Heather B. Roman

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.

The Cysteine Dioxgenase Knockout Mouse: Altered Cysteine Metabolism in Nonhepatic Tissues Leads to Excess H2S/HS− Production and Evidence of Pancreatic and Lung Toxicity

AIMS To define the consequences of loss of cysteine dioxygenase (CDO) on cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS(-) (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO(-/-) mice that were fed either a taurine-free or taurine-supplemented diet. RESULTS CDO(-/-) mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of cysteine, consistent with the loss of CDO. CDO(-/-) mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS(-). Accumulation of cystathionine and lanthionine appeared to result from cystathionine β-synthase (CBS)-mediated cysteine desulfhydration. Very high levels of hypotaurine in pancreas of wild-type mice and very high levels of cystathionine and lanthionine in pancreas of CDO(-/-) mice were observed, suggesting a unique cysteine metabolism in the pancreas. INNOVATION The CDO(-/-) mouse model provides new insights into tissue-specific cysteine metabolism, particularly the role of pancreas in metabolism of excess cysteine by CBS-catalyzed reactions, and will be a useful model for studying the effects of excess endogenous production of H2S/HS(-). CONCLUSION The CDO(-/-) mouse clearly demonstrates that H2S/HS(-) production in tissues can exceed the capacity of the animal to oxidize sulfide to sulfate and demonstrates that pancreas and lung are more susceptible to toxicity from endogenous H2S/HS(-)production than are liver and kidney.

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 bodys 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.

Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate-cysteine ligase subunits

Abstract Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate–cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser51 phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent.

Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice.

To gain further insights into the effects of elevated cysteine levels on energy metabolism and the possible mechanisms underlying these effects, we conducted studies in cysteine dioxygenase (Cdo1)–null mice. Cysteine dioxygenase (CDO) catalyzes the first step of the major pathway for cysteine catabolism. When CDO is absent, tissue and plasma cysteine levels are elevated, resulting in enhanced flux of cysteine through desulfhydration reactions. When Cdo1‐null mice were fed a high‐fat diet, they gained more weight than their wild‐type controls, regardless of whether the diet was supplemented with taurine. Cdo1‐null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl‐CoA desaturase 1 (SCD1) compared to wild‐type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated cysteine levels with greater weight gain and with elevated hepatic Scd1 expression are also seen in the Cdo1‐null mouse model. Hepatic accumulation of acylcarnitines suggests impaired mitochondrial β‐oxidation of fatty acids in Cdo1‐null mice. The strong associations of elevated cysteine levels with excess H2S production and impairments in energy metabolism suggest that H2S signaling could be involved.

Insights into Taurine Synthesis and Function Based on Studies with Cysteine Dioxygenase ( CDO1 ) Knockout Mice

Cysteine is metabolized to cysteinesulfinate by cysteine dioxygenase (CDO), which is encoded by the Cdo1 gene. This CDO-mediated pathway is the dominant pathway for biosynthesis of taurine in mammals. Studies with Cdo1 −/− mice offer new insights about taurine synthesis and function. Taurine and hypotaurine levels in 8-week old Cdo1 −/− mice, which had access to taurine only from their heterozygous (Cdo1 +/− ) dams during gestation and suckling, were very low compared to levels in wild-type mice fed the same taurine-free diet. On the other hand, hepatocyte-specific disruption of Cdo1 did not result in taurine or hypotaurine depletion in liver; and plasma, kidney and pancreas hypotaurine levels were actually significantly increased along with increased abundance of CDO in these extrahepatic tissues, demonstrating the capacity of extrahepatic tissues to compensate for the loss of hepatic CDO. Unexpectedly, hypotaurine levels varied markedly among tissues in wild-type mice and were particularly high in the pancreas; hypotaurine levels in the pancreas were 65-times those in liver, and hypotaurine as a percentage of taurine content of the same tissue (on a molar basis) was 60 % in pancreas but less than 1 % in liver. Lack of CDO and low tissue taurine levels were also associated with increases in unconjugated and glycine-conjugated bile acid concentrations and increases in concentrations of other potential organic osmolytes.

Identification of Taurine-Responsive Genes in Murine Liver Using the Cdo1-Null Mouse Model

The cysteine dioxygenase (Cdo1)-null mouse is unable to synthesize hypotaurine and taurine by the cysteine/cysteine sulfinate pathway and has very low taurine levels in all tissues. The lack of taurine is associated with a lack of taurine conjugation of bile acids, a dramatic increase in the total and unconjugated hepatic bile acid pools, and an increase in betaine and other molecules that serve as organic osmolytes. We used the Cdo1-mouse model to determine the effects of taurine deficiency on expression of proteins involved in sulfur amino acid and bile acid metabolism. We identified cysteine sulfinic acid decarboxylase (Csad), betaine:homocysteine methytransferase (Bhmt), cholesterol 7α-hydroxylase (Cyp7a1), and cytochrome P450 3A11 (Cyp3a11) as genes whose hepatic expression is strongly regulated in response to taurine depletion in the Cdo1-null mouse. Dietary taurine supplementation of Cdo1-null mice restored hepatic levels of these four proteins and their respective mRNAs to wild-type levels, whereas dietary taurine supplementation had no effect on abundance of these proteins or mRNAs in wild-type mice.

Cysteine dioxygenase is essential for mouse sperm osmoadaptation and male fertility

Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles. Cysteine dioxygenase (CDO) is a critical enzyme for taurine synthesis. A previous study reported that male CDO−/− mice exhibit idiopathic infertility, prompting us to investigate the functions of CDO in male fertility. Immunoblotting and quantitative reverse transcription‐polymerase chain reaction analysis of epididymal segments showed that androgen‐dependent CDO expression was highest in the caput epididymidis. CDO−/− mouse sperm demonstrated a severe lack of in vitro fertilization ability. Acrosome exocytosis and tyrosine phosphorylation profiles in response to stimuli were normal, suggesting normal functioning of pathways associated with capacitation. CDO−/− sperm had a slight increase in head abnormalities. Taurine and hypotaurine concentrations in CDO−/− sperm decreased in the epididymal intraluminal fluid and sperm cytosol. We found no evidence of antioxidant protection against lipid peroxidation. However, CDO−/− sperm exhibited severe defects in volume regulation, swelling in response to the relatively hypo‐osmotic conditions found in the female reproductive tract. Our findings suggest that epididymal CDO plays a key role in post‐testicular sperm maturation, enabling sperm to osmoregulate as they transition from the male to the female reproductive tract, and provide new understanding of the compartmentalized functions of the epididymis.