Elsevier

Methods in Enzymology

Volume 588, 2017, Pages 133-153
Methods in Enzymology

Chapter Eight - Magnetic Resonance Spectroscopy to Study Glycolytic Metabolism During Autophagy

https://doi.org/10.1016/bs.mie.2016.09.078Get rights and content

Abstract

Cancer cells undergoing starvation- and treatment-induced autophagy were found to exhibit reduced intracellular lactate, reduced rates of steady-state lactate excretion and reduced real-time pyruvate–lactate exchange rates, indicating that glycolytic metabolism was altered in autophagic cells. In this chapter, we describe the technical details of the use of 1H-magnetic resonance spectroscopy (MRS) to measure endogenous cellular concentrations of lactate and glucose in autophagic cells and tissues, how to measure the rate of steady-state lactate excretion and glucose uptake by 1H-MRS in autophagic cells, and details of the real-time measurement of [1-13C] pyruvate to lactate exchange in autophagic cells by 13C-MRS-DNP (dynamic nuclear polarization).

Introduction

Glucose is metabolized by cells via glycolysis to produce pyruvate. The efficiency of this process is dependent on the rate of glucose uptake which is controlled by the glucose transporters (GLUTs) and the activity of the various enzymes along the glycolytic pathway. Pyruvate is then converted to acetyl-CoA by pyruvate dehydrogenase (PDH) or converted to oxaloacetate by pyruvate carboxylase (PC) and enters the TCA cycle to produce substrates for oxidative phosphorylation in the mitochondria. Alternative metabolic fates for pyruvate include transamination to form alanine or reduction in the cytosol to form lactate. The rate of reduction of pyruvate to lactate is dependent on the expression level and activity of lactate dehydrogenase (LDH) and cofactors such as oxidized (NAD+) and reduced nicotinamide adenine dinucleotide (NADH) (Fig. 1), as well as the monocarboxylate (MCT)-mediated transport of substrates into and out of the cell. If the availability of oxygen is restricted, then preferential conversion of pyruvate to lactate and concomitant transport of lactate out of the cell regenerates NAD+ in the cytosol, which allows glycolysis to continue even in the absence of oxygen, such as in ischemia or acute exercise.

A shift from oxidative phosphorylation to glycolysis and the conversion of pyruvate to lactate for energy production is favored by cancer cells, even in the presence of freely available oxygen. In cancer, there is increased transcriptional regulation of a number of glycolytic and mitochondrial enzymes, including lactate dehydrogenase-A (LDH-A), pyruvate dehydrogenase kinase, among many others. This reprogramming of energy metabolism is called the “Warburg effect” and has become a widely accepted hallmark of cancer (Hanahan and Weinberg, 2000, Hanahan and Weinberg, 2011, Warburg, 1956).

Glycolytic metabolism was found to be altered in starvation (amino acid and serum deprived)- and treatment (PI103 and dichloroacetate)-induced autophagic cancer cells (Lin, Andrejeva, et al., 2014, Lin, Hill, et al., 2014). In these experiments, induction of autophagy was confirmed in colorectal HT29 and HCT116 Bax-ko cancer cells by overexpression of LC3II in Western blots and the presence of double-membrane autophagic vesicles by electron microscopy, while the absence of apoptosis was confirmed by the lack of cleaved poly(ADP-ribose) polymerase (PARP) or change in caspase 3 expression. Reductions in the rate of lactate excretion and intracellular lactate were observed, as well as reductions in pyruvate–lactate exchange kinetics measured in real time. It was also shown that by replacing the amino acid- and serum-deprived media with full media, or by stopping the treatment, there was a reversal of this phenomenon, which reported on cellular recovery from autophagy (Lin, Andrejeva, et al., 2014, Lin, Hill, et al., 2014). In these studies, magnetic resonance spectroscopy (MRS) methods were used to examine the rates of lactate production or exchange in steady state and in real time, respectively. 1H-MRS analysis was used to measure cellular lactate levels in cell extracts and lactate secretion and glucose uptake in cell culture media. Dynamic nuclear polarization (DNP) and 13C-MRS of 13C-labeled pyruvate were used to measure the rate of pyruvate exchange with lactate in vitro and in vivo in real time. The mechanisms behind these changes were found to be associated with decreased LDH activity in starvation-induced autophagy and an increased NAD+/NADH ratio in treatment-induced autophagy, suggesting a possible shift in glycolytic metabolism during autophagy (Lin, Andrejeva, et al., 2014, Lin, Hill, et al., 2014).

