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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1890-1895
Department of Nutritional Sciences and the Nutritional Sciences Program, University of Arizona, Tucson, AZ 85721
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The partitioning of cysteine metabolism between sulfate and taurine biosynthetic pathways may be regulated in part by the activity of cysteine sulfinic acid decarboxylase (CSAD). CSAD activity is repressed by high-protein feeding, and we have previously reported that changes in CSAD activity are correlated with changes in CSAD protein. We conducted experiments to determine the relative expression of CSAD mRNA in rats fed 18 or 60% casein diets. In rats fed a 60% casein diet for 1 wk, hepatic CSAD activity and CSAD protein were 16 and 36%, respectively, of the values measured in rats fed the 18% casein diet. CSAD mRNA abundance in rats fed the 60% casein diet was 14% of the CSAD mRNA abundance in rats fed an 18% casein diet. The time course of the change in CSAD activity and mRNA abundance was examined in rats fed 18 or 60% casein diets for 48 h. Within 6 h of switching rats to a 60% casein diet, CSAD activity was decreased by 20% and after 48 h, activity was decreased 47% compared to activity measured at baseline. CSAD mRNA abundance was decreased 54% within 12 h of feeding rats a high-protein diet and remained depressed at 48 h. In a parallel group of rats fed the 18% casein diet, CSAD activity and CSAD mRNA were not significantly different from baseline values at 48 h. The decreased expression of CSAD mRNA in rats fed a high-protein diet is consistent with decreases in both CSAD enzyme activity and CSAD protein. Our results suggest dietary protein may regulate CSAD at the level of mRNA.
KEY WORDS: mRNA · cysteine sulfinate · carboxy-lyase · taurine · rats
Cysteine sulfinic acid (CSA)5 is at a branch-point in the CSA-dependent pathway of taurine biosynthesis. Transamination of CSA leads to formation of pyruvate and sulfate, whereas decarboxylation of CSA by cysteine sulfinic acid decarboxylase (CSAD) (EC 4.1.1.29) ultimately leads to the biosynthesis of taurine (Huxtable 1986 Cysteine dioxygenase catalyzes the conversion of cysteine to CSA and commits cysteine to the CSA pathway of cysteine catabolism and taurine biosynthesis. Although cysteine dioxygenase activity increases in livers of rats fed a high-protein diet (Bagley and Stipanuk 1994 To study the mechanisms underlying regulation of CSAD by nutritional and hormonal status, we purified CSAD from rat liver and produced polyclonal antibodies against CSAD (Jerkins and Steele 1991a Animals and experimental diets.
Male, Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) were used in all experiments. The animal protocols were reviewed and approved by the University of Arizona Institutional Animal Care and Use Committee and complied with National Institutes of Health guidelines for laboratory animal care. The animals were individually housed in wire-mesh cages in a room with a 12-h light/dark cycle and were allowed free access to food and water, unless otherwise noted. The dark cycle was from 1800 to 0600 h. Body weight was measured daily during the course of the experiments. All rats were adapted to an 18% casein diet, based on American Institute of Nutrition (AIN)-76 formulation (AIN 1977), which was previously reported (Jerkins et al. 1989 Experimental Design
Effect of dietary protein on steady-state CSAD.
The effect on CSAD activity, protein and mRNA abundance of feeding moderate (18% casein) or high (60% casein) protein diets was examined in rats weighing 100-125 g at the start of the experiment. Twelve rats were fed an 18% casein-based diet for 7-10 d and then randomly assigned (six rats per group) to either the 18 or 60% casein-based diet that was fed for 7 d. Food intakes were determined on d 4-6 of feeding the experimental diets. After 7 d the rats were killed by decapitation, between 0800 and 0900 h, and livers assayed for CSAD activity, enzyme protein and mRNA abundance.
Time course of change in CSAD with feeding a high-protein diet.
