Metabolic Determinants of Cardiac Bioactive Sphingolipids in Lipid Overload

描述（由申请人提供）：肥胖和代谢综合征是身体开始在包括心脏在内的非脂肪细胞中储存多余脂肪的两种重叠情况。这种异位脂质储存导致胰岛素抵抗和脂肪毒性，部分原因是这些脂质可以作为合成一类被称为鞘脂的信号脂质的底物。最近，研究表明，由不同种类的脂肪制成的鞘脂会启动不同的细胞信号程序。因此，了解哪种脂肪优先用于鞘脂合成，以及不同膳食脂肪如何调节鞘脂的水平和生产是很重要的。神经酰胺是一种重要的神经鞘脂信号分子，与糖尿病性心肌病和胰岛素抵抗的发生有关。神经酰胺含有两条酰基链，由丝氨酸棕榈酰转移酶（SPT）和（二氢）神经酰胺合成酶（CerS）依次添加。底物利用的同型特异性模式允许合成含有不同酰基链长度的鞘脂，它们可以具有不同的作用。本研究的目的是通过调节SPT组成、CerS表达和底物供应来确定脂质过载对组织特异性鞘脂谱的影响。为此，我们将首先分析小鼠心脏SPT复合物在体外替代底物的利用，并将通过质谱分析该酶在心脏中的亚基组成。接下来，我们将量化cer异构体对由这些替代底物制成的鞘脂的活性。之后，我们将确定外源性脂肪酸如何控制SPTLC亚基和所有六种CerS亚型的表达。最后，我们将测量鞘脂谱，以确定饮食脂肪对鞘脂水平的影响。这一系列研究将揭示过量膳食脂肪组成与心脏鞘脂水平的关系，并将确定具有潜在生物学相关性的脂质种类。拟议的研究将支持一个强有力的研究培训计划，包括课程作业、在科学会议上的演讲，以及与其他科学家的结构化和非正式互动。课程将讨论特定的生物化学和生物医学主题以及统计方法，它们将补充之前关于拨款写作，科学伦理以及动物和人类受试者的利用的指导。此外，学生将参加和参加一个期刊俱乐部和部门和项目赞助的系列研讨会。最后，学生将在东南地区脂质会议和国际查尔斯顿神经酰胺会议等会议上发表演讲或海报，并提交作品在同行评审的期刊上发表。

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. PUBLIC HEALTH RELEVANCE: Type II diabetes and obesity are both characterized by elevated levels of lipids (fats and oils) in the blood plasma. The purpose of this study is to determine how the body regulates the incorporation of those plasma lipids into signaling molecules in the heart. Since production of these molecules may lead to heart disease, the findings of this study will help researchers to understand how diets high in specific kinds of fat can initiate the development of heart disease in diabetes and obesity.