Mass spectrometry-based stable-isotope tracing uncovers metabolic alterations in pyruvate kinase-deficient Aedes aegypti mosquitoes.,Insect Biochemistry and Molecular Biology

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Mass spectrometry-based stable-isotope tracing uncovers metabolic alterations in pyruvate kinase-deficient Aedes aegypti mosquitoes.
Insect Biochemistry and Molecular Biology ( IF 3.2 ) Pub Date : 2020-04-07 , DOI: 10.1016/j.ibmb.2020.103366
Natthida Petchampai ₁ , Jun Isoe ₂ , Thomas D Horvath ₃ , Shai Dagan ₄ , Lin Tan ₃ , Philip L Lorenzi ₃ , David H Hawke ₅ , Patricia Y Scaraffia ₁

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A recent in vitro characterization of a recombinant pyruvate kinase (PK) from Aedes aegypti mosquitoes demonstrated that the enzyme is uniquely regulated by multiple allosteric effectors. Here, we further explored PK gene and protein expression, and enzymatic activity in key metabolic tissues of mosquitoes maintained under different nutritional conditions. We also studied the metabolic effects of PK depletion using several techniques including RNA interference and mass spectrometry-based stable-isotope tracing. Transcriptional analysis showed a dynamic post-feeding PK mRNA expression pattern within and across mosquito tissues, whereas corresponding protein levels remained stable throughout the time course analyzed. Nevertheless, PK activity significantly differed in the fat body of sucrose-, blood-fed, and starved mosquitoes. Genetic silencing of PK did not alter survival in blood-fed females maintained on sucrose. However, an enhanced survivorship was observed in PK-deficient females maintained under different nutritional regimens. Our results indicate that mosquitoes overcame PK deficiency by up-regulating the expression of genes encoding NADP-malic enzyme-1, phosphoenolpyruvate carboxykinase-1, phosphoglycerate dehydrogenase and glutamate dehydrogenase, and by decreasing glucose oxidation and metabolic pathways associated with ammonia detoxification. Taken together, our data demonstrate that PK confers to A. aegypti a metabolic plasticity to tightly regulate both carbon and nitrogen metabolism.

中文翻译：

基于质谱的稳定同位素追踪揭示了丙酮酸激酶缺陷的埃及伊蚊的代谢变化。

最近对埃及伊蚊重组丙酮酸激酶 (PK) 的体外表征表明，该酶受到多种变构效应子的独特调节。在这里，我们进一步探讨了不同营养条件下蚊子关键代谢组织中的PK基因和蛋白质表达以及酶活性。我们还使用多种技术研究了 PK 消耗的代谢影响，包括 RNA 干扰和基于质谱的稳定同位素示踪。转录分析显示蚊子组织内和跨蚊子组织的动态喂食后 PK mRNA 表达模式，而相应的蛋白质水平在整个分析过程中保持稳定。然而，蔗糖蚊子、吸血蚊子和饥饿蚊子的脂肪体中 PK 活性存在显着差异。 PK 的基因沉默不会改变以蔗糖维持的血饲雌性的存活率。然而，在不同营养方案下维持的 PK 缺乏的雌性中观察到存活率提高。我们的研究结果表明，蚊子通过上调编码 NADP-苹果酸酶-1、磷酸烯醇丙酮酸羧激酶-1、磷酸甘油酸脱氢酶和谷氨酸脱氢酶的基因的表达，并通过减少葡萄糖氧化和与氨解毒相关的代谢途径来克服 PK 缺陷。综上所述，我们的数据表明 PK 赋予埃及伊蚊一种代谢可塑性，可以严格调节碳和氮代谢。

更新日期：2020-04-08

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