The time course of the change in CSAD activity and mRNA abundance in rats fed a high-protein diet was followed in rats weighing 75-99 g at the start of the experiment. Rats were individually housed in wire-mesh cages in a room with a 16 h/8 h light/dark cycle and adapted to a controlled feeding schedule for 3 wk. Food was available from 0800 to 1600 h during the 8-h dark cycle and uneaten food was removed at the beginning of the light cycle. Fifty-six rats were fed the 18% casein diet during the 3-wk adaptation period. The feeding schedule was adapted to ensure consumption of the high-protein diet and to minimize oscillations in metabolism (Potter et al. 1968 CSAD activity.
All rats used in these experiments were killed by decapitation. Livers were rapidly removed and portions homogenized in ice-cold 50 mmol/L of potassium phosphate buffer, pH 6.8 containing 3 g/L of Triton X-100, to prepare a 400 g/L of homogenate. After centrifugation for 30 min at 20,000 × g at 4°C, the supernatants were assayed for CSAD activity using a modification of the method of Daniels and Stipanuk (1982) Western blot analysis.
Samples of 3 µg of liver supernatant protein from rats fed 18 or 60% casein diets were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Laemmli (1970) Northern blot analysis.
At the end of the experimental period, rats were killed and livers quickly excised. Portions of liver were frozen in liquid N2 and stored at Protein assay.
Protein in liver supernatants was measured by the Bradford method (1976) using bovine-gammaglobulin (Sigma Chemical Co., St. Louis, MO) as the standard.
Statistical methods.
Data were analyzed using a one-way analysis of variance (ANOVA) (steady-state) or one-way ANOVA and Duncan's multiple range test (time course) using the SAS statistical package (SAS/STAT Version 6.12 for Windows 95, SAS Institute, Cary, NC). Values of P < 0.05 were accepted as statistically significant (Steel and Torrie 1960 Effect of dietary protein on steady-state CSAD.
In rats fed a high-protein diet, CSAD activity in liver was 84% lower than activity in rats fed the moderate protein (18% casein) diet (Table 1). The abundance of CSAD mRNA was determined in livers analyzed for CSAD enzyme activity. A single species of mRNA was detected, with an apparent size of 2.5 kb (Fig. 1). After correction for loading and transfer of total RNA using the amount of 18S rRNA, hepatic CSAD mRNA was 86% lower in rats fed the 60% casein diet for 1 wk compared to rats fed 18% casein (Table 1, Fig. 1). The magnitude of the difference closely paralleled that in hepatic CSAD activity; these differences in CSAD activity and mRNA were similar whether expressed per unit protein and total RNA, respectively, or on a total body weight basis (Table 1). Immunologically detected CSAD protein in rats fed the 60% casein diet was 36% of that in rats fed the 18% casein diet (Fig. 2). No differences were observed in the food intake of rats fed the diets; however, rats fed the 18% casein diet weighed 15% more (P < 0.05) than rats fed the 60% casein diet (data not shown). Liver weight as a percentage of body weight (relative liver size) was 23% greater (P < 0.05) in rats fed 60% casein than in rats fed an 18% casein diet (data not shown). Differences in CSAD activity, protein and mRNA abundance were specific and not attributable to differences in food intake.
Time course of change in CSAD with feeding a high-protein diet.
A time-course study was conducted to determine how rapidly CSAD activity and mRNA abundance decreased in response to feeding rats a high-protein diet (Fig. 3). Within 6 h after switching rats from the 18% to the 60% casein diet, enzyme activity decreased 20% (P
When the intake of dietary protein exceeds the requirement for synthesis, the rate of amino acid degradation rises, and this is typically accompanied by increased activity of enzymes involved in amino acid catabolism. Such increases are documented for serine dehydratase (Ogawa et al. 1991
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). The decarboxylation of CSA has been well-studied in rat liver. Feeding rats a high-protein (60% casein) diet for at least 1 wk was reported to significantly decrease CSAD activity in rat liver (Loriette et al. 1979). We reported that CSAD activity decreases progressively and significantly when rats are fed 18, 30, 45 or 60% casein diets for 1 wk (Jerkins et al. 1989). CSAD is the only enzyme of the cysteine catabolic pathway that is repressed by high-protein feeding. The physiological importance of CSAD regulation by dietary protein is not understood but may be related to the partitioning of CSA between sulfate and taurine synthesizing pathways (Bagley and Stipanuk 1994). Partitioning of CSA toward pyruvate and sulfate, as a result of repressed CSAD activity, conserves the cysteine carbon skeleton for further utilization in energy-yielding metabolic pathways, whereas, taurine formation and excretion produce a loss of the potential energy in the cysteine carbon skeleton (Bagley and Stipanuk 1995, Bella and Stipanuk 1996).
