Dysregulated branched chain amino acid (BCAA) metabolism has emerged as a key metabolic feature associated with the obese insulin resistant state, and adipose BCAA catabolism is decreased in this context. BCAA catabolism is upregulated early in adipogenesis, but the impact of suppressing this pathway on the broader metabolic functions of the resultant adipocyte remains unclear. Here, we use CRISPR/Cas9 to decrease BCKDHA in 3T3-L1 and human pre-adipocytes, and ACAD8 in 3T3-L1 pre-adipocytes to induce a deficiency in BCAA catabolism through differentiation. We characterize the transcriptional and metabolic phenotype of 3T1-L1 cells using RNAseq and 13C metabolic flux analysis within a network spanning glycolysis, tricarboxylic acid (TCA) metabolism, BCAA catabolism, and fatty acid synthesis. While lipid droplet accumulation is maintained in Bckdha-deficient adipocytes, they display a more fibroblast-like transcriptional signature. In contrast, Acad8 deficiency minimally impacts gene expression. Decreased glycolytic flux emerges as the most distinct metabolic feature of 3T3-L1 Bckdha-deficient cells, accompanied by a ∼40% decrease in lactate secretion, yet pyruvate oxidation and utilization for de novo lipogenesis are increased to compensate for loss of BCAA carbon. Deletion of BCKDHA in human adipocyte progenitors also led to a decrease in glucose uptake and lactate secretion, however these cells did not upregulate pyruvate utilisation and lipid droplet accumulation and expression of adipocyte differentiation markers was decreased in BCKDH knockout cells. Overall our data suggest that human adipocyte differentiation may be more sensitive to the impact of decreased BCKDH activity than 3T3-L1 cells, and that both metabolic and regulatory cross-talk exists between BCAA catabolism and glycolysis in adipocytes. Suppression of BCAA catabolism associated with metabolic syndrome may result in a metabolically compromised adipocyte.

中文翻译：

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脂肪细胞分化过程中支链氨基酸 （BCAA） 分解代谢受损会降低糖酵解通量。


支链氨基酸 （BCAA） 代谢失调已成为与肥胖胰岛素抵抗状态相关的关键代谢特征，在这种情况下，脂肪 BCAA 分解代谢减少。BCAA 分解代谢在脂肪生成早期上调，但抑制该途径对所得脂肪细胞更广泛的代谢功能的影响仍不清楚。在这里，我们使用 CRISPR/Cas9 减少 3T3-L1 和人前脂肪细胞中的 BCKDHA，并使用 3T3-L1 前脂肪细胞中的 ACAD8 通过分化诱导 BCAA 分解代谢缺陷。我们在涵盖糖酵解、三羧酸 （TCA） 代谢、BCAA 分解代谢和脂肪酸合成的网络中使用 RNAseq 和 13C 代谢通量分析来表征 3T1-L1 细胞的转录和代谢表型。虽然脂质液滴积累在 Bckdha 缺陷的脂肪细胞中得以维持，但它们显示出更像成纤维细胞的转录特征。相比之下，Acad8 缺陷对基因表达的影响最小。糖酵解通量降低是 3T3-L1 Bckdha 缺陷细胞最明显的代谢特征，伴随着乳酸分泌减少约 40%，但丙酮酸氧化和从头脂肪生成的利用增加以补偿 BCAA 碳的损失。人脂肪细胞祖细胞中 BCKDHA 的缺失也导致葡萄糖摄取和乳酸分泌减少，但这些细胞没有上调丙酮酸利用，脂滴积累和脂肪细胞分化标志物的表达在 BCKDH 敲除细胞中降低。 总体而言，我们的数据表明，人类脂肪细胞分化可能比 3T3-L1 细胞对 BCKDH 活性降低的影响更敏感，并且脂肪细胞中的 BCAA 分解代谢和糖酵解之间存在代谢和调节串扰。抑制与代谢综合征相关的 BCAA 分解代谢可能导致脂肪细胞代谢受损。