RONALD C. SIMPSON, FREDRIC W. HILL AND RICHARD A. FREEDLAND Department of Physiological Sciences, School of Veterinarii Medicine and Department of Nutrition, University of California, Davis, California 95616 ABSTRACT The effects of dietary and hormonal treatments on the rate of gluconeogenesis from L-cysteine have been investigated in the perfused rat liver in situ. In order to demonstrate gluconeogenesis from L-cysteine, rats were fed either a 90% casein diet, or this diet with 2 or 4% cysteine, added in place of casein, and perfused in the fed state; or fed stock diet and starved 48 or 72 hours; or fasted and injected with cortisol. The net rate of gluconeogenesis (in /umoles/min/g liver) from cysteine in rats fed 4% cysteine was 0.24; in 48-hour starved rats it was 0.10; in 72-hour starved rats it was 0.16; and in the cortisol injected rats it was 0.23. When [U-"C]cysteine plus carrier cysteine ( 10 imi ) was added as the substrate for gluconeogenesis in 72-hour starved rats; 3.9% of the label appeared in glucose. The above dietary and hormonal treat ments stimulated gluconeogenesis from L-cysteine. J. Nutr. 105: 379-384, 1975. INDEXING cortisol

KEY WORDS

gluconeogenesis

•L-cysteine

•liver perfusion

diets, and cysteine and cortisol injections all increase the activity of the enzyme cysteine desulfhydrase (L-cysteine hydro gen sulfide-lyase, deaminating, EC 4.4.1.1) in the liver of chickens. Yamagucchi et al. (9) have shown that both cortisol and cysteine injections increase the activity of cysteine oxidase (EC 1.8) in the rat liver. In the present studies the effects of starva tion, high protein and high cysteine diets, and cortisol injections on gluconeogenesis from L-cysteine in the perfused rat liver have been investigated. To verify that the net increase in glucose production was the result of the conversion of cysteine to glucose, 3-day starved rats were perfused with [U-14C]cysteine,3 and the radioactivity in glucose was measured.

Cysteine is an amino acid that should be readily glucogenic according to the known pathways of intermediary metab olism (1). However, the in vivo investiga tions of the glucogenic nature of cysteine have produced inconsistent results. Dakin (2) and Hess (3) in separate reports both found cysteine to be glucogenic, whereas Butts et al. (4) were unable to demon strate a glucogenic response from cysteine. The studies of gluconeogenesis in the per fused liver (5), perfused kidney (6), and kidney cortex slices (7) have not shown cysteine to be one of the glucogenic amino acids. It is possible that the activities of the catabolic enzymes involved in the conver sion of cysteine to pyruvate are very low under the conditions normally employed in both the in vivo and in vitro investigations of gluconeogenesis from cysteine. If so, it would be probable that treatments that in crease the activity of these catabolic en zymes would also increase the rate of glu cose production from cysteine. Goswami et al. (8) have shown that periods of starva tion of 3 to 9 days, feeding high protein

METHODS

AND MATERIALS

Male Sprague-Dawley * rats weighing 110-160 g were used in all experiments. Received for publication October 21, 1074. 1Supported by Grant USPHS AM-04732 anil a Procter and Gamble Fellowship. 2A preliminary report was presented at the 58th Anniial Meeting of the Federation of American So cieties of Experimental Biology, 7-12 April 1974, Atlantic City, N.J. Federation Proc. 33, 2001. (Abstr.) 3Amersham/Searle Corp., Arlington Heights, 111. 4Hilltop Lab Animals, Scottdale, Pa. 379

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Gluconeogenesis from L-Cysteine in the Perfused Rat Liver1'2

380

SIMPSON, HILL AND FREEDLAND

TABLE 2 Efffd of starvation on glucose production from cysleine in the perfused rat liver Group Diet1

starvedhr484S7272PerfusionsubstrateConcn

glucose productionnmolea/min/g Glucose productionNet

liverNoneCysteineNoneCysteine0.11

(15)2lOmM

StockStock2 StockStockTime

7)10 mm

±0.02' 3)0.35 0.21 ±0.04" ( ±0.03 ( 0.51 ±0.04° (12)0.100.16

All animals were injected with phloridzin 3 hours before perfusion. ' Mean ±SKM. >Number of animals per treatment. 1Means with superscripts are significantly different from controls (P < 0.05).

