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Positron Emission Tomography Probe Demonstrates a Striking Concentration of Ribose Salvage in the Liver

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Positron Emission Tomography Probe Demonstrates a Striking Concentration of Ribose Salvage in the Liver Positron emission tomography probe demonstrates a striking concentration of ribose salvage in the liver Peter M. Clarka,1, Graciela Floresb,c, Nikolai M. Evdokimovb,d, Melissa N. McCrackenb, Timothy Chaia, Evan Nair-Gillb, Fiona O’Mahonye, Simon W. Beavene, Kym F. Faullf,g, Michael E. Phelpsb,c,1, Michael E. Jungd, and Owen N. Wittea,b,h,i,1 Departments of aMicrobiology, Immunology, and Molecular Genetics, bMolecular and Medical Pharmacology, dChemistry and Biochemistry, and gPsychiatry and Biobehavioral Sciences, cCrump Institute for Molecular Imaging, eDivision of Digestive Diseases, fPasarow Mass Spectrometry Laboratory, Semel Institute for Neuroscience and Human Behavior, hEli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and iHoward Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095 Contributed by Michael E. Phelps, June 5, 2014 (sent for review March 10, 2014) PET is a powerful technique for quantifying and visualizing bio- zyme extracts (2, 7). Additionally, fasting serum ribose concen- chemical pathways in vivo. Here, we develop and validate a trations (∼100 μM) in humans are similar to plasma and serum novel PET probe, [18F]-2-deoxy-2-fluoroarabinose ([18F]DFA), for concentrations of other gluconeogenic substrates, including pyru- in vivo imaging of ribose salvage. DFA mimics ribose in vivo and vate (∼50 μM) and glycerol (∼100 μM) (8, 9). Finally, the con- accumulates in cells following phosphorylation by ribokinase and 18 version of ribose to glucose requires less energy and reducing further metabolism by transketolase. We use [ F]DFA to show power than either pyruvate or glycerol (1). that ribose preferentially accumulates in the liver, suggesting a Other data suggest that ribose may have alternative and dis- striking tissue specificity for ribose metabolism. We demonstrate tinct roles different from those of glucose in the body. Fasting that solute carrier family 2, member 2 (also known as GLUT2), a glu- ∼ cose transporter expressed in the liver, is one ribose transporter, but blood ribose concentrations are 50-fold lower than blood glu- we do not know if others exist. [18F]DFA accumulation is attenuated cose concentrations (3). With few exceptions, cells and tissues – in several mouse models of metabolic syndrome, suggesting an as- cannot survive with ribose as their sole carbohydrate source (10 sociation between ribose salvage and glucose and lipid metabolism. 12). Additionally, RBKS is substrate-inhibited at ribose con- These results describe a tool for studying ribose salvage and sug- centrations of >0.5 mM, suggesting that cells may limit the gest that plasma ribose is preferentially metabolized in the liver. amount of ribose they salvage (6). PET is used for imaging and quantifying changes in whole- molecular imaging | sugar metabolism | Slc2a2 body metabolism through the use of radiolabeled substrates (13). The potential utility of new PET probes is best exemplified by ibose is a naturally occurring monosaccharide whose me- the radiolabeled glucose analog [18F]2-fluoro-2-deoxyglucose Rtabolites contribute to DNA, RNA, and energy production ([18F]FDG). [18F]FDG has been extensively used both preclini- (1). Most studies and textbooks focus on the intracellular syn- cally and clinically to study glucose metabolism in vivo, includ- thesis of ribose-5-phosphate from glucose via the oxidative and ing to identify metabolic changes during brain development, to nonoxidative pentose phosphate pathways (1) (Fig. 1A). How- monitor myocardial viability, and to image and identify tumors in ever, a few studies suggest that cells can also salvage extracellular vivo (13–15). We reasoned that a PET probe for measuring and ribose for further intracellular metabolism (2). Ribose is likely the imaging ribose salvage could provide unique and novel infor- second most abundant carbohydrate in blood, being present at mation on this understudied pathway in vivo. ∼100 μM in human fasting serum (3). Ribose is also present in the diet and is absorbed through the intestines into the blood stream Significance (3–5). However, despite the abundance of blood ribose, little is known about ribose salvage, including its biodistribution and regulation. The saccharide ribose is naturally present in food and circulates During salvage, ribose is transported across the cell mem- in the blood. Previous studies suggest that cells internalize ri- brane and phosphorylated by ribokinase (RBKS) to ribose-5- bose directly from the extracellular space, but how, why, and phosphate (6). Ribose-5-phosphate can be incorporated into where this occurs in the body are not well understood. Here, we developed a new PET probe to monitor this process