Metabolic Determinants of Cardiac Bioactive Sphingolipids in Lipid Overload

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DESCRIPTION (provided by applicant): Obesity and the metabolic syndrome are two overlapping conditions in which the body begins to store excess fat in non-adipose cells, including the heart. This ectopic lipid storage leads to insulin resistance and lipotoxicity, in part because these lipids can serve as substrate for synthesis of a class of signaling lipids known as sphingolipids. Recently, it has been shown that sphingolipids made using different kinds of fat initiate distinct cellular signaling programmes. It is thus important to understand what kinds of fat are preferentially used for sphingolipid synthesis, as well as how sphingolipid levels and production are regulated by different dietary fats. Ceramide, a key sphingolipid signaling molecule, has been implicated in the development of diabetic cardiomyopathy and insulin resistance. Ceramide contains two acyl chains, which are added sequentially by the enzymes serine palmitoyltransferase (SPT) and (dihydro)ceramide synthase (CerS). Isoform-specific patterns of substrate utilization allow synthesis of sphingolipids incorporating different acyl chain lengths, which can have distinct roles. The purpose of the proposed study is to determine the effect of lipid overload on tissue-specific sphingolipid profiles, via the regulation of SPT composition and CerS expression and substrate supply. To do this, we will first analyze in vitro alternative substrate utilization by the murine cardiac SPT complex, and we will analyze the subunit makeup of the enzyme in heart by mass spectrometry. Next, we will quantify the activity of the CerS isoforms toward sphingolipids made from these alternative substrates. Afterward, we will determine how exogenous fatty acids govern the expression of the SPTLC subunits and all six CerS isoforms. Finally, we will measure sphingolipid profiles to ascertain the impact of dietary fat on sphingolipid levels. This series of studies will reveal the relationship of excess dietary fat composition to sphingolipid levels in the heart and will identify lipid species with potential biological relevance. The proposed studies will support a robust research training plan incorporating coursework, presentations at scientific meetings, and both structured and informal interactions with other scientists. Classes will address specific biochemical and biomedical topics as well as statistical methods, and they will complement previous instruction on grant writing, scientific ethics, and utilization of animal and human subjects. Furthermore, the student will attend and participate in a journal club and both departmental and program-sponsored seminar series. Finally, the student will present talks or posters at meetings such as the Southeastern Regional Lipid Conference and the International Charleston Ceramide Conference and submit work for publication in peer-reviewed journals.