QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cresenzi, C. L. Articles by Stipanuk, M. H. Search for Related Content PubMed PubMed Citation Articles by Cresenzi, C. L. Articles by Stipanuk, M. H.

---

Biochemical and Molecular Actions of Nutrients

Cysteine Is the Metabolic Signal Responsible for Dietary Regulation of Hepatic Cysteine Dioxygenase and Glutamate Cysteine Ligase in Intact Rats¹

Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853

²To whom correspondence should be addressed. E-mail: mhs6{at}cornell.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Cysteine, rather than a precursor or metabolite of cysteine, appears to mediate the upregulation of cysteine dioxygenase (CDO) and the downregulation of glutamate cysteine ligase (GCL) in cultured primary rat hepatocytes. However, similar experiments in intact rats have not been performed to confirm in vivo that changes in hepatic cysteine levels are associated with the regulation of CDO or GCL activity. Therefore, rats were fed a low protein basal diet (100 g casein/kg diet) with or without supplemental sulfur amino acids (8 g cystine, 9 g homocystine or 10 g methionine/kg diet) and with or without propargylglycine (PPG, 1 mmol/kg), an irreversible inhibitor of cystathionine -lyase. Rats were fed the assigned diet for 2 full days and up until the mid-point of the dark cycle on d 3, at which time they were killed for collection of liver. Rats fed the PPG-containing diets had hepatic cystathionine -lyase activities that were 16% of the uninhibited level. PPG treatment reduced CDO activity by 50 and 54%, increased GCL activity by 41 and 61% and lowered total cysteine concentration by 33 and 64% in liver of the homocystine and methionine-supplemented groups, respectively, but not in the cystine-supplemented groups or unsupplemented groups. Glutathione levels were not affected by PPG treatment in any groups. These experiments are consistent with a role for cysteine, rather than a precursor or metabolite of cysteine, in the metabolic signaling responsible for diet-induced regulation of CDO and GCL.

KEY WORDS: • cysteine • cysteine dioxygenase • glutamate cysteine ligase • glutathione • homocysteine • methionine

In addition to utilization for protein synthesis, cysteine is incorporated into the tripeptide glutathione (GSH)² and catabolized to cysteinesulfinate, taurine, pyruvate and inorganic sulfur (Fig. 1). These products of cysteine metabolism play important roles in synthetic reactions, detoxification processes, osmotic regulation, nervous system function and antioxidative processes. Cysteine metabolism is also necessary for removal of excess cysteine, permitting its carbon chain to be used as a fuel and its N and S atoms to be excreted. Methionine is an important precursor of cysteine sulfur. Methionine is metabolized by the transmethylation pathway to homocysteine, and the homocysteine is either remethylated to methionine or condensed with serine to form cystathionine and ultimately cysteine, -ketobutyrate and ammonia.

View larger version (22K): [in this window] [in a new window]   FIGURE 1 Major pathways of sulfur amino acid metabolism, showing the conversion of methionine and homocystine to cysteine and the metabolism of cysteine. The steps catalyzed by cysteine dioxygenase (CDO) and -glutamate cysteine ligase (GCL) and the reaction catalyzed by cystathionine -lyase and blocked by DL-propargylglycine (PPG) are indicated.

Normal regulation of cysteine catabolism plays an important role in health because elevated levels of cysteine have been shown to be both cytotoxic and neurotoxic, and increased levels of homocysteine, a precursor of cysteine in the methionine transsulfuration pathway, have been associated with increased risk for cardiovascular disease and with the occurrence of neural tube defects (1–4). In addition, high plasma total cysteine concentrations have been associated with risk of preeclampsia, premature delivery, low birthweight and cardiovascular disease (5,6). Although cysteine levels must be high enough to satisfy the body’s needs for GSH and protein synthesis, it is clear that they must also be kept low so as to not harm the tissues. We have demonstrated that cysteine levels are tightly regulated by both hepatic CDO and GCL activities, which respond rapidly to changes in the amount of dietary protein or sulfur amino acids. These two enzymes respond in a reciprocal manner, with CDO activity increasing and GCL activity decreasing in response to an increase in protein or sulfur amino acid level (7–10). When dietary sulfur amino acids are high, high CDO and low GCL activities act to somewhat limit the rate of GSH synthesis and favor the catabolism of cysteine to taurine and sulfate. On the other hand, when dietary sulfur amino acids are low, high GCL and low CDO activities appear to ensure that cysteine is efficiently used for GSH synthesis, rather than being catabolized.

