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Differential effects of 2-oxo acids on pyruvate utilization and fatty acid synthesis in rat brain.

ABSTRACT: 1. The effects of 2-oxo-4-methylpentanoate, 2-oxo-3-methylbutanoate and 2-oxo-3-methylpentanoate on the activity of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase, (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old rats were investigated. 2. The pyruvate dehydrogenase enzyme activity was competitively inhibited by 2-oxo-3-methylbutanoate with respect to pyruvate with a K(i) of 2.04mm but was unaffected by 2-oxo-4-methylpentanoate or 2-oxo-3-methylpentanoate. 3. The citrate synthase activity was inhibited competitively (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~7.2mm) and 2-oxo-3-methylbutanoate (K(i)~14.9mm) but not by 2-oxo-3-methylpentanoate. 4. The acetyl-CoA carboxylase activity was not inhibited significantly by any of the 2-oxo acids investigated. 5. The fatty acid synthetase activity was competitively inhibited (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~930mum) and 2-oxo-3-methylpentanoate (K(i)~3.45mm) but not by 2-oxo-3-methylbutanoate. 6. Preliminary experiments indicate that 2-oxo-4-methylpentanoate and 2-oxo-3-phenylpropionate (phenylpyruvate) significantly inhibit the ability of intact brain mitochondria from 14-day-old rats to oxidize pyruvate. 7. The results are discussed with reference to phenylketonuria and maple-syrup-urine disease. A biochemical mechanism is proposed to explain the characteristics of these diseases.

SUBMITTER: Clark JB

PROVIDER: S-EPMC1167967 | biostudies-other | 1974 Apr

REPOSITORIES: biostudies-other

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Project description:BackgroundPyruvate carboxylase (PC) is an important anaplerotic enzyme in the tricarboxylic acid cycle (TCA) in cancer cells. Although PC overexpression has been observed in thyroid cancer (TC), the mechanisms involved in the carcinogenic effects of PC are still unclear.MethodsBioinformatics analysis and clinical specimens were used to analyze the relationship of PC expression with clinicopathological variables in TC. Fatty acid synthesis was monitored by LC/MS, Nile red staining, and triglyceride analysis. Mitochondrial oxygen consumption was evaluated by the Seahorse XF Mito Cell Stress Test. The correlation of PC with FASN and SREBP1c was assessed by qRT-PCR and IHC in 38 human TC tissues. Western blotting was used to evaluate the protein expression of PC, FASN, and SREBP1c and members of the AKT/mTOR and EMT pathways in TC cell lines. Wound-healing, CCK-8, and Transwell assays and a nude mouse xenograft model were used to verify the regulatory effects of PC and SREBP1c on thyroid tumor cell proliferation, migration and invasion.ResultsWe demonstrated that PC increased fatty acid synthesis, which then promoted TC progression and metastasis. Analysis of GEO data showed that the overexpression of PC in papillary thyroid cancer (PTC) was associated with PTC invasion and the fatty acid synthesis pathway. Analysis of clinical tissue specimens from PTC patients revealed that PC was more highly expressed in specimens from PTC patients with lymph node metastasis than in those from patients without metastasis. Multiple genes in the fatty acid synthesis signaling pathway, including FASN and SREBP1c, were downregulated in PC-knockdown TC cells compared to control cells. Lipid levels were also decreased in the PC-knockdown TC cells. Moreover, the ability of cells to grow, invade, and metastasize was also suppressed upon PC knockdown, suggesting that PC-mediated lipogenesis activation increases the aggressiveness of TC cells. In addition, PC was found to activate the AKT/mTOR pathway, thus improving FASN-mediated de novo lipogenesis in TC cells by upregulating SREBP1c expression. Studies in a nude mouse xenograft model showed that PC knockdown decreased tumor weight, but this effect was attenuated by forced expression of SREBP1c.ConclusionsOur results demonstrate that PC is strongly involved in the tumor aggressiveness of TC via its stimulation of fatty acid synthesis.

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Differential effects of 2-oxo acids on pyruvate utilization and fatty acid synthesis in rat brain.

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