High cysteine diet reduces insulin resistance in SHR-CRP rats.

Increased plasma total cysteine (tCys) has been associated with obesity and metabolic syndrome in human and some animal studies but the underlying mechanisms remain unclear. In this study, we aimed at evaluating the effects of high cysteine diet administered to SHR-CRP transgenic rats, a model of metabolic syndrome and inflammation. SHR-CRP rats were fed either standard (3.2 g cystine/kg diet) or high cysteine diet (HCD, enriched with additional 4 g L-cysteine/kg diet). After 4 weeks, urine, plasma and tissue samples were collected and parameters of metabolic syndrome, sulfur metabolites and hepatic gene expression were evaluated. Rats on HCD exhibited similar body weights and weights of fat depots, reduced levels of serum insulin, and reduced oxidative stress in the liver. The HCD did not change concentrations of tCys in tissues and body fluids while taurine in tissues and body fluids, and urinary sulfate were significantly increased. In contrast, betaine levels were significantly reduced possibly compensating for taurine elevation. In summary, increased Cys intake did not induce obesity while it ameliorated insulin resistance in the SHR-CRP rats, possibly due to beneficial effects of accumulating taurine.

Cysteine dioxygenase: a robust system for regulation of cellular cysteine levels.

Cysteine catabolism in mammals is dependent upon cysteine dioxygenase (CDO), an enzyme that adds molecular oxygen to the sulfur of cysteine, converting the thiol to a sulfinic acid known as cysteinesulfinic acid (3-sulfinoalanine). CDO is one of the most highly regulated metabolic enzymes responding to diet that is known. It undergoes up to 45-fold changes in concentration and up to 10-fold changes in catalytic efficiency. This provides a remarkable responsiveness of the cell to changes in sulfur amino acid availability: the ability to decrease CDO activity and conserve cysteine when cysteine is scarce and to rapidly increase CDO activity and catabolize cysteine to prevent cytotoxicity when cysteine supply is abundant. CDO in both liver and adipose tissues responds to changes in dietary intakes of protein and/or sulfur amino acids over a range that encompasses the requirement level, suggesting that cysteine homeostasis is very important to the living organism.

Surprising insights that aren't so surprising in the modeling of sulfur amino acid metabolism.

The modeling of whole organism sulfur amino acid flux control has been aided in recent years by advancements in proteomics and mass spectroscopy-based metabolite analysis. The convergence of these two fields and their respective techniques, as demonstrated by a new study using yeast by Lafaye et al., has shown that researchers seeking to model whole cell/organism metabolism should give careful consideration to the relationships connecting enzyme concentration, enzyme activity, substrate concentration, and metabolic flux. In this paper, we outline some of the fundamental concepts for modeling sulfur amino acid metabolism and how they relate to our current understanding of mammalian sulfur amino acid metabolism.

Evidence for expression of a single distinct form of mammalian cysteine dioxygenase.

Cysteine dioxygenase (CDO) plays a critical role in the regulation of cellular cysteine concentration. Because multiple forms of CDO ( approximately 23 kDa, approximately 25 kDa, and approximately 68 kDa) have been claimed based upon separation and detection using SDS-PAGE/western blotting (with antibodies demonstrated to immunoprecipitate CDO), we further investigated the possibility of more than one CDO isoform. Using either rabbit antibody raised against purified rat liver CDO or against purified recombinant his(6)-tagged CDO (r-his(6)-CDO) and using 15% (wt/vol) polyacrylamide for the SDS-PAGE, we consistently detected the approximately 25 kDa band, but never detected a approximately 68 kDa band, in rat liver, kidney, lung and brain. Nondenatured gel electrophoresis of r-his(6)-CDO yielded a molecular mass estimate of 25.7 kDa and no evidence of dimerization. Mass spectrometry of r-his(6)-CDO yielded two peaks with molecular masses of 24.1 kDa and 24.3 kDa. Anion-exchange FPLC of r-his(6)-CDO also gave two peaks, with the first containing CDO that was 7.5-times as active as the more anionic form that eluted second. When the two peaks recovered from FPLC were run on SDS/PAGE, the first (more active) CDO fraction yielded two bands (perhaps as an artifact of SDS/PAGE), whereas the second (less active) CDO fraction yielded only the approximately 23 kDa band. We conclude that the physiologically active form of CDO is the approximately 25 kDa (i.e., 23.5 kDa based on mass spectrometry) monomer and that this active form is probably derived by post-translational modification of the 23 kDa gene product.

