实时超极化13 C磁共振检测到丙酮酸脱氢酶激酶2 / 4-双敲除小鼠肝脏中丙酮酸的氧化增加。,Scientific Reports

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实时超极化13 C磁共振检测到丙酮酸脱氢酶激酶2 / 4-双敲除小鼠肝脏中丙酮酸的氧化增加。
Scientific Reports ( IF 3.8 ) Pub Date : 2019-11-11 , DOI: 10.1038/s41598-019-52952-6
Gaurav Sharma ₁ , Cheng-Yang Wu ₁ , R Max Wynn _{2,

3} , Wenjun Gui ₂ , Craig R Malloy _{1,

3,

4} , A Dean Sherry _{1,

4,

5} , David T Chuang _{2,

3} , Chalermchai Khemtong _{1,

4}

Affiliation

丙酮酸脱氢酶复合物（PDH）严格调节碳水化合物的代谢。丙酮酸脱氢酶激酶1-4（PDK1-4）之一将PDH磷酸化，可减少碳水化合物进入TCA循环的通量。抑制PDK可增加碳水化合物的氧化代谢，因此靶向PDK已成为控制各种代谢疾病的重要治疗方法。因此，非常需要开始建立用于啮齿动物模型中PDH通量的非侵入性测量的成像工具。在这项研究中，我们使用超极化（HP）13C磁共振波谱研究了PDK2 / PDK4双敲除（DKO）对瘦肉和饮食诱发的肥胖（DIO）小鼠灌注肝脏中丙酮酸代谢的影响，并通过高剂量验证了HP观察值。提取物的13 C-核磁共振（NMR）光谱和稳态同位素分析。我们观察到，与年龄匹配的对照肝脏相比，PDK缺陷型肝脏从HP- [1-13C]丙酮酸盐中产生的HP-碳酸氢盐更多。对组织提取物的稳态13C-NMR异构体分析证实，在PDK缺乏的肝脏中，通过PDH的通量率以及丙酮酸羧化酶和丙酮酸循环活性显着更高。免疫印迹实验证实，由HP- [1-13C]丙酮酸盐生成的HP-碳酸氢盐可减少肝脏组织中PDHE1α亚基（pE1α）的磷酸化。我们的发现表明，结合使用实时超极化13C NMR光谱学和13C同位素异构体分析，可以定量了解PDK敲除小鼠的中间代谢。我们建议这种方法将有助于评估代谢性疾病的状态，并开发出改善PDH通量的疗法。

"点击查看英文标题和摘要"

Real-time hyperpolarized 13C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4-double knockout mouse livers.

The pyruvate dehydrogenase complex (PDH) critically regulates carbohydrate metabolism. Phosphorylation of PDH by one of the pyruvate dehydrogenase kinases 1-4 (PDK1-4) decreases the flux of carbohydrates into the TCA cycle. Inhibition of PDKs increases oxidative metabolism of carbohydrates, so targeting PDKs has emerged as an important therapeutic approach to manage various metabolic diseases. Therefore, it is highly desirable to begin to establish imaging tools for noninvasive measurements of PDH flux in rodent models. In this study, we used hyperpolarized (HP) 13C-magnetic resonance spectroscopy to study the impact of a PDK2/PDK4 double knockout (DKO) on pyruvate metabolism in perfused livers from lean and diet-induced obese (DIO) mice and validated the HP observations with high-resolution 13C-nuclear magnetic resonance (NMR) spectroscopy of tissue extracts and steady-state isotopomer analyses. We observed that PDK-deficient livers produce more HP-bicarbonate from HP-[1-13C]pyruvate than age-matched control livers. A steady-state 13C-NMR isotopomer analysis of tissue extracts confirmed that flux rates through PDH, as well as pyruvate carboxylase and pyruvate cycling activities, are significantly higher in PDK-deficient livers. Immunoblotting experiments confirmed that HP-bicarbonate production from HP-[1-13C]pyruvate parallels decreased phosphorylation of the PDH E1α subunit (pE1α) in liver tissue. Our findings indicate that combining real-time hyperpolarized 13C NMR spectroscopy and 13C isotopomer analysis provides quantitative insights into intermediary metabolism in PDK-knockout mice. We propose that this method will be useful in assessing metabolic disease states and developing therapies to improve PDH flux.

更新日期：2019-11-11

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