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

项目总结 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。 将能量转化为热能。在冷暴露或β3-肾上腺素能受体(β3AR)的药物刺激下， BAT从循环中吸收了大量不同的底物(如脂肪酸、葡萄糖和氨基酸)。 然而，蝙蝠如何协调各种底物的代谢利用来产生热量仍然是个谜。 这个项目的目标是阐明在BAT中协调复杂底物利用的机制。 对寒冷或β3AR刺激的反应。苹果酸-天冬氨酸穿梭(MAS)由胞质和胞质组成。 线粒体酶GOT1/2和MDH1/2是一种促进胞浆净转运的生化系统。 糖酵解产生NADH到线粒体电子传输链(ETC)，同时再生NAD+。 胞浆以维持糖酵解。对于这种转移，MAS利用氨基酸和代谢物的子集作为 穿梭衬底。在初步研究中，我们发现，与其他组织不同，GOT1的表达非常低 在蝙蝠里。然而，冷暴露或药物性β3-肾上腺素能激动剂显著诱导GOT1的表达 在蝙蝠体内，其他酶保持不变。我们的初步研究为GOT1提供了强有力的证据- BAT依赖的MAS激活及其对底物利用的影响：I)GOT1在棕色中过表达 脂肪细胞随着线粒体呼吸的增加而增加胞浆NADH氧化；ii)转基因 在BAT中过表达GOT1增加了小鼠的能量消耗，提高了小鼠的耐寒性；以及iii) 令人惊讶的是，GOT1在BAT中的过表达增强了脂肪酸的氧化，而GOT1的缺失导致了 冷刺激的FA氧化。在这个项目中，我们将测试冷诱导的GOT1作为一个 分子开关开启MAS，MAS反过来作为协调冷刺激的机制 BAT的糖酵解、底物穿梭和FA氧化支持产热。为了检验这一假设，我们 将使用一种将动物生理学与细胞和分子技术相结合的综合方法。在目标1中， 我们将通过稳定同位素示踪、NADH生物传感器Peredox和 高分辨率呼吸测定法。在目标2中，我们将阐明GOT1增强脂肪酸的机制 氧化。在目标3中，我们将描述β3AR信号增强GOT1活性的机制。这项研究 结果将确定Got1是将冷刺激的β3AR信号连接到MAS的关键节点，其中 协调蝙蝠对寒冷的糖酵解、氨基酸利用和FA氧化。

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.