Extrahepatic tissues compensate for loss of hepatic taurine synthesis in mice with liver-specific knockout of cysteine dioxygenase.

Because hepatic cysteine dioxygenase (CDO) appears to play the major role in controlling cysteine catabolism in the intact rat, we characterized the effect of a lack of hepatic CDO on the regulation of cysteine and its metabolites at the whole body level. In mice with liver-specific deletion of CDO expression, hepatic and plasma cysteine levels increased. In addition, in mice with liver-specific deletion of CDO expression, the abundance of CDO and the proportion of CDO existing as the mature, more active isoform increased in extrahepatic tissues that express CDO (kidney, brown fat, and gonadal fat). CDO abundance was also increased in the pancreas, where most of the enzyme in both control and liver CDO-knockout mice was in the more active isoform. This upregulation of CDO concentration and active-site cofactor formation were not associated with an increase in CDO mRNA and thus presumably were due to a decrease in CDO degradation and an increase in CDO cofactor formation in association with increased exposure of extrahepatic tissues to cysteine in mice lacking hepatic CDO. Extrahepatic tissues of liver CDO-knockout mice also had higher levels of hypotaurine, consistent with increased metabolism of cysteine by the CDO/cysteinesulfinate decarboxylase pathway. The hepatic CDO-knockout mice were able to maintain normal levels of glutathione, taurine, and sulfate. The maintenance of taurine concentrations in liver as well as in extrahepatic tissues is particularly notable, since mice were fed a taurine-free diet and liver is normally considered the major site of taurine biosynthesis. This redundant capacity for regulation of cysteine concentrations and production of hypotaurine/taurine is additional support for the body's robust mechanisms for control of body cysteine levels and indicates that extrahepatic tissues are able to compensate for a lack of hepatic capacity for cysteine catabolism.

Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide.

Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO(+/-) mice) were crossed to generate CDO(-/-), CDO(+/-), and CDO(+/+) mice. CDO(-/-) mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO(-/-) mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO(-/-) mice than in CDO(+/-) or CDO(+/+) mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO(-/-) mice. H(2)S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H(2)S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H(2)S/sulfane sulfur levels and facilitate the use of H(2)S as a signaling molecule.

Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur.

Synthesis of cysteine as a product of the transsulfuration pathway can be viewed as part of methionine or homocysteine degradation, with cysteine being the vehicle for sulfur conversion to end products (sulfate, taurine) that can be excreted in the urine. Transsulfuration is regulated by stimulation of cystathionine ß-synthase and inhibition of methylene tetrahydrofolate reductase in response to changes in the level of S-adenosylmethionine, and this promotes homocysteine degradation when methionine availability is high. Cysteine is catabolized by several desulfuration reactions that release sulfur in a reduced oxidation state, generating sulfane sulfur or hydrogen sulfide (H2S), which can be further oxidized to sulfate. Cysteine desulfuration is accomplished by alternate reactions catalyzed by cystathionine ß-synthase and cystathionine γ-lyase. Cysteine is also catabolized by pathways that require the initial oxidation of the cysteine thiol by cysteine dioxygenase to form cysteinesulfinate. The oxidative pathway leads to production of taurine and sulfate in a ratio of approximately 2:1. Relative metabolism of cysteine by desulfuration versus oxidative pathways is influenced by cysteine dioxygenase activity, which is low in animals fed low-protein diets and high in animals fed excess sulfur amino acids. Thus, desulfuration reactions dominate when cysteine is deficient, whereas oxidative catabolism dominates when cysteine is in excess. In rats consuming a diet with an adequate level of sulfur amino acids, about two thirds of cysteine catabolism occurs by oxidative pathways and one third by desulfuration pathways. Cysteine dioxygenase is robustly regulated in response to cysteine availability and may function to provide a pathway to siphon cysteine to less toxic metabolites than those produced by cysteine desulfuration reactions.

3T3-L1 adipocytes and rat adipose tissue have a high capacity for taurine synthesis by the cysteine dioxygenase/cysteinesulfinate decarboxylase and cysteamine dioxygenase pathways.

