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Carbon dioxide fixation during hibernation and arousal from hibernation
Comp. Biochem. Physiol., 1968, P'ol. 25, pp. 363 to 366. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION CARBON D I O X I D E F I X A T I O N D U R I N G H I B E R N A T I O N AND AROUSAL FROM H I B E R N A T I O N GEORGE J. K L A I N and BERTWELL K. W H I T T E N U.S. Army Medical Research and Nutrition Laboratory, Fitzsimons General Hospital, Denver, Colorado 80240 (Received 30 October 1967) Abstract--1. In vitro studies indicate that hepatic tissue preparations from hibernating and arousing ground squirrels have a greater capacity for incorporation of Cx'OI into glucose and glycogen than similar preparations from normothermic animals. 2. An increase in COs fixation was observed both at 37 and 6"C. INTRODUCTION Pm~vIous studies from this laboratory have indicated that accelerated gluconeogenic processes are closely associated with the ability of the hibernator to maintain blood glucose levels during hibernation and during immediate arousal from hibernation (Burlington & Klain, 1967). These authors found that kidney cortex slices from hibernating and arousing ground squirrels had a greater capacity for gluconeogenesis from several glucogenic precursors than corresponding preparations from normothermic animals. In addition, activities of hepatic or kidney glutamicpyruvic and glutamic-oxalacetic transaminase and of lactic dehydrogenase were markedly increased during hibernation or arousal when compared with the normothermic controls. It has been established that reversal of glycolysis from 3-carbon glucogenic precursors to phosphoenolpyruvate proceeds primarily via oxalacetate as an intermediate. Thus, Topper & Hastings (1949) reported that molecular distribution of C 14 in glucose formed by liver slices incubated with pyruvate-2-C 14 is consistent with the carboxylation of pyruvate to form malate or oxalacetate, and equilibration of the label within the dicarboxylic acid shuttle, prior to glucose formation by reversal of glycolysis. Similar observations have been made on the conversion of CX4-1abeled lactate to glucose in the intact animal (Lorber et al., 1950). Furthermore, under conditions where increased hepatic glucose production would be expected, e.g. prior treatment with cortisone, incorporation of C 14 from pyruvate-2-C 1~ and C14Os into blood glucose is increased to a comparable extent (Landau et al., 1962). Incorporation of labeled carbon from C140~ into blood glucose is also increased in alloxan-diabetic rats (Wagle & Ashmore, 1963). In view of the above considerations, in vitro CO S fixation was studied in liver slices from hibernating, arousing and normothermic thirteen-lined ground squirrels (Citellus tridecemlineatus). 363
364
GEORGE J. KLAIN AND BERTWELL K. WHtTTEN MATERIALS AND METHODS
Pregnant ground squirrels were trapped during May in the vicinity of Aurora, Colorado, and the resultant male and female offspring were used for this study. The young squirrels remained with their mothers in nesting cages for approximately 40 days. Thereafter, they were transferred to individual cages, kept at 26 + I°C and maintained on a diet of Purina Rat Chow, fresh carrots and water ad lib. In November, hibernation was induced by placing the animals in a dark room at 5 + 1°C. At least 60 days were allowed to elapse before hibernating animals were removed from the cold room and at the time of sacrifice they were between 250 and 300 days old. Three groups of animals were utilized: normothermic squirrels (150+ 15 g) which were removed from the cold room and maintained for at least 30 days at 26 + 1°C with food and water ad lib. ; hibernating squirrels (100 + 20 g) which were sacrificed immediately after removal from the cold room; and hibernating squirrels (100 + 20 g) which were removed from the cold room and allowed to arouse from hibernation for 2 hr at 26 + I°C with water but no food available. The animals were sacrificed by rapid decapitation, livers were quickly removed and chilled in ice-cold isotonic saline. Liver slices were prepared with a Stadie-Riggs microtome (maximum thickness 