, Benjamin and Steele 1986, Hosokawa et al. 1988, Kohashi et al. 1978), taurine production does not parallel the increase in cysteine dioxygenase activity (Bagley and Stipanuk 1994). Taurine production from CSA is limited in rats fed a high-protein diet, whereas sulfate production is favored as a result of decreased CSAD activity (Bagley and Stipanuk 1994). In studies utilizing primary hepatocytes, Bagley and Stipanuk (1994) demonstrate the net result of this reciprocal regulation of cysteine dioxygenase and CSAD activities by dietary protein is that taurine production is restricted or prevented, whereas sulfate production is markedly increased in hepatocytes isolated from rats fed higher levels of dietary protein.
). We also documented that alterations in CSAD activity caused by hormonal status are positively correlated with enzyme protein concentration (Jerkins and Steele, 1991a and 1992). In addition, we cloned the CSAD cDNA and demonstrated that the repression of CSAD activity and protein concentration by thyroid hormone is established through a reduction of CSAD mRNA (Kaisaki et al. 1995). To date, no studies have been carried out to examine the nutritional regulation of CSAD gene expression. In the present studies, we examine the effect of feeding moderate and high-protein diets on steady-state CSAD activity, enzyme protein and mRNA abundance and on the time course of change in these variables to determine if decreased CSAD activity in response to dietary protein is associated with decreased CSAD enzyme protein and CSAD mRNA. The results suggest that changes in the abundance of CSAD mRNA account for a portion of the regulation of hepatic CSAD expression after feeding rats a high-protein diet.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
) except that 50 g/kg cellulose (Teklad Test Diets, Madison, WI) was added to the diet at the expense of cerelose. The 60% casein diet was prepared by increasing the vitamin-free casein to 600 g/kg and by reducing the cornstarch and cerelose to 151.5 and 96.5 g/kg of the diet, respectively. To facilitate monitoring of food intake, dry diets were mixed 1:1 with a boiling 10 g/L agar solution and fed in agar gel form. The agar diet was kept at 4°C and used within 10 d.
). For determination of baseline (t = 0) CSAD activity and mRNA abundance, eight rats fed 18% casein were killed at 0800 h. The remaining 48 rats were then randomly assigned to treatment groups and either switched to the 60% casein diet (32 rats) or allowed to continue to consume the 18% casein diet (16 rats). Eight rats fed 60% casein and four rats fed 18% casein were killed after 6, 12, 24 and 48 h, and enzyme activity and mRNA were determined.
as previously described (Benjamin and Steele 1986). The reaction was initiated with the addition of CSA containing 1.11 kBq of L-[14C(U)]CSA to each assay tube. 14CO2 was trapped on potassium hydroxide-saturated filter paper in center wells suspended from serum stoppers. The assay was found to be linear with up to 200 µL of supernatant over a 60-min incubation period. L-[14C(U)]CSA was prepared from L-[14C(U)]cystine (NEN, Boston, MA) using a modified method of Daniels and Stipanuk (1982). CSAD activity was calculated from the specific activity of added L-CSA. Radioactivity in the carboxyl carbon of L-[14C(U)]CSA was determined by the ninhydrin method of Rosen (1957).