Our studies of the regulation of CDO and GCL activities in rat liver by dietary sulfur amino acid intake indicated that increases in CDO activity were due predominantly to parallel increases in enzyme concentration, whereas decreases in GCL activity were accounted for both by decreases in GCL-catalytic subunit concentration and by decreases in its activity state (8,9,11). Upregulation of CDO concentration is due to an inhibition of CDO polyubiquitination and degradation by the 26S proteasome; either cysteine or cysteamine, but not mercaptoethanol or cysteinesulfinate, inhibited CDO degradation (12). Studies in rat hepatocytes have clearly shown that cysteine, not methionine or transmethylation/transsulfuration intermediates, is essential for upregulation of CDO activity and downregulation of GCL activity (13). Furthermore, sulfate and taurine, metabolites of cysteine, had no effect on CDO or GCL activities in hepatocytes (13). Additionally, inhibition of GSH synthesis with buthionine sulfoximine had no effect on CDO activity; GCL gene transcription was upregulated by the decline in GSH status of the hepatocytes, making it difficult to rule out an effect of cysteine via GSH in regulation of GCL in response to nutrient supply at the cellular level (13).

This study was performed to determine whether inhibition of the transsulfuration pathway would block the upregulation of CDO and downregulation of CDO in rats fed diets supplemented with methionine, or homocystine, but not in those supplemented with cystine. We used propargylglycine, an irreversible inhibitor of cystathionine -lyase (also known as cystathionase, EC 4.4.1.1), to inhibit the cleavage of cystathionine to cysteine and, thus, block the transsulfuration pathway (Fig. 1). We also measured hepatic cysteine and GSH concentrations (at the mid-point of the feeding period) to ascertain relationships of thiol concentrations to the regulation of CDO and GCL activities in intact rats.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and dietary treatments.

A semipurified low protein (LP, 100 g casein/kg) diet was prepared by Dyets (Bethlehem, PA) in powdered form and lacking 25 g sucrose per kg. The diet premix was based on the AIN 93A formulation (14) and contained (g/975g): 100 vitamin-free casein, 507.5 cornstarch, 155 dextrinized cornstarch, 75 sucrose, 50 Solka-Floc, 40 soybean oil, 35 AIN 93-MX mineral mix, 10 AIN-93-MX vitamin mix, 2.5 choline bitartrate, and 0.008 tert-butylhydroquinone. We used this premix to prepare the eight different experimental diets described in Table 1. L-Cystine (C) (8 g/kg diet), L-homocystine (H) (9 g/kg diet), or L-methionine (M) (10 g/kg diet) and DL-propargylglycine (PPG) (1 mmol/kg diet) were added to the premixed diet with the appropriate amount of sucrose. These experimental diets were then mixed with an equal volume (1 L/kg diet) of a hot agar solution (3 g/L). Diets were cooled, refrigerated and cut into cubes for feeding.

Rats were fed the assigned experimental diets, with fresh diet being given at the beginning of each dark cycle. Food intake and body weight were measured over the experimental period. At the mid-point (between 1250 and 1420 h) of the feeding or dark cycle on d 3, rats were anesthetized with CO₂ and killed by decapitation for collection of liver samples. Rats were killed in order of assigned weight blocks; within blocks, rats from various treatment groups were killed in random order. The protocol was approved by the Cornell University Institutional Animal Care and Use Committee.

Livers were rapidly removed, rinsed with ice-cold saline, blotted, weighed and frozen in liquid nitrogen. Frozen tissues were stored at -140°C until analyses were performed. Preliminary studies demonstrated that enzyme activity was not affected by freezing and storing tissues for up to 2 wk.

Enzyme assays.

Portions of frozen tissue were homogenized in ice-cold 0.05 mol/L 2-(N-morpholino)ethanesulfonic acid buffer, pH 6.0, or 0.03 mol/L potassium phosphate buffer, pH 6.9, for assay of CDO and cystathionine -lyase activities, respectively. For assay of GCL activity, portions of frozen tissue were homogenized in 20 mmol/L Tris buffer, pH 8.2, and the 100,000 x g supernatant fraction was obtained. Assay of CDO activity was conducted as described previously (10). Assay of cystathionine -lyase activity was carried out as described by Gaitonde et al. (15) and Viña et al. (16). Assay of GCL activity was as described by Yan and Huxtable (17) except that both -glutamylcysteine and GSH were measured as products because some of the -glutamylcysteine was further converted to GSH using glycine present in the tissue supernatant. GSH and -glutamylcysteine were quantified by the HPLC method of Fariss and Reed (18) as modified by Stipanuk et al. (19). Protein concentration was determined by the method of Smith et al. (20).