This chapter describes the technical details of how to measure endogenous cellular lactate and glucose in autophagic cells and tissues by 1H-MRS, how to measure the rate of lactate secretion and glucose uptake in autophagic cells by 1H-MRS, and the measurement of the real-time exchange of 13C-labeled pyruvate to lactate in cells by 13C-MRS-DNP and the derivation of simple kinetic parameters from these measurements. The methods described here are generally applicable in cells and tissues to investigate cellular processes, cellular metabolism alterations following genetic perturbations or in disease state, and monitoring treatment response (Chung et al., 2015, Chung and Griffiths, 2011).

MRS is limited by low signal strength at thermal equilibrium due to low spin polarization. Nevertheless, MRS and MRS imaging offer chemically specific analysis of metabolite concentrations in body fluids, cell or tissue extracts, intact tissues, or in vivo. In combination with metabolomics and statistical methods such as principal component analysis, MRS has become a widely used tool to measure a wide range of metabolites in cell extracts, whole cells, or tissue biopsy samples (Beckonert et al., 2007, Chung et al., 2015, Chung and Griffiths, 2011). Furthermore, the ability to perform metabolic imaging in vivo using MRS (de Graaf, 2007) and the translation of these techniques clinically in humans allow the development of molecular biomarkers of response to therapeutics that act directly on metabolic enzymes and transporters or through the inhibition of cell signaling pathways that transcriptionally control and regulate metabolic enzymes.

MRS exploits an intrinsic property of the nucleus known as spin. A wide range of biologically important isotopes possess a nonzero spin, including 1H, 13C, 31P, 23Na, and 15N, among others. When placed in an external magnetic field of the spectrometer, the spin states (angular momentum) of these nuclei become quantized. When excited by a radiofrequency pulse, a characteristic spectrum is generated depending on the gyromagnetic ratio of the nucleus studied and the electronic environment of the molecule. Thus, unique spectral peaks with distinct resonance frequencies known as chemical shift correspond to unique molecular environments. In addition, MRS is quantitative at thermal equilibrium in the sense that the peak integral is proportional to molecular concentration and can be measured directly via the addition of a reference compound of known concentration. Despite limited sensitivity, 1H MRS is able to measure metabolite concentrations within the micromole range. In addition, incorporation of stable isotopes such as 13C can report on the steady-state activities and fluxes through different metabolic pathways. 13C isotopomer incorporation can be measured by MRS or mass spectrometry and is dependent on the respective labeling positions in starting substrates and their relative rates of incorporation through different pathways into reaction products and intermediates (Buescher et al., 2015, DeBerardinis et al., 2007).

The sensitivity limitations of MRS can be overcome by using hyperpolarization techniques to transiently increase the spin polarization and thereby increase sensitivity. Techniques such as DNP enable significant MRS signal enhancements by a factor greater than 10,000 for low gyromagnetic ratio nuclei such as 13C and 15N in a range of endogenous biological metabolites (Ardenkjaer-Larsen et al., 2003). The ability to distinguish parent and downstream metabolites by virtue of a difference in chemical shift allows the measurement of the interconversion of metabolites in real time in cellular systems, whole organ preparations, as well as in vivo, and thereby reports on the activity of endogenous enzymes and membrane transporters that catalyze their kinetic interconversion (Day et al., 2007). For a more extensive discussion of DNP, including some of the challenges and limitations of the technique, a number of excellent reviews in this area have recently been published (Chaumeil et al., 2015, Comment and Merritt, 2014, Kurhanewicz et al., 2011).

