Abstract DRAYTON, JOSEPHINE BENTLEY. Methylated Medium- and Long-Chain Fatty Acids as Novel Sources of Anaplerotic Carbon for Exercising Mice. (Under the direction of Dr. Jack Odle and Dr. Lin Xi.) We hypothesized that methylated fatty acids (e.g. 2-methylpentanoic acid (2MeP), phytanic acid or pristanic acid) would provide a novel source of anaplerotic carbon and thereby enhance fatty acid oxidation, especially under stressed conditions when tricarboxylic acid (TCA) cycle intermediates are depleted. The optimal dose of 2MeP, hexanoic acid (C6) and pristanic acid for increasing in vitro [1-14C]-oleic acid oxidation in liver or skeletal muscle homogenates from fasted mice was determined using incremental doses of 0, 0.25, 0.5, 0.75, or 1.0 mM. The 0.25 mM amount maximally stimulated liver tissue oxidation of 14 14 14 [1- C]-oleate to CO2 and [ C]-acid soluble products (ASP; P < 0.05). Similar incubations of 0.25 mM 2MeP, C6, palmitate, phytanic acid, or pristanic acid or 0.1 mM malate or propionyl-CoA were conducted with liver or skeletal muscle homogenates from exercised or sedentary mice. In vitro oxidation of [1-14C]-oleic acid in liver homogenates with 2MeP 14 14 increased mitochondrial CO2 accumulation (P <0.05), but no change in [ C]-ASP accumulation as compared to C6 (P > 0.05). Phytanic acid treatment increased [14C]-ASP accumulation in liver tissue as compared to palmitate (P < 0.05). Exercise increased [14C] accumulations (P < 0.05). Results were consistent with our hypothesis that methyl-branched fatty acids (2-MeP, phytanic and pristanic acids) provide a novel source of anaplerotic carbon and thereby stimulate in vitro fatty acid oxidation in liver and skeletal muscle tissues. Next, serum concentrations of β-hydroxybutyrate (BHB) were measured in mice at 0, 1, 2 and 4 h after gavaging 0.110 mL (~13 umol/kg body weight) of one of three triglycerides: tri-hexanoate (t-C6), tri-2-methylpentanoate (t-2MeP) or t-C6 + t-2MeP (mixed). Serum concentrations of BHB peaked after one hour; however, the peak from the mixed triglyceride was less than from t-C6 by 49% (2.52 versus 4.927 mM, respectively; P < 0.05). No change in BHB was observed from t-2MeP (P < 0.05). After two hours, BHB levels decreased in the t-C6 and mixed gavaged mice, but were not different from one hour concentrations (P < 0.05). After four hours, mice dosed with t-C6 still experienced elevated BHB levels as compared to t-2MeP (P < 0.05). Consistent with our hypothesis, an oral bolus of t-C6 induced ketogenesis whereas t-2MeP did not. Finally, 47 mice were blocked for body weight and randomly allocated to a basal control diet or the basal diet with an additional 8% soybeanoil, 8% t-2MeP, 8% t-C6 or mixed diet (4% t-C6 + 4% t-2MeP). Over the course of 9 weeks, feed intake and body weights were measured weekly. At the end of the trial, tissue weights and serum were collected. Feed intake was not affected by dietary treatment (P > 0.05). However, by week 3, mice fed t-2MeP experienced decreased body weights (P < 0.05). By week 4, all mice fed medium-chain triglycerides weighed less than soyoil fed mice (P < 0.05). Mice fed t-2MeP had increased liver weights (P < 0.05). Mice fed t-2MeP had elevated fasting blood urea nitrogen, alkaline phosphatase and BHB but decreased triglyceride levels as compared to soyoil or t-C6 fed mice (P < 0.05). Serum albumin and nonesterified fatty acids were decreased in t-2MeP fed mice as compared to soyoil fed mice (P < 0.05). We found that feeding t-2MeP decreased body weight gain, increased liver weight and affected serum metabolite profiles as compared to t-C6 or soyoil feeding (P < 0.05). Overall, these results are consistent with our hypothesis that the anaplerotic structure of methyl-branched fatty acids affects metabolism and produces a unique physiological response. Methylated Medium- and Long-Chain Fatty Acids as Novel Sources of Anaplerotic Carbon for Exercising Mice by Josephine Bentley Drayton A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Animal Science Raleigh, North Carolina 2012 APPROVED BY: ______________________ ______________________ Dr. R. Andrew Shanely Dr. Michael McIntosh ______________________ ______________________ Dr. Jack Odle Dr. Lin Xi Committee Co-Chair Committee Co-Chair Biography Josie is from Baltimore, Maryland. She received a Bachelor of Science degree in Animal Science with an emphasis in companion animals from the University of California, Davis in 2006. While there, she worked in a nutrition lab and became interested in nutritional biochemistry. In 2010, Josie began working towards a Master of Science degree, studying Animal Science and Nutrition under the guidance of Dr. Jack Odle and Dr. Lin Xi. Josie will be pursuing a Doctor of Veterinary Medicine degree at the