Cysteine metabolism in periportal and perivenous hepatocytes: perivenous cells have greater capacity for glutathione production and taurine synthesis but not for cysteine catabolism.

Hepatocyte preparations highly enriched in cells from either the periportal or the perivenous zone of the liver acinus were prepared using a digitonin/collagenase perfusion method. Five enzymes of cysteine metabolism were assayed in both periportal and perivenous preparations. The ratios of periportal to perivenous activity were 0.76, 0.60, 0.81, 1.62, and 1.01 for cysteine dioxygenase, cysteinesulfinate decarboxylase, gamma-glutamylcysteine synthetase, cystathionase, and asparate (cysteinesulfinate) aminotransferase, respectively. Only cysteinesulfinate decarboxylase activity was significantly different between periportal and perivenous cells. In incubations with 2 mmol/L [(35)S]cysteine, total cysteine catabolism ([(35)S]taurine plus [(35)S]sulfate) between periportal and perivenous cells was not different, which is consistent with the observation of similar cysteine dioxygenase activity across the hepatic acinus. Consistent with the lower cysteinesulfinate decarboxylase activity in periportal cells, 16% of the total catabolism of [(35)S]cysteine in periportal cells resulted in taurine synthesis compared to 28% in perivenous cells. A lower rate of [(35)S]glutathione synthesis was observed in periportal cells compared to perivenous cells, but gamma-glutamylcysteine synthetase activity was not significantly different between perivenous and periportal cells. Cysteinesulfnate decarboxylase can be added to the list of enzymes whose activities are markedly enriched in perivenous cells.

Murine cysteine dioxygenase gene: structural organization, tissue-specific expression and promoter identification.

The murine gene encoding cysteine dioxygenase (CDO; EC 1.13.11.20), a key enzyme of L-cysteine metabolism, was isolated and characterized, and the proximal promoter was identified. A bacterial artificial chromosome mouse library was screened and a single clone containing the entire CDO gene was isolated. The murine CDO gene contains five exons and spans about 15 kb. The open reading frame is encoded within all five exons. All intron/exon splice junctions and all intron sizes are conserved with the rat CDO gene and are very similar to those of the human CDO gene. The primary transcriptional initiation site is located 213 bp upstream of the initiation ATG codon. The nucleotide sequence of the 5'-promoter region is highly conserved between the mouse and rat genes and contains a TATA-box-like sequence and GC boxes. A variety of consensus cis-acting elements were also identified in the 5'-flanking region. These included HNF-3 beta, HFH-1, HFH-2, HFH-3, C/EBP, and C/EBP beta, all of which are consistent with the tissue-specific expression profiles of the gene. Gene reporter studies of the CDO 5'-region indicated the presence of an active promoter within the first 223 bp upstream of the transcriptional initiation site and the possible presence of repressor elements upstream of bp -223. Northern blot analyses indicated that the CDO gene displays tissue-specific expression, with the highest mRNA level present in liver and with detectable levels found in kidney, lung, brain and small intestine. Western blot analyses indicated that CDO protein levels parallel mRNA levels. These results are consistent with the known function of CDO in whole-body cysteine homeostasis.

Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes.

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.

Cysteine dioxygenase and gamma-glutamylcysteine synthetase activities in primary cultured hepatocytes respond to sulfur amino acid supplementation in a reciprocal manner.

Hepatocytes were cultured for 3 days as spheroids (aggregates) or as monolayers in basal medium and in sulfur amino acid-supplemented media. Cultured hepatocytes had low levels of cysteine dioxygenase (CDO) activity and normal levels of gamma-glutamylcysteine synthetase (GCS) and cysteine-sulfinate decarboxylase (CSDC) activities compared to freshly isolated cells. CDO activity increased and GCS activity decreased in a dose-response manner in cells cultured in either methionine- or cysteine-supplemented media. CSDC activity was not significantly affected by methionine supplementation. Changes in CDO and GCS were associated with changes in cysteine catabolism to taurine plus sulfate and in synthesis of glutathione, respectively. These responses are similar to those observed in liver of intact rats fed diets supplemented with sulfur amino acids. A near-maximal response of CDO or GCS activity was observed when the medium contained 1.O mmol/L of methionine plus cyst(e)ine. Changes in CDO and GCS activities did not appear to be mediated by changes in the intracellular glutathione concentration. Cultured hepatocytes offer a useful model for further studies of cysteine metabolism and its regulation in response to sulfur amino acid availability.

