Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

BACKGROUND:Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear. METHODOLOGY/PRINCIPAL FINDINGS:This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects. CONCLUSIONS/SIGNIFICANCE:The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

<p>Abstract</p> <p>Background</p> <p>Fungal β-<it>N</it>-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-<it>N</it>-acetylhexosaminidase. The fungal β-<it>N</it>-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from <it>Aspergillus oryzae </it>was purified and its sequence was determined.</p> <p>Results</p> <p>The complete primary structure of the fungal β-<it>N</it>-acetylhexosaminidase from <it>Aspergillus oryzae </it>CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the <it>N</it>-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.</p> <p>Conclusion</p> <p>Whereas the intracellular bacterial β-<it>N</it>-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-<it>N</it>-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and <it>N</it>-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys⁴⁴⁸with Cys⁴⁸³stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.</p