Dietary egg intervention protects liver cancer induced by a long term high fat diet in C57BL/6J mice. Mol Nutr Food Res (potential). (In submission)
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Xin Guo*, Lei Wu*, Yi Lyu, Winyoo Chowanadisai, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Hui He, Weiqun Wang, Denis M Medeiros, Dingbo Lin. 2017. Ablation of B,B-carotene -9, 10-oxygenase 2 remodels the hypothalamic metabolome leading to metabolic disorders in mice. J Nutr Biochem (accepted). * equal contribution
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Lei Wu, Xin Guo, Steven Hartson, Abby Davis, Hui He, Denis M Medeiros, Weiqun Wang, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Johannes von Lintig, Dingbo Lin. 2016. Lack of B,B-carotene -9, 10-oxygenase leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice. Mol Nutr Food Res. DOI: 10.1002/mnfr.201600576. [Epub ahead of print]
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Lei Wu, Xin Guo, Wenqun Wang, Denis M Medeiros, Stephen L Clarke, Edralin A Lucas, Brenda J Smith, Dingbo Lin. 2016. Molecular aspects of B, B-carotene-9, 10-oxygenase 2 in carotenoid metabolism and diseases. Exp Biol Med. 241(17):1879-1887
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Xin Guo, Lei Wu, Weiqun Wang, Denis M Medeiros, Stephen Clarke, Edralin Lucas, Brenda J Smith, Winyoo Chowanadisai, Dingbo Lin. 2016. Hypothalamic mitochondria in energy homeostasis and obesity. Integrative Molecular Medicine. 3(2):590-599. DOI: 10.15761/IMM.1000209
  • Type: Theses/Dissertations Status: Other Year Published: 2018 Citation: Lei Wu, Ph.D. Dissertation: Carotenoid metabolism and BCO2 in mitochondrial function and inflammation. Fall 2018
  • Type: Theses/Dissertations Status: Other Year Published: 2017 Citation: Xin Guo, M.S. Thesis: The impact of beta,beta-carotene 9,10-oxygenase 2 (BCO2) on mitochondrial function and hypothalamic metabolism, Spring 2017


Progress 03/01/16 to 02/28/17

Outputs
Target Audience:Scientists and individuals in the relevant fields. It also includes the industrial personnel interested in health promotion and disease prevention. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant partially supported two graduate students (Lei Wu and Xin Guo) to complete their M.S. and/or Ph.D. thesis/dissertation. The PI and graduate students had presented the results in national nutrition conference and local diabetes research symposium. How have the results been disseminated to communities of interest?Informal discussion and communication during the Experimental Biology conferences, USDA NIFA review panelists, and other small group meetings. What do you plan to do during the next reporting period to accomplish the goals?Complete the data analysis, submit two-three more manuscripts for publication.