Post-transcriptional regulation of cysteine dioxygenase in rat liver.

Changes in hepatic cysteine dioxygenase (CDO) activity in response to diet play a dominant role in regulation of cysteine catabolism and taurine synthesis. We have conducted several studies of the molecular regulation of CDO activity in rat liver and rat hepatocytes. Compared to levels observed in liver of rats fed a basal 10% casein diet, up to 180-fold higher levels of CDO activity and protein were observed in liver of rats fed diets that contained additional protein, complete amino acid mixture, methionine, or cystine. Neither CDO activity nor CDO protein was induced by excess non-sulfur amino acids alone. Excess sulfur amino acids or protein did not significantly increase the concentration of hepatic CDO mRNA. Preliminary studies indicate that the polysome profile for association of CDO mRNA with polysomes is not altered by an increase in dietary protein level, suggesting that regulation may be posttranslational and possibly involve a decrease in the rate of CDO degradation. In primary cultures of rat hepatocytes, CDO mRNA, protein, and activity all virtually disappeared by 12 to 24 h of culture in standard medium whereas CDO protein, but not CDO mRNA, accumulated markedly between 12 and 24 h in hepatocytes cultured in medium with excess methionine or cyst(e)ine. These observations are also consistent with a limited role of transcriptional or translational regulation of CDO in response to diet.

Effects of nonsulfur and sulfur amino acids on the regulation of hepatic enzymes of cysteine metabolism.

To determine the role of nonsulfur vs. sulfur amino acids in regulation of cysteine metabolism, rats were fed a basal diet or diets supplemented with a mixture of nonsulfur amino acids (AA), sulfur amino acids (SAA), or both for 3 wk. Hepatic cysteine-sulfinate decarboxylase (CSDC), cysteine dioxygenase (CDO), and gamma-glutamylcysteine synthetase (GCS) activity, concentration, and mRNA abundance were measured. Supplementation with AA alone had no effect on any of these measures. Supplementation of the basal diet with SAA, with or without AA, resulted in a higher CDO concentration (32-45 times basal), a lower CSDC mRNA level (49-64% of basal), and a lower GCS-heavy subunit mRNA level (70-76%). The presence of excess SAA and AA together resulted in an additional type of regulation: a lower specific activity of all three enzymes was observed in rats fed diets with an excess of AA and SAA. Both SAA and AA played a role in regulation of these three enzymes of cysteine metabolism, but SAA had the dominant effects, and effects of AA were not observed in the absence of SAA.

Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo.

Little is known about mechanisms of regulation of cysteine dioxygenase (CDO), gamma-glutamylcysteine synthetase (GCS), and cysteine-sulfinate decarboxylase (CSDC) in response to diet. Enzyme activity and Western and Northern or dot blot analyses were conducted on liver samples from rats fed a basal low-protein diet or diets with graded levels of protein or methionine for 2 wk. Higher levels of CDO activity and CDO protein but not of CDO mRNA were observed in liver of rats fed methionine or protein-supplemented diets, indicating that CDO activity is regulated by changes in enzyme concentration. Lower concentrations of the heavy or catalytic subunit of GCS (GCS-HS) mRNA and protein, as well as a lower activity state of GCS-HS in rats fed methionine- or protein-supplemented diets, indicated that dietary regulation of GCS occurs by both pretranslational and posttranslational mechanisms. Lower CSDC activity, CSDC protein concentration, and CSDC mRNA concentration were found in rats fed the highest level of protein, and regulation appeared to involve changes in mRNA concentration. Regulation of key enzymes of cysteine metabolism in response to diet determines the use of cysteine for synthesis of its essential metabolites.

Variations in dietary protein but not in dietary fat plus cellulose or carbohydrate levels affect cysteine metabolism in rat isolated hepatocytes.