Cysteine and GSH concentrations.

For analysis of total cysteine and GSH (thiol plus disulfide forms), 100–200 mg of frozen tissue was pulverized with a mortar and pestle sitting on dry ice. The powdered tissue was weighed and added to 1 mL of 1 mol/L perchloric acid containing 1 mmol/L bathophenanthroline disulfonate and 1 mmol/L bathocuproine disulfonate. The sample mixture was sonicated with two 20-s bursts separated by a cooling period, and the sonicated mixture was frozen at -70°C. For further analysis, samples were thawed and centrifuged at 15,000 x g for 10 min to obtain the acid extract. As an internal standard, -glutamylglutatmate was added to the acid supernatant to yield a final concentration of 0.5 mmol/L. The sample/internal standard mixture was then derivatized as described by Fariss and Reed (18). The 2,4-dinitrophenyl derivatives of acidic amino acids, including those of the derivatized thiols (S-carboxymethylcysteine and S-carboxymethylglutathione), were separated by reversed-phase ion-exchange HPLC and quantified as described previously (19).

Statistics.

Data were analyzed by two-way ANOVA and Tukey’s -procedure using Minitab 13.1. A mean separation procedure was necessary, in addition to ANOVA, because of the strong interaction between sulfur amino acid supplement and PPG, which was anticipated. Values for CDO activity were transformed to log₁₀ before analysis to meet the assumption of equal variance. Differences were considered significant at P 0.05. Although there were fewer rats in the homocystine-fed groups, variances of the means were similar to those for other groups. The presence of fewer rats in these groups would decrease the likelihood of observing a significant effect.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Weight gain, food intake and liver weight.

The mean weight of rats at the start of the dietary treatment period was 227 g. Rats fed diets supplemented with cystine, homocystine or methionine gained more weight during the 2.5-d experimental period than those in the LP group, and rats fed diets supplemented with PPG gained significantly less weight than rats fed the same diet without PPG (Table 2). Nevertheless, because of the short treatment period, the final body weights of the rats did not differ among the diet groups. Food intake was similar for rats fed the LP, LP+C and LP+M diets but was greater for those fed the LP+H diet. Rats fed the LP, LP+H or LP+M diets with PPG consumed less diet than did those fed the same diet without PPG; rats fed the LP+C diet with or without PPG had similar food intakes. Absolute and relative liver weights differed among groups with the LP+C and LP+M groups (-PPG) having the largest mean liver weights and groups fed the LP diet (±PPG) having the lowest mean liver weights. Results for other measurements are expressed on a concentration basis (per g tissue or per mg protein) and therefore do not reflect the contribution of changes in liver weight to metabolic capacity or metabolite concentrations. However, increases in liver weight would magnify the effect of protein or sulfur amino acid supplementation on CDO but would somewhat compensate for the effects of diet on GCL, whether metabolic capacity is expressed as total capacity per rat or capacity per 100 g body.

Hepatic cystathionine-lyase activity.

Rats fed diets that contained PPG had significantly lower cystathionine -lyase activity than the corresponding non-PPG groups. The LP, LP +C, LP+H and LP+M groups had 5-, 6-, 4- and 5-times greater cystathionine -lyase activity than the LP+PPG, LP+C+PPG, LP+H+PPG and LP+M+PPG groups, respectively (Table 3). Overall, cystathionine -lyase activity was reduced to 16% of its uninhibited level.

View this table: [in this window] [in a new window]   TABLE 3 Cystathionine -lyase, cysteine dioxygenase (CDO) and glutamate cysteine ligase (GCL) activities and cysteine and glutathione (GSH) concentrations in liver of rats fed low protein (LP) diets with or without supplemental cystine (C), homocystine (H) or methionine (M) and with or without propargylglycine (PPG)1

CDO activity in liver of rats fed the LP diet was only 0.32 ± 0.13 nmol/(min · mg protein), but CDO activity was 6-, 7-, and 6.5-fold higher in the LP+C, LP+H and LP+M groups (Table 3). PPG treatment reduced (P 0.05) CDO activity in the homocystine- and methionine-supplemented groups to levels that were about half those in the rats fed the same diet without PPG. In contrast, PPG did not affect CDO activity in rats fed the LP and LP+C diets.