on 4.95 and 10% stacking and separating gels, respectively. Protein transfers onto nitrocellulose were carried out for 2 h at 114 V at 4°C using a buffer of 200 mL/L of methanol, 192 mmol/L of glycine and 25 mmol/L of Tris. The complete transfer of proteins was confirmed by staining gels with Coomassie blue. After blocking for 1 h with 2.5 g/L of gelatin PBS-Tween 20, blots were washed with PBS polyethylene sorbitan monolaurate (Tween 20) and incubated at room temperature for 1 h with rabbit antirat CSAD antibody (Jerkins and Steele 1991a) diluted 1:25,000 in PBS-Tween 20. Antigen-antibody complexes were detected by incubating blots with goat antirabbit immunoglobulin G conjugated to horseradish peroxidase (Life Technologies, Grand Island, NY) diluted 1:5000 in PBS-Tween 20 and washed. Autoradiography was performed utilizing enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham, Arlington Heights, IL). The blot was exposed to Fuji RX film preflashed using a Sensitize RPN 2051 modified flash unit (Amersham) calibrated to raise the film optical density 0.1 to 0.2 optical density units above that of standard film. CSAD was quantified using laser densitometry and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
70°C. Total cellular RNA was isolated from frozen tissue using TRIzol reagent (Life Technologies, Grand Island, NY), a monophasic solution of phenol and guanidine isothiocyanate, developed to complement the single-step method of Chomczynski and Sacchi (1987). Absorbance at 260 and 280 nm was used to quantify and assess the RNA purity. Isolated RNA was stored at 70°C until Northern blot analysis. For Northern blot analysis 15-20 µg of total RNA, isolated from livers of adult male rats fed 18 or 60% casein diets, was denatured and size-fractionated on 10 g/L of agarose gels in 2.2 mol/L of formaldehyde, 20 mmol/L of 3-(N-morpholino) propanesulfonic acid, pH 7.0, and 1 mmol/L of EDTA, stained with ethidium bromide and transferred to Hybond-N membrane (Amersham). Following prehybridization for 4 h in 500 mL/L of formamide, 50 mmol/L of sodium phosphate, pH 7.5, 5× saline sodium citrate, 5× Denhardt's, 5 g/L sodium dodecyl sulfate and 100 mg/L of salmon sperm DNA at 42°C, RNA blots were hybridized for 16-20 h under identical conditions with the addition of at least 1 × 109 cpm/L of 32P random-primed (Feinberg and Vogelstein 1983) CSAD cDNA as probe (Kaisaki et al. 1995). The 694 base pair-labeled cDNA fragment spanned nucleotides 539 to 1232 of the CSAD cDNA (Kaisaki et al. 1995). Washed filters were exposed with an intensifying screen to Fuji RX film. The density of the signal from CSAD was determined by laser densitometry. To correct for differences in RNA loading onto gels or in RNA transfer to membranes, membranes were stripped of CSAD probe, checked overnight by exposure to film and rehybridized to a 32P-labeled 18S rRNA probe (Omiecinski et al. 1990). The signal densities for each were corrected for the amount of total size-fractionated RNA; the corrected signal density for CSAD was divided by that for 18S.
). Values represent means ± sem.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Cysteine sulfinic acid decarboxylase (CSAD) activity and mRNA abundance in rats fed 18 or 60% casein diets for 7 d1
View larger version (69K):
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Fig 1.
Northern blot of cysteine sulfinic acid decarboxylase (CSAD) mRNA from liver of rats fed 18 or 60% casein diets for 7 d. Total RNA (15 µg) was size-fractionated on a 1% denaturing agarose gel and transferred to nylon membrane. The membrane was hybridized to a 32P-labeled CSAD cDNA fragment. To correct for differences in RNA loading or transfer to membrane, the membrane was also hybridized to a 32P-labeled 18S rRNA probe. Laser densitometric scanning of autoradiograms was used to quantify the relative amounts of mRNA. The signal densities for each were corrected for the amount of total RNA size-fractionated, and the corrected signal density for CSAD was divided by that for 18S. The abundance of 18S rRNA in rats fed 18% casein (236 ± 29 AU/µg total RNA, n = 6) was not significantly different from that in rats fed 60% casein (220 ± 59 AU/µg total RNA, n = 6). Lanes 1 and 3, 60% casein; lanes 2 and 4, 18% casein. AU = arbitrary absorbance units.
