EFFECT OF SUCCINATE, FUMARATE, AND OXALACETATE ON KETONE BODY PRODUCTION BY LIVER SLICES FROM NON-DIABETIC AND DIABETIC RATS*

EFFECT OF SUCCINATE, FUMARATE, AND OXALACETATE ON KETONE BODY PRODUCTION BY LIVER SLICES FROM NON-DIABETIC AND DIABETIC RATS* BY CLARISSA H. BEATTY, EDWARD S. WEST, AND ROSE MARY BOCEK (From the Department of Biochemistry, University of Oregon Medical School, Portland, Oregon) (Received for publication, September 17, 1957) The metabolism of fatty acids involves the production of acetyl coenzyme A and its condensation with oxalacetate for complete oxidation in the citric acid cycle. The availability of oxalacetate can determine the extent of metabolism via the Krebs cycle (1). In the presence of a low level of oxalacetate in the liver, fatty acid metabolism should produce an excess of ketone bodies. Previously (2) we have demonstrated that the administration of various precursors of oxalacetic acid (succinic acid, malic acid, a-ketoglutaric acid, etc.) and oxalacetic acid itself decreases total urinary ketone body excretion in non-diabetic rats made ketotic by the administration of butyric acid. We have also shown (3) that the administration of succinic and malic acids in doses sufficient to produce increased glucosuria has no effect on the ketonuria of alloxan-diabetic rats fed Wesson oil. Furthermore, the administration of small amounts of insulin plus succinic or malic acid to our insulin-deficient preparation caused a larger decrease in ketonuria than when insulin alone was given. In the present investigation we have compared the effects of oxalacetic acid precursors such as fumarate on the production of ketone bodies by liver slices from normal and diabetic rats. Changes in oxygen consumption and in glucose production were also measured. EXPERIMENTAL Female Sprague-Dawley rats, 180 to 220 gm., were used throughout these experiments. Rats were made diabetic by the subcutaneous injection of 7 to 8 mg. per 100 gm. of recrystallized alloxan monohydrate (10 per cent solution) and were not used less than 3 weeks after alloxan administration. All the alloxan-diabetic rats had 20 hour fasting blood sugar levels of 300 *This work was supported in part by research grant No. A-213 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service. Presented in part at the Federation of the American Societies for Experimental Biology, March, 1956. 725

726 KETONE PRODUCTION BY LIVER SLICES mg. per cent or more. Another group of rats was made diabetic by removing 95 per cent or more of the pancreas. These animals had non-fasting blood sugar concentrations above 300 mg. per cent in 7 days and 20 hour fasting blood sugar levels of 180 to 330 mg. per cent in 4 weeks. They were maintained on a diet of Purina chow plus 3 per cent pancreatin. The rats were lightly anesthetized with 3 mg. per 100 gm. of Nembutal and decapitated; the livers were quickly excised and dampened with medium. Slices were immediately cut with a Stadie-Riggs slicer and placed in a 25 ml. Warburg flask containing 3 ml. of modified Krebsphosphate-buffered medium at ph 7.4. The flasks were then incubated in 100 per cent oxygen at 37 with constant shaking at 120 strokes per minute. Ketone body concentration in the medium was determined by the method of Greenberg and Lester (4) and glucose by the procedure of Somogyi (5). Nitrogen was determined by digestion and vacuum distillation (6). In order to correct for the amounts of substances present in the slice before incubation, determinations of ketone bodies and glucose in the medium were made after incubation for 15 minutes and after 75 minutes (7). The differences between these concentrations are taken as measures of ketone body and glucose production, respectively. The substrates were tipped in following the 15 minute control period. In view of the work by Hastings and Buchanan (8) on the incubation of liver slices in a high potassium medium, O2 consumption and ketone body production were investigated over 1 and 2 hour periods in media high in either Kf or Naf. As the results on 02 consumption and ketone body production were very similar in both media, the experiments were continued with the high Naf medium. The Ca++ level was decreased to one-half of the value recommended by Krebs in order to eliminate precipitation in the medium. This change did not affect either 02 consumption or ketone body production. RESULTS AND DISCUSSION As can be seen in Table I, liver slices from rats fasted for 24 hours produced an average of 89.6 y per 100 mg. per hour of ketone bodies while slices from rats fasted for 14 hours produced only 43.4 or 34.4 y per 100 mg. per hour. These values agree well with those of others (7, 9). Over the same experimental periods glucose was also produced. The addition of succinate or fumarate decreased the production rate of ketone bodies. We have no explanation for the fact that Weinhouse and Millington (10) could show no effect of fumarate on acetoacetate oxidation by liver slices from fasted rats. However, they state that the effect of fumarate could have been missed owing to the trapping of Cl3 in the large excess of

