NMR IN BIOMEDICINE NMR Biomed. 2004;17:51–59 Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/nbm.860 Compartmentation of glycolysis and glycogenolysis in the perfused rat heart Nick Anousis, 1 Rui A. Carvalho, 1–3 Piyu Zhao, 1 Craig R. Malloy 3,4 and A. Dean Sherry 1,3 * 1 Department of Chemistry, University of Texas at Dallas, Richardson, TX 75083-0688, USA 2 Department of Biochemistry and Center of Neurosciences and Cellular Biology, University of Coimbra, Apartado 3126, 3001-401 Coimbra, Portugal 3 The Mary Nell and Ralph B. Rogers Magnetic Resonance Center, Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9085, USA 4 Department of Internal Medicine, University of Texas Southwestern Medical Center and Department of Veterans Affairs Medical Center, Dallas, TX 75216, USA Received 4 September 2003; Revised 16 October 2003; Accepted 19 November 2003 ABSTRACT: Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using 13 C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.5 mM [U- 13 C]glucose plus 50 mU/mL insulin until newly synthesized glycogen recovered to new steady-state levels (  60% of pre-depleted values). After a short wash-out period, the perfusate glucose was then switched to [1- 13 C]glucose, and glycolysis and glycogenolysis were stimulated by addition of glucagon (1 mg/ml). A 13 C NMR multiplet analysis of the methyl resonance of lactate provided an estimate of pyruvate derived from glucose vs glycogen while a multiplet analysis of the C4 resonance of glutamate provided an estimate of acetyl-CoA derived from glycolytic pyruvate vs glycogenolytic pyruvate. These two indices were not equivalent and their difference was further magnified in the presence of insulin during the stimulation phase. These combined observations are consistent with functional compartmentation of glycolytic and glycogenolytic enzymes that allows pyruvate generated by these two processes to be distinguished at the level of lactate and acetyl-CoA. Copyright # 2004 John Wiley & Sons, Ltd. KEYWORDS: metabolic compartmentation; glycolysis; glycogenolysis; NMR isotopomer analysis INTRODUCTION Experimental evidence has been accumulating for func- tional organization of enzymes within the cytosol of cells. 1 One example of possible functional compartmen- tation came from the early work of Mowbray and Ottaway, 2–5 who first presented evidence for multiple, metabolic pools of pyruvate. Most early evidence for compartmentation came from differences in 14 C specific activities of alanine vs lactate, metabolites that share pyruvate as a 3-carbon precursor. 6 Because of differences in activity and regulation of alanine transaminase vs lactate dehydrogenase, it is not unusual to find differen- tial alanine- and lactate-specific activities prior to meta- bolic steady state, 7 but significant differences between the specific activities of these two metabolites at meta- bolic steady state has often been used as evidence for functional compartmentation of pyruvate. 8 Peuhkurinen et al. 9 proposed that one pool of pyruvate in the myocyte is associated more closely with glycolysis and tissue lactate, while a second ‘peripheral’ pool is in close communication with extracellular pyruvate and mito- chondrial pyruvate. Tissue alanine is thought to be a better reflection of the second pyruvate pool than the first. 6 This phenomenon was reevaluated in more recent 13 C NMR studies of the heart. 8,10–16 Hardin and Kushmerick 15 demonstrated in smooth skeletal muscle that glycolytic intermediates derived from glucose do not fully mix with glycolytic intermediates derived from glycogen during simultaneous stimulation of both glyco- lysis and glycogenolysis. Furthermore, they observed that pyruvate derived from glycogen is oxidized in the citric Copyright # 2004 John Wiley & Sons, Ltd. NMR Biomed. 2004;17:51–59 *Correspondence to: A. D. Sherry, The Mary Nell and Ralph B. Rogers Magnetic Resonance Center, Department of Radiology, UT Southwestern Medical Center, 5801 Forest Park Road, Dallas, TX 75235-9085, USA. E-mail: Dean.Sherry@UTSouthwestern.edu Contract grant sponsor: NCR Biomedical Research Technology Pro- gram; contract grant number: P41-RR02584. Contract grant sponsor: NIH; contract grant number: HL-34557. Contract grant sponsor: Portuguese Foundation for Science and Technology; contract grant number: POCTI/CBO/38611/01. Abbreviations used: C3S, singlet component of the methyl carbon resonance of lactate; C3D, doublet component of the methyl carbon resonance of lactate; C4S, singlet component of the carbon-4 reso- nance of glutamate; C4D34, doublet component of the carbon-4 resonance of glutamate representing spin–spin coupling between carbon-3 and carbon-4; C4D45, doublet component of the carbon-4 resonance of glutamate representing spin–spin coupling between carbon-4 and carbon-5; C4DQ, quartet component of the carbon-4 resonance of glutamate representing spin–spin coupling between carbon-4 and both carbon-3 and carbon-5; [1- 13 C]glucose, glucose enriched with 13 C at the C1 carbon; gww, gram wet weight; HR, heart rate; KHB, Krebs–Henseleit bicarbonate; MSD rats, male Sprague– Dawley rats; PDH, pyruvate dehydrogenase complex; TCA cycle, tricarboxylic acid cycle; [U- 13 C]glucose, glucose enriched with 13 C in all carbons.