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Effect of Fasting and Fasting and Refeeding on Hepatic and Renal Gluconeogenic Enzymes in the Chicken
Effect of Fasting and Fasting and Refeeding on Hepatic and Renal Gluconeogenic Enzymes in the Chicken C. S. SHEN and S. P. MISTRY
Laboratory of Nutritional Biochemistry, Department of Animal Science, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (Received for publication September 12, 1978)
INTRODUCTION The chicken maintains not only a relatively high blood glucose level but also a normal glucose turnover rate during fasting (Belo et al, 1976; Pearce, 1971; Sarkar, 1971). Several studies have shown that the chicken is unable to synthesize glucose from a number of substrates which are readily utilized by the rat (Brady et al, 1978a; Sarkar, 1971; Schultz and Mistry, 1978; Soling et al, 1973). Thus, the process of gluconeogenesis and its regulation appears to be different in the chicken than in mammals, particularly in the rat. Pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6diphosphatase (FDPase), and glucose-6phosphatase (G6Pase) are key gluconeogenic enzymes. It is known that the activities of these enzymes in the liver and the kidney of the rat and several other species change in response to various nutritional and hormonal treatments, such as fasting, fasting and refeeding, diabetes and Cortisol administration, conditions which are known to influence gluconeogenesis (Exton, 1972; Krebs, 1963; Soling and Kleineke, 1976). However, only few such studies have been made with the chicken (Allred and Boehrig, 1970; Brady et al, 1978b). Furthermore, with the demonstrated presence of both cytosolic and mitochondrial PEPCK in the chicken liver and the kidney (Shen and Mistry, 1978), it is important to know whether one or both of these enzymes can be induced or repressed under suitable conditions.
In the present study, the effect of fasting and fasting and refeeding on the activities of the key hepatic and renal gluconeogenic enzymes in the chicken have been investigated.
MATERIALS AND METHODS Animals. Newly hatched female crossbred chicks (Gallus domesticus: New Hampshire male X Columbian female) were fed ad libitum a corn-based diet (Shen and Mistry, 1977) until six 'weeks of age. Sixteen chickens weighing between 600 and 650 g were divided into four groups. Group A served as the control and the animals were sacrificed without further treatment. Groups B and C were fasted for 2 and 4 days, respectively. Group D was fasted for 4 days and refed the same diet for 4 days before sacrifice. Water was provided at all times. Enzyme Preparations. Chickens were killed by cervical dislocation. Livers and kidneys were removed, blotted, weighed quickly, and then dropped into an ice-cold homogenizing medium containing .25M sucrose and 1 mM EDTA. Tissues were homogenized in nine volumes of the above medium with a Potter-Elvehjem homogenizer for 3 min. The homogenates were centrifuged at 100,000 X g for 1 hr in a Beckman preparative ultracentrifuge, model L2-65. The supernatant fraction was used for the assay of FDPase and cytosolic PEPCK. The sediment was suspended in the homogenizing medium and lyophilized. The lyophilized samples were resuspended in 890
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ABSTRACT The effect of fasting and fasting and refeeding on hepatic and renal gluconeogenic enzyme activities were studied in six-week-old chickens (Gallus domesticus: New Hampshire male X Columbian female). Hepatic pyruvate carboxylase appeared not to be affected by fasting, but the renal enzyme activity increased in four-day fasted chickens. The hepatic mitochondrial and cytosolic phosphoenolpyruvate carboxykinases were essentially not affected by fasting. The renal mitochondrial phosphoenolpyruvate carboxykinase showed a slight increase in activity only after a four-day fast, but the cytosolic enzyme activity increased markedly already after a two-day fast. Also, the activities of the hepatic and renal fructose-1,6-diphosphatase and glucose-6-phosphatase increased markedly on fasting. Refeeding for four days after a four-day fast returned these enzyme activities to near control values. 1979 Poultry Science 58:890-895
GLUCONEOGENIC ENZYMES IN THE CHICKEN
RESULTS AND DISCUSSION