C. H. BEATTY, E. S. WEST, AND R. M. BOCEK 727 fumarate. In control experiments without added substrate the O2 consumption decreased 0.12 f 0.03 (the standard error) ~1. per mg. over the 1 hour experimental period. The addition of succinate caused an approximate doubling of 02 uptake, while fumarate merely abolished the drop which occurred in 02 consumption in the control flasks (+0.02 f 0.05 ~1. per mg. per hour). TABLE Production of Ketone Bodies* and Glucose (Micrograms per 100 Mg. per Hour Wet Weight) by Liver Slices from Fasted Control Rats in Krebs-Phosphate-Buffered Medium, ph 7.4 to 7.6, with and without Substrate No substrate (1O)t..... A + succinate$.... P... No substrate (8).... A + succinate$... P... No substrate (8).... A + fumaratet... P... I Production of ketone bodies 24 hr. fast Production of glucose 89.6 53.8-15.2 f 3.50 +31.9 f 15.9s <O.Ol >O.lO 14 hr. fast.. 43.4.. -13.6 f 3.9.. <0.02... 34.4... -11.2 i 1.3.. <O.Ol * Expressed as acetone. t The figures in parentheses represent the number of experiments. $ Final concentration, 20 mmoles per liter. Q Standard error of the difference. 63.8-0.4 i 14.6 >O.lO 117 +57.0 f 19.0 >0.025 When succinate was added to liver slices from fasted alloxan-diabetic rats, no decrease was observed in ketone production (Table 11). This is in agreement with our previous observations in viva (3). However, there were an approximate doubling of O2 consumption and an increase in glucose production. When fumarate was added to liver slices from fasted alloxan-diabetic rats, a significant decrease in ketone production occurred (Table II). This is in agreement with Stadie et al. (9), who demonstrated a decrease in ketone formation by liver slices from two diabetic cats following the addition of fumarate. The difference in results with fumarate

728 KETONE PRODUCTION BY LIVER SLICES and succinate may be due to the fact that succinate has alternative pathways of metabolism apart from the citric acid cycle (11-13). Although no alloxan-diabetic rats were used sooner than 3 weeks after alloxan administration, the fact that liver slices from 14 hour-fasted diabetic rats produced no more ketones than did slices from controls fasted for 14 hours (Table IV) raised the question as to whether or not these TABLE Production of Ketone Bodies* and Glucose (Micrograms per 100 Mg. per Hour Wet Weight) by Liver Slices from Rats Fasted for 14 Hours The liver slices were incubated in Krebs-phosphate-buffered medium, ph 7.4 to 7.5, with and without substrate. No substrate (9)t... A + succinatet:.... P... No substrate (9).... A + fumarate j.... P... II Production of ketone bodies Alloxan-diabetic rats Production of glucose 38.4 254.0-2.6 f 2.2$ +92 f 9.6s >O.lO <O.Ol 43.8 231.6-12.6 f 2.6 $44.8 f 47.4 <O.Ol >O.lO Alloxan-diabetic adrenalectomized rats]/ No substrate (5). 40.0 129.0 A + fumaratel. _. _. _. _. -13.2 f 2.5 +283 f 79 P <O.Ol <0.025 * Expressed as acetone. t The figures in parentheses represent the number of experiments. $ Final concentration, 20 mmoles per liter. 5 Standard error of the difference. 11 Fasting blood sugar values, 150 to 400 mg. per cent before adrenalectomy. diabetic livers were in good physiological condition. Longer periods of fasting increased ketone production by liver slices from control but not from alloxan-diabetic rats. No histological evidence of liver damage was observed in the fasted diabetic series, and the per cent dry weight, per cent nitrogen, and 02 consumption were similar for both series (Table V). Morita and Orten (14) have shown that adrenalectomy decreases the high liver glycogen levels found in the fasting alloxan-diabetic rat. If the low ketone production in the fasted diabetic rat is due to a high liver glycogen concentration, adrenalectomy should increase ketosis. However,