Hepatic GCL activity.

GCL activity in liver of rats fed the LP diet was 8.9 nmol/(min · mg protein), which was less than half that in liver of rats fed the LP diet supplemented with cystine, methionine or homocystine (Table 3). Addition of PPG did not affect GCL activity in rats fed the LP or LP+C diets, but increased it (P 0.05) in the LP+M groups and tended to increase it (P 0.10) in the LP+H groups. The decrease in GCL activity when sulfur amino acids are abundant may limit the high rate of GSH synthesis that results from elevated intracellular cysteine concentration, thus facilitating the degradation of cysteine by CDO.

Hepatic cysteine and GSH concentrations.

Total cysteine concentration was 0.024 µmol/g liver in the LP group, and the LP+C, LP+H and LP+M groups had cysteine concentrations that were 2-, 3- and 5- times that of the LP group, respectively (Table 3). Rats fed the LP and LP+C diets with and without PPG had similar cysteine concentrations. The LP+H+PPG and LP+M+PPG groups had hepatic cysteine concentrations that were 67 and 36% those of the LP+H and LP+M groups, respectively.

The hepatic total GSH concentration of rats fed the LP diet was 1.3 µmol/g. Rats fed diets supplemented with one of the sulfur-containing amino acids had higher GSH concentrations (4.4 µmol/g liver). The hepatic GSH concentration was not affected by the addition of PPG to any of the diets.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Cellular cysteine levels must be closely regulated to prevent cysteine toxicity while maintaining adequate levels of cysteine for synthesis of protein, GSH and coenzyme A. Previous work in our laboratory demonstrated that hepatic CDO and GCL play critical roles in regulating cysteine metabolism (8,9). Liver also is the major tissue involved in the transsulfuration of dietary methionine to form cysteine, largely because of the presence in liver of an isozyme of methionine adenosyltransferase (EC 2.5.1.6) that has a high K_m for methionine (21). Cystathionine -lyase catalyzes the final step in the transsulfuration pathway, resulting in cleavage of cystathionine to yield cysteine, -ketobutyrate and ammonia. Essentially all methionine sulfur is transferred to serine to form cysteine before oxidation of the sulfur atom (22,23).

In this study, PPG was used to inhibit cystathionine -lyase activity and, hence, flux through the transsulfuration pathway, thereby reducing cysteine formation from methionine or homocysteine. This was clearly effective because hepatic cystathionine -lyase activity was reduced by 84% in rats fed diets containing 1 mmol PPG/kg diet. Also, the addition of PPG reduced hepatic cysteine concentration in rats fed the LP+H or LP+M diets to 67 or 36%, respectively, of the level in rats fed the same diet without PPG. Nevertheless, the inhibition of transsulfuration was not complete, and residual cystathionine -lyase activity allowed livers of rats fed the LP+H+PPG or LP+M+PPG diets to maintain cysteine and GSH concentrations that were higher than those in rats fed the unsupplemented LP diet.

It is not surprising that cystathionine -lyase activity was not completely inhibited in this study. In work done in vitro with rat hepatocytes, cystathionine -lyase activity was reduced by 99% by 1 mmol/L PPG (24). However, the amount of PPG used in this study was restricted to avoid severe adverse effects on the animals, including markedly reduced food intake, and the amount of PPG consumed by rats (50 µmol over 2.5 d per 230-g rat) clearly could not have produced a concentration approaching that used in the in vitro studies.

Because CDO and GCL activities have been shown to markedly respond to dietary protein or sulfur amino acid supply over a range of 0.5- to 2-times the requirement, diets were designed to be nutritionally limiting or excessive in sulfur amino acids. The LP diet provided 3.3 g methionine equivalents/kg diet and was inadequate in total sulfur amino acid content compared with the NRC recommendation that sulfur amino acids comprise 6 g/kg diet (25). The LP+C, LP+H and LP+M diets each provided 13.2 g methionine equivalents/kg and thus an excess of sulfur amino acids. During the 2.5-d experimental period, although rats fed the LP diet consumed the same amount of food as rats fed the methionine- and cystine-supplemented diets, they gained less weight, demonstrating that the LP diet clearly was limiting in sulfur amino acids. Rats fed diets containing PPG consistently gained less weight than those fed the same diet without PPG, but rats fed the cystine-supplemented diet with PPG gained significantly more weight than those in the other groups given PPG. Food intake was significantly reduced in all groups given PPG except for the cystine-supplemented groups. Thus, both food intake and the adequacy of cysteine supply affected weight gain. Because we demonstrated previously that CDO and GCL activities respond only to the protein or sulfur amino acid content of the diet and are not affected by changes in macronutrient composition (26), it is unlikely that differences in food intake of the magnitude observed in this study would have much effect on CDO or GCS activity.

