Real-Time Estimates of Hepatic Gluconeogenesis 1 Real-Time Detection of Hepatic Gluconeogenic and Glycogenolytic States Using Hyperpolarized [2- C]Dihydroxyacetone*

Glycogenolysis and gluconeogenesis are sensitive to nutritional state and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized (HP) carbon-13 magnetic resonance (MR) is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or glycogenolytic state. Unexpectedly we found that [2-C]DHA was metabolized within a few seconds to the common intermediates and end-products of both glycolysis and gluconeogenesis under both conditions, including [2,5-C]glucose, [2C]glycerol-3-phosphate, [2C]phosphoenolpyruvate (PEP), [2-C]pyruvate, [2-C]alanine, and [2-C]lactate. [2C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis was monitored in functioning tissue for the first time. Observation of [2-C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers. Gluconeogenesis from glycerol, lactate, propionate and amino acids is required for survival during prolonged fasting, and many disorders such as type 2 diabetes are associated with abnormal regulation of gluconeogenesis. Consequently numerous methods have been developed to monitor gluconeogenesis in the liver (1-7). Hyperpolarization of C-labeled precursors offers a new approach for investigating metabolism that combines detailed chemical information about the products of metabolism coupled with the practical advantages of stable isotopes (8). A method to noninvasively detect and conceivably image http://www.jbc.org/cgi/doi/10.1074/jbc.M114.613265 The latest version is at JBC Papers in Press. Published on October 28, 2014 as Manuscript M114.613265 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on July 5, 2017 hp://w w w .jb.org/ D ow nladed from Real-Time Estimates of Hepatic Gluconeogenesis 2 hepatic gluconeogenesis would have a major impact. One approach could be to monitor the rate of appearance of hyperpolarized (HP) CO2 and HP [C]bicarbonate, which may be sensitive to gluconeogenesis. HP CO2 is generated during gluconeogenesis from HP [1-C]pyruvate via the following pathway: carboxylation to [1C]oxaloacetate followed by scrambling in the fumarate and succinate pool to generate [4C]oxaloacetate and subsequent decarboxylation at phosphoenolpyruvate carboxykinase. However, decarboxylation of [1-C]pyruvate via pyruvate dehydrogenase also generates CO2, so the contribution of flux through phosphoenolpyruvate carboxykinase to the total rate of production of HP CO2 and HP [C]bicarbonate is uncertain(9,10). Dihydroxyacetone (DHA), like pyruvate, is avidly consumed as a substrate for gluconeogenesis in the liver (11). DHA is phosphorylated rapidly to dihydroxyacetone phosphate (DHAP) which exchanges readily with glycerol-3 phosphate and glycerol. Like glycerol, the DHA carbon backbone enters gluconeogenesis in the triose phosphate pool (Figure 1). In this study, HP [2-C]DHA was investigated as an agent for unambiguously detecting conversion of a gluconeogenic precursor to hexoses, in real time. We tested the hypothesis that HP [2-C]DHA would be rapidly metabolized to hexoses in the gluconeogenic but not glycogenolytic state. Conditions were chosen to stimulate gluconeogenesis from 3-carbon precursors by studying livers from fasted animals provided with excess pyruvate (gluconeogenic state) compared to livers taken from fed animals where glucose production is predominantly by glycogenolysis (glycogenolytic state). The distribution of H in glucose produced by the liver in the presence of H2O was used to independently confirm that exported glucose arose primarily from glycogenolysis or gluconeogenesis. Conventional C NMR spectroscopy of effluent glucose provided independent confirmation of metabolism of [2-C]DHA to its various products. Generation of phosphoenolpyruvate was monitored continuously in functioning liver for the first time. The functional state of the liver – glycogenolytic vs. gluconeogenic – was distinguished in real time using hyperpolarized [2-C]DHA. This is the first case where gluconeogenesis from a 3-carbon precursor has been directly detected using a HP imaging agent. We found that the intermediates and end products of both glycolysis and gluconeogenesis were rapidly (on the time scale of ~10 seconds) enriched under dramatically different nutritional conditions. Earlier studies using Henriched glucose and C-enriched glycerol also found simultaneous flux through both glycolysis and gluconeogenesis in isolated hepatocytes. This finding was interpreted as compartmentalization of glycolysis and gluconeogenesis in a cell. Similar results could be observed if two populations of hepatocytes are present, one associated with glycolytic properties and predisposed to metabolize H-enriched glucose, and the other associated with gluconeogenic properties and predisposed to metabolize C-enriched glycerol (12). Since a single labeled compound was used in the current study, the results cannot be due to preferential uptake of one tracer or another, and provide further support for bi-directional flux in gluconeogenic and glycolytic pathways. EXPERIMENTAL PROCEDURES [2-C]Dihydroxyacetone-dimer (99 % enriched) was purchased from Isotec Laboratories (Miamisburg, OH) and used without further purification. The trityl radical, tris[8-carboxyl2,2,6,6-tetra-[2-(1-hydroxyethyl)]-benzo-(1,2d:4,5-d)-bis-(1,3)-dithiole-4-yl]-methyl sodium salt, was purchased from Oxford Molecular Biotools Ltd. (Abingdon, Oxfordshire, UK) and used without further purification. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) at the highest quality available. Female C57BL/6 mice (20–25 g) were obtained from Charles River Laboratories (Wilmington, MA). Fasted animals were fasted overnight (~12-15 hours) and fed animals were fed ad libitum prior to experimentation. The studies were performed under a protocol approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center. Hyperpolarization. An Oxford HyperSense (Abingdon, U.K.) dynamic nuclear polarization (DNP) hyperpolarizer was used to hyperpolarize [2C]dihydroxyacetone-dimer (DHA-dimer). An 8.0 by gest on July 5, 2017 hp://w w w .jb.org/ D ow nladed from Real-Time Estimates of Hepatic Gluconeogenesis 3 M solution of [2-C]DHA in a (2:1) water:DMSO mixture was doped with 15 mM stable trityl free radical (Oxford Molecular Biotools) and 1.0 mM Prohance. The frozen sample was cooled to 1.05 K in a pumped helium bath inside the magnetic field (3.35 T) of the HyperSense, and the microwave irradiation was turned on. When the final polarization was reached (~1.5 – 2 hours) the irradiation was turned off and the sample was rapidly dissolved with 4 mL hot, > 190 C, PBS (10 mM, pH ~ 7.4) and transferred to an 89 mm vertical 9.4 T NMR spectrometer for transfer into the perfusate chamber and spectral acquisition. The level of hyperpolarization can be estimated in separate experiments for each compound by measuring the NMR signal intensity after hyperpolarization and comparing it to a standard using the same NMR spectrometer. Spin-lattice relaxation of [2-C]DHA. The spinlattice relaxation, T1, time of carbon–2 for [2C]DHA was determined by the fitted exponential decay of the hyperpolarized compound using the following equation (13): S(τ) = Mpcosθ τ TR ⁄ ∗ sinθ −τ T1 ⁄ where S() is the signal intensity, Mp is the z magnetization at time = 0,  is the pulse width,  is time, and TR is the total repetition time. The T1 relaxation time at 9.4 T was determined to be 32