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NaHCOa, 0.1 ml of sodium heparin,10 and was brought to a final volume of 100 ml with Krebs-Ringer bicarbonate solution ( 14 ). Cysteine was added to the perfusion medium at a final concentration of 10 mM, diet880205040104%cysteine86040so4010 37 minutes after the start of the perfusion. The period between the start of the per base2Corn fusion and the addition of substrate al oil3Mineral lowed for complete glycogenolysis. Sam premix4Vitamin premix5compositionHighprotein000—5040102%cysteineg/kff ples of the perfusate (0.5 ml) were taken at 10-minute intervals, between 40 and 80 1 Purchased from Nutritional Biochemicals Corp., Cleveland, minutes. The samples were deproteinized Ohio. 'Sigma Chemical Co., St. Louis, Mo. J Mazola com oil. Best Foods, Englewood Cliffs, N.J. «SeeRef. (10). with 9 volumes of 2% HC1O4. The de «SeeRef. (11). proteinized samples were neutralized with 3 M KoCOs and 0.5 M triethanolamine9 and were analyzed for glucose by the The rats were subjected to one of the fol lowing experimental treatments : ( 1 ) fed method of Krebs et al. (7). laboratory stock diet5 and starved for 48 To verify that cysteine was converted or 72 hours before being perfused; (2) fed to glucose, an experiment using [U-14C]a high protein diet (table 1) and perfused cysteine plus unlabeled cysteine at a final without prior starvation; (3) fed a 2% concentration of 10 mM was carried out. cysteine diet (table 1) and perfused with Samples of the perfusate (4.0 ml) were out prior starvation; (4) fed a 4% cysteine taken at the end of the perfusion and de diet ( table 1 ) and perfused without prior proteinized with 1.0 ml of 17.5% HC1O4. starvation; or (5) fed laboratory stock diet, The deproteinized samples were neutral injected with hydrocortisone acetate8 (5 ized with 3 M K2CO3 and 0.5 M triethanola mg/day/animal ) for 4 days, and starved mine. The neutralized samples were intro for 48 hours before being perfused. The duced first into a cation-exchange column " experimental diets in table 1 were fed for and then into a anion-exchange column.12 9 days prior to perfusion. On the day of The eluates were evaporated to dryness, the perfusion, the rats were injected with and glucose pentaacetate was derived acphloridzin 7 or phloridzin plus glucagon 8 5 Purina laboratory chow, Ralston Purina Co., St. between 9 and 10 AM and perfused be Mo. tween 12 and 1 PM. The phloridzin or Louis, •Invenex Pharmaceuticals, Chagrin Falls, Ohio mg/ml ). phloridzin plus glucagon treatment was (50'Phloridzin. K & K Laboratories, Hollywood, Calif. (60-70 mg/100 g body weight). used to completely eliminate endogenous 8 Glucagon. Eli Lilly & Co., Indianapolis, Ind. glycogen ( 12 ). The technique of liver per (5 mg/100 g body weight). "Sigma Chemical Co.. St. Louis, Mo. (fraction V). fusion in situ as described by Hems et al. ">Invenex Pharmaceuticals, Chagrin Falls, Ohio (13) was used in all experiments. The per (1,000 U.S.P. units/ml). «Bio-Had Laboratories. Richmond, Calif. (2 cm X fusion medium contained 4 ml of washed cm columns. AG 50X8, 200-400 mesh H- form). whole red cells of human blood, 2.6 g of 10 12Bio-Rad Laboratories, Richmond, Calif. (2 cm X bovine serum albumin,9 1.0 ml of l M 10 cm columns, AG 1-X8, 100-200 mesh CI- forni). 1DietIngredienteCasein1L-Cysteine-free TABLE

GLUCONEOGENESIS

FROM L-CYSTEINE

381

Group

Diet fedPerfusion

glucose productionNet productionttmoles/min/g

substrateConcnGlucose

gaing/dav5.5±0.404.0 wt

liver1

(4)20.3!) ±0.05'»

casein2 90% 88% +2%casein cysteine3

±0.04«(4) (6)0.37 0.28 ±0.03°

NoneCysteine

mmlOniM0.28 ±0.05°(5) (3)0.59 0.35±0.02° 86% +4%casein NoneCysteinelOniM10 cysteineNoneCysteine ±0.06* (4)0.110.0»0.24Av

±0.331.8±0.25

All animala were injected with glucagon and phloridzin 3 hours before perfusion. >Mean ±SEU. *Number ot animals per treatment. a-fcMeans not sharing common superscripts are significantly different (P < 0.01).

