BETA, BETA-CAROTENE 9’,10’-OXYGENASE 2 IN HEPATIC MITOCHONDRIAL FUNCTION AND OBESITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006403
• Project Start Date: Jul 1, 2015
• Project End Date: Jun 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078

Performing Department
Human Sciences

Non Technical Summary
The obesity epidemic is one of most significant health challenges to the State of Oklahoma. In the rural areas of Oklahoma, lack of access to affordable fresh fruits and vegetables leads to an even higher level of obesity, compared to that in the urban areas. Such a poor diet lacking fruits and vegetables typically contains a high amount of fat. Thus, to improve the quality of life of individuals in the rural areas, there is an urgent need to understand why increased intake of a high fat diet with low or even no greens results in a higher level of obesity and what the underlying molecular mechanism is.Fresh fruits and vegetables contain a variety of bioactive components, including but not limited to carotenoids that have significant potentials to reduce the incidence of obesity (1). There are two carotenoid metabolic enzymes identified so far. β,β-carotene 9',10'-oxygenase 2 (BCO2) is a newly identified enzyme that catalyzes the asymmetric cleavage of carotenoids in mammals, whereas β,β-carotene 15,15'-monooxygenase 1 (BCMO1) symmetrically cleaves only pro-vitamin A carotenoids (2). Carotenoid cleavage products by BCMO1 and BCO2 may serve as activating ligands or antagonists for homodimeric or heterodimeric nuclear receptor complexes such as retinoid X receptor (RXR)/RXR, retinoic acid receptor (RAR)/RXR, and RXR/PPARs (3).

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
703	7010	1010	100%

Knowledge Area
703 - Nutrition Education and Behavior;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

?

?-carotene 9’

10’-oxygenase 2

mitochondrial function

obesity

Goals / Objectives
The long-term goal of the research project is to better understand how BCO2 regulated by carotenoid-enriched diets contributes to the integrity of mitochondrial function in the development of obesity, and to develop dietary interventions and treatment strategies against obesity. Because the liver is the center of nutrient metabolism, this seed grant will be focused on the fundamental function of BCO2 in the integrity of hepatic mitochondrial function in the liver of healthy and obese mice.Our hypothesis for this seed grant is that a high fat diet-suppression of hepatic BCO2 protein expression causes disturbed integrity of mitochondrial function and subsequently triggers cellular oxidative stress and inflammation. Complete ablation of BCO2 causes those knockout mice to be more susceptible to a high fat diet-induced obesity due to more severe proton leak and subsequent damage to hepatic mitochondria.