The effects of fasting and fasting and refeeding on body weight, liver weight and kidney weight are shown in Table 1. As expected, the chickens lost weight during the fast which was regained after refeeding. Liver weight relative to body weight decreased by about 10% as a result of either a 2-day or a 4-day fast. Refeeding for 4 days after a 4-day fast increased the liver weight even more than that of the fed control chickens. Similarly, the weight of the kidney decreased by about 10% as a consequence of fasting; refeeding brought the weight back to the normal level. The alterations in liver weights observed in the present study were essentially in accord with previous reports (Feigenbaum and Fisher, 1963; Leveille, 1969). They were primarily due to changes in glycogen and water content, as noted for the chicken (Leveille, 1969) and the rat (Leveille and Chakrabarty, 1967). However, the increase in the liver
weight observed after fasting and refeeding might have been in part due to an increase in the lipid content. Feigenbaum and Fisher (1963) and Leveille (1969) have reported that fasting did not change the liver fat content in the chick, but subsequent refeeding did increase the lipid content. The reason for the decrease in the kidney weight seen on fasting is not clear, since analytical data are not available. The effect of fasting and fasting and refeeding on hepatic and renal PC activities of growing chickens are shown in Figure 1. Hepatic PC appeared to be unaffected by either a 2 day or a 4 day fast, but the activity in the fasted and refed chickens was significantly lower than in the controls. Renal PC was also not affected by a 2-day fast but the activity increased after a 4-day fast; refeeding restored the activity to that of the control. There is no agreement concerning the effect of fasting on hepatic (Filsell et al, 1969; Krebs, 1966; Soling et al., 1973) and renal (Filsell et al., 1969; Achuta Murthy et al, 1977) PC in the rat. The activity was reported to increase in the liver of the fasted sheep (Filsell et al, 1969; Taylor et al, 1971), the guinea pig (Soling et al., 1970; Soling et al, 1973), the cow (Ballard et al, 1968), the pig (Swiatek et al, 1970), and in the kidney of the fasted sheep (Taylor et al, 1971). In contrast, hepatic PC remained unchanged in the fasted rabbit (Garber and Hanson, 1971; Ray, 1976). Corresponding changes in the hepatic and renal PEPCK activities are shown in Figure 2. The hepatic mitochondrial or the cytosolic enzyme was virtually unaffected by fasting; refeeding after a 4-day fast significantly decreased the mitochondrial activity. The renal mitochondrial enzyme activity increased slightly only after a 4-day fast, but the cytosolic activity more than doubled after a 2-day fast; refeeding decreased both activities to slightly lower levels than those of the controls. The increased cytosolic PEPCK activity in the kidney was not due to the leakage of the enzyme from the mitochondria into the cytosol. As seen in Figure 3, while the renal PEPCK activity in the cytosol increased from 35% in the fed chickens to 56% in the 2-day fasted chickens, the activity of the mitochondrial marker enzyme, GDH, remained unchanged in the cytosol. In agreement with an earlier report, both hepatic and renal mitochondrial PEPCK activities in the chicken did not fluctuate markedly during fasting (Brady et al, 1978b).
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distilled water and used for the assay of PC, mitochondrial PEPCK, and G6Pase. Glutamate dehydrogenase (GDH) was used as a mitochondrial marker enzyme; its activity was assayed in both fractions. Enzyme Assays. PC and PEPCK were assayed by the radiochemical carboxylation assay as described by Deodhar and Mistry (1969) and Ballard and Hanson (1967), respectively. In both assays the incorporation of 14 C-bicarbonate into oxalacetate was measured as citrate and malate, respectively. After termination of the reaction and centrifugation, the residual 1 C-bicarbonate was removed from the proteinfree supernates by repeated additions of small pieces of dry ice over a period of 1 hr. Aliquots were then counted in a Packard liquid scintillation counter after adding 15 ml of Aquasol. G6Pase was assayed according to Baginski et al. (1974); FDPase was determined spectrophotometrically in a coupled reaction system as described by Latzko and Gibbs (1974). GDH was determined spectrophotometrically as described by Schmidt (1974). PC, PEPCK, and G6Pase were determined at 37 C, whereas F6Pase and GDH were measured at 30 C. Enzyme activities were expressed as Mtnoles of substrate consumed or product formed per minute per gram of wet tissue. The appropriate dilution which gave a linear relationship of enzymatic activity with respect to time and protein concentration was determined beforehand for each enzyme assay.