C. H. BEATTY, E. S. WEST, AND R. M. BOCEK 729 liver slices from fasted, adrenalectomized, alloxan-diabetic rats (Table II) produced no more ketone bodies than liver slices from fasted control animals (Table I). These diabetic rats had fasting blood sugar values between 150 and 400 mg. per cent before adrenalectomy. They were used ap- TABLE Production of Ketone Bodies and Glucose (Micrograms per 100 Mg. per Hour Wet Weight) by Liver Slices from Fed Diabetic Rats in Krebs-Phosphate-Bufered Medium with and without Substrate III Production of ketone bodies Production of glucose No substrate (9)*.... A -+ fumaratet... P... Alloxan-diabetic rats, ph of medium 7.4 to 7.5 39.6 303.0-8.7 f 2.41 +94 f 541: <O.Ol >O.lO No substrate (7).... 37.6 324.0 A + oxalacetates... -6.5 f 2.0-49 i 31 P... <0.02 >O.lO Depancreatised rats, ph of medium 7.4 to 7.5 No substrate (6).... 59.6 223.3 A + fumaratet... -11.8 f 2.8-51.7 ZIZ 32.8 P... <O.Ol >o. 10 Depancreatized rats, ph of medium 7.0 No substrate (6).... 40.2 195.0 A + fumaratet... +1.0 f 5.5-23.2 f 19 P... >o. 10 >o. 10 * The figures in parentheses represent the number of experiments. t Final concentration, 20 mmoles per liter. $ Standard error of the difference. $ Final concentration, 5 mmoles per liter. proximately 3 weeks following bilateral adrenalectomy and were tested for the presence of functioning adrenal tissue 2 days before the experiment by a water tolerance test previously developed in this laboratory (15). However, adrenalectomy itself might mask the effect of the lower liver glycogen. Petersen and Lotspeich (16) showed that, while liver slices from adrenalectomized rats produced ketones at the same rate as liver slices from control rats, the adrenals were necessary to demonstrate the ketotic effect of growth hormone.

730 KETONE PRODUCTION BY LIVER SLICES Since ketone production per 100 mg. of liver was low in the slices from fasted diabetic rats (Table IV) and it was thought possible that these animals might be in a semiterminal condition, experiments were made on TABLE IV Production of Ketone Bodies by Rat Liver Slices in Krebs-Phosphate-Bufered Medium, ph 7.4 to 7.6 y per 100 mg. per hr. (wet weight) Mg. per 100 gm. of rat* per hr. 24 hr. fast No substrate Control (10) t 90.0 f 6.11 No substrate Control (10) Alloxan-diabetic (10) Depancreatieed (2) Plus butyrate, 10 mm Control (9) Alloxan-diabetic (5) No substrate Alloxan-diabetic]/ (8) Depancreatiaed (6) Plus butyrate, 10 mm Alloxan-diabetic7 (8) Depancreatized (6) 14 hr. fast 37.9 f 6.5 41.3 f 5.5 29, 29 Non-fasting 1.1 2.0 312s f 26 9.3 218 f 22 10.7 43.6 i 3.7 2.4 59.6 i 4.2 3.0 181 f 15 10.3 267 f 25 13.3 * Calculated on the observed liver size (see Table V). t The figures in parentheses represent the number of rats. $ Standard error. $ Ketone production over and above production without substrate. 11 Alloxan versus depancreatized, P = <0.02. T[ Alloxan versus depancreatized, P = <O.Ol. liver slices from fed diabetic rats (Table III). The addition of either fumarate or oxalacetate itself to these preparations caused a decrease in ketone production. These decreases in ketone production were probably not caused by a conversion of substrate to glucose with a subsequent glucose effect because no significant change in the glucose concentration of the medium was observed.