View larger version (56K):
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Fig 2.
Western blot of cysteine sulfinic acid decarboxylase (CSAD) protein in liver of rats fed 18 or 60% casein diets for 7 d. Rat liver supernatant proteins (3 µg) were separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with rabbit anti-CSAD serum and visualized as described in text. Lane M, molecular weight markers; lanes 1 and 3, rat liver supernatant protein from rats fed 60% casein; lanes 2 and 4, rat liver supernatant protein from rats fed 18% casein. CSAD migrated as a 52-53 kDa protein.
0.05; Fig. 3B). After 48 h, CSAD activity was 47% lower (P < 0.05) than at baseline (Fig. 3B). CSAD mRNA abundance decreased 54% (P < 0.05) within 12 h of switching rats from an 18 to 60% casein diet and remained depressed for the remainder of the 48-h experiment (Fig. 3). In control rats fed the 18% casein diet, CSAD activity and mRNA at 48 h were not significantly different from baseline; however, the transient increases in CSAD activity and mRNA abundance observed within 12 h of initiating the experiment may have been due to diurnal variation (Fig. 3B).
View larger version (48K):
[in a new window]
Fig 3.
Time course of response of cysteine sulfinic acid decarboxylase (CSAD) activity and mRNA abundance in liver of rats fed 18 or 60% casein diets for 48 h. Rats were fed an 18% casein diet for 3 wk. At baseline (time 0), rats were killed to measure CSAD activity and mRNA, and the remaining rats were either switched to a 60% casein diet or fed the 18% casein diet. Rats were killed after 6, 12, 24 and 48 h. A: Northern blot analysis of CSAD mRNA and 18S rRNA in rat liver. Total liver RNA was isolated from rats killed at the indicated times. Total RNA (20 µg) was separated on denaturing gels, transferred to nylon membranes and successively hybridized to 32P-labeled CSAD and 18S rRNA probes as described in text. B: CSAD activity (upper panel) and mRNA abundance (lower panel) in response to dietary protein. CSAD activity and CSAD mRNA were measured as described in text. The signal density for CSAD mRNA was divided by that for 18S rRNA. Each point represents mean ± SEM (n = 4-8 rats). Means in a panel with no letters in common were significantly different, P < 0.05. AU = arbitrary absorbance units.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
), ornithine decarboxylase (Farwell et al. 1977, Kameji et al. 1987) and glutaminase (Watford et al. 1985). Cysteine dioxygenase catalyzes the conversion of cysteine to CSA and commits cysteine to the cysteine sulfinic acid pathway of cysteine catabolism and taurine biosynthesis. CSA serves as a branch point in the CSA pathway. Transamination of CSA leads to pyruvate and sulfate formation, whereas decarboxylation of CSA, via CSAD, leads ultimately to the biosynthesis of taurine. Cysteine dioxygenase activity increases in livers of rats fed a high-protein diet (Bagley and Stipanuk 1994, Benjamin and Steele 1986, Hosokawa et al. 1988, Kohashi et al. 1978). In contrast to cysteine dioxygenase, CSAD activity is repressed by high-protein feeding (Bagley and Stipanuk 1994, Jerkins et al. 1989, Loriette et al. 1979). This reciprocal regulation of cysteine dioxygenase and CSAD by dietary protein alters the partitioning of cysteine between sulfate and taurine synthesizing pathways (Bagley and Stipanuk 1994). Although cysteine dioxygenase activity increases with dietary casein, taurine production does not parallel cysteine dioxygenase activity. Taurine production from CSA is limited at high dietary protein (60% casein), and sulfate production is favored as a result of decreased CSAD activity (Bagley and Stipanuk 1994). In studies utilizing primary hepatocytes, Bagley and Stipanuk demonstrated that the net result of this reciprocal regulation of cysteine dioxygenase and CSAD activities by dietary protein is that taurine production is restricted or prevented, whereas sulfate production increases markedly in hepatocytes isolated from rats fed higher levels of dietary protein (Bagley and Stipanuk 1994).