891
892
SHEN AND MISTRY TABLE 1. Effect of fasting and fasting and refeeding on body weight, liver weight, and kidney weight of growing chickens*b
Group A Fed Body weight, g
629
± 13
1.71 ±
.08
Kidney weight, g/lOOgbody wt
.88 ±
.06
Group C Fasted for 4 days
Group D Fasted for 4 days and refed for 4 days
530 ± 18 P<.01 1.49 ± .06 P<.025
435 ±5 P<.005 1.47 ± .10 P<.05
701 ± 29 P<.05 2.28 ± .13 P-C.025
.76 ± P<.05
.77 ± .04 P<.05
.04
.81 ± NS
.04
Each value represents the mean ± SEM of four chickens. Significance of difference (P) from the t-test was calculated by testing the mean of the fasted and fasted and refed chickens against the control (Group A). "NS" denotes not significant, i.e., P>.05.
T h a t fasting increased the cytosolic b u t n o t t h e m i t o c h o n d r i a l hepatic PEPCK activity has b e e n s h o w n for t h e rat (Filsell et al., 1 9 6 9 ; Hanson and Garber, 1 9 7 2 ; Shrago et al., 1 9 6 3 ) ; t h e guinea pig ( L a r d y et al, 1 9 6 4 ; Soling et al, 1970), and t h e rabbit (Johnson et al, 1 9 7 0 ; Ray, 1976). In sheep (Taylor et al, 1971) and pig (Swiatek et al, 1 9 7 0 ) liver, fasting m a r k e d ly increased b o t h t h e m i t o c h o n d r i a l and t h e cytosolic e n z y m e activities b u t t h e increase in
t h e cytosolic activity was in part due t o t h e leakage of t h e e n z y m e from t h e m i t o c h o n d r i a since t h e y were m o r e fragile in t h e fasting state and, hence, m o r e susceptible t o breakage during h o m o g e n i z a t i o n and subcellular fractionation (Taylor et al, 1 9 7 1 ) . This was also t h e case for
H e p a t i c PEPCK cytosolic
H e p a t i c PEPCK mitochondrial 20-
ft
m o o
rh
P<0
\
rh
_
<0.05
-
l
15-
5 -
a.
n
1 o _ Renal S 15
PEPCK mitochondrial
_ Renal PEPCK cytosolic
£ N
FIG. 1. Effect of fasting and fasting and refeeding on pyruvate carboxylase of the chicken. Abbreviations: A, fed control; B, 2-day-fasted; C, 4-day-fasted; and D, 4-day-fasted and 4-day refed chickens. Data are expressed as the mean of four animals ± SEM (vertical lines). Significance of the difference (P) from the t-test was calculated by testing the mean values of the fasted and fasted and refed chickens against the control.
10
r h
,.i
m Q ov a. r-S-
0 B
C
m o q o a.
n n
S g v ?, §v
£°
rh
V 0.
a.
B
£
q
n D
FIG. 2. Effect of fasting and fasting and refeeding on phosphoenolpyruvate carboxykinase of the chicken. Abbreviations and other details are the same as in the legend to Figure 1.
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Liver weight, g/lOOgbody wt
Group B Fasted for 2 days
GLUCONEOGENIC ENZYMES IN THE CHICKEN
Hepatic PEPCK
Hepatic GDH
Hepatic
1 00
10 in
o o d v a.
80
^FDPase o o d v a.
o d v a.
40
o o 6 v a.
o o d v
a. a a E3
4 PTl E3Renal GDH
Particulate Cytosol
ih £
>> 0
B
Q_
60
^ 40
D
FIG. 4. Effect of fasting and fasting and refeeding on fructose-l,6-diphosphatase of the chicken. Abbreviations and other details are the same as in the legend to Figure 1.