To date, the most widely employed metabolic substrate for hyperpolarized 13C-MR imaging has been [1-13C] pyruvate (Golman, in't Zandt, Lerche, Pehrson, & Ardenkjaer-Larsen, 2006). This is largely due to the very favorable spin lattice relaxation time (T1) of the C1 and C2 carbonyl carbons, which is reported to be in the range of 40–70 s depending on the magnetic field strength (Keshari & Wilson, 2014), as well as the fast rates of MCT-mediated entry of pyruvate into the cell. Pyruvate is located at the end point of glycolysis being subject to a number of metabolic fates, including LDH-mediated exchange with lactate, alanine transaminase-mediated exchange with alanine, one-way decarboxylation mediated by PDH to form CO2 or one-way carboxylation mediated by PC to form oxaloacetate. The ability to probe the metabolic fates of hyperpolarized pyruvate, as well as its associated kinetic rates and metabolic fluxes, is therefore of great current interest for imaging metabolic processes in vivo and is complementary to steady-state metabolomics-type techniques. The apparent exchange rate constant of hyperpolarized [1-13C] pyruvate to lactate (kPL) has previously been shown to provide a potential metabolic biomarker for diagnosis and for assessing treatment response (Golman, in't Zandt, et al., 2006b, Golman and Petersson, 2006, Park et al., 2010, Ward et al., 2010). This apparent rate has been shown to decrease following drug-induced cell death, attributed to apoptosis with the activation of PARP and depletion of the cofactors NAD(H) (Day et al., 2007).

Apparent rate constants have also been shown to be dependent on NAD+/NADH ratios (Christensen, Karlsson, Winther, Jensen, & Lerche, 2014), the expression and activity of LDH (Ward et al., 2010), as well as on the activity of the MCT transporter family (Harris, Eliyahu, Frydman, & Degani, 2009), which mediate pyruvate and lactate transport into and out of the cell. The rates of exchange-mediated pyruvate–lactate conversion have further been shown to correlate with FDG uptake following etoposide treatment in an EL4 tumor model with concomitant decreases in NADH levels and GLUT3 expression (Witney et al., 2009). Kinetic assays using hyperpolarized pyruvate are therefore sensitive to a range of physicochemical properties of the cell that report on glycolytic fluxes, as well as being complementary to more conventional imaging techniques for probing glycolysis, such as FDG PET imaging.

Section snippets

Measurement of Intracellular Lactate and Glucose Levels in Autophagic Cells or Tumor Extracts

Intracellular lactate and glucose metabolites can be extracted from cultured cells or tissues using a dual-phase extraction method detailed by Tyagi, Azrad, Degani, and Salomon (1996).

Preparation of Media Samples for 1H-MRS Analysis

  • (1)

    Put 500 μL of the culture medium (see Section 2.1, step 2) into a 5-mm NMR tube.

  • (2)

    Add 50 μL of D2O and 50 μL of 0.75% TSP in D2O in the sample.

1H-MRS of Media Samples

  • (1)

    1H-MR spectra of the media samples are acquired at 25°C using a pulse-acquired MR sequence with water suppression (a 1D NOESY presat sequence) in a BBI NMR probe on a 500- or 600-MHz NMR system. For cell culture media samples, the typical NMR acquisition parameters are 7500 Hz spectral width, 32,768 time domain points, 2.7 s repetition time, and 64 scans. NMR

Hyperpolarization Methods for Dissolution DNP Using Pyruvic Acid

DNP exploits the high spin polarization of unpaired electrons in the form of stable-free radicals at a very low temperature. When placed in a strong magnetic field and cooled in liquid helium to temperatures < 4.2 K, unpaired electrons are polarized to near unity owing to their large gyromagnetic ratio. By irradiating the sample close to the electron paramagnetic resonance frequency (ωe ± ωC) (where ωe and ωC are the Larmor frequencies of the electron and carbon, respectively, at a field strength

Discussion

We have described in this chapter the use of in vitro steady-state 1H-MRS methods and real-time hyperpolarized 13C-MRS for probing glycolytic metabolism in cells undergoing autophagy. The methods report on the steady-state rates of glucose uptake and lactate excretion, endogenous concentration of lactate, and the rate constants for the exchange of pyruvate to lactate. In turn, these measurements are sensitive to a range of cellular parameters, including GLUT, downstream rates of glycolysis, LDH

Acknowledgments

This work is supported by funding received from the CR-UK Cancer Imaging Centre, in association with the MRC and Department of Health (England) Grant C1060/A10334, C1060/A16464, and NHS funding to the NIHR Biomedical Research Centre.

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