Branched-chain Keto-acids and Aerobic Glycolysis in Vascular Smooth Muscle Cells

项目摘要。合成表型的血管平滑肌细胞（VSMCs）满足其能量 主要是通过有氧糖酵解。缺氧诱导因子-1 α（Hif-1α）诱导一种复合物 这是一个转录程序，在低氧环境下促进糖酵解。在常氧条件下，HIF-1α 通过脯氨酰羟基化和泛素化进行蛋白酶体降解。在从事以下活动的VSMC中， 然而，有氧糖酵解，HIF-1α是稳定的机制仍不清楚。初步数据 提供了对Hif-1α常氧稳定机制的深入了解，表明Hif-1α依赖性 有氧糖酵解仍然是ATP的主要来源;从VSMCs获得的条件培养基 含有稳定Hif-1α的低分子量因子;这些因子的初步鉴定 表明它们包括支链酮酸（BCKAs）、α-酮异己酸（KIC）、α- 酮基-β-甲基戊酸（KMV）和α-酮基异戊酸（KIV），源自其母体支链 必需氨基酸（BCAA）、亮氨酸、缬氨酸和异亮氨酸。鉴于这些初步 结果，该建议的中心假设是合成的VSMCs参与有氧糖酵解 通过BCKAs对HIF-1α稳定性的影响。为了解决这一假设，我们提出以下建议 具体目标：1）我们将研究VSMCs中BCKA合成的决定因素及其调节; 2） 我们将研究BCKAs对HIF-1α稳定性的影响，并探索潜在的潜在分子机制。 机制; 3）我们将研究BCKAs对VSMC表型的影响， 表型转换和代谢重编程之间的关系。BCKAs对VSMC的作用 还将在肺动脉高压的动物模型中研究表型和代谢。结果 这些研究将为深入了解HIF-1α稳定性的分子机制提供有用的信息 和有氧糖酵解，BCKA在这一过程中的作用，以及BCKA- 依赖有氧糖酵解和VSMC表型和病理表型。

Project Summary. Vascular smooth muscle cells (VSMCs) of a synthetic phenotype meet their energy requirements largely via aerobic glycolysis. Hypoxia-inducible factor-1α (Hif-1α) induces a complex transcriptional program that facilitates glycolysis in the setting of low oxygen tension. In normoxia, Hif-1α undergoes proteasomal degradation via prolyl hydroxylation and ubiquitination. In VSMCs engaged in aerobic glycolysis, however, Hif-1α is stabilized by mechanisms that remain unclear. Preliminary data provide insight into the mechanism of this normoxic stabilization of Hif-1α, showing that Hif-1α-dependent aerobic glycolysis remains the primary source of ATP; that conditioned media obtained from VSMCs contains low-molecular-weight factors that stabilize Hif-1α; and that initial identification of these factors indicates that they comprise the family of branched-chain keto-acids (BCKAs), α-ketoisocaproate (KIC), α- keto-β-methylvalerate (KMV), and α-ketoisovalerate (KIV), derived from their parent branched-chain essential amino acids (BCAAs), leucine, valine, and isoleucine, respectively. Given these preliminary results, the central hypothesis of this proposal is that synthetic VSMCs engage in aerobic glycolysis through the effect of BCKAs on Hif-1α stabilization. To address this hypothesis, we propose the following specific aims: 1) we will examine the determinants of BCKA synthesis in VSMCs and their regulation; 2) we will examine the effect of BCKAs on Hif-1α stabilization and explore potential underlying molecular mechanisms; and 3) we will examine the effect of BCKAs on VSMC phenotype and examine the relationship between phenotype switching and metabolic re-programming. The role of BCKAs on VSMC phenotype and metabolism will also be studied in animal models of pulmonary hypertension. The results of these studies should provide useful insight into molecular mechanisms underlying Hif-1α stabilization and aerobic glycolysis in VSMCs, the role of BCKAs in that process, and the relationship between BCKA- dependent aerobic glycolysis and VSMC phenotype and pathophenotype.