Regulation of brown fat fuel utilization by the malate-aspartate shuttle

项目摘要 啮齿动物和人类的棕色脂肪组织（BAT）具有很高的氧化营养物质和转化营养物质的能力。 把能量转化为热能。在冷暴露或药物刺激β3肾上腺素能受体（β 3 AR）时， BAT从循环中吸收大量不同的底物（例如脂肪酸、葡萄糖和氨基酸）。 然而，BAT如何协调各种底物的代谢利用以产生热量仍然难以捉摸。 本项目的目标是阐明协调BAT中复杂底物利用的机制， 对冷或β3AR刺激的反应。苹果酸-天冬氨酸穿梭体（MAS）由胞浆和胞浆内的蛋白质组成， 线粒体酶GOT 1/2和MDH 1/2是一种生物化学系统，促进细胞溶质的净转移， 糖酵解中产生的NADH进入线粒体电子传递链（ETC），同时在线粒体中再生NAD+。 细胞质以维持糖酵解。对于这种转移，MAS利用氨基酸和代谢物的子集， 穿梭基质。在初步研究中，我们发现，与其他组织不同，Got 1的表达非常低 在BAT然而，冷暴露或药理学β3-肾上腺素能激动剂显著诱导Got 1表达， 在BAT中，而其他酶保持不变。我们的初步研究为GOT 1- 依赖MAS激活及其对BAT中底物利用的影响：i）Got 1在棕色细胞中的过表达 脂肪细胞增加胞质NADH氧化沿着线粒体呼吸的增加; ii）转基因 BAT中Got 1的过表达增加了小鼠的能量消耗并提高了小鼠的耐冷性;和iii） 令人惊讶的是，Got 1在BAT中的过表达增强了脂肪酸氧化，而Got 1缺失导致了脂肪酸氧化水平的下降。 冷刺激FA氧化。在这个项目中，我们将测试冷诱导的GOT 1作为一个假设， 分子开关打开MAS，这反过来又作为一种机制，以协调冷刺激 BAT中的糖酵解、底物穿梭和FA氧化以支持产热。为了验证这个假设，我们 将使用一种综合的方法，将动物生理学与细胞和分子技术相结合。在目标1中， 我们将通过使用稳定同位素示踪，NADH生物传感器Peredox， 高分辨率呼吸测定法在目标2中，我们将阐明GOT 1增强脂肪酸的机制， 氧化在目标3中，我们将描述β3AR信号增加GOT 1活性的机制。研究 结果将确定GOT 1作为一个关键节点，将冷刺激的β3AR信号与MAS联系起来， 协调BAT中的糖酵解、氨基酸利用和FA氧化以响应寒冷。

PROJECT SUMMARY Brown adipose tissue (BAT) in rodents and humans has a high capacity to oxidize nutrients and convert nutrient- derived energy into heat. During cold exposure or pharmacologic stimulation of β3-adrenergic receptor (β3AR), BAT takes up large amounts of diverse substrates (e.g. fatty acids, glucose and amino acids) from the circulation. However, it remains elusive how BAT orchestrates metabolic utilization of various substrates for heat production. The goal of this project is to elucidate the mechanism that coordinates complex substrate utilization in BAT in response to cold or β3AR stimulation. The malate-aspartate shuttle (MAS), which is composed of cytosolic and mitochondrial enzymes GOT1/2 and MDH1/2, is a biochemical system that facilitates the net transfer of cytosolic NADH produced in glycolysis to the mitochondrial electron transport chain (ETC) while regenerating NAD+ in the cytosol to maintain glycolysis. For this transfer, the MAS utilizes a subset of amino acids and metabolites as shuttling substrates. In the preliminary study, we found that, unlike in other tissues, Got1 expression is very low in BAT. However, cold exposure or pharmacological β3-adrenergic agonism markedly induces Got1 expression in BAT while other enzymes remain unchanged. Our preliminary study provides strong evidence for GOT1- dependent MAS activation and its impact on substrate utilization in BAT: i) Got1 overexpression in brown adipocytes increases cytosolic NADH oxidation along with an increase in mitochondrial respiration; ii) Transgenic overexpression of Got1 in BAT increases energy expenditure and improves cold tolerance in mice; and iii) Surprisingly, Got1 overexpression in BAT enhances fatty acid oxidation while Got1 deletion leads to a decline in cold-stimulated FA oxidation. In this project, we will test the hypothesis that cold-induced GOT1 acts as a molecular switch turning on the MAS, which in turn serves as a mechanism to coordinate cold-stimulated glycolysis, substrate shuttling, and FA oxidation in BAT to support thermogenesis. To test this hypothesis, we will use an integrative approach that combines animal physiology with cell and molecular techniques. In Aim 1, we will define the role of GOT1 in MAS activation by using stable isotope tracing, NADH biosensor Peredox, and high-resolution respirometry. In Aim 2, we will elucidate the mechanism by which GOT1 enhances fatty acid oxidation. In Aim 3, we will delineate the mechanism by which β3AR signaling increases GOT1 activity. The study outcomes will identify GOT1 as a critical node that links cold-stimulated β3AR signaling to the MAS, which coordinates glycolysis, amino acid utilization, and FA oxidation in BAT in response to cold.