To determine if previously observed effects of dietary protein on hepatic cysteine metabolism were due specifically to increases in dietary protein or to the accompanying decreases in dietary carbohydrate, two experiments were conducted. In one experiment, rats were fed diets that contained different levels of protein vs. an isocaloric mixture of fat + cellulose and a constant amount of carbohydrate. In the other, rats were fed diets that contained a constant amount of protein but different levels of carbohydrate vs. an isocaloric mixture of fat+cellulose. Diets were fed for 2-3 wk and hepatocytes were then isolated. Hepatic cysteine dioxygenase activity increased and cysteinesulfinate decarboxylase and gamma-glutamylcysteine synthetase activities decreased in a stepwise manner when protein was added to the diet at the expense of fat + cellulose. Changes in cysteine dioxygenase, cysteinesulfinate decarboxylase and gamma-glutamylcysteine synthetase activities were consistent with changes in rates of cysteine catabolism, taurine production and glutathione synthesis, respectively, by intact hepatocytes incubated with 0.2 mmol/L cysteine. When the carbohydrate to fat+ cellulose ratio was varied, but the protein level was held constant, little or no change in enzyme activities or levels of metabolite production was observed. Regulation of the activities of enzymes involved in cysteine metabolism is predominantly due to changes in dietary protein intake and not to the associated changes in intake of other dietary macronutrients.

High levels of dietary protein or methionine have different effects on cysteine metabolism in rat hepatocytes.

This study clearly indicates that relatively high levels of both CDO and CSAD activity are needed for substantial taurine synthesis and that protein and methionine supplementation, at equimolar sulfur amino acid levels, are not equivalent in terms of their effects on cysteine catabolic enzyme activities and cysteine metabolism in hepatocytes. Evidence for a reciprocal regulation of cysteine catabolism (or CDO activity) and GSH synthesis (or gamma-glutamylcysteine synthetase activity) in rat liver was also obtained. Although very high levels of protein and methionine were fed in this study, previous studies with lower levels of protein or methionine showed similar changes in cysteine metabolism. Several questions regarding regulation of cysteine metabolism remain unanswered. Beyond sulfur amino acid availability, animals fed high protein diets appear to have other signals for regulation of CDO and CSAD activities. These signals may be related to the different hormonal and metabolic state of these animals. Furthermore, little is known about the molecular mechanisms involved in the observed changes in CDO and CSAD activities. The association between CDO activity and CDO protein has not been evaluated. Jerkins and Steele, using immunochemical detection and quantification of CSAD protein in rat liver, showed that changes in CSAD protein concentration were correlated to changes in CSAD activity. The exact mechanisms or direct effectors which bring about changes in CDO and CSAD activities have yet to be determined. Further exploration of these potential regulatory mechanisms needs to be conducted to better understand the response of cysteine sulfinate-dependent cysteine catabolism to high levels of dietary protein or sulfur amino acids.

Effects of protein, methionine, or chloride on acid-base balance and on cysteine catabolism.

Metabolism of cysteine to sulfate results in production of H+, whereas metabolism of cysteine to taurine does not. Rats were fed a basal low-protein diet or a diet with excess protein, methionine, or chloride for 2-3 wk, and effects of these treatments on acid-base homeostasis and on cysteine metabolism were determined. Hepatocytes from rats fed diets with excess methionine, but not from rats fed diets with excess protein or chloride, catabolized a high proportion of cysteine to taurine (32% vs. 4-7% for other groups), and intact rats fed excess methionine excreted more sulfur as taurine (51% of total sulfur vs. 1-6% for other groups). The formation of taurine vs. sulfate as the end product of cysteine catabolism provides a metabolic compensation that minimizes the acid load in rats fed excess sulfur amino acids. However, increased production of taurine vs. sulfate is not a general adaptive response to acidogenic diets.

Evaluation and modification of an assay procedure for cysteine dioxygenase activity: high-performance liquid chromatography method for measurement of cysteine sulfinate and demonstration of physiological relevance of cysteine dioxygenase activity in cysteine catabolism.

Conflicting reports in the literature of appropriate assay procedures for measurement of cysteine dioxygenase activity led us to evaluate the procedure for assay of cysteine dioxygenase activity in rat liver preparations. Cysteine dioxygenase activity was largely in the soluble fraction of liver and was stimulated by addition of NAD+ and Fe2+. The pH optimum of the enzyme was 6.1. Addition of an inhibitor of pyridoxal 5-phosphate-dependent enzymes was necessary to prevent rapid removal of the reaction product cysteine sulfinate. Cysteine sulfinate and cysteic acid were separated by anion-exchange HPLC on a polymer-based column with trimethylamino active groups, and the reaction products were quantitated by measurement of 35S radioactivity or by formation and measurement of fluorescent derivatives. This assay of cysteine dioxygenase under optimal conditions provides a physiologically relevant measure of cysteine dioxygenase activity in liver and hepatocytes based on the observation that this activity was highly correlated with the capacity for cysteine catabolism and taurine production by isolated hepatocytes.

Rats fed a low protein diet supplemented with sulfur amino acids have increased cysteine dioxygenase activity and increased taurine production in hepatocytes.

