Abstract
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.
Highlights
The pyruvate dehydrogenase (PDH) complex plays a central role in metabolic regulation[1,2]
pyruvate dehydrogenase complex (PDH) kinases modulate the metabolic state of the liver and have become key drug targets for metabolic diseases such as type 2 diabetes and hepatic steatosis
Tao et al reported that the deficiency of both PDK2 and PDK4 (PDK2/4−/−) improves insulin sensitivity in double knockout (DKO) mice[31]
Summary
The pyruvate dehydrogenase (PDH) complex plays a central role in metabolic regulation[1,2]. Increased PDK expression in obesity contributes to lower PDH activity and reduced oxidation of carbohydrates[10] This suggests that inhibition of PDK 2 and PDK 4 may reverse adverse metabolic effects by increasing pyruvate oxidation. A number of studies have shown that targeting PDK activity is promising for the treatment of obesity-related metabolic conditions[11,12,13,14] Small molecules such as dichloroacetate inhibit PDKs and www.nature.com/scientificreports administration is associated with improved glucose tolerance in rodent models[13,14,15,16]. The current study was designed to determine whether the appearance of [13C]bicarbonate after administration of [1-13C]pyruvate can be used as a reliable indicator of PDH flux in diet-induced obesity. The correlation between the appearance of HP 13C-bicarbonate and the knockout of hepatic PDK enzymes is important for translating HP 13C-MRS as a noninvasive imaging tool for the treatment and management of chronic liver diseases
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