Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line - PubMed (original) (raw)

Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line

Hyuntae Yoo et al. J Biol Chem. 2008.

Abstract

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 alpha-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-(13)C]glutamine or [5-(13)C]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 alpha-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.

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Figures

FIGURE 1.

FIGURE 1.

Mass isotopomer distribution of palmitate isolated from WT brown adipocytes under incubation in medium containing 4 mm [5-13C]glutamine from day 2 to day 6 (mean ± S. E.;n = 3). Contribution of [5-13C]glutamine to palmitate synthesis (D value) and fractional new synthesis of palmitate (g value) determined by ISA.

FIGURE 2.

FIGURE 2.

Effect of oxalomalate on palmitate synthesis from [U-13C]glutamine. Shown is the mass isotopomer distribution of palmitate from WT brown adipocytes on day 4 under 6 h of incubation in medium containing 4 m

m

[U-13C]glutamine and 0, 5, or 10 m

m

oxalomalate (Error bars, mean ± S.E.; n = 3).

FIGURE 3.

FIGURE 3.

Effect of increasing concentration of oxalomalate (A) and 2-methylisocitrate (B) on D(Gln) and g(6 h) values of palmitate synthesis from [U-13C]glutamine and from [U-13C]glucose (C). Asterisk (*) indicates significant difference compared with the control condition without inhibitor (p < 0.005). Error bars, mean ± S.E.

FIGURE 4.

FIGURE 4.

Effect of increasing concentration of oxalomalate on labeling patterns of key intermediates of CAC. Mass isotopomer distributions of glutamate (A) and citrate (B) from WT brown adipocytes on day 4 under 6 h of incubation in medium containing 4 m

m

[U-13C]glutamine and 0, 5, or 10 m

m

oxalomalate. Data were corrected for natural isotope enrichments. Error bars, mean ± S.E.

FIGURE 5.

FIGURE 5.

Flux from [U-13C]glucose to key intermediates of glycolysis and CAC. Mass isotopomer distributions of pyruvate and lactate (A) and malate and aspartate (B) from WT brown adipocytes on day 4 under 6 h of incubation in medium containing 25 m

m

[U-13C]glucose and 4 m

m

unlabeled glutamine. Data were corrected for natural isotope enrichments. Error bars, mean ± S.E.

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References

    1. Newsholme, E. A., Crabtree, B., and Ardawi, M. S. (1985) Biosci. Rep. 5 393–400 - PubMed
    1. Fasshauer, M., Klein, J., Kriauciunas, K. M., Ueki, K., Benito, M., and Kahn, C. R. (2001) Mol. Cell. Biol. 21 319–329 - PMC - PubMed
    1. Yoo, H., Stephanopoulos, G., and Kelleher, J. K. (2004) J. Lipid Res. 45 1324–1332 - PubMed
    1. Mazurek, S., and Eigenbrodt, E. (2003) Anticancer Res. 23 1149–1154 - PubMed
    1. Board, M., Humm, S., and Newsholme, E. A. (1990) Biochem. J. 265 503–509 - PMC - PubMed

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