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

Gaurav Sharma, Cheng Yang Wu, R. Max Wynn, Wenjun Gui, Craig R. Malloy, A. Dean Sherry, David T. Chuang, Chalermchai Khemtong

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number16480
JournalScientific reports
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2019

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Pyruvate Dehydrogenase Complex
Pyruvic Acid
Knockout Mice
Magnetic Resonance Spectroscopy
Liver
Tissue Extracts
Metabolic Diseases
Carbohydrate Metabolism
Bicarbonates
Pyruvate Dehydrogenase (Lipoamide)
Phosphorylation
Pyruvate Carboxylase
Obese Mice
Immunoblotting
pyruvate dehydrogenase (acetyl-transferring) kinase
Rodentia
Carbohydrates
Diet
Therapeutics

ASJC Scopus subject areas

  • General

Cite this

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title = "Real-time hyperpolarized 13C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4–double knockout mouse livers",
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.",
author = "Gaurav Sharma and Wu, {Cheng Yang} and Wynn, {R. Max} and Wenjun Gui and Malloy, {Craig R.} and Sherry, {A. Dean} and Chuang, {David T.} and Chalermchai Khemtong",
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T1 - Real-time hyperpolarized 13C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4–double knockout mouse livers

AU - Sharma, Gaurav

AU - Wu, Cheng Yang

AU - Wynn, R. Max

AU - Gui, Wenjun

AU - Malloy, Craig R.

AU - Sherry, A. Dean

AU - Chuang, David T.

AU - Khemtong, Chalermchai

PY - 2019/12/1

Y1 - 2019/12/1

N2 - 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.

AB - 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.

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