Impacts
What was accomplished under these goals? We successfully completed the egg dietary interventions in two separate animal studies, including the long-term, high-fat diet induced obese/diabetic C57BL/6J (B6) and BCO2/B6 BCO2 knockout mice, and the db/db type 2 diabetic mice. In both studies, consumption of egg decreased plasma pro-inflammatory cytokines levels, and increased anti-inflammatory cytokine IL10 level, especially in BCO2 knockout mice, suggesting that intact egg-derived carotenoids, might be superior in prevention of inflammation compared to their metabolites by cleavage of BCO2. Most significantly, egg consumption for 8 weeks significantly improved the diabetic parameters in db/db type 2 diabetic mice, e.g., lowered blood glucose, insulin, ghrelin, but increased glucagon-like peptide 1 (GLP-1). However, we did find unexpected results in cholesterol metabolism in those experimental mouse models. Egg dietary intervention caused the elevation of total blood cholesterol, HDL, LDL, and plasminogen activator inhibitor (PAI-1), but decreased the ratio of HDL/LDL. That might indicate that those mice might have increased risks of thrombosis and atherosclerosis. Further biochemical and molecular assessments will be performed in the final year of the funding period.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dingbo Lin, Lei Wu, Xin Guo, Yi Lyu, Stephen Clarke, Edralin Lucas, Brenda J Smith. Deficiency of ?,?-carotene-9,10-oxygenase 2 links to dyslipidemina and glucose intolerance. Harold Hamm Diabetes Center Research Symposium, Oct. 28, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lei Wu, Xin Guo, Abby Davis, Traces Petchdee Soh, Stephen Clarke, Edralin Lucas, Brenda Smith, Weiqun Wang, Denis Medeiros, Dingbo Lin. Ablation of BCO2 leads to increased susceptibility to high fat diet-induced metabolic disorders in mice. Experimental Biology 2016, San Diego (oral presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Xin Guo, Lei Wu, Traces Soh, Winyoo Chowanadisai, Stephen Clarke, Edralin Lucas, Brenda Smith, Weiqun Wang, Denis Medeiros, Dingbo Lin. Altered fatty acid metabolism remodels the hypothalamic metabolome to stimulate feeding behavior in BCO2 knockout mice. Experimental Biology 2016, San Diego (oral presentation)
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Xin Guo*, Lei Wu*, Yi Lyu, Winyoo Chowanadisai, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Hui He, Weiqun Wang, Denis M Medeiros, Dingbo Lin. 2017. Ablation of ?,?-carotene -9, 10-oxygenase 2 remodels the hypothalamic metabolome leading to metabolic disorders in mice. J Nutr Biochem (accepted). * equal contribution
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Lei Wu, Xin Guo, Steven Hartson, Abby Davis, Hui He, Denis M Medeiros, Weiqun Wang, Stephen L. Clarke, Edralin A. Lucas, Brenda J. Smith, Johannes von Lintig, Dingbo Lin. 2016. Lack of ?,?-carotene -9, 10-oxygenase leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice. Mol Nutr Food Res. DOI: 10.1002/mnfr.201600576. [Epub ahead of print]
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Lei Wu, Xin Guo, Wenqun Wang, Denis M Medeiros, Stephen L Clarke, Edralin A Lucas, Brenda J Smith, Dingbo Lin. 2016. Molecular aspects of ?, ?-carotene-9, 10-oxygenase 2 in carotenoid metabolism and diseases. Exp Biol Med. 241(17):1879-1887
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Xin Guo, Lei Wu, Weiqun Wang, Denis M Medeiros, Stephen Clarke, Edralin Lucas, Brenda J Smith, Winyoo Chowanadisai, Dingbo Lin. 2016. Hypothalamic mitochondria in energy homeostasis and obesity. Integrative Molecular Medicine. 3(2):590-599. DOI: 10.15761/IMM.1000209
  • Type: Theses/Dissertations Status: Other Year Published: 2017 Citation: Xin Guo, M.S. Thesis: The impact of beta,beta-carotene 9,10-oxygenase 2 (BCO2) on mitochondrial function and hypothalamic metabolism, Spring 2017


Progress 03/01/15 to 02/29/16

Outputs
Target Audience:Scientist and lay people in nutrition and metabolism Changes/Problems:We are repeating the 38-week high fat-dietary study due to loss of 2 cages of mice (1 cage (4) of BCO2 knockout and 1 cage (4) B6). What opportunities for training and professional development has the project provided?A M.S. and a Ph.D. students worked and trained in this project. How have the results been disseminated to communities of interest?The results are being used for a publication in the Journal of Nutrition (in revision). What do you plan to do during the next reporting period to accomplish the goals?Follow the timeline and objectives as proposed.

Impacts
What was accomplished under these goals? We have completed the two dietary studies #1, whole eggs in high fat-induced diabetic C57BL/6J (B6) and KO mice of AMPKα2 and BCO2; Study # 2, Egg in db/db diabetic mice. Blood, liver and muscle tissues were collected and stored at -80 °C freezer. Lab analysis was partially completed. Cytokines of blood samples were determined by the Bio-rad Bio-Plex. The results showed that Whole eggs lowered blood TNF-α, IL-6, IL-1β, and elevated IL-10, suggesting that consumption of eggs attenuated systemic inflammation in diet-induced diabetic mice, and db/db mice, but not AMPKα2 or BCO2 KO mice, though all of the whole eggs treated mice showing higher liver lutein concentrations. We also assessed the mitochondrial autophagy and mitochondrial respiratory activity in diabetic db/db mice that consumption of whole eggs significantly enhanced hepatic mitochondrial biogenesis and potentiated mitophagy, and activated mitochondrial respisratory activities in the electron transport chain complex I and II in db/db diabetic mice. Further, dietary whole eggs did not impact blood glucose control in either diet-induced diabetic or db/db mice. Problem: During the 38-week high fat dietary intervention, a cage (4) of B6 mice and a cage (4) of BCO2 knockout mice mice were not as healthy as others. They were terminated, therefore that caused decreases in the animal group size. I am planning to repeat the the dietary study in year 2.

Publications