Identifying and characterizing pathways of amino acid metabolism and signaling

In muscle cells a balanced usage of nutrients such as glucose, fatty acids, ketones, amino acids and lactate is a prerequisite for optimal muscle growth. The longstanding view is that generation of energy within the power house of the cells (mitochondria) primarily from glucose and fatty acids stimulates muscle growth and survival. Amino acids particularly branched-chain amino acids (BCAA) such as leucine, isoleucine, and valine are mostly used for manufacturing proteins for growth within muscle but are also used for generating energy. However our understanding of how BCAA are utilized for generating energy within muscle cells is still lacking. Goals of our research are to determine the enzymes mediating this process, how these enzymes are regulated and the impact on the cell when BCAA are used as an energy source. We want to know whether BCAA compete with other nutrients for being consumed by the cell for energy and which pathways regulate the competition process. What is known so far is that, once inside the cell, BCAA are chemically converted to branch chain alpha ketoacids (BCKA) by virtue of the enzymes branch chain amino transferase (BCAT), branch chain ketodehydrogenase (BCKDH) and ketodehydrogenase kinase (BCKDK) to allow BCKA to enter mitochondria for use as an energy source. Despite the widespread presence of amino acid metabolizing enzymes in different tissues their impact on mitochondrial energy generation and function remains unexplored. Using cell biology, genetic and whole body approaches in zebrafish and mouse my research program will focus specifically on addressing two conceptual puzzles. Puzzle 1) to uncover how the nutrient triad glucose, fatty acid and BCAA biochemically communicate with each other resulting in nutrient transport and utilization within the mitochondria; Puzzle 2) to decipher whether within the cell different biomolecules such as ions, hormones, lipids, sugars and neurotransmitters employ the services of amino acids to govern cellular growth and survival. For puzzle 1, studies will be undertaken in zebrafish and mouse evaluating the effect of alterations in concentration and types of BCAA and BCKA on mitochondrial function in skeletal and cardiac muscle tissues. A method for analysing breakdown products of BCAA will be developed. In addition, conventional cell and molecular biology methodologies will be employed to evaluate the effects of altered BCAA and BCKA on murine and zebrafish amino acid metabolic enzymes (BCAT, BCKDH and BCKDK). To establish the contribution of these enzymes to cellular homeostasis, genetically mutated versions of these enzymes tagged with light emitting probes (fluorescence) will be engineered and expressed in murine cells and zebrafish. For resolving puzzle 2, cellular organelles from murine and zebrafish cells and/or tissues subjected to BCAA and BCKA treatment will be collected and subjected to discovery of genes and proteins influencing cellular size, proliferation and survival. To address how amino acids exert physiological effects of hormones, ions, sugars, and neuronal factors, mice and zebrafish will be administered biomolecules to mirror biological effects and cells and tissues harvested for molecular and physiological analysis. This research program will identify and characterize previously unrecognized proteins and novel molecular networks of cellular amino acid signaling, metabolism and physiology. The research will provide opportunities for graduate and undergraduate trainees to directly contribute to expanding our knowledge of amino acid biology, knowledge that will have direct impacts in the fields of mitochondrial biology, food science and human nutrition offering tremendous benefit to Canadian discovery science, education and innovation.