Hyperpolarized 13C Magnetic Resonance and Its Use in Metabolic Assessment of Cultured Cells and Perfused Organs

Lloyd Lumata*,1; Chendong Yang†; Mukundan Ragavan‡; Nicholas Carpenter‡; Ralph J. DeBerardinis†,1; Matthew E. Merritt‡,1    * Department of Physics, University of Texas at Dallas, Richardson, Texas, USA
† Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA
‡ Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA
1 Corresponding authors: email address: lloyd.lumata@utdallas.edu, ralph.deberardinis@utsouthwestern.edu, matthew.merritt@utsouthwestern.edu

1 Introduction: Importance of Developing Methods to Observe Metabolic Flux in Disease States

Metabolism is at the root of essentially all physiological processes (DeBerardinis & Thompson, 2012). The production and expenditure of energy, storage and breakdown of macromolecules, disposal of waste, and many other processes are subserved by thousands of enzymatic reactions at work in human cells. Recent work has produced insights that greatly extend the influence of metabolism and metabolites to include such seemingly disparate processes as signal transduction, posttranslational modification of proteins, and epigenetic effects on gene expression (Choudhary, Weinert, Nishida, Verdin, & Mann, 2014; Kaelin & McKnight, 2013; Ward & Thompson, 2012). These observations further emphasize the principle that metabolism is inexorably intertwined with cellular function and tissue homeostasis. In short, normal tissue function cannot occur unless metabolism is properly regulated.

For most of the past century, metabolism research has been dominated by studies in organs like the liver, one of whose major functions is to maintain metabolic homeostasis for the entire body, and the skeletal muscle, heart, and brain, whose normal function involves energetically demanding processes. However, many other tissues are equally dependent on a broad complement of metabolic activities. As an example, an emerging theme in cell biology research is the importance of acute metabolic changes for enabling physiological cell growth and proliferation (Metallo & Vander Heiden, 2013; Plas & Thompson, 2005). Because growth and proliferation are viewed as metabolically demanding in terms of the need for energy and macromolecular synthesis, there has been interest in understanding how the signals that stimulate punctuated bursts of cell proliferation engage the metabolic network to satisfy these demands. T-cell activation is one of the many processes now known to require several specific metabolic activities to support cell growth and proliferation (Gerriets et al., 2015).

Given the many links between metabolism and cellular function, it is unsurprising that most diseases feature abnormal metabolism at the cellular level (DeBerardinis & Thompson, 2012). Of the hundreds of monogenic diseases caused by mutations in single enzymes (the so-called inborn errors of metabolism), a high fraction affects the liver, heart, muscle, and brain. Many other monogenic metabolic diseases involve poorly defined effects on growth, either because of systemic metabolic imbalances or perhaps effects on specific populations of cells whose function is required for normal growth. Importantly, common diseases also involve altered metabolism. Among the most common causes of death in the United States, heart disease, cancer, stroke, and diabetes can all be viewed as involving altered metabolism at the level of the cell and/or organ. Thus, we need better tools to understand metabolic regulation in diseased tissues. Preferably, some of these tools will support metabolic analysis in intact tissue.

Metabolic dysregulation is a prominent and clinically relevant feature of cancer biology. As early as the 1920s, Warburg demonstrated that malignant cells have a propensity to take up excess amounts of glucose and convert it into lactate, even when oxygen availability was sufficient to oxidize glucose completely to carbon dioxide (Warburg, 1956b). The successful use of 18fluoro-2-deoxyglucose (FDG) as a radiotracer for positron emission tomography (PET) studies in cancer patients has validated the clinical importance of glucose uptake in human tumors (Gallamini, Zwarthoed, & Borra, 2014). FDG-PET is commonly used to image the distribution of malignant tissue and to monitor the effects of therapy. Other imaging techniques have also been employed to observe altered metabolic states in tumors. Proton magnetic resonance spectroscopy (1H MRS) enables the detection and quantitation of abundant metabolites, some of which have prognostic or diagnostic value. To date, this technique has been used most extensively in brain tumors and other central nervous system diseases (Oz et al., 2014). An example of a recent technical development is the use of 1H MRS to detect 2-hydroxyglutarate, an “oncometabolite” produced by mutant forms of the metabolic enzymes isocitrate dehydrogenase-1 and -2 in brain tumors (Andronesi et al., 2012; Choi et al., 2012; Elkhaled et al., 2012; Pope et al., 2012). After a considerable amount of research over the past decade, metabolic reprogramming as a consequence of tumorigenic mutations in oncogenes or tumor suppressor genes is now considered to be one of the major biological hallmarks of cancer (Hanahan & Weinberg, 2011).

Stable isotope tracers like 13C are widely used to investigate metabolism in living systems. Transfer of 13C from a parent molecule (e.g., glucose) into downstream metabolites reports the activity of metabolic pathways, providing an important complement to measurements of steady-state metabolite levels. Because 13C is a nonradioactive tracer that is effectively monitored using analytical techniques like nuclear magnetic resonance or mass spectrometry, administering 13C to animals or human subjects is safe. A typical experiment involves the administration of one or more isotope-labeled nutrients to the subject, followed by periodic or endpoint acquisition of tissues of interest (e.g., blood, urine, tumor), extraction of informative metabolites, and analysis of isotope enrichment patterns to infer metabolic pathway activity. This type of approach has been used successfully in mice and humans to probe metabolic alterations in cancer and other metabolic disorders (Busch, Neese, Awada, Hayes, & Hellerstein, 2007; Maher et al., 2012; Marin-Valencia et al., 2012; Sunny, Parks, Browning, & Burgess, 2011; Ying et al., 2012; Yuneva et al., 2012).

Each of these methods has only a limited ability to report on the metabolic network. Stable isotope approaches are invasive and destructive; that is, they require tissue sampling and metabolite extraction in order to obtain information about metabolism. In addition, metabolic activity is inferred from 13C enrichment rather than by observing metabolism directly through an imaging technique. On the other hand, while PET supports direct, noninvasive detection of a labeled metabolic probe, the resulting information is limited to anatomic localization of probe uptake and accumulation, with essentially no information about downstream metabolic...