As was reported previously, supplementation of the diet with cystine or methionine markedly increased hepatic CDO activity and markedly decreased hepatic GCL activity. In this study, methionine, homocystine and cystine were equally effective. CDO and GCL activities in liver of rats fed the cystine-supplemented diet were not affected by inhibition of cystathionine -lyase activity with PPG, i.e., CDO activity remained high and GCL activity remained low. Hepatic cysteine and GSH concentrations also were unaffected by PPG. On the other hand, inhibition of cystathionine -lyase activity diminished the effectiveness of homocystine and/or methionine in upregulation of CDO and downregulation of GCL, i.e., CDO was lower (P 0.05) in rats fed the LP+H or LP+M diet with PPG, and GCL was higher (P 0.05) in rats fed the LP+M diet with PPG. This indicates that formation of cysteine from homocyst(e)ine or methionine is necessary for these sulfur amino acids to affect CDO or GCL activity. Hepatic cysteine concentration in rats fed the LP+H or LP+M diet was decreased by 50% in rats given PPG, but GSH concentration was not affected. No effect of PPG on CDO and GCL activities occurred in rats fed the LP diet because the sulfur amino acid content of the diet was much lower than the requirement for adequate protein synthesis and was insufficient to elevate cellular cysteine content even in the absence of PPG. These observations in intact rats are consistent with our observation in cultured rat hepatocytes that cysteine, rather than methionine or a transmethylation/transsulfuration pathway intermediate, plays a key role in mediating changes in CDO and GCL activities (13). In addition, these studies in intact rats demonstrate that both CDO and GCL activities respond to a change in cysteine concentration in the absence of any change in GSH concentration.

Unpredictably, hepatic cysteine concentration was higher in rats fed the diets supplemented with homocystine or methionine than in those fed the diets supplemented with cystine, but this was not associated with differences in CDO or GCL activity. Consequently, there was an apparent lack of association of CDO or GCL activity with hepatic cysteine concentration among the sulfur amino acid–supplemented groups, which was also observed in rats fed diets supplemented with protein vs. sulfur amino acids (11). A likely explanation is that the time course for uptake and metabolism of homocystine and methionine by the liver results in a different pattern of fluctuation in cysteine concentration in the liver that is not apparent from our single time point measurement. Steady-state activities of CDO and GCL likely result from the level of cysteine exposure over a period of hours or days.

Because cysteine is a substrate for GSH synthesis, hepatic GSH levels tend to parallel hepatic cysteine concentrations, at least when intakes are in the low-to-normal range. This was observed in this study in that GSH levels were much higher in liver of rats fed the sulfur amino acid–supplemented diets than in liver of rats fed the unsupplemented low protein diet. Interestingly, hepatic GSH concentration was not decreased by the addition of PPG to the various diets, but this may be explained by the previous observation that hepatic GSH concentration remains relatively constant when cysteine concentration is >0.04 µmol/g (11) and also by the higher priority of GSH synthesis than cysteine catabolic pathways for cysteine when its supply is limiting (27). However, the combination of high GSH concentration with low weight gain in rats given both supplemental sulfur amino acid and PPG seems inconsistent with the higher priority of protein synthesis than GSH synthesis for available cysteine (27). In fact, rats fed the LP diet (-PPG) gained more weight than did rats fed the LP+H+PPG or LP+M+PPG diet (but not the LP+C+PPG diet) and yet had much lower cysteine and GSH concentrations. Thus, it appears that much of the effect of PPG on weight gain must be attributed to effects of PPG other than its effect on food intake or on methionine/homocysteine conversion to cysteine for use in protein synthesis.