20
FIG. 3. Comparison of the subcellular distribution of phosphoenolpyruvate carboxykinase and glutamate dehydrogenase of the chicken. The 100,000 X g sediment and supernate are described as particulate and cytosol fractions, respectively. Abbreviations are the same as in the legend to Figure 1.
t h e hepatic m i t o c h o n d r i a from fasted (Walter, 1 9 7 6 ) and diabetic (Hall et al, I 9 6 0 ; Matsub u r a and T o c h i n o , 1969) rats. Fasting m a r k e d l y increased t h e hepatic and renal FDPase activities in the chicken (Figure 4 ) ; t h e activities increased by a b o u t 8 0 and 90%, respectively, after a 4-day fast. U p o n refeeding, t h e activities r e t u r n e d t o near control levels. Starvation was also reported t o increase t h e e n z y m e activity in t h e liver and t h e kidney of t h e sheep (Filsell et al, 1 9 6 9 ) . In contrast, fasting had n o effect on hepatic FDPase in t h e rat (Filsell et al, 1 9 6 9 ; Soling etal, 1 9 7 0 ) , t h e rabbit (Ray, 1976), and t h e guinea pig (Soling etal, 1970). Both hepatic and renal G6Pase activities increased b y 15 and 30% respectively in t h e 4 day fasted chickens and r e t u r n e d t o near c o n t r o l values u p o n refeeding (Figure 5). Increase in G6Pase activity during starvation has been observed in t h e liver and t h e k i d n e y of t h e rat (Nordlie etal, 1 9 6 8 ; Soling et al, 1970) and t h e sheep (Filsell et al, 1 9 6 9 ) and in t h e liver of t h e pig (Swiatek et al, 1 9 7 0 ) and
t h e guinea pig (Soling et al, 1970). T h e increased G6Pase activity in fasted animals was in p a r t due t o an increase in glycogenolysis (Nordlie and Jorgenson, 1975). ACKNOWLEDGMENTS This study was s u p p o r t e d in p a r t b y United States Public Health Service, National Institutes of Health research grant A M - 0 8 3 7 3 . We t h a n k A. Frick for taking care of t h e animals.
A
B
C
D
A
B
C
D
FIG. 5. Effect of fasting and fasting and refeeding on glucose-6-phosphatase of the chicken. Abbreviations and other details are the same as in the legend to Figure 1.
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80
in
ci-
Renal PEPCK
^ 100 "
Renal FDPase
in
60
20
893
894
SHEN AND MISTRY
REFERENCES
Latzko, E., and M. Gibbs, 1974. Alkaline C, -fructose1,6-diphosphatase. Page 881—884 in Methods of enzymatic analysis. Vol. 2. H. U. Bergmeyer, ed. Academic Press, New York and London. Leveille, G. A., 1969. In vivo fatty acid and cholesterol synthesis in fasted and fasted-refed chicks. J. Nutr. 98:367-372. Leveille, G. A., and K. Chakrabarty, 1967. Diurnal variation in tissue glycogen and liver weight of meal-fed rats. J. Nutr. 93:546—554. Matsubura, T., and Y. Tochino, 1969. Depression of respiratory activities in the liver mitochondria of diabetic rats and the restorative action of insulin. J. Biochem. 66:397-404. Nordlie, R. C , W. J. Arion, T. L. Hanson, J. R. Gilsdrof, and R. N. Home, 1968. Biological regulation of liver microsomal inorganic pyrophosphateglucose phosphotransferase, glucose-6-phosphatase and inorganic pyrophosphatase. J. Biol. Chem. 243:1140-1146. Nordlie, R. C , and R. A. Jorgenson, 1975. Glucose-6phosphatase. Page 4 6 5 - 4 9 1 in The enzymes of biological membranes. Vol. 2. A Martonosi, ed. Plenum Press, New York. Pearce, J., 1971. Carbohydrate metabolism in the domestic fowl. Proc. Nutr. Soc. 30:254-259. Ray, P. D., 1976. Hepatic gluconeogenesis in the rabbit. Page 293—333 in Gluconeogenesis: Its regulation in mammalian species. R. W. Hanson and M. A. Mehlman, ed. John Wiley and Sons, New York and London. Sarkar, N. K., 1971. Gluconeogenesis