C. H. BEATTY, E. S. WEST, AND R. M. BOCEK 731 These experiments seem to indicate that there is no block in the utilization of the various members of the citric acid cycle in the livers of alloxandiabetic rats. However, all the experiments reported here were made in a Krebs-phosphate-buffered medium at ph 7.4 to 7.5, an environment that may be quite different from that of an acidotic diabetic rat liver in vivo. The Krebs medium is sufficiently buffered so that the drop in ph during TABLE Comparison of Per Cent Dry Weight and Nitrogen, Liver Size, and Oxygen Uptake of Rat Liver Slices from Fed, Fasted Control, and Diabetic Rats V I Control Alloxan-diabetic* Depancreatized* 14 hr. fast Dry weight, %.. 30.2 f 0.2t (14)$ 30.6 Z!Z 0.3t (lo)$ N, %.........._ 3.46 xk 0.07 (11) 3.36 f 0.11 (8) Body weight, %$.... 2.9 f 0.01 (6) 4.9 f 0.2 (7)/j Qo, fl.. 1.80 * 0.03 (22) 1.90 f 0.05 (10) Non-fasting Dry weight, %.. 27.5 f 0.3 (8) 31.6 f 0.5 (9)11 30.4 i 0.3 (5)1/ N, %........... 3.26 f 0.04 (8) 3.78 f 0.1 (8)** 3.56 f 0.1 (5) Body weight, %. 4.1 i 0.1 (10) 5.6 f 0.3 (9)ll 5.1 f 0.3 (5)1/ Qo,....... 1.95 f 0.1 (9) 1.89 zk 0.3 (5) * The average body weight of this diabetic series did not change from the prediabetic value during the period of diabetes. t Standard error. $ The figures in parentheses represent the number of rats. $ Weight of liver in gm. per total body weight in gm. X 100. 11 P for difference control versus diabetic, <O.Ol. 7 Microliters per hour per mg. wet weight. ** The dehydration in the livers of the fed alloxan-diabetic rats as compared to the fed controls was sufficient to account for the increase in per cent nitrogen. the experimental period was only 0.10 to 0.15 ph unit. Frohman and Orten (17) have demonstrated that increasing the ph of diabetic rats by administering large doses of bicarbonate orally or intraperitoneally corrects the block in acetate utilization present in the livers of alloxan-diabetic rats. Therefore we repeated our experiments at ph 7.0 in an effort to duplicate the more acidotic conditions present in vivo in the diabetic rat. The diabetic animals used in this experiment were depancreatized instead of alloxanized in an effort to eliminate any possible direct toxic effect of

732 KETONE PRODUCTION BY LIVER SLICES alloxan on the liver that might alter ketone body production. In paired experiments (Table III) made on liver slices from the same rat, the addition of fumarate at ph 7.0 caused no change in ketone body production, although at ph 7.4 to 7.5 fumarate decreased ketone production. The O2 uptake during the 15 minute control period was 0.16 ~1. per mg. per hour less at ph 7.0 than at ph 7.5 (P <0.05 >0.025). There was no difference between the two series in the decrease in 02 consumption over the experimental period. This indicates that this ph was compatible with cellular respiration. We consider that our results are in agreement with those of Frohman and Orten (17) and indicate that the block in metabolism at the citric acid cycle in diabetic rat liver is ph-dependent, at least in part. We are currently investigating the effect of ph changes on citric acid cycle activity in muscle preparations from control and diabetic rats. Liver slices from our fasted diabetic rats produced approximately the same amount of ketone bodies per 100 mg. of liver as those from fasted control rats (Table IV). However, these diabetic rats fed ad libitum had a Lwork hypertrophy of the liver (Table V), and, when the average ketone body production per 100 gm. of rat was calculated on the basis of liver size for the two series, the 14 hour-fasted diabetic rats produced more ketones than the 14 hour-fasted controls (Table IV). It was thought that the low ketone body production of liver slices from fasted alloxan-diabetic rats might be explained on the basis of substrate lack. However, results from the addition of butyrate to the liver slices of alloxan-diabetic rats do not support this theory (Table IV). The data also indicate that ketone production by liver slices with and without butyrate is greater for fed pancreatectomized rats than for fed alloxan-diabetic animals (Table IV). This also seemed to be true for the output per 100 gm. of rat. However, no statistical analyses were made on the latter figures since they are calculated values. The temperature of our rat room usually increases 2-4 during the summer months. Cori and Cori (18) have shown that ketone body production is influenced by the season of the year, and this observation has been confirmed in our laboratory. Therefore, no comparisons can be made between ketone production in different groups of rats unless they are carefully paired. This was done in comparison of the fed alloxandiabetic and depancreatized series. The difference in ketone production of the alloxan and depancreatized series might be explainable by means of either a toxic effect of alloxan on the liver or the presence of hyperglycemic factor in the alloxan-diabetic rats (19, 20). The data on fed as well as fasted rats show a significant increase in the per cent of body weight represented by liver in diabetics as compared to controls. These diabetic rats had not been given insulin, and therefore the hypertrophy of the liver was different from that reported by Osborn