Project Methods
Obesity mouse models, diets, and examination of obesity status and body temperature: The breeding colonies of both strains, the genetic background wild type 129S6 (WT) and BCO2 knockout (KO), are maintained in the Animal Resources Facility at Oklahoma State University. Male WT and KO mice at 6 weeks of age will be used in this study. Each animal group will have 12 mice as determined by the statistical power test. Mice will be group-housed (3 mice/cage) in the Animal Resources Facility, according to the institutional approved IACUC protocol. Mice will be fed 2 types of diet, e.g., a control diet (CD, 10 % kCal from fat, catalog # D12450B, purchased from the Research Diets, Inc) and a high fat diet (HD, 45 % kCal from fat, catalog # D12451, purchased from the Research Diets, Inc). Duration of dietary treatments is 28 weeks, e.g., a total of 48 mice will be used (2 strains, 2 diets, 12 mice/group) for 28 weeks. The animals at 6 weeks of age without any dietary intervention will be used as a basal line (i.e., Time 0, a total of 24 mice). At termination of study, animals will be euthanized by CO2 after fasting for 6 hours. Blood and liver tissues will be collected for laboratory analysis. Animal body fat and obesity status will be determined using a Lunar PIXI (GE Healthcare). Mouse body temperature will be measured by use of a typical mouse thermometer.Blood metabolic parameter determination: Blood fats (free fatty acid and triglycerides), inflammatory biomarkers (IL-6, IL-18, and IL-1β), and hormones (leptin and insulin) will be measured by ELISA and/or spectrophotometry, respectively (13).ROS level, ATP production, and Western blot: Mitochondrial ROS production will be measured using a JC-1 assay. Mitochondrial ATP production will be measured by the CellTiter-Glo Cell Viability Kit (Promega, Madison, WI). Altered protein expression will be determined by Western blot using specific antibodies (14).Hepatic mitochondrial function analysis-coupling and electron flow assay: Fresh liver tissues will be subjected to isolation of live mitochondria by traditional homogenization and centrifugation (14). Isolated mitochondrial density will be measured by a BCA protein concentration assay. Mitochondria at 4µg/well will be subjected to mitochondrial function analysis by measuring the oxygen consumption rate (OCR) in the Seahorse XFe96 mitochondrial metabolic analyzer. Specifically, two different assays, coupling and electron flow will be conducted. Coupling assay is to examine the degree of coupling between the electron transport chain (ETC), and the oxidative phosphorylation machinery (OXPHOS), and to distinguish between ETC and OXPHOS with respect to mitochondrial function/dysfunction, such as altered proton leak, basal OCR, and maximum capacity of respiration rate. Electron flow assay is to examine sequential electron flow through different complexes of the ETC (Complexes I, II, III, and IV) which can identify the mechanism of mitochondrial dysfunction or modulation specified in an individual ETC complex. The Seahorse XFe96 Mitochondrial Metabolic Analyzer is newly installed in our laboratories. It is the only equipment on campus and the third in the State of Oklahoma.Global metabolomic profiling: To examine the functional impact of BCO2 on global metabolic phenotype and status of cellular oxidative stress and inflammation in the liver of mice, a global metabolomic profiling assay will be conducted in the Metabolon, Inc (Durham, NC). The Metabolon is a pioneer and industry leader in metabolomics. The Metabolon has helped us to identify altered metabolite profiles in BCO2 KO vs WT mice (see preliminary data above). In brief, the metabolites of liver samples will be identified by ultrahigh performance liquid chromatography-tandem mass spectroscopy and gas chromatography-mass spectroscopy. Multiple bioinformatic analysis methods will be used, including T-test, three-way ANOVA, Hotelling's T2 test, random forest, hierarchical clustering, and principal components analysis. Two types of statistical analysis are usually performed to analyze the collected data: (1) significance tests and (2) classification analysis.Statistical analysis: Overall, our study has three factors: animal strain, diet, and time period of dietary intervention. The first factor, animal strain, has two genotypes: the WT (129S6), and BCO2 KO. The second factor, diet, has two treatments: CD and HD. The third factor, time period, has two treatments: 0 wks and 28 wks. Thus the results can be analyzed by the three-way ANOVA. Since we expect that the HD will have different effects for different animal strains, we will include all interactions between factors in the ANOVA model. Then, all of hypothesis can be performed based on the statistical analysis of ANOVA. The ANOVA method can provide better statistical efficiency and more information than performing many individual student's t-tests, since ANOVA model allows to pool all information together. The overall effects of the three factors can be tested based on the significance of the main effects. Some multiple comparison adjustments, such as Tukey, Bonferroni, and Scheffe adjustments, would be made. The experiments, but not the dietary intervention, will be repeated at least 3 times. Results will be expressed as mean ± S.D. of each group. The statistical significance will be set at p<0.05. The variables of metabolomic data will be measured as described in the previous section.