). Within 3 h of feeding a high-protein meal, CSAD activity decreased and continued to decrease for 2 wk (Jerkins et al. 1989). These data suggested that decreased CSAD activity may be due to protein inactivation; however, in subsequent studies examining sulfur amino acid regulation of CSAD, immunochemical detection and quantification of CSAD protein in rat liver indicated that CSAD protein concentration paralleled changes in enzyme activity (Jerkins and Steele 1991b). In liver samples from rats fed sulfur and nonsulfur amino acids for 14 d, a significant linear correlation between CSAD activity and CSAD protein was observed for all groups from the lowest activity (ethionine-supplemented) to the highest activity (S-methyl-cysteine-supplemented) (Jerkins and Steele 1991b). Enzyme activity may be altered through changes in the rate of synthesis and/or degradation or through changes in the activation state of existing protein. The observed linear relationship between CSAD protein and CSAD activity (Jerkins and Steele 1991b) suggests that CSAD does not undergo posttranslational modification in response to dietary manipulation. While our previous studies demonstrated that decreases in CSAD activity and protein were parallel (Jerkins and Steele 1991a, 1991b, 1992), in the present study, steady-state CSAD activity and CSAD protein in rats fed 60% casein diets were 16 and 36%, respectively, of the values measured in rats fed 18% casein diets (Table 1, Fig. 2). Although the proportional reductions in CSAD activity and protein were not the same, the changes were concomitant. Our current studies utilized ECL detection and autoradiography, while previous experiments utilized 5-bromo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium detection and reflectance densitometry. The discrepancy in the correlation between CSAD activity and CSAD protein between past and current studies may be related to the detection systems employed.
). However, the decrease in CSAD activity was slower than the decrease in CSAD mRNA (Fig. 3B). Significant reductions in CSAD activity were not observed until 48 h after feeding the high-protein diet; whereas, a rapid decrease in CSAD mRNA was observed within 12 h of feeding rats a 60% casein diet (Fig. 3). This rapid decrease in CSAD mRNA may be due to an immediate attenuation of CSAD gene transcription in response to feeding a high-protein diet. Therefore, the rapid decrease in CSAD mRNA abundance after the diet switch may reflect CSAD mRNA half-life. By using the equation t1/2 = 0.693/[2.303 × the slope of the line (log10 mRNA vs. time)], a CSAD mRNA half-life of 8.7 h was estimated for rats fed a 60% casein diet.
). Evidence from our previous work with adrenalectomized rats suggests that adrenal hormones do not mediate methionine-induced depression of CSAD activity and protein, as CSAD depression in response to methionine feeding in rats was not alleviated in the absence of adrenal glands (Jerkins and Steele 1992). In these studies, CSAD activity was depressed by 60% due to adrenalectomy and by another 60% when adrenalectomized rats were fed a methionine-supplemented diet (Jerkins and Steele 1992). In adrenalectomized rats fed a methionine-supplemented diet, CSAD activity and protein were only 17 and 16%, respectively, of the values determined in sham-operated control rats fed a basal diet (Jerkins and Steele 1992).
, Jerkins and Steele 1991a, 1991b, 1992, Loriette et al. 1979). Strong repression of CSAD may have physiological advantages. In a recent report by Kishimoto and colleagues (1996), CSAD protein and mRNA expression were stimulated in rats during hepatocarcinogenesis. Further investigation will determine if decreased expression of CSAD mRNA in response to feeding rats a high-protein diet is due to decreased CSAD gene transcription, decreased CSAD mRNA stability or a combination of these factors. We are currently conducting experiments to determine the mechanisms responsible for decreased abundance of CSAD mRNA in response to nutritional manipulation.
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FOOTNOTES |
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Manuscript received 26 March 1998. Initial reviews completed 16 June 1998. Revision accepted 16 July 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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