The mammalian liver tightly regulates its free cysteine pool, and intracellular cysteine in rat liver is maintained between 20 and 100 nmol/g even when sulfur amino acid intakes are deficient or excessive. By keeping cysteine levels within a narrow range and by regulating the synthesis of glutathione, which serves as a reservoir of cysteine, the liver addresses both the need to have adequate cysteine to support normal metabolism and the need to keep cysteine levels below the threshold of toxicity. Cysteine catabolism is tightly regulated via regulation of cysteine dioxygenase (CDO) levels in the liver, with the turnover of CDO protein being dramatically decreased when intracellular cysteine levels increase. This occurs in response to changes in the intracellular cysteine concentration via changes in the rate of CDO ubiquitination and degradation. Glutathione synthesis also increases when intracellular cysteine levels increase as a result of increased saturation of glutamate-cysteine ligase (GCL) with cysteine, and this contributes to removal of excess cysteine. When cysteine levels drop, GCL activity increases, and the increased capacity for glutathione synthesis facilitates conservation of cysteine in the form of glutathione (although the absolute rate of glutathione synthesis still decreases because of the lack of substrate). This increase in GCL activity is dependent on up-regulation of expression of both the catalytic and modifier subunits of GCL, resulting in an increase in total catalytic subunit plus an increase in the catalytic efficiency of the enzyme. An important role of cysteine utilization for coenzyme A synthesis in maintaining cellular cysteine levels in some tissues, and a possible connection between the necessity of controlling cellular cysteine levels to regulate the rate of hydrogen sulfide production, have been suggested by recent literature and are areas that deserve further study.

The NAD+-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic sensor of the Sirtuin family that mediates homeostatic responses to certain physiological stresses such as nutrient restriction. Previous reports have implicated fluctuations in intracellular NAD+ concentrations as the principal regulator of SIRT1 activity. However, here we have identified a cAMP-induced phosphorylation of a highly conserved serine (S434) located in the SIRT1 catalytic domain that rapidly enhanced intrinsic deacetylase activity independently of changes in NAD+ levels. Attenuation of SIRT1 expression or the use of a non-phosphorylatable SIRT1 mutant prevented cAMP-mediated stimulation of fatty acid oxidation and gene expression linked to this pathway. Overexpression of SIRT1 in mice significantly potentiated the increases in fatty acid oxidation and energy expenditure caused by either pharmacological β-adrenergic agonism or cold exposure. These studies support a mechanism of Sirtuin enzymatic control through the cAMP/PKA pathway with important implications for stress responses and maintenance of energy homeostasis.

Summary Hepatic glucose production (HGP) maintains blood glucose levels during fasting but can also exacerbate diabetic hyperglycemia. HGP is dynamically controlled by a signaling/transcriptional network that regulates the expression/activity of gluconeogenic enzymes. A key mediator of gluconeogenic gene transcription is PGC-1α. PGC-1α’s activation of gluconeogenic gene expression is dependent upon its acetylation state, which is controlled by the acetyltransferase GCN5 and the deacetylase Sirt1. Nevertheless, whether other chromatin modifiers—particularly other sirtuins—can modulate PGC-1α acetylation is currently unknown. Herein we report that Sirt6 strongly controls PGC-1α acetylation. Surprisingly, Sirt6 induces PGC-1α acetylation and suppresses HGP. Sirt6 depletion decreases PGC-1α acetylation and promotes HGP. These acetylation effects are GCN5 dependent: Sirt6 interacts with and modifies GCN5, enhancing GCN5’s activity. Leprdb/Leprdb mice, an obese/diabetic animal model, exhibit reduced Sirt6 levels; ectopic re-expression suppresses gluconeogenic genes and normalizes glycemia. Activation of hepatic Sirt6 may therefore be therapeutically useful for treating insulin-resistant diabetes.

Insulin constitutes a major evolutionarily conserved hormonal axis for maintaining glucose homeostasis1-3; dysregulation of this axis causes diabetes2,4. PGC-1α links insulin signaling to the expression of glucose and lipid metabolic genes5-7. GCN5 acetylates PGC-1α and suppresses its transcriptional activity, whereas SIRT1 deacetylates and activates PGC-1α8,9. Although insulin is a mitogenic signal in proliferative cells10,11, whether components of the cell cycle machinery contribute to insulin’s metabolic action is poorly understood. Herein, we report that insulin activates cyclin D1-CDK4, which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high throughput chemical screen, we identified a CDK4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK3β signaling induces cyclin D1 protein stability via sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 mRNA transcripts. Activated cyclin D1-CDK4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycemia. In diabetic models, cyclin D1-CDK4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division.

Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the ␥-sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactorcontaining enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe 2؉ ) and O 2 . Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by ϳ10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.Recent biochemical and crystallographic studies have revealed that some enzymes contain unusual modifications to the amino acid residues residing within their active sites. Also known as amino acid-derived cofactors, these altered residues represent a new and exciting area for research in the field of protein post-translational modifications. Unlike other posttranslational modifications, which are made by attaching extrinsic molecules onto target proteins through reactions that are specifically catalyzed by third party enzymes, amino acid cofactors are generated directly from the amino acids within the proteins that contain them and are required for the production of fully competent enzymes (1, 2). From a biological perspective, amino acid-derived cofactors are significant because they can create novel structural motifs within the enzyme active site as well as alter the chemical properties of the unmodified parent amino acid residues and thus expand the otherwise limited range of catalytic reactions in which the common amino acids can participate.One recent addition to the list of enzymes known to contain an amino acid cofactor is cysteine dioxygenase (CDO).3 CDO is an iron (Fe 2ϩ )-dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cys...

In metazoa and fungi, the catabolic dissimilation of cysteine begins with its sulfoxidation to cysteine sulfinic acid by the enzyme cysteine dioxygenase (CDO). In these organisms, CDO plays an important role in the homeostatic regulation of steady-state cysteine levels and provides important oxidized metabolites of cysteine such as sulfate and taurine. To date, there has been no experimental evidence for the presence of CDO in prokaryotes. Using PSI-BLAST searches and crystallographic information about the active-site geometry of mammalian CDOs, we identified a total of four proteins from Bacillus subtilis, Bacillus cereus, and Streptomyces coelicolor A3(2) that shared low overall identity to CDO (13 to 21%) but nevertheless conserved important active-site residues. These four proteins were heterologously expressed and purified to homogeneity by a single-step immobilized metal affinity chromatography procedure. The ability of these proteins to oxidize cysteine to cysteine sulfinic acid was then compared against recombinant rat CDO. The kinetic data strongly indicate that these proteins are indeed bona fide CDOs. Phylogenetic analyses of putative bacterial CDO homologs also indicate that CDO is distributed among species within the phyla of Actinobacteria, Firmicutes, and Proteobacteria. Collectively, these data suggest that a large subset of eubacteria is capable of cysteine sulfoxidation. Suggestions are made for how this novel pathway of cysteine metabolism may play a role in the life cycle of the eubacteria that have it.Cysteine is an indispensable amino acid for all forms of life. It serves as a precursor for protein synthesis, and the unique chemistry of its free thiol group forms the active moiety of several essential metabolites such as coenzyme A, glutathione and its derivatives, and mycothiol. Because many basic cellular processes are dependent upon a steady supply of cysteine, there has been extensive work to identify in both prokaryotes and eukaryotes the pathways that ultimately determine the steady-state free intracellular cysteine pool (26). The generic pathways that increase the levels of free intracellular cysteine are those of de novo biosynthesis and transport of preformed cysteine from the extracellular milieu. On the other hand, the pathways responsible for depleting free cysteine include those that incorporate cysteine into other molecules and those that physically degrade the cysteine molecule.Among the specific pathways that contribute to the metabolic economy of intracellular cysteine, one of the major differences between eukaryotes and prokaryotes has been believed to be the capacity to degrade cysteine to cysteine sulfinic acid (21). This reaction, catalyzed by the Fe 2ϩ -dependent enzyme cysteine dioxygenase (CDO; EC 1.13.11.20), irreversibly oxidizes the sulfhydryl group of cysteine (Fig. 1). CDO shows a high degree of specificity for cysteine since structurally related thiols are neither substrates for oxidation nor competitive inhibitors of activity (31). In mammals-eukaryotes for wh...

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Mammals possess an intricate regulatory system for controlling flux through fuel utilization pathways in response to the dietary availability of particular macronutrients. Under fasting conditions, for instance, mammals initiate a whole body metabolic response that limits glucose utilization and favors fatty acid oxidation. Understanding the underlying mechanisms by which this process occurs will facilitate the development of new treatments for metabolic disorders such as type II diabetes and obesity. One of the recently identified components of the signal transduction pathway involved in metabolic reprogramming is PGC-1α. This transcriptional coactivator is able to coordinate the expression of a wide array of genes involved in glucose and fatty acid metabolism. The nutrientmediated control of PGC-1α activity is tightly correlated with its acetylation state. In this review, we evaluate how the nutrient regulation of PGC-1α activity squares with the regulation of its acetylation state by the deacetylase Sirt1 and the acetyltransferase GCN5. We also propose an outline of additional experimental directives that will help to shed additional light on this very powerful transcriptional coactivator.