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J. Biol. Chem., Vol. 283, Issue 30, 20621-20627, July 25, 2008

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Quantifying Reductive Carboxylation Flux of Glutamine to Lipid in a Brown Adipocyte Cell Line^*

Hyuntae Yoo¹², Maciek R. Antoniewicz¹³, Gregory Stephanopoulos, and Joanne K. Kelleher⁴

From the Department of Chemistry and Department of Chemical Engineering, Bioinformatics and Metabolic Engineering Laboratory, Massachusetts Institute of Technology, Cambridge Massachusetts 02139

We previously reported that glutamine was a major source of carbon for de novo fatty acid synthesis in a brown adipocyte cell line. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from -ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-¹³C]glutamine or [5-¹³C]glutamine and analyzed the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution to fluxes. We also investigated inhibitors of NADP-dependent isocitrate dehydrogenase and mitochondrial citrate export. The results indicated that one third of glutamine entering the citric acid cycle travels to citrate via reductive carboxylation while the remainder is oxidized through succinate. The reductive carboxylation flux accounted for 90% of all flux of glutamine to lipid. The inhibitor studies were compatible with reductive carboxylation flux through mitochondrial isocitrate dehydrogenase. Total cell citrate and -ketoglutarate were near isotopic equilibrium as expected if rapid cycling exists between these compounds involving the mitochondrial membrane NAD/NADP transhydrogenase. Taken together, these studies demonstrate a new role for glutamine as a lipogenic precursor and propose an alternative to the glutaminolysis pathway where flux of glutamine to lipogenic acetyl-CoA occurs via reductive carboxylation. These findings were enabled by a new modeling tool and software implementation (Metran) for global flux estimation.

Received for publication, August 6, 2007 , and in revised form, March 25, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant DK075850. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ Both authors contributed equally to this work.

² Present address: Inst. for Systems Biology, Seattle, WA 98103. E-mail: hyoo{at}systemsbiology.org.

³ Present address: Dept. of Chemical Engineering, University of Delaware, Newark, DE 19716. E-mail: mranton{at}udel.edu.

⁴ To whom correspondence should be addressed: Inst. of Technology, 77 Massachusetts Ave., Rm. 66-401, Cambridge, MA 02139. Tel.: 617-253-3178; Fax: 617-253-3122; E-mail: jkk{at}mit.edu.

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