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

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 ␣-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 ␣-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.Glutamine is utilized at a high rate by rapidly growing cells, including almost all cultured cell lines, where it is required at super-physiological concentrations of 2-4 mM for optimal growth (1). Recently, we evaluated the role of glutamine as a substrate for lipogenesis in a transformed wild type (WT) 5 and IRS-1 knock-out brown adipose cell lines developed by Kahn and co-workers (2). Using isotopomer spectral analysis (ISA) we found that WT cells utilized glutamine for over 40% of their lipogenic acetyl-CoA (3). Glutamine was the largest precursor for lipogenic carbon, supplying more acetyl-CoA units than glucose or any other single source. This unexpected result led us to investigate glutamine metabolism in brown fat cell lines in more detail.The pathway for glutamine utilization in rapidly dividing cell is generally described as "glutaminolysis" where glutamine enters the citric acid cycle (CAC) as ␣-ketoglutarate traversing the cycle to oxaloacetate and exits as pyruvate or aspartate (1, 4). Pyruvate then could be converted to acetyl-CoA and citrate in the lipogenic pathway. A major argument for glutaminolysis is the need for a large supply of anaplerotic substrates for rapidly growing cells, which explains the high rate of glutamine utilization (5). An alternative to glutaminolysis is the reductive carboxylation path...

Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole

“…Loss of NAD + -dependent IDH is shared with dinoflagellates, and therefore the switch to the NADP + -dependent form likely occurred early, although the reasons for this are unknown [9]. This activity is unusual because, in mammals, NADP + -dependent IDH classically operates in the reductive (or reverse) direction, allowing TCA cycle regulation [55,56], although the oxidative direction can occur when under oxidative stress [57].…”

Metabolic Pathway Alterations that Support Cell Proliferation

“…Whereas the reductive metabolism of glutamine was known to occur in some differentiated tissues (Yoo et al 2008), recent evidence using isotope-labeled carbon tracing suggests that the reductive metabolism of glutamine can be a major pathway for lipid production when cells are hypoxic . Reductive glutamine metabolism is a minor source of lipogenic acetyl-coA when oxygen levels are high, but glutamine becomes the major source of carbon for lipid synthesis when cells are proliferating under low-oxygen conditions (Fig.…”

“…Many proliferating cells synthesize lipids de novo (Ookhtens et al 1984;Menendez and Lupu 2007), and glucose is a major source of acetylcoA in cells (Hatzivassiliou et al 2005;Wellen et al 2009). However, glutamine can also be a source of carbon for acetyl-coA (Yoo et al 2004(Yoo et al , 2008DeBerardinis et al 2007), and it may be the principle source of this important biosynthetic precursor under some physiological conditions .…”

“…This route of glutamine metabolism, termed glutaminolysis, can fuel mitochondrial ATP generation, serve as a source of NADPH by the action of malic enzyme, and supply anapleurotic carbon to the TCA cycle (DeBerardinis et al 2007;Dang 2010). However, glutamine can also be used to generate cytosolic acetyl-coA via a reductive pathway involving direct conversion of a-ketoglutarate and CO 2 to citrate (Yoo et al 2008;Metallo et al 2011). This citrate can then be used as a source of acetyl-coA in the cytosol (Fig.…”

Distinct mTORC1 pathways for transcription and cleavage of SREBP-1c

“…Because the synthesis of fatty acids from glucose depends on flux through PDH, it is difficult to understand why a coordinated lipogenic signal by mTORC1 would include inhibition of this enzyme, which is typically stimulated by insulin. It is possible to generate fatty acids from glutamate by reductive carboxylation in the liver, adipose tissue, and some tumors by a pathway not requiring PDH (18)(19)(20), but the quantitative contribution of this pathway to normal and pathological lipid accumulation has not been defined. Interestingly, mouse livers, which have constitutive accumulation of HIF-2 by virtue of tissue-specific deletion of von Hippel-Lindau protein, develop steatosis, consistent with HIF-2 being a positive regulator of triglyceride accumulation (21).…”