The metabolism of cysteine and cysteinesulfinate and the activities of key enzymes in cysteine catabolic pathways were investigated in hepatocytes isolated from rats fed a basal (100 g casein/kg) diet or the diet supplemented with L-methionine (3 or 10 g/kg diet) or the sulfur equivalent as L-cystine (2.4 or 8 g/kg diet). Cysteine dioxygenase activity was higher in hepatocytes from rats fed diets with the higher level of sulfur amino acid supplementation, and the higher enzyme activity was paralleled by a greater total catabolite production (taurine + sulfate) from cysteine. Taurine production as a percentage of total cysteine catabolism was significantly greater in hepatocytes from rats fed the diet with excess methionine or cystine (basal, 22%; excess methionine, 61%, excess cystine, 49%). Glutathione production was markedly lower in hepatocytes from rats fed excess sulfur amino acids such that total cysteine utilization was similar for all dietary treatments. Cysteinesulfinate decarboxylase activity and catabolism of cysteinesulfinate by hepatocytes were unaffected by the dietary supplementations. Results are in contrast to previous studies in which increased dietary protein resulted in decreased cysteinesulfinate decarboxylase activity and decreased partitioning of cysteinesulfinate to taurine vs. sulfate. Thus, sulfur amino acids may be less effective than protein in decreasing cysteinesulfinate decarboxylase activity and may result in a pattern of sulfur catabolite production from cysteine that favors taurine production.

The activities of rat hepatic cysteine dioxygenase and cysteinesulfinate decarboxylase are regulated in a reciprocal manner in response to dietary casein level.

The catabolism of cysteine and cysteinesulfinate, the activities of key enzymes in cysteine catabolic pathways, and the effects of inhibitors of specific enzymes on cysteine catabolism were investigated in hepatocytes isolated from rats fed low (100 g casein/kg diet), moderate (300 g casein/kg diet) or high (600 g casein/kg diet) levels of dietary protein. Cysteine was catabolized predominantly by cysteinesulfinate-dependent pathways. Cysteine dioxygenase activity increased with increases in dietary casein level, and the higher enzyme activity was paralleled by a greater total catabolite production (taurine + hypotaurine + sulfate) from cysteine. However, taurine production did not closely follow cysteine dioxygenase activity. Taurine production doubled with an increase in dietary casein from 100 to 300 g/kg but did not increase with a further increase in dietary casein to 600 g/kg. Taurine production as a percentage of total catabolism decreased progressively with the increases in dietary casein and closely paralleled observed decreases in cysteinesulfinate decarboxylase activity. Thus, taurine production was limited at high protein levels by the decrease in cysteinesulfinate decarboxylase activity such that sulfate production from cysteinesulfinate was favored. D-Cysteinesulfinate inhibited cysteinesulfinate-dependent catabolism of cysteine, but inhibition of cysteinesulfinate decarboxylase was not specific.

The utilization of N-acetylcysteine and 2-oxothiazolidine-4-carboxylate by rat hepatocytes is limited by their rate of uptake and conversion to cysteine.

N-Acetyl-L-cysteine (NAC) and L-2-oxothiazolidine-4-carboxylate (OTC) are converted enzymatically to cysteine and have been used to stimulate hepatic glutathione synthesis. Using hepatocytes isolated from male Sprague-Dawley rats and 35S-labeled substrates, the uptake and metabolism of these cysteine precursors was measured and compared with those for cells provided with an equimolar amount of cysteine. Cysteine was utilized more rapidly than NAC or OTC for sulfate and taurine production and more rapidly than OTC for glutathione production. N-Acetyl-L-cysteine itself was taken up slowly by hepatocytes, but deacetylation of NAC to cysteine seemed to occur extracellularly. Utilization of OTC seemed to be limited by a low rate of uptake and slow intracellular conversion to cysteine. The rate of accumulation of [35S]glutathione from OTC was low compared to that from other substrates, but glutathione production accounted for 78% of the measured OTC metabolism. Although the rate of accumulation of [35S]glutathione was similar for hepatocytes incubated with [35S]cysteine or [35S]NAC, glutathione synthesis accounted for a higher percentage of NAC metabolism than of cysteine metabolism (62-81% vs. 46%). The apparent preferential distribution of OTC and NAC to glutathione vs. taurine and sulfate can be partly explained by a lower rate of substrate availability, but another unknown mechanism also appears to favor the conversion of NAC to glutathione.