Progress 07/01/15 to 06/30/17

Outputs
Target Audience:Scientist and lay people in nutrition andmetabolism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Theproject partially supportedgraduate studentsto 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, and USDA PI meetings, USDA review panelists, and other small group meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This is a basic nutritional biochemistry study by targeting the novel role of β, β-carotene 9', 10'-oxygenase 2 (BCO2) in the development of obesity. We discovered that BCO2 is critical to the mitochondrial respiration activity and production of reactive oxygen species (ROS). Deficiency of BCO2 caused mitochondrial dysfunction and impaired energy metabolism in the liver, hypothalamic neurons, and skeletal muscles, resulting in insulin resistance and obesity. The findings are new, leading to the development of NIH R15 proposal (first submission impact scores of 42).Wepresented our updated research findings in the scientific conferences, such as American Society for Nutrition (ASN) annual conferences, and local scientific meetings. In addition to those specified audiences, findings werealso used to develop the slides and assignments for undergraduates and graduates classes, NSCI4143 and NSCI5043 respectively.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: 1. 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: 2017 Citation: 2. 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. 46:74-82
• Type: Journal Articles Status: Published Year Published: 2016 Citation: 3. 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: 4. 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: Journal Articles Status: Under Review Year Published: 2016 Citation: 3. Xin Guo*, Lei Wu*, Winyoo Chowanadisai, Denis M Medeiros, Weiqun Wang, Stephen Clarke, Edralin Lucas, Deana Hildebrand, Brenda Smith, Dingbo Lin. 2016. The role of ?,?-carotene -9, 10-oxygenase 2 in the hypothalamic mitochondrial proteome and inflammation in mice. Exp Biol Med (under review). * equal contribution
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: 1. Lei Wu, Xin Guo, Stephen Clarke, Edralin Lucas, Brenda Smith, Deana Hildebrand, Dingbo Lin. 2017. ?, ?-carotene -9, 10-oxygenase 2 deficiency-associated mitochondrial respiratory chain dysfunction promotes systemic inflammation and initiates dyslipidemia and glucose intolerance. Nature Communication (in submission)
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: 2. Lei Wu, Xin Guo, Sandy Peterson, Deana Hildebrand, Stephen Clarke, Edralin Lucas, Brenda Smith, Weiqun Wang, Denis M. Medeiros, Deana Hildebrand, Dingbo Lin. 2017. Metabolomics reveals the importance of a single functional copy of BCO2 gene in maintenance of hepatic metabolic homeostasis in mice. Scientific Reports (under revision review)

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientist and lay people in nutrition and energy metabolism 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, and USDA PI meetings, USDA 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 more manuscripts for publication

Impacts
What was accomplished under these goals? We conducted the high fat dietary intervention in mouse models for 28 weeks. Histology, biochemistry, transcriptomics, metabolomics, and real time PCR were performed in samples of liver tissues and blood from the mice before and after dietary intervention. The results showed that deficiency of beta-carotene 9,10- oxygenase 2 (BCO2) caused activation of natural killer cells and macrophages, chronic inflammation, dyslipidemia, and relative glucose intolerance. Overnutrition for 28 weeks by fed a high fat diet (45 % kcal from fat, similar to many fast foods provided in the United States of American) made the metabolomics disorders much severe, in particularly inflammation and liver fibrosis. And even cancer. In addition to those findings, we found that increased consumption of carotenoids-enriched diet promoted BCO2 gene expression in the healthy, wild type mice. Metabolomics studies revealed that a large number of known metabolites related to metabolomic disorders were elevated in BCO2 knockout mice even before the dietary intervention. Both objectives were met and well done. The study would be beneficial to the public by providing the possible link between consumption of vegetables and fruits rich in carotenoids and mitochondrial function in mammals.

Publications

• Type: Conference Papers and Presentations Status: Submitted Year Published: 2016 Citation: 2. 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: Submitted Year Published: 2016 Citation: 3. 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: Published Year Published: 2016 Citation: 4. 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: 5. 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. (in press)
• Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: 1. Xin Guo, The impact of beta,beta-carotene 9,10-oxygenase 2 (BCO2) on mitochondrial function and hypothalamic metabolism. M.S. Thesis. 2016

Progress 07/01/15 to 09/30/15

Outputs
Target Audience:Scientist and lay people in nutrition and metabolism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A M.S. student 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. 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 determined whether genetic ablation of BCO2 leads to liver oxidative stress by targeting mitochondrial function in mice at 6 weeks of age with no dietary intervention. The results reveal that compared to the WT, KO mice had significant higher food intake, but displayed lower body weight. The mitochondrial basal respiratory rate, proton leak, and ETC complex II capacity were significantly elevated in the liver of KO compared to the WT. Mitochondrial proteomic profiling results demonstrated that knockout of BCO2 caused altered expression of enzymes and/or proteins involved in fatty acid β-oxidation, the tricarboxylic acid cycle, and ETC/OXPHOS, indicative of mitochondrial dysfunction. The ROS level was elevated in the BCO2 KO liver homogenates. Global metabolomics analyses revealed that the levels of glutathione conjugates e.g., S-lactoylglutathione and 4-hydroxy-nonenal-glutathione were elevated, keeping with cellular oxidative stress in KO mice as more glutathione is required to neutralize ROS. Ablation of BCO2 leads to elevated cellular oxidative stress by altering hepatic mitochondrial function. Our findings indicate that intact BCO2 appears to be critical for proper hepatic mitochondrial function in mice.

Publications