Taurine is the most abundant free amino acid in the body and is synthesized in mammals by 2 pathways. Taurine is synthesized either from the oxidation of cysteine via cysteine dioxygenase (CDO), which generates cysteinesulfinate that is decarboxylated by cysteinesulfinic acid decarboxylase (CSAD), or from the oxidation of cysteamine by cysteamine (2-aminoethanethiol) dioxygenase (ADO). Both pathways generate hypotaurine, which is oxidized to taurine. To determine whether these pathways for taurine synthesis are present in the adipocyte, we studied 3T3-L1 cells during their adipogenic conversion and fat from rats fed diets with varied sulfur-amino acid content. CDO, CSAD, and ADO protein levels increased during adipogenic differentiation of 3T3-L1 cells and all of these enzymes were significantly increased when cells achieved a mature adipocyte phenotype. Furthermore, these changes were accompanied by an increased hypotaurine and taurine production, particularly when cells were treated with cysteine or cysteamine. CDO mRNA levels also responded robustly to cysteine or cysteamine treatment in adipocytes but not in undifferentiated 3T3-L1 cells. Furthermore, CDO protein and activity were greater in adipose tissue from rats fed a high protein or cystine-supplemented low protein (LP) diet than in adipose tissue from rats fed a LP diet. Overall, our results demonstrate that CDO is regulated at both the level of enzyme abundance and the level of mRNA in mature adipocytes.

The acid-labile subunit is required for full effects of exogenous growth hormone on growth and carbohydrate metabolism.

Normal postnatal growth is dependent in part on overlapping actions of GH and IGF-I. These actions reflect GH stimulation of IGF-I production in liver and extrahepatic tissues, representing respectively the endocrine and autocrine/paracrine arms of the IGF system. Recent experiments in genetically modified mice show that each source of IGF-I can compensate for absence of the other but do not resolve their relative role in postnatal growth. In an effort to address this issue, we studied the GH responsiveness of mice harboring a null mutation of the acid-labile subunit (ALS). Null ALS mice have a substantial reduction in endocrine IGF-I but, unlike other models of plasma IGF-I deficiency, have no obvious additional endocrine defects. Wild type and null ALS mice of both sexes received daily sc injections of saline or recombinant bovine GH between d 35 and 63 of postnatal age. The GH-stimulated body weight gain of null ALS mice was reduced by more than 30% relative to wild type mice, irrespective of sex. Reductions in GH responsiveness were also seen for kidney and linear growth. Absence of ALS eliminated the ability of GH to increase plasma IGF-I despite intact GH-dependent stimulation of IGF-I expression in liver, adipose tissue, and skeletal muscle. GH treatment was also less efficient in antagonizing insulin action in null ALS mice. Overall, these results suggest that the GH effects mediated by endocrine IGF-I depends on ALS, and accordingly null ALS mice are less responsive to exogenous GH therapy.

Measurement of Cysteine Dioxygenase Activity and Protein Abundance.

Cysteine dioxygenase is an iron (Fe(2+))-dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cysteine to generate 3-sulfinoalanine (commonly called cysteinesulfinic acid). Cysteine dioxygenase activity is routinely assayed by measuring cysteinesulfinate formation from substrate L-cysteine at pH 6.1 in the presence of ferrous ions to saturate the enzyme with metal cofactor, a copper chelator to diminish substrate oxidation, and hydroxylamine to inhibit pyridoxal 5'-phosphate-dependent degradation of product. The amount of cysteine dioxygenase may be measured by immunoblotting. Upon SDS-PAGE, cysteine dioxygenase can be separated into two major bands, with the upper band representing the 23-kDa protein and the lower band representing the mature enzyme that has undergone formation of an internal thioether cross link in the active site. Formation of this cross link is dependent upon the catalytic turnover of substrate and produces an enzyme with a higher catalytic efficiency and catalytic half-life.

Enzymes of the taurine biosynthetic pathway are expressed in rat mammary gland.

Taurine is the most abundant free amino acid in the body and is present at high concentrations during development and in the early milk. It is synthesized from cysteine via oxidation of cysteine to cysteinesulfinate by the enzyme cysteine dioxygenase (CDO), followed by the decarboxylation of cysteinesulfinate to hypotaurine, catalyzed by cysteine sulfinic acid decarboxylase (CSAD). To determine whether the taurine biosynthetic pathway is present in mammary gland and whether it is differentially expressed during pregnancy and lactation, and also to further explore the possible regulation of hepatic taurine synthesis during pregnancy and lactation, we measured mammary and hepatic CDO and CSAD mRNA and protein concentrations and tissue, plasma and milk taurine concentrations. CDO and CSAD mRNA and protein were expressed in mammary gland and liver regardless of physiological state. Immunohistochemistry demonstrated the expression of CDO in ductal cells of pregnant rats, but not in other mammary epithelial cells or in ductal cells of nonpregnant rats. CDO was also present in stromal adipocytes in mammary glands of both pregnant and nonpregnant rats. Our findings support an upregulation of taurine synthetic capacity in the mammary gland of pregnant rats, based on mammary taurine and hypotaurine concentrations and the intense immunohistochemical staining for CDO in ductal cells of pregnant rats. Hepatic taurine synthetic capacity, particularly CSAD, and taurine concentrations were highest in rats during the early stages of lactation, suggesting the liver may also play a role in the synthesis of taurine to support lactation or repletion of maternal reserves.