and the factors that control the process in chickens. Life Sci. 10(2):293-300. Schmidt, E., 1974. Glutamate dehydrogenase. Page 650—656 in Methods of enzymatic analysis. Vol. 2. H. U. Bergmeyer, ed. Academic Press, New York and London. Schultz, P., and S. P. Mistry, 1978. A study of gluconeogenesis in isolated liver cells. Page 319 in American Society of Animal Science, 70th Annu. Mtng., East Lansing, MI. (Abstr.) Shen, C. S., and S. P. Mistry, 1977. Activities of pyruvate and propionyl coenzyme A carboxylase in chicken tissues during normal growth and biotin deficiency. Poultry Sci. 56:1900-1903. Shen, C. S., and S. P. Mistry, 1978. Phosphoenolpyruvate carboxylkinase (PEPCK) of avian kidney: Intracellular location and the effect of prolonged fasting on the enzyme activity. Fed. Proc. 37:594. (Abstr.) Shrago, E., H. A. Lardy, R. C. Nordlie, and D. O. Foster, 1963. Metabolic and hormonal control of phosphoenolpyruvate carboxykinase and malic enzyme in rat liver. J. Biol. Chem. 238:3188-3192. Soling, H.-D., and J. Kleineke, 1976. Species dependent regulation of hepatic gluconeogenesis in higher animals. Page 369—462 in Gluconeogenesis: Its regulation in mammalian species. R. W. Hanson and M. A. Mehlman, ed. John Wiley and Sons, New Y6rk and London. Soling, H.-D., J. Kleineke, B. Willms, G. Janson, and A. Kuhn, 1973. Relationship between intracellular distribution of phosphenolpyruvate carboxykinase, regulation of gluconeogenesis, and energy cost of glucose formation. Europ. J. Biochem. 37:233— 243.
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Achuta Murthy, P. N., C. S. Shen, and S. P. Mistry, 1977. Effect of fasting and refeeding high-carbohydrate, high-fat and high-protein diets on the activities of some enzymes of carbohydrate metabolism in rat kidney. Federation Proc. 36:1172. (Abstr.) Allred, J. B., and K. L. Boehrig, 1970. Hepatic gluconeogenesis and glycolysis in chickens fed "carbohydrate free" diets. J. Nutr. 100:615-622. Baginski, E. S., P. P. Foa, and B. Zak, 1974. Page 876—880 in Methods of enzymatic analysis. Vol. 2. H. U. Bergmeyer, ed. Academic Press, New York and London. Ballard, F. J., and R. W. Hanson, 1967. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver. Biochem. J. 104:866—871. Ballard, F. J., R. W. Hanson, D. S. Kronfeld, and F. Raggi, 1968. Metabolic changes in liver associated with spontaneous ketosis and starvation in cows. J. Nutr. 94:160-172. Belo, P. S., D. R. Romsos, and G. A. Levielle, 1976. Blood metabolites and glucose metabolism in the fed and fasted chicken. J. Nutr. 106:1135-1143. Brady, L. J., D. R. Romsos, P. S. Brady, W. G. Bergen, and G. A. Leveille, 1978b. The effect of fasting on body composition, glucose turnover, enzymes and metabolites in the chick. J. Nutr. 108:648-657. Brady, L., D. R. Romsos, G. A. Leveille, and E. R. Miller, 1978a. Gluconeogenesis in isolated hepatocytes. Federation Proc. 37:677. (Abstr.) Deodhar, A. D., and S. P. Mistry, 1969. Gluconeogenesis in biotin deficiency: in vitro synthesis of pyruvate holocarboxylase in biotin-deficient rat liver. Biochem. Biophys. Res. Comm. 34:755-759. Exton, J. H., 1972. Gluconeogenesis. Metabolism 2 1 : 945-990. Feigenbaum, A. S., and H. Fisher, 1963. Changes in fatty acid composition in nutritional fatty degeneration of the liver. 