C. H. BEATTY, E. S. WEST, AND R. M. BOCEK 733 et al. (21) in fed alloxan-diabetic rats after insulin administration. The per cent body weight represented by liver was higher in our fed diabetic rats than in those of Osborn et al. However, their animals were males of the Long-Evans strain and ours were females of the Sprague-Dawley strain. SUMMARY 1. Liver slices from fasted control rats show a decrease in ketone body production after the addition of succinate and fumarate, while slices from fasted diabetic rats show a drop in ketone production with fumarate but not with succinate (ph 7.4 to 7.5). 2. Ketone body production by liver slices from fed diabetic rats is decreased by the addition of fumarate and oxalacetate at ph 7.5 to 7.4, but no change occurs in ketone production with the addition of fumarate at ph 7.0. We are grateful to Dr. Frank B. Queen of the Department of Pathology for the histological examinations. BIBLIOGRAPHY 1. Potter, V. R., and Recknagel, R. O., Federation Proc., 9, 215 (1950). 2. Beatty, C. H., and West, E. S., J. Biol. Chem., 190, 603 (1951). 3. Beatty, C. H., and West, E. S., J. Biol. Chem., 216, 661 (1955). 4. Greenberg, L. A., and Lester, D., J. Biol. Chem., 164, 177 (1944). Lester, D., and Greenberg, L. A., J. Biol. Chem., 174, 903 (1948). 5. Somogyi, M., J. BioZ. Chem., 196, 19 (1952). 6. Rinehart, R. E., Grondahl, R. D., and West, E. S., Arch. Biochem., 2, 163 (1943). 7. Bondy, P. K., and Wilhelmi, A. E., J. BioZ. Chem., 186, 245 (1950). 8. Hastings, A. B., and Buchanan, J. M., Proc. Nat. Acad. SC., 28, 478 (1942). 9. Stadie, W. C., Zapp, J. A., Jr., and Lukens, F. D. W., J. BioZ. Chem., 132, 423 (1940). 10. Weinhouse, S., and Millington, R. H., J. BioZ. Chem., 193, 1 (1951). 11. Topper, Y. J., and Stetten, D., Jr., J. BioZ. Chem., 209, 63 (1954). 12. Frohman, C. E., and Orten, J. M., J. BioZ. Chem., 206, 717 (1953). 13. Seaman, G. R., and Naschke, M. D., J. BioZ. Chem., 217, 1 (1955). 14. Morita, Y., and Orten, J. M., Am. J. Physiol., 161, 545 (1950). 15. Beatty, C. H., Bocek, R. M., and Peterson, R. D., Am. J. Physiol., 177,287 (1954). 16. Petersen, V. P., and Lotspeich, W. D., Am. J. Physiol., 182, 273 (1955). 17. Frohman, C. E., and Orten, J. M., J. BioZ. Chem., 220, 315 (1956). 18. Cori, G. T., and Cori, C. F., J. BioZ. Chem., 72, 615 (1927). 19. Stewart, R. D., and Roitman, E., Endocrinology, 63, 192 (1953). 20. Beatty, C. H., and Peterson, R. D., Am. J. Physiol., 183, 325 (1955). 21. Osborn, M. J., Felts, J. M., and Chaikoff, I. L., J. BioZ. Chem., 203, 173 (1953).

EFFECT OF SUCCINATE, FUMARATE, AND OXALACETATE ON KETONE BODY PRODUCTION BY LIVER SLICES FROM NON-DIABETIC AND DIABETIC RATS Clarissa H. Beatty, Edward S. West and Rose Mary Bocek J. Biol. Chem. 1958, 230:725-733. Access the most updated version of this article at http://www.jbc.org/content/230/2/725.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/230/2/725.citation.full.ht ml#ref-list-1