cording to the method of Jones ( 15 ). The glucose pentaacetate was dissolved in 15 ml of liquid scintillation solution,13 and radioactivity was counted in a liquid scin tillation counter." Means were compared with the Student's f test (16). RESULTS The effect of starvation on glucose pro duction from cysteine in the perfused liver is shown in table 2. Rats starved for 48 and 72 hours produced glucose at a signifi cantly higher rate when cysteine was added to the perfusate than when no substrate was added. The net rate of glucose produc tion from cysteine in the 48-hour starved rats was about 10% of the rate found with lactate (5) under similar conditions. The net rate of gluconeogenesis from cysteine in the 72-hour starved rats was 60% higher than that of the 48-hour starved rats. The effects of feeding high protein and high cysteine diets on gluconeogenesis from cysteine in the perfused liver are shown in table 3. Neither the high protein (group 1) nor the 2% cysteine diet (group

2) had any significant effect on the rate of gluconeogenesis from cysteine in the per fused liver. However, the 4% cysteine diet (group 3) had a marked effect on glucose production from cysteine. The livers from these rats produced glucose at a signifi cantly higher rate when cysteine was added than when no substrate was added during liver perfusion. The effect of added dietary cysteine on growth is also shown in table 3. As has been previously reported (17), excessive dietary cysteine severely depressed the rate of growth. The daily rate of weight gain in the rats fed the 4% cysteine diet was only about one-third that of the rats fed the 90% casein diet. The effect of cortisol injections on glu coneogenesis is shown in table 4. Cortisol injections caused a significant increase in the rate of glucose production from cysteine in the perfused liver, above the rate found when no substrate was added. Since the rats injected with cortisol were also starved for 48 hours prior to perfusion, it is im portant to compare the results from these 13Aqnnsol. New England Nuclear. Boston. Mass. 14Packard Tri-Carb liquid scintillation counter.

TABLE 4 Glucose production in the perfused liver of rats treated with cortisol Group

Diet

Time starved

perfusion substrate

JConcn

Glucose production

Net glucose niñeóse production

liverNone

1 2

Stock Stockhr48

48limole*/min/fl CysteinelOniM0.32

±0.03' (5)2 0.55±0.03°(6)0.23

All animals were injected with 5 mg of hydrocortisone acetate for 4 days prior to the day of perfusion and each animal was injected with glucagun and phloridzin 3 hours before time of perfusion. ' Mean ±SEM. * Number of animals per treatment. • Means with superscripts are significantly different from control (P < 0.005).

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TABLE 3 Glucose production in the perfused rat liver

382

SIMPSON, HILL AND FHEEDLAND TABLE 6 Glucose production from dietary cysteine and methionine High protein diet

4% cysteine diet

(g)Net liver weight Average production(jimoles/min/g glucose liver)Potential glucoseproduction total day(mg)Cysteine per toproduce required potentialglucose (mg)Average intake(g/day)Average food methionineplus intake(mg/day)7.30.10127170155406.20.2324833311.5855 cysteine

vincingly established cysteine as a gluconeogenic amino acid. In the present ex periments, it has been shown that the starved rat will, in fact, produce net glu cose from cysteine. Also it is shown that increasing the period of starvation from 48 to 72 hours caused a 60% increase in the rate of gluconeogenesis. This increased con version of cysteine to glucose is consistent with the work of Goswami et al. (8) who showed that progressive starvation in creased the activity of cysteine desulfhydrase, an important enzyme in the con version of cysteine to pyruvate. The increased rate of gluconeogenesis observed in the rats fed the 4% cysteine diet might be accounted for by the sub strate inducibility of the enzymes associ ated with cysteine catabolism. It has been DISCUSSION reported that the activities of both cysteine The results from earlier investigations desulfhydrase (8) and cysteine oxidase using the intact animal (2-4) have not con- (9) in intact animals were enhanced by cysteine injections. Kredich et al. ( 19 ) TABLE 5 have also shown that when the bacterium Glucose production from various substrates Salmonella typhimurium is grown on a during liver perfusion in the rat cysteine-rich medium, the activity of the rate of enzyme cysteine desulfhydrase was 1,000 PerfusionsubstrateLactate1Serine1CysteineLac Klucose times greater than when cysteine was starvedhr4848487272Net productionnmolett/min/gliver1.090.610.101.020.16 omitted from the medium. The increased rate of gluconeogenesis in the cortisoltreated rats was also consistent with the IHM10 HIM10 results from the studies on the activities IHMlOniM10 of the enzymes associated with cvsteine tat«CysteineConcn10 catabolism (8, 9). The absence of a re IHMTime sponse from the rats fed the high protein 1 Data previously reported from this laboratory (20). diet might be explained by the low cysteine