1. Effect of starvation. Brit. J. Nutr. 17:31-37. Filsell, O. H., I. G. Jarrett, P. H. Taylor, and D. B. Keech, 1969. Effects of fasting, diabetes and glucocorticoids on gluconeogenic enzymes in the sheep. Biochim. Biophys. Acta 184:54—63. Garber, A. J., and R. W. Hanson, 1971. The control of phosphoenolpyruvate formation by rabbit liver mitochondria. J. Biol. Chem. 246:5555-5562. Hall, J. C , L. A. Sordahl, and P. L. Stefko, 1960. The effect of insulin on oxidative phosphorylation in normal and diabetic mitochondria. J. Biol. Chem. 235:1536-1539. Hanson, R. W., and A. J. Garber, 1972. Phosphoenolpyruvate carboxykinase I. Its role in gluconeogenesis. Amer. J. Clin. Nutr. 25:1010-1021. Johnson, D. C , K. A. Ebert, and P. D. Ray, 1970. Hepatic gluconeogenesis of the rabbit. Biochem. Biophys. Res. Comm. 39:750-756. Krebs, H. A., 1963. Renal gluconeogenesis. Adv. Enz. Reg. 1:385-400. Krebs, H. A., 1966. The regulation of the release of ketone bodies by the liver. Adv. Enz. Reg. 4:339— 353. Lardy, H. A., D. O. Foster, E. Shargo, and P. D. Ray, 1964. Metabolic and hormonal regulation of phosphopyruvate synthesis. Adv. in Enzyme Reg. 2: 39-47.
GLUCONEOGENIC ENZYMES IN THE CHICKEN Soling, H.-D., B. Willms, J. Kleineke, and M. Gehloff, 1970. Regulation of gluconeogenesis in guinea pig liver. Europ. J. Biochem. 16:289-302. Swiatek, K. R., K. L. Chao, H. L. Chao, M. Cornblath, and T. Tildon, 1970. Distribution of phosphoenolpyruvate carboxykinase in pig liver. Biochim. Biophys. Acta 2 0 6 : 3 1 6 - 3 2 1 . Taylor, P. H., J. C. Wallace, and D. B. Keech, 1971. Gluconeogenic enzymes in sheep liver. Intracellu-
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lar localization of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in normal, fasted and diabetic sheep. Biochim. Biophys. Acta 237: 179-191. Walter, P., 1976. Pyruvate carboxylase: Intracellular localization and regulation. Page 239—265 in Gluconeogenesis: Its regulation in mammalian species. R. W. Hanson and M. A. Mehlman, ed. John Wiley and Sons, New York and London.
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Laboratory of Nutritional Biochemistry, Department of Animal Science, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (Received for publication September 12, 1978)
INTRODUCTION The chicken maintains not only a relatively high blood glucose level but also a normal glucose turnover rate during fasting (Belo et al, 1976; Pearce, 1971; Sarkar, 1971). Several studies have shown that the chicken is unable to synthesize glucose from a number of substrates which are readily utilized by the rat (Brady et al, 1978a; Sarkar, 1971; Schultz and Mistry, 1978; Soling et al, 1973). Thus, the process of gluconeogenesis and its regulation appears to be different in the chicken than in mammals, particularly in the rat. Pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6diphosphatase (FDPase), and glucose-6phosphatase (G6Pase) are key gluconeogenic enzymes. It is known that the activities of these enzymes in the liver and the kidney of the rat and several other species change in response to various nutritional and hormonal treatments, such as fasting, fasting and refeeding, diabetes and Cortisol administration, conditions which are known to influence gluconeogenesis (Exton, 1972; Krebs, 1963; Soling and Kleineke, 1976). However, only few such studies have been made with the chicken (Allred and Boehrig, 1970; Brady et al, 1978b). Furthermore, with the demonstrated presence of both cytosolic and mitochondrial PEPCK in the chicken liver and the kidney (Shen and Mistry, 1978), it is important to know whether one or both of these enzymes can be induced or repressed under suitable conditions.
In the present study, the effect of fasting and fasting and refeeding on the activities of the key hepatic and renal gluconeogenic enzymes in the chicken have been investigated.
