Simultaneous Steady-state and Dynamic 13C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells

Yang, Chendong; Harrison, Crystal; Jin, Eunsook S.; Chuang, David T.; Sherry, A. Dean; Malloy, Craig R.; Merritt, Matthew E.; DeBerardinis, Ralph J.

doi:10.1074/jbc.m113.543637

Cited by 49 publications

(53 citation statements)

References 47 publications

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“…Alternatively, procedures that measure pyruvate-driven oxygen consumption in real time do not represent a direct quantification of the activity of MPC since many steps are required for pyruvate oxidation following its import into the mitochondria. Furthermore, flux metabolomics (Hiller and Metallo, 2013), including the use of hyperpolarized 13 C-enriched substrates (Yang et al, 2014a) that define a precise metabolic phenotype of cells, do not allow direct assessment of the MPC activity. In addition, all of these different approaches are time and cell consuming.…”

Section: Designmentioning

confidence: 99%

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

et al. 2015

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The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types.

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Section: Designmentioning

confidence: 99%

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

et al. 2015

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“…Future work will attempt to use HP DHA for studying in vivo metabolism. In addition, optimization of the detection scheme to use shaped pulses for detection of downstream metabolites would enhance the sensitivity significantly as rectangular pulses were used for excitation of the resonances (29). The broadband excitation results in unneeded destruction of polarization in DHA.…”

Section: Metabolism Of Hp [2-mentioning

confidence: 99%

Real-time Detection of Hepatic Gluconeogenic and Glycogenolytic States Using Hyperpolarized [2-13C]Dihydroxyacetone

Moreno

Satapati

DeBerardinis

et al. 2014

Journal of Biological Chemistry

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Background: Gluconeogenesis is sensitive to nutritional state and disease. Results: After injection of hyperpolarized [2-13 C]dihydroxyacetone in the perfused mouse liver, the ratio of 3-carbon intermediates and hexoses provides an index of gluconeogenesis. Conclusion: Glycogenolysis versus gluconeogenesis correlates with metabolism of hyperpolarized dihydroxyacetone. Significance: Hyperpolarized [2-13 C]dihydroxyacetone detects gluconeogenesis in living tissues, in real time.

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“…Pyruvate Decarboxylation Assay-Pyruvate decarboxylation assay was performed essentially as described elsewhere (47). A piece of chromatography paper (0.6 ϫ 1 cm) was put on top of microbridges (Hampton Research) fitted in wells of a 24-well plate.…”

Section: Preparation Of Lysates From Mouse Tissues and Spermmentioning

confidence: 99%

MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells

Vanderperre¹,

Cermakova²,

Escoffier

et al. 2016

Journal of Biological Chemistry

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Selective transport of pyruvate across the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) is a fundamental step that couples cytosolic and mitochondrial metabolism. The recent molecular identification of the MPC complex has revealed two interacting subunits, MPC1 and MPC2. Although in yeast, an additional subunit, MPC3, can functionally replace MPC2, no alternative MPC subunits have been described in higher eukaryotes. Here, we report for the first time the existence of a novel MPC subunit termed MPC1-like (MPC1L), which is present uniquely in placental mammals. MPC1L shares high sequence, structural, and topological homology with MPC1. In addition, we provide several lines of evidence to show that MPC1L is functionally equivalent to MPC1: 1) when co-expressed with MPC2, it rescues pyruvate import in a MPC-deleted yeast strain; 2) in mammalian cells, it can associate with MPC2 to form a functional carrier as assessed by bioluminescence resonance energy transfer; 3) in MPC1 depleted mouse embryonic fibroblasts, MPC1L rescues the loss of pyruvate-driven respiration and stabilizes MPC2 expression; and 4) MPC1-and MPC1L-mediated pyruvate imports show similar efficiency. However, we show that MPC1L has a highly specific expression pattern and is localized almost exclusively in testis and more specifically in postmeiotic spermatids and sperm cells. This is in marked contrast to MPC1/MPC2, which are ubiquitously expressed throughout the organism. To date, the biological importance of this alternative MPC complex during spermatogenesis in placental mammals remains unknown. Nevertheless, these findings open up new avenues for investigating the structure-function relationship within the MPC complex.The metabolic balance between glycolysis and oxidative phosphorylation (OXPHOS) 2 is of crucial importance in determining cell function and fate. Rapidly proliferating cells rely preferentially on glycolysis despite its lower yield of ATP, because glycolytic intermediates efficiently meet the anabolic needs for sustained cell growth. In rapidly dividing cells, this occurs even in the presence of oxygen, a process called aerobic glycolysis, or the Warburg effect (1, 2). Differentiated cells rely more heavily on OXPHOS to meet the energy demands associated with their specialization. In addition, a shift from glycolytic to oxidative metabolism is necessary for the differentiation process to occur (2-4), and conversely, a hallmark of cancer lies in the rewiring of cellular metabolism toward increased glycolytic flux (5). Pyruvate, the end product of glycolysis, is imported into the mitochondrial matrix where it fuels the TCA cycle and thereby provides electrons and reducing equivalents to the respiratory chain. Pyruvate-derived acetyl-CoA can also be used as an anabolic substrate for the synthesis of lipids and amino acids. Thus, elucidating the molecular mechanisms that determine the intracellular fate of pyruvate is important for understanding the control of cell metabolism and ultimately of cell funct...

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Simultaneous Steady-state and Dynamic 13C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells

Abstract: Background:13 C hyperpolarization sensitively and non-destructively detects pyruvate-lactate exchanges in cancer cells.

Cited by 49 publications

References 47 publications

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

Real-time Detection of Hepatic Gluconeogenic and Glycogenolytic States Using Hyperpolarized [2-13C]Dihydroxyacetone

MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells

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