Overall, it appears that PPG only partially blocked the conversion of methionine and homocysteine to cysteine and that rats fed these diets with PPG still had an adequate supply of cysteine as judged by intracellular cysteine and GSH concentrations. Nevertheless, PPG did impair methionine transsulfuration, as demonstrated by the decrease in hepatic cysteine concentration and association of the lower cysteine concentration with lower CDO and higher GCL activities. Additionally, PPG did not affect hepatic cysteine concentration nor did it affect CDO or GCL activities in rats fed a cystine-supplemented diet. Thus, the results of this study in intact rats support previous work with hepatocytes that suggested the regulation of CDO and GCL in response to cysteine, rather than methionine or GSH concentration.

Recent work on the regulation of CDO in hepatocytes has provided more direct evidence for a role for cysteine (12). Cysteine appears to act directly on CDO or on the E2-E3 complex involved in ubiquitination of CDO to block degradation of CDO by the ubiquitin-proteasome system. The mechanism by which cysteine downregulates GCL activity has not been elucidated but appears to be distinct from the well-known transcriptional regulation in response to oxidative stress and from the allosteric inhibition of the enzyme by GSH. Further work on the biological mechanisms involved in the dietary regulation of both of these enzymes by cysteine is required to confirm and clarify the role of cysteine in mediating its own metabolism.

	FOOTNOTES

 
¹ Supported by grant DK56649 from the National Institutes of Health.

³ C, L-Cystine; CDO, cysteine dioxygenase; GCL, glutamate cysteine ligase; GSH, glutathione; H, L-homocystine; LP, low protein; M, L-methionine; PPG, DL-propargylglycine.

Manuscript received 21 April 2003. Initial review completed 24 May 2003. Revision accepted 17 June 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Karlsen, R. L., Grofova, I., Malthe-Sorenssen, D. & Fonnum, F. (1981) Morphological changes in rat brain induced by L-cysteine injection in newborn animals. Brain Res. 208:167-180.[Medline]

2. Mathisen, G. A., Fonnum, F. & Paulsen, R. E. (1996) Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells. Neurochem. Res. 21:293-298.[Medline]

3. Pedersen, O. O. & Karlsen, R. L. (1980) The toxic effect of L-cysteine on the rat retina. A morphological and biochemical study. Investig. Ophthalmol. Vis. Sci. 19:886-892.[Free Full Text]

4. Sandberg, M., Orwar, O. & Hehmann, A. (1991) L-Cysteine toxicity: effects on non-sulfur amino acids, sulfur-containing excitatory amino acids and -glutamyl peptides in the immature brain. J. Neurochem. 57:S152.

5. El-Khairy, L., Vollset, S. E., Refsum, H. & Ueland, P. M. (2003) Plasma total cysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine Study. Am. J. Clin. Nutr. 77:467-472.[Abstract/Free Full Text]

6. El-Khairy, L., Ueland, P. M., Nygård, O., Refsum, H. & Vollset, S. E. (1999) Lifestyle and cardiovascular disease risk factors as determinants of total cysteine in plasma: the Hordaland Homocysteine Study. Am. J. Clin. Nutr. 70:1016-1024.[Abstract/Free Full Text]

7. Bagley, P. J. & Stipanuk, M. H. (1995) Rats fed a low protein diet supplemented with sulfur amino acids have increased cysteine dioxygenase activity and increased taurine production in hepatocytes. J. Nutr. 125:933-940.

8. Bella, D. L., Hahn, C. & Stipanuk, M. H. (1999) Effects of non-sulfur and sulfur amino acids on the regulation of hepatic enzymes of cysteine metabolism. Am. J. Physiol. 277:E144-E153.

9. Bella, D. L., Hirschberger, L. L., Hosokawa, Y. & Stipanuk, M. H. (1999) The mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo. Am. J. Physiol. 276:E326-E335.

10. Bella, D. L. & Stipanuk, M. H. (1995) Effects of protein, methionine, or chloride on acid-base balance and on cysteine catabolism. Am. J. Physiol. 269:E910-E917.

11. Stipanuk, M. H., Londono, M., Lee, J. I., Hu, M. & Yu, A. F. (2002) Enzymes and metabolites of cysteine metabolism in nonhepatic tissues of rats show little response to changes in dietary protein or sulfur amino acid levels. J. Nutr. 132:3369-3378.[Abstract/Free Full Text]

12. Stipanuk, M. H., Londono, M & Hirschberger, L. L. (2003) Cysteine-responsive regulation of hepatic cysteine dioxygenase by the ubiquitin-proteasome system. FASEB J. 17:A737 (abs.).

13. Kwon, Y. H. & Stipanuk, M. H. (2001) Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes. Am. J. Physiol. 280:E804-E815.

14. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.

15. Gaitonde, M. K. (1967) A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J. 104:627-633.[Medline]

16. Viña, J., Gimenez, A., Puertes, I. R., Gasco, E. & Viña, J. (1992) Impairment of cysteine synthesis from methionine in rats exposed to surgical stress. Br. J. Nutr. 68:421-429.[Medline]

17. Yan, C. C. & Huxtable, R. J. (1996) Effects of monocrotaline, a pyrrolizidine alkaloid, on glutathione metabolism in the rat. Biochem. Pharmacol. 51:375-379.[Medline]

18. Fariss, M. W. & Reed, D. J. (1987) High performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol. 143:101-109.[Medline]

19. Stipanuk, M. H., Bagley, P. J., Coloso, R. M. & Banks, M. F. (1992) Metabolism of cysteine to taurine by rat hepatocytes. Adv. Exp. Med. Biol. 315:413-421.[Medline]

20. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85.[Medline]

21. Sullivan, D. M. & Hoffman, J. L. (1983) Fractionation and kinetic properties of rat liver and kidney methionine adenosyltransferase isozymes. Biochemistry 22:1641-1645.[Medline]

22. Stipanuk, M. H. (1986) Metabolism of sulfur-containing amino acids. Annu. Rev. Nutr. 6:179-209.[Medline]

23. Stipanuk, M. H. (1999) Homocysteine, cysteine, and taurine. Shils, M. E. Olson, J. A. Shike, M. Ross, A. C. eds. Modern Nutrition in Health and Disease 9th ed. 1999:543-558 Williams and Wilkins Baltimore, MD. .

24. Drake, M. R., De La Rosa, J. & Stipanuk, M. H. (1987) Metabolism of cysteine in rat hepatocytes. Biochem. J. 244:279-286.[Medline]

25. National Research Council (1978) Nutrient Requirements of Laboratory Animals 1978:13-16 National Academy of Sciences Washington, DC.

26. Bella, D. L., Kwon, Y. H. & Stipanuk, M. H. (1996) Variations in dietary protein but not in dietary fat plus cellulose or carbohydrate levels affect cysteine metabolism in rat isolated hepatocytes. J. Nutr. 126:2179-2187.

27. Stipanuk, M. H., Coloso, R. M., Garcia, R.A.G. & Banks, M. F. (1992) Cysteine concentration regulates cysteine metabolism to glutathione, sulfate and taurine in rat hepatocytes. J. Nutr. 122:420-427.

This article has been cited by other articles:

				J.-I. Lee, J. E. Dominy Jr., A. K. Sikalidis, L. L. Hirschberger, W. Wang, and M. H. Stipanuk HepG2/C3A cells respond to cysteine deprivation by induction of the amino acid deprivation/integrated stress response pathway Physiol Genomics, April 1, 2008; 33(2): 218 - 229. [Abstract] [Full Text] [PDF]

				J. E. Dominy Jr., J. Hwang, and M. H. Stipanuk Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E62 - E69. [Abstract] [Full Text] [PDF]

				S. Ye, X. Wu, L. Wei, D. Tang, P. Sun, M. Bartlam, and Z. Rao An Insight into the Mechanism of Human Cysteine Dioxygenase: KEY ROLES OF THE THIOETHER-BONDED TYROSINE-CYSTEINE COFACTOR J. Biol. Chem., February 2, 2007; 282(5): 3391 - 3402. [Abstract] [Full Text] [PDF]

				M. Reid, A. Badaloo, T. Forrester, and F. Jahoor In vivo rates of erythrocyte glutathione synthesis in adults with sickle cell disease Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E73 - E79. [Abstract] [Full Text] [PDF]

				M. H. Stipanuk, J. E. Dominy Jr., J.-I. Lee, and R. M. Coloso Mammalian Cysteine Metabolism: New Insights into Regulation of Cysteine Metabolism J. Nutr., June 1, 2006; 136(6): 1652S - 1659S. [Abstract] [Full Text] [PDF]

				M. H. Stipanuk, L. L. Hirschberger, M. P. Londono, C. L. Cresenzi, and A. F. Yu The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver Am J Physiol Endocrinol Metab, March 1, 2004; 286(3): E439 - E448. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cresenzi, C. L. Articles by Stipanuk, M. H. Search for Related Content PubMed PubMed Citation Articles by Cresenzi, C. L. Articles by Stipanuk, M. H.