Metabolic Determinants of Cardiac Bioactive Sphingolipids in Lipid Overload

描述(申请人提供)：肥胖和代谢综合征是两种重叠的情况，身体开始将多余的脂肪储存在非脂肪细胞中，包括心脏。这种异位的脂肪储存会导致胰岛素抵抗和脂肪毒性，部分原因是这些脂类可以作为合成一类称为神经鞘脂脂的信号脂的底物。最近，有研究表明，由不同种类的脂肪制成的鞘脂启动了不同的细胞信号程序。因此，重要的是要了解哪种脂肪优先用于鞘脂合成，以及不同的膳食脂肪如何调节鞘脂的水平和产量。神经酰胺是一种关键的鞘磷脂信号分子，与糖尿病心肌病和胰岛素抵抗的发生密切相关。神经酰胺含有两个酰基链，由丝氨酸棕榈酰转移酶(SPT)和(二氢)神经酰胺合成酶(CERs)依次加成。底物利用的异构体特定模式允许合成含有不同酰链长度的鞘磷脂，这可能具有不同的作用。本研究的目的是通过调节SPT的组成、CERs的表达和底物的供应来确定脂质过载对组织特异性鞘磷脂的影响。为此，我们将首先分析小鼠心脏SPT复合体对替代底物的体外利用，并通过质谱仪分析心脏中酶的亚单位组成。接下来，我们将量化CERs亚型对由这些替代底物制成的鞘磷脂的活性。然后，我们将确定外源脂肪酸如何控制SPTLC亚基和所有六种CERs亚型的表达。最后，我们将测量鞘脂的分布，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饮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健全的研究培训计划，包括课程作业、在科学会议上的陈述以及与其他科学家的有组织的和非正式的互动。课程将涉及特定的生化和生物医学主题以及统计方法，它们将补充先前关于拨款撰写、科学伦理以及动物和人类科目的利用的教学。此外，学生还将参加一个期刊俱乐部，以及部门和项目赞助的系列研讨会。最后，学生将在东南地区脂肪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议的期刊上发表。

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.