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rats with the results from the 48-hour starved rats shown in table 2. The rate of gluconeogenesis from cysteine in the cortisol-treated rats was more than twice the rate of the 48-hour starved rats, whereas there was no difference in final body weights between these two groups of rats. To verify that the net glucose production resulted from the conversion of cysteine to glucose, radioactive cysteine was used as the substrate for gluconeogenesis. Approxi mately 4 /¿Ciof [U-14C]cysteine was added along with the unlabeled cysteine ( 10 HIM) to the perfusion medium during six sepa rate perfusions. The average amount of radioactivity that appeared in glucose after a 40-minute perfusion was 3.9% of the original activity added to the perfusate as [U-14C]cysteine. The average total glucose produced dur ing a 40-minute perfusion with 10 m\i cysteine in the perfusate, was 26 /¿moles. From the radioactivity recovered in glu cose, it was calculated that 39 /¿molesof cysteine was converted to glucose. How ever, because of the loss of radioactivity due to randomization of carbon atoms dur ing the conversion of pyruvate to phosphoenolpyruvate (18), the amount of cysteine converted to glucose based on radioactivity is underestimated by onesixth. Therefore, 46.8 /¿molesof cysteine was actually converted to glucose or 23.4 /¿molesof glucose was derived from cys teine. From this information, it was calcu lated that 90% of the net glucose produced came from cysteine.

GLUCONEOGENESIS

383

that would be available from muscle pro tein catabolism and the capacity for con verting cysteine to glucose, it is apparent that all of the available cysteine plus me thionine could be converted to glucose by the starving rat. However, it should be noted that even though the capability for converting cysteine and methionine quan titatively to glucose probably exists, it is unlikely that glucose is the major end prod uct of cysteine catabolism. This is partly because of the low circulating levels of cysteine and methionine but more im portantly because of the high demand on cysteine for taurine synthesis. The capacity for converting methionine and cysteine to glucose may be even greater than expected in view of the fact that the urinary excretion of nitrogen gradually decreases during the first 4 days of starvation (21). Suggesting that even though the capacity to produce glucose from amino acids is increased during a similar period of starvation (23), the actual catabolism of amino acids is less than in animals fed a moderate level of protein (15-20%) in the diet. Gluconeogenesis from cysteine alone probably does not significantly contribute to total glucose production of the animal. However, when the glucose produced from cysteine is combined with the glucose pro duced from other partially gluconeogenic substrates (glycine, ornithine, etc.) their overall contribution to gluconeogenesis is probably important. LITERATURE CITED 1. Schepartz, B. (1973) Regulation of Amino Acid Metabolism in Mammals, W. B. Saunders Co., Philadelphia. 2. Dakin, H. D. (1913) Studies on the inter mediary metabolism of amino acids. J. Biol. Chem. 14, 321-333. 3. Hess, W. C. (1949) The rates of absorption and the formation of liver glycogen by methi onine, cystine and cysteine. J. Biol. Chem. 181, 23-30. 4. Butts, J. H., Blunden, H. & Dunn, M. S. (1938) Studies in amino acid metabolism. V. The metabolism of L-cystine and dZ-serine in the normal rat. J. Biol. Chem. 124, 709714. 5. Ross, B. D., Hems, R. & Krebs, H. A. (1967) The rate of gluconeogenesis from various pre cursors in the perfused rat liver. Biochem. J. 120, 942-951. 6. Nishiitsulsuje-UWO, J. M., Ross, B. D. & Krebs, H. A. (1967) Metabolic activities of

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content of casein. The 2% cysteine diet may also provide insufficient substrate for enzymatic induction. The transfer of radioactivity from fU-14C]cysteine to glucose in livers from 72-hour starved rats and the net glucose production from cysteine after dietary and hormonal treatments of the rats indicates that cysteine is a glucogenic amino acid. Although cysteine is definitely glucogenic, it may not be quantitatively very important. The rates of glucose production for other precursors of glucose, as well as cysteine, are shown in table 5. The rate of gluconeogenesis from lactate is about 8 to 10 times that of cysteine, and serine is ap proximately 6 times as glucogenic as cys teine. Nutritionally it was of interest to calcu late what portion of the dietary cysteine and methionine could potentially be con verted to glucose. The methionine was in cluded in this calculation because for every mole of methionine catabolized, 1 mole of cysteine is produced from the transsulfuration between methionine and serine. These calculations were made for the rats fed the high protein and 4% cysteine diets based on the data shown in table 6. The rats fed the high protein diet would be potentially capable of converting 170 mg of cysteine to glucose per day or 31% of the daily intake of dietary cysteine plus methionine in the high protein diet to glucose. The rats fed the 4% cysteine diet would be potentially capable of converting 333 mg of cysteine to glucose per day, or 39% of the daily in take of dietary cysteine plus methionine in the 4% cysteine diet to glucose. If one assumed that after a period of starvation that muscle protein provides a majority of the amino acids catabolized, then based on urinary nitrogen excretion ( 21 ) and the average amino acid composi tion of muscle protein ( 22 ), it was possible to calculate that 0.26 mmole of cysteine plus methionine per animal per day would be available for gluconeogenesis. From the net rates of gluconeogenesis with cysteine reported for starved rats in table 2, it was further calculated that 1.15 (48-hour starved) and 1.84 (72-hour starved) mmoles of cysteine per animal per day could be converted to glucose. Considering the amount of cysteine plus methionine

