Quantitative analysis of carbon fluxes for fat biosynthesis in wild-type and IRS-1 knockout brown adipocytes

Abstract

Excessive fat synthesis and the subsequent dysregulation of lipid metabolism constitute the major pathological factors of obesity and type 2 diabetes through triggering insulin resistance. Thus, controlling fat synthesis by identifying key sites for regulation of lipogenesis and modulating the lipogenic fluxes may provide novel approaches to intervention of the diseases. As a first step to quantitative investigation of lipogenic fluxes from various carbon sources as related to insulin signaling, relative contribution of glucose, glutamine, and acetoacetate to fat biosynthesis in wild-type (WT) and insulin receptor substrate-i knockout (IRS-1 KO) brown adipocytes were analyzed by stable-isotope labeling, GC/MS, and flux estimation. Glutamine contributed more to fatty acid synthesis than glucose in WT cells while glucose's contribution was heavier in IRS-1 KO cells. Unlike the straightforward pathway for lipogenesis from glucose, two possibilities for glutamine's route to fatty acid synthesis have been proposed: glutaminolysis pathway through conventional tricarboxylic acid cycle and a pathway via reductive carboxylation of a-ketoglutarate to isocitrate. These pathways were integrated into a metabolic network model for quantitative estimation of individual lipogenic fluxes. Incubation of the cells with [U-13C] glutamine for 6 hrs led to metabolic and isotopic steady state where individual fluxes of the model were estimated with 95% confidence by least-square fit method.

(cont.) Dose dependent repression of estimated net flux of reductive carboxylation by specific inhibition of NADP+-dependent isocitrate dehydrogenase and the subsequent reduction in glutamine's contribution to fatty acid synthesis in WT cells strongly indicated that reductive carboxylation is an important site of regulating glutamine's lipogenic flux. Abolition of this net flux, reduction in glutamine's lipogenic contribution, and concurrent rise in glucose's lipogenic contribution in IRS-1 KO cells were consistent with the importance of reductive carboxylation. Differential effects of lack of insulin signaling on individual lipogenic fluxes suggested that there might be specific sites at which insulin signaling regulates lipogenic utilization of carbon sources. These results revealed the importance of other carbon sources such as glutamine in fat synthesis and the means by which the flux of these carbon sources to fat synthesis can be controlled.