in vivo. nucleic acids via the de novo nucleotide synthesis pathway or Using this probe and [14C]ribose, we show that ribose salvage is metabolized to glycolytic intermediates via the nonoxidative concentrated in the liver. We identify that solute carrier family 2, pentose phosphate pathway (1). Enzymes in the nonoxidative member 2 is one of potentially several ribose transporters. We pentose phosphate pathway include transketolase (TKT) and demonstrate that ribose salvage is down-regulated during meta- transaldolase 1 (TALDO1), sequential enzymes that transfer bolic syndrome. This work raises the possibility that ribose is an carbons between phosphorylated carbohydrate intermediates. important sugar for whole-body metabolism. An additional enzyme in the nonoxidative pentose phosphate pathway is ribose-5-phosphate isomerase (RPIA), which cat- Author contributions: P.M.C., G.F., N.M.E., F.O., S.W.B., K.F.F., M.E.P., M.E.J., and O.N.W. alyzes the interconversion of ribose-5-phosphate and ribulose- designed research; P.M.C., G.F., N.M.E., M.N.M., T.C., E.N.-G., and F.O. performed research; 5-phosphate (1). Alternatively, the first step to incorporate P.M.C., G.F., and N.M.E. contributed new reagents/analytic tools; P.M.C., G.F., N.M.E., M.N.M., T.C., K.F.F., M.E.P., M.E.J., and O.N.W. analyzed data; and P.M.C. and O.N.W. ribose-5-phosphate into nucleic acids is phosphorylation by wrote the paper. phosphoribosyl pyrophosphate synthetase 1 or 2 (PRPS1 or PRPS2) A Conflict of interest statement: P.M.C., G.F., N.M.E., M.E.J., and O.N.W. are coauthors on (1) (Fig. 1 ). a patent application that covers the PET probe described in this paper. Several lines of evidence suggest that blood ribose and ribose Freely available online through the PNAS open access option. salvage could be linked to glucose metabolism. Cells intercon- 1 To whom correspondence may be addressed. Email: [email protected], mphelps@ vert ribose-5-phosphate and glucose-6-phosphate via the non- mednet.ucla.edu, or [email protected]. oxidative pentose phosphate pathway (1). Ribose is metabolized This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to glucose and glucose-6-phosphate in liver slices and liver en- 1073/pnas.1410326111/-/DCSupplemental. E2866–E2874 | PNAS | Published online June 30, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1410326111 Downloaded by guest on October 1, 2021 PNAS PLUS P De novo nucleotide O O synthesis pathway A HO HO OH OH P O O Glucose-6- O P O P OH OH phosphate Phosphoribosyl Pentose phosphate pyrophosphate synthetase 1 Phosphoribosyl pathway and 2 (PRPS1/2) pyrophosphate HO P O O OH O OH Ribokinase Non-oxidative pentose phosphate pathway OH OH (RBKS) OH OH Ribose OH OH OH OH Ribose-5- P O HO O HO P Ribose-5-phosphate phosphate Transketolase (TKT) + O OH OH O OH isomerase (RPIA) xylulose-5-phosphate Sedoheptulose Fructose-6- -7-phosphate phosphate Non-oxidative pentose + Transaldolase 1 + phosphate pathway (TALDO1) O OH O OH O P O P O O P OH OH OH OH Ribulose-5- Glyceraldehyde- Erythrose-4- phosphate 3-phosphate phosphate B [18 ([18F]-DFA) C [18F]-DFA [18F]-DFA + [19F]-DFA standard Electrochemical Electrochemical detector detector intensity intensity Relative signal Relative signal Gamma Gamma detector detector intensity intensity Relative signal Relative signal 012 3456789 10 0345678912 10 Time (min) Time (min) D E [18F]-DFA plus n plasma, 0 minutes C/mm *1000 g o TLC i g e r 5 r F O R 4 intensity Relative signal /mm 3 3 [18F]-DFA plus 2 plasma, 180 minutes Counts x 10 1 intensity 0 Relative signal 0 20 40 60 80 Position (mm) 03456789112 0 Time (min) Fig. 1. [18F]DFA can be prepared in 99% radiochemical purity. (A) Ribose salvage and metabolic pathway. (B)[18F]DFA synthetic scheme. (C) HPLC analysis of [18F]DFA with and without [19F]DFA standard coinjection. In all cases, the signal is normalized to the maximum signal in each chromatogram. (D) Radio-TLC analysis of [18F]DFA. (E) Radio-HPLC analysis of [18F]DFA incubated in plasma for 0 and 180 min. 18 Here, we report and validate a ribose-based PET probe, [ F] Results MEDICAL SCIENCES 18 2-deoxy-2-fluoroarabinose ([ F]DFA), for studying ribose sal- [18F]DFA Is a PET Probe for Ribose Salvage. To understand ribose vage. Using this probe, we show that ribose salvage is concen- salvage in vivo better, we developed a PET probe that mimics trated in the liver. We also demonstrate that solute carrier family the structure of ribose. An earlier study showed that human 2, member 2 (Slc2a2) is one among potentially several ribose RBKS can metabolize ribose and, with lower affinity, arabinose transporters. Finally, we show that ribose salvage is attenuated in (6). This suggested to us that ribose derivatized at the 2-position mouse models of metabolic syndrome. Our studies suggest that with a positron-emitting nuclide could be metabolized through [18F]DFA is a powerful imaging probe for studying ribose salvage the ribose salvage pathway.
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