FROM L-CYSTEINE

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8.

9.

10.

11.

12.

13. 14.

the isolate perfused rat kidney. Biochem. J. 103, 852-862. Krebs, H. A., Bennett, D. A. H., De Gasquet, P., Gascoyne, T. & Yosida, T. (1963) Renal gluconeogenesis. Biochem. J. 86, 22-27. Coswami, M. N. D., Robblee, A. R. & McElroy, L. W. ( 1959 ) Further observations on fac tors affecting L-cysteine desulfhydrase activity in chick liver. J. Nutr. 68, 671-682. Yamaguchi, K., Sakakibara, S., Kooa, K. & Ueda, I. (1971) Induction and activation of cysteine oxidase of rat liver. Biochim. Biophys. Acta 237, 502-512. Phillips, P. H. & Hart, E. B. (1935) The effect of organic dietary constituents upon chronic fluorine toxicosis in the rat. J. Biol. Chem. 109, 657-663. Benevenga, N. J., Stielau, W. J. & Freedland, R. A. (1964) Factors affecting the activity of pentose phosphate-metabolizing enzymes in rat liver. J. Nutr. 84, 345-350. Ross, B. D., Hems, R., Freedland, R. A. & Krebs, H. A. (1967) Carbohydrate metab olism of the perfused rat liver. Biochem. J. 105, 869-875. Hems, R., Ross, B. D., Berry, M. N. fit Krebs, H. A. ( 1966 ) Gluconeogenesis in the per fused rat liver. Biochem. J. 201, 284-292. Krebs, H. in A. the & Henseleit, K. Hoppe-Seyler's (1932) Urea formation animal body.

Z. Physiol. Chem. 210, 33-66. 15. Jones, G. B. (1965) Determination of the specific activity of labeled blood glucose by liquid scintillation using glucose pentaacetate. Anal. Biochem. 12, 249-259.

16. Steel, R. G. D. & Torrie, J. H. ( 1960) Prin ciples and Procedures of Statistics, McGrawHill Book Co., Inc., New York. 17. Harper, A. E., Benevenga, N. J. & Wohlhueter, R. M. (1970) Effect of ingestión of dispro portionate amounts of amino acids. Physiol. Rev. 50, 428-558. 18. Lorber, U., Lifson, N., Wood, H. G., Sakami, W. & Shreeve, W. W. (1950) Conversion of lactate to liver glycogen in the intact rat, studied with isotopie lactate. J. Biol. Chem. 183, 517-529. 19. Kredich, N. M., Keenen, B. S. & Foote, L. J. ( 1972 ) The purification of cysteine desulf hydrase from Salmonella typhimurium. J. Biol. Chem. 247, 7157-7162. 20. Chan, T. M. & Freedland, R. A. ( 1971 ) The role of L-serine dehydratase in the metab olism of L-serine in the perfused rat liver. Biochim. Biophys. Acta 237, 99-106. 21. Shapiro, I. (1935) Studies on ketosis V. The comparative glycogenic and ketolytic action of glucose and carbohydrate intermediates. J. Biol. Chem. 108, 373-387. 22. Blaxter, K. L. (1964) Protein metabolism and requirements in pregnancy and lactation. In: Mammalian Protein Metabolism (Munro, H. N. & Allison, J. B., eds.), pp. 173-223, Academic Press, New York. 23. Freedland, R. A. & Szepesi, B. (1971) Con trol of enzyme activity: nutritional factors. In: Enzyme Synthesis and Degradation in Mam malian Systems (Recheigl, M., éd.),pp. 103140. S. Karger, Basel.

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7.

SIMPSON, HILL AND FREEDLAND

Gluconeogenesis from L-cysteine in the perfused rat liver.

The effects of dietary and hormonal treatments on the rate of gluconeogenesis from L-cysteine have been investigated in the perfused rat liver in situ...
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