MATERIALS AND METHODS Animals. Newly hatched female crossbred chicks (Gallus domesticus: New Hampshire male X Columbian female) were fed ad libitum a corn-based diet (Shen and Mistry, 1977) until six 'weeks of age. Sixteen chickens weighing between 600 and 650 g were divided into four groups. Group A served as the control and the animals were sacrificed without further treatment. Groups B and C were fasted for 2 and 4 days, respectively. Group D was fasted for 4 days and refed the same diet for 4 days before sacrifice. Water was provided at all times. Enzyme Preparations. Chickens were killed by cervical dislocation. Livers and kidneys were removed, blotted, weighed quickly, and then dropped into an ice-cold homogenizing medium containing .25M sucrose and 1 mM EDTA. Tissues were homogenized in nine volumes of the above medium with a Potter-Elvehjem homogenizer for 3 min. The homogenates were centrifuged at 100,000 X g for 1 hr in a Beckman preparative ultracentrifuge, model L2-65. The supernatant fraction was used for the assay of FDPase and cytosolic PEPCK. The sediment was suspended in the homogenizing medium and lyophilized. The lyophilized samples were resuspended in 890
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ABSTRACT The effect of fasting and fasting and refeeding on hepatic and renal gluconeogenic enzyme activities were studied in six-week-old chickens (Gallus domesticus: New Hampshire male X Columbian female). Hepatic pyruvate carboxylase appeared not to be affected by fasting, but the renal enzyme activity increased in four-day fasted chickens. The hepatic mitochondrial and cytosolic phosphoenolpyruvate carboxykinases were essentially not affected by fasting. The renal mitochondrial phosphoenolpyruvate carboxykinase showed a slight increase in activity only after a four-day fast, but the cytosolic enzyme activity increased markedly already after a two-day fast. Also, the activities of the hepatic and renal fructose-1,6-diphosphatase and glucose-6-phosphatase increased markedly on fasting. Refeeding for four days after a four-day fast returned these enzyme activities to near control values. 1979 Poultry Science 58:890-895
GLUCONEOGENIC ENZYMES IN THE CHICKEN
RESULTS AND DISCUSSION
The effects of fasting and fasting and refeeding on body weight, liver weight and kidney weight are shown in Table 1. As expected, the chickens lost weight during the fast which was regained after refeeding. Liver weight relative to body weight decreased by about 10% as a result of either a 2-day or a 4-day fast. Refeeding for 4 days after a 4-day fast increased the liver weight even more than that of the fed control chickens. Similarly, the weight of the kidney decreased by about 10% as a consequence of fasting; refeeding brought the weight back to the normal level. The alterations in liver weights observed in the present study were essentially in accord with previous reports (Feigenbaum and Fisher, 1963; Leveille, 1969). They were primarily due to changes in glycogen and water content, as noted for the chicken (Leveille, 1969) and the rat (Leveille and Chakrabarty, 1967). However, the increase in the liver
weight observed after fasting and refeeding might have been in part due to an increase in the lipid content. Feigenbaum and Fisher (1963) and Leveille (1969) have reported that fasting did not change the liver fat content in the chick, but subsequent refeeding did increase the lipid content. The reason for the decrease in the kidney weight seen on fasting is not clear, since analytical data are not available. The effect of fasting and fasting and refeeding on hepatic and renal PC activities of growing chickens are shown in Figure 1. Hepatic PC appeared to be unaffected by either a 2 day or a 4 day fast, but the activity in the fasted and refed chickens was significantly lower than in the controls. Renal PC was also not affected by a 2-day fast but the activity increased after a 4-day fast; refeeding restored the activity to that of the control. There is no agreement concerning the effect of fasting on hepatic (Filsell et al, 1969; Krebs, 1966; Soling et al., 1973) and renal (Filsell et al., 1969; Achuta Murthy et al, 1977) PC in the rat. The activity was reported to increase in the liver of the fasted sheep (Filsell et al, 1969; Taylor et al, 1971), the guinea pig (Soling et al., 1970; Soling et al, 1973), the cow (Ballard et al, 1