University of Minnesota in the fall of 2012. ii Acknowledgements Thank you first and foremost to Dr. Jack Odle for all of the guidance, inspiration and patience. I am so thankful you took me on as a student. Two years went by quickly but I know the “Odle-isms” about details, “re-search” and tenacity will be lessons I take with me in my future endeavors. Thank you for this opportunity- you are an outstanding mentor. Thank you to Dr. Lin Xi for the instruction and encouragement. You are always willing to explain and teach, and troubleshoot and laugh. Thank you so much for the many roles you play in the lab- you have made it a fun and an educational experience. Thank you to Dr. Shanley for sharing your wealth of knowledge on mouse exercise. Thank you Dr. Sean Adams for your patience and forgiveness as I first learned my way around the lab- I sincerely appreciate that you believed in me and helped me continue my education in research. Dr. Tina Herfel & Dr. Sheila Jacobi- thank you for welcoming me into the lab and always being there for me. I am fortunate to have had such excellent role models and teachers. Thank you Dr. McIntosh, Bai Xiumei, Ahmad, Ericca & Heerangi. Amir, Emily, Kolberg and Frances- thank you for the support!! Gampie and Grandad- thank you for always believing in me, supporting me and encouraging me to follow my dreams. I would like to acknowledge the Kannapolis Scholars program, supported in part by a fellowship provided by Agriculture and Food Research Initiative Grant no. 2010-65200- 20354 from the USDA National Institute of Food and Agriculture. iii Table of Contents List of Tables .................................................................................................................x List of Figures ............................................................................................................. xii Chapter 1: Literature Review .........................................................................................1 Triglyceride and fatty acid structure .........................................................................1 Lipid digestion and absorption ..................................................................................2 Beta-oxidation ...........................................................................................................5 Regulation of β-oxidation ..........................................................................................6 Peroxisomal fatty acid oxidation ...............................................................................8 Ketogenesis .............................................................................................................10 Measurement of triglyceride and fatty acid metabolism in animal models ............11 Anaplerosis ..............................................................................................................12 Anaplerotic reactions ...............................................................................................14 Differences between rodent and human exercise physiology .................................16 Evaluation of exercise endurance in mice ...............................................................17 Regulation of fuel selection during exercise ...........................................................18 Anaplerotic methyl-branched fatty acids ................................................................20 Toxicological evaluation of medium chain triglycerides ........................................22 Figures .....................................................................................................................25 References ...............................................................................................................36 Chapter II: Methyl-branched fatty acids provide anaplerotic carbon to stimulate in vitro fatty acid oxidation in exercised mice .................................................................43 iv Abstract ...................................................................................................................43 Introduction .............................................................................................................44 Materials & Methods ...............................................................................................47 Experimental design............................................................................................47 Tissue homogenates ............................................................................................47 14 14 Total fatty acid oxidation: collection of CO2 & [ C]-acid soluble products ...49 Oxygen consumption ..........................................................................................51