0.5 mm). After rinsing with isotonic saline, approximately 0.5 g of wet slices were distributed in 25-ml incubation flasks containing 6 ml Krebs-Henseleit buffer (pH 7.4) and 5/~c of C 14 bicarbonate. The slices were subsequently incubated for 1 hr at 37°C or 6°C under 95% 02-5% CO2. The 6°C incubation temperature was chosen to approximate the body temperature during hibernation. After incubation, the slices were removed from the flasks, rinsed with distilled water and digested in boiling 30% KOH. The dissolved glycogen was precipitated by addition of 2 ml 95~o ethanol. After centrifugation, the glycogen was dissolved in warm water, reprecipitated as before and washed with absolute alcohol followed by successive washings with acetone and finally with ether. A weighed quantity of the isolated glycogen was dissolved in 1 ml water in scintillation vials. Fifteen ml of Bray's medium containing 4 per cent of Cab-O-Sil (Packard Instrument Co., Downers Grove, Illinois) were added to the vials and the glycogen was assayed for radioactivity. Glucose isolation from the incubation medium was carried out essentially according to the procedure of Nadkarni et al. (1960). Briefly, 2 ml of 0.5 N Ba(OH)2 solution, followed by 2 ml of 5% ZnSO4 solution, were added to the incubation medium to precipitate bicarbonate and proteins. The solution was centrifuged, and the resulting supernatant was poured through an MB-3 Amberlite column, previously treated with 4 N HC1. After adjusting the pH to 7"2 with diluted NaOH, glucose concentration in the eluate was determined by the glucose oxidase method (Washko & Rice, 1961). One-ml aliquots were transferred into scintillation vials, 15 ml of Bray's solution were added and the samples were assayed for radioactivity in a Packard Scintillation spectrometer. The results were expressed as disintegrations/min per mg of glucose in the medium or per mg of glycogen in the liver slices (dpm/mg).
CARBONDIOXIDEFIXATIONDURINGAROUSALFROMHIBERNATION
365
Statistical evaluation of the data was accomplished by calculating the mean and standard error of the mean for each of the measurements and Fisher's t-test was used to compare the control and experimental means. RESULTS AND DISCUSSION The data in Table 1 show that tissue preparations from hibernating and aroused ground squirrels have a greater capacity for incorporation of C1402 into glucose or glycogen than similar preparations from normothermic animals. Such an TABLE 1--INcoRPORATION OF C140~1 INTO GLYCOGEN AND GLUCOSE BY LIVER SLICES FROM HIBERNATING, AROUSED AND NORMOTHERMIC GROUND SQUIRRELS
Hibernating
Aroused
Norrnothermic
37°C 27,200 + 2,100"
30,100+ 2,800
16,800+ 1,200
Glucose (dpm/mg) 6°C 37°C
7100+ 998 329+32
6700 + 836 308+_24
99 + 24
81 + 16
2100 + 510 115+_16
Glycogen (dpm/mg) 6°C
22 + 5
* .~+ S.E. n = 6 . Values for hibernating and aroused animals are all significant from normothermic values at P < 0.05. increase in CO 2fixation was observed both at 37 and 6°C. These results demonstrate that a capacity for gluconeogenesis is retained and elevated in the liver during extensive periods of hibernation. It is apparent that the rate of gluconeogenesis would be limited by decreased temperature in hibernation. However, during arousal an increased rate of gluconeogenesis would enhance the return of blood glucose to normal. The data presented herein are similar to those of Burlington & Klain (1967), who reported that kidney cortex slices from hibernating or aroused ground squirrels incubated at 40 or 6°C synthesized more glucose from glucogenic intermediates than the corresponding tissue preparations from normothermic animals. The nature of the control mechanisms responsible for increased CO~ fixation during hibernation or arousal remains undefined. However, adrenal corticosteroids have been shown to stimulate CO2 fixation both in vivo (Ashmore et al., 1961) and in vitro (Uete & Ashmore, 1963). A marked increase in the concentrations of circulating blood corticosteroids in hamsters arousing from hibernation has also been reported (Denyes & Horwood, 1960). Recent data of McLean & Gurney (1963) demonstrate that activities of enzymes of the urea cycle are increased in the livers of rats chronically treated with glucocorticoids and decreased following adrenalectomy. Such an increase would be consistent with the effect of these
366
GEORGE J. KLAIN AND BERTWELLK. WmTTEN
hormones on protein catabolism and gluconeogenesis. It would appear that an increase in CO2 fixation in hibernating and, in particular, in arousing ground squirrels is due, in part, to an increase in protein catabolism leading to an increased formation of metabolic intermediates capable of adding CO2. Indeed, recent findings in this laboratory indicate that an arousal from hibernation is associated with protein catabolism (Whitten & Klain, 1968). REFERENCES ASHMORE J., S~UCXSR F., L o w W. C. & KILSHEIM~ G. (1961) Corticol stimulation of glycogen synthesis in fasted rats. Endocrinology 68, 599--606. BURLINGTONR. F. & KLAIN G. J. (1967) Gluconeogenesis during hibernation and arousal from hibernation. Comp. Biochem. Physiol. 22, 701-708. D F _ ~ s A. & HOnWOOD R. H. (1960) A comparison of free adrenal corticol steroids in the blood of a hibernating and non-hibernating mammal. Can.ft. Biochem. Physiol. 38, 1479. LANDAU B. R., MAmam R., ASHMOREJ., ELWYN D., HASTINGSA. B. & ZoTru S. (1962) Cortisone and the regulation of hepatic gluconeogenesis. Endocrinology 70, 47-53. Lom3Ea V., LI~oN N., WOOD H. G., SAK~It W. & Smum~ W. W. (1950) Conversion of lactate to liver glycogen in the intact rat, studied with isotopic lactate, j~. biol. Chem. 183, 517-529. McLEAN P. & Gtm,'q~ M. W. (1963) Effects of adrenalectomy and growth hormone on enzymes concerned with urea synthesis in rat liver. Biochem. 3. 87, 96-104. NADKARNI G. B., Fnnm~,cN B. & WEINHOUSE S. (1960) Gluconeogenesis from glycine and serine in the rat. ~t. biol. Chem. 235, 420-425. TOPPER Y. J. & H^sTINcs A. B. (1949) A study of the chemical origins of glycogen by use of C 14 labeled carbon dioxide, acetate and pyruvate. ~. biol. Chem. 179, 1255-1264. Uma~ T. & AaHMO~ J. (1963) Effects of triamcinolone on carbohydrate synthesis by rat liver slices..~, biol. Chem. 238, 2906-2912. WAGLE S. R. & Asmvlom~J. (1963) Studies on experimental diabetes--II. Carbon dioxide fixation. ~t. biol. Chem. 238, 17-21. WASHKO M. E. & RzCE E. W. (1961) Determination of glucose by an improved enzymatic procedure. Clin. Chem. 7, 542-545. W m T T ~ B. K. & EmAIN G. J. (1968) Protein metabolisn in hibernating, aroused and normothermic ground squirrels (Citellus tridecemlineatus). Am. jr. Physiology. (Submitted.)
SHORT COMMUNICATION CARBON D I O X I D E F I X A T I O N D U R I N G H I B E R N A T I O N AND AROUSAL FROM H I B E R N A T I O N GEORGE J. K L A I N and BERTWELL K. W H I T T E N U.S. Army Medical Research and Nutrition Laboratory, Fitzsimons General Hospital, Denver, Colorado 80240 (Received 30 October 1967) Abstract--1. In vitro studies indicate that hepatic tissue preparations from hibernating and arousing ground squirrels have a greater capacity for incorporation of Cx'OI into glucose and glycogen than similar preparations from normothermic animals. 2. An increase in COs fixation was observed both at 37 and 6"C. INTRODUCTION Pm~vIous studies from this laboratory have indicated that accelerated gluconeogenic processes are closely associated with the ability of the hibernator to maintain blood glucose levels during hibernation and during immediate arousal from hibernation (Burlington & Klain, 1967). These authors found that kidney cortex slices from hibernating and arousing ground squirrels had a greater capacity for gluconeogenesis from several glucogenic precursors than corresponding preparations from normothermic animals. In addition, activities of hepatic or kidney glutamicpyruvic and glutamic-oxalacetic transaminase and of lactic dehydrogenase were markedly increased during hibernation or arousal when compared with the normothermic controls. It has been established that reversal of glycolysis from 3-carbon glucogenic precursors to phosphoenolpyruvate proceeds primarily via oxalacetate as an intermediate. Thus, Topper & Hastings (1949) reported that molecular distribution of C 14 in glucose formed by liver slices incubated with pyruvate-2-C 14 is consistent with the carboxylation of pyruvate to form malate or oxalacetate, and equilibration of the label within the dicarboxylic acid shuttle, prior to glucose formation by reversal of glycolysis. Similar observations have been made on the conversion of CX4-1abeled lactate to glucose in the intact animal (Lorber et al., 1950). Furthermore, under conditions where increased hepatic glucose production would be expected, e.g. prior treatment with cortisone, incorporation of C 14 from pyruvate-2-C 1~ and C14Os into blood glucose is increased to a comparable extent (Landau et al., 1962). Incorporation of labeled carbon from C140~ into blood glucose is also increased in alloxan-diabetic rats (Wagle & Ashmore, 1963). In view of the above considerations, in vitro CO S fixation was studied in liver slices from hibernating, arousing and normothermic thirteen-lined ground squirrels (Citellus tridecemlineatus). 363
364
GEORGE J. KLAIN AND BERTWELL K. WHtTTEN MATERIALS AND METHODS
Pregnant ground squirrels were trapped during May in the vicinity of Aurora, Colorado, and the resultant male and female offspring were used for this study. The young squirrels remained with their mothers in nesting cages for approximately 40 days. Thereafter, they were transferred to individual cages, kept at 26 + I°C and maintained on a diet of Purina Rat Chow, fresh carrots and water ad lib. In November, hibernation was induced by placing the animals in a dark room at 5 + 1°C. At least 60 days were allowed to elapse before hibernating animals were removed from the cold room and at the time of sacrifice they were between 250 and 300 days old. Three groups of animals were utilized: normothermic squirrels (150+ 15 g) which were removed from the cold room and maintained for at least 30 days at 26 + 1°C with food and water ad lib. ; hibernating squirrels (100 + 20 g) which were sacrificed immediately after removal from the cold room; and hibernating squirrels (100 + 20 g) which were removed from the cold room and allowed to arouse from hibernation for 2 hr at 26 + I°C with water but no food available. The animals were sacrificed by rapid decapitation, livers were quickly removed and chilled in ice-cold isotonic saline. Liver slices were prepared with a Stadie-Riggs microtome (maximum thickness 0.5 mm). After rinsing with isotonic saline, approximately 0.5 g of wet slices were distributed in 25-ml incubation flasks containing 6 ml Krebs-Henseleit buffer (pH 7.4) and 5/~c of C 14 bicarbonate. The slices were subsequently incubated for 1 hr at 37°C or 6°C under 95% 02-5% CO2. The 6°C incubation temperature was chosen to approximate the body temperature during hibernation. After incubation, the slices were removed from the flasks, rinsed with distilled water and digested in boiling 30% KOH. The dissolved glycogen was precipitated by addition of 2 ml 95~o ethanol. After centrifugation, the glycogen was dissolved in warm water, reprecipitated as before and washed with absolute alcohol followed by successive washings with acetone and finally with ether. A weighed quantity of the isolated glycogen was dissolved in 1 ml water in scintillation vials. Fifteen ml of Bray's medium containing 4 per cent of Cab-O-Sil (Packard Instrument Co., Downers Grove, Illinois) were added to the vials and the glycogen was assayed for radioactivity. Glucose isolation from the incubation medium was carried out essentially according to the procedure of Nadkarni et al. (1960). Briefly, 2 ml of 0.5 N Ba(OH)2 solution, followed by 2 ml of 5% ZnSO4 solution, were added to the incubation medium to precipitate bicarbonate and proteins. The solution was centrifuged, and the resulting supernatant was poured through an MB-3 Amberlite column, previously treated with 4 N HC1. After adjusting the pH to 7"2 with diluted NaOH, glucose concentration in the eluate was determined by the glucose oxidase method (Washko & Rice, 1961). One-ml aliquots were transferred into scintillation vials, 15 ml of Bray's solution were added and the samples were assayed for radioactivity in a Packard Scintillation spectrometer. The results were expressed as disintegrations/min per mg of glucose in the medium or per mg of glycogen in the liver slices (dpm/mg).
CARBONDIOXIDEFIXATIONDURINGAROUSALFROMHIBERNATION
365
Statistical evaluation of the data was accomplished by calculating the mean and standard error of the mean for each of the measurements and Fisher's t-test was used to compare the control and experimental means. RESULTS AND DISCUSSION The data in Table 1 show that tissue preparations from hibernating and aroused ground squirrels have a greater capacity for incorporation of C1402 into glucose or glycogen than similar preparations from normothermic animals. Such an TABLE 1--INcoRPORATION OF C140~1 INTO GLYCOGEN AND GLUCOSE BY LIVER SLICES FROM HIBERNATING, AROUSED AND NORMOTHERMIC GROUND SQUIRRELS
Hibernating
Aroused
Norrnothermic
37°C 27,200 + 2,100"
30,100+ 2,800
16,800+ 1,200
Glucose (dpm/mg) 6°C 37°C
7100+ 998 329+32
6700 + 836 308+_24
99 + 24
81 + 16
2100 + 510 115+_16
Glycogen (dpm/mg) 6°C
22 + 5
* .~+ S.E. n = 6 . Values for hibernating and aroused animals are all significant from normothermic values at P < 0.05. increase in CO 2fixation was observed both at 37 and 6°C. These results demonstrate that a capacity for gluconeogenesis is retained and elevated in the liver during extensive periods of hibernation. It is apparent that the rate of gluconeogenesis would be limited by decreased temperature in hibernation. However, during arousal an increased rate of gluconeogenesis would enhance the return of blood glucose to normal. The data presented herein are similar to those of Burlington & Klain (1967), who reported that kidney cortex slices from hibernating or aroused ground squirrels incubated at 40 or 6°C synthesized more glucose from glucogenic intermediates than the corresponding tissue preparations from normothermic animals. The nature of the control mechanisms responsible for increased CO~ fixation during hibernation or arousal remains undefined. However, adrenal corticosteroids have been shown to stimulate CO2 fixation both in vivo (Ashmore et al., 1961) and in vitro (Uete & Ashmore, 1963). A marked increase in the concentrations of circulating blood corticosteroids in hamsters arousing from hibernation has also been reported (Denyes & Horwood, 1960). Recent data of McLean & Gurney (1963) demonstrate that activities of enzymes of the urea cycle are increased in the livers of rats chronically treated with glucocorticoids and decreased following adrenalectomy. Such an increase would be consistent with the effect of these
366
GEORGE J. KLAIN AND BERTWELLK. WmTTEN
hormones on protein catabolism and gluconeogenesis. It would appear that an increase in CO2 fixation in hibernating and, in particular, in arousing ground squirrels is due, in part, to an increase in protein catabolism leading to an increased formation of metabolic intermediates capable of adding CO2. Indeed, recent findings in this laboratory indicate that an arousal from hibernation is associated with protein catabolism (Whitten & Klain, 1968). REFERENCES ASHMORE J., S~UCXSR F., L o w W. C. & KILSHEIM~ G. (1961) Corticol stimulation of glycogen synthesis in fasted rats. Endocrinology 68, 599--606. BURLINGTONR. F. & KLAIN G. J. (1967) Gluconeogenesis during hibernation and arousal from hibernation. Comp. Biochem. Physiol. 22, 701-708. D F _ ~ s A. & HOnWOOD R. H. (1960) A comparison of free adrenal corticol steroids in the blood of a hibernating and non-hibernating mammal. Can.ft. Biochem. Physiol. 38, 1479. LANDAU B. R., MAmam R., ASHMOREJ., ELWYN D., HASTINGSA. B. & ZoTru S. (1962) Cortisone and the regulation of hepatic gluconeogenesis. Endocrinology 70, 47-53. Lom3Ea V., LI~oN N., WOOD H. G., SAK~It W. & Smum~ W. W. (1950) Conversion of lactate to liver glycogen in the intact rat, studied with isotopic lactate, j~. biol. Chem. 183, 517-529. McLEAN P. & Gtm,'q~ M. W. (1963) Effects of adrenalectomy and growth hormone on enzymes concerned with urea synthesis in rat liver. Biochem. 3. 87, 96-104. NADKARNI G. B., Fnnm~,cN B. & WEINHOUSE S. (1960) Gluconeogenesis from glycine and serine in the rat. ~t. biol. Chem. 235, 420-425. TOPPER Y. J. & H^sTINcs A. B. (1949) A study of the chemical origins of glycogen by use of C 14 labeled carbon dioxide, acetate and pyruvate. ~. biol. Chem. 179, 1255-1264. Uma~ T. & AaHMO~ J. (1963) Effects of triamcinolone on carbohydrate synthesis by rat liver slices..~, biol. Chem. 238, 2906-2912. WAGLE S. R. & Asmvlom~J. (1963) Studies on experimental diabetes--II. Carbon dioxide fixation. ~t. biol. Chem. 238, 17-21. WASHKO M. E. & RzCE E. W. (1961) Determination of glucose by an improved enzymatic procedure. Clin. Chem. 7, 542-545. W m T T ~ B. K. & EmAIN G. J. (1968) Protein metabolisn in hibernating, aroused and normothermic ground squirrels (Citellus tridecemlineatus). Am. jr. Physiology. (Submitted.)