The purpose of this application is to further Dr. Adalberto Gallegos' transition from the physical sciences into biomedical research relevant to fatty acid metabolism, diabetes, and obesity. In this proposal, the applicant will be encouraged to take advantage of his strong background in chemical engineering and physical chemistry in order to develop novel approaches to investigating the metabolism of fatty acids in living cells. The applicant will be mentored in this transition by experts in both biophysical (Schroeder) and biological (Kier) aspects of biomedical research who are experienced in this training of physical scientists. Specifically, the short-term objectives will be to address the intracellular trafficking of cholesteryl ester derived fatty acids. It is well known that cells derive most of their exogenous cholesterol from cholesteryl esters associated primarily with low density lipoprotein (LDL) and less so with high density lipoprotein (HDL). However, intracellular hydrolysis releases not only cholesterol but also fatty acids from these lipoprotein derived cholesteryl esters. While the intracellular targeting of the cholesterol component of cholesteryl esters is becoming increasing well understood, almost nothing is known regarding the intracellular trafficking and metabolic channeling of the fatty acid portion of this molecule. The immediate goals will be to: (i) Synthesize a series of labeled cholesteryl esters wherein the fatty acid and/or sterol moieties are fluorescent; (ii) Incorporate these fluorescent lipids into lipoproteins (LDL and HDL); (iii) Examine the uptake and intracellular trafficking/targeting of these lipoprotein cholesteryl ester-derived fluorescent fatty acids in primary hepatocytes cultured from mouse liver; and (iv) Determine the role of the liver fatty acid binding protein, L-FABP, in cholesteryl ester-derived fluorescent fatty acid targeting to intracellular organelles for metabolism (mitochondria, peroxisomes, endoplasmic reticulum) or gene regulation (PPARalpha in the nucleus).
These aims will allow the applicant to also be trained in recent advances in imaging technologies for examining synthetic and naturally-occurring fluorescent fatty acid trafficking in living cells by laser scanning confocal microscopy (LSCM) and multiphoton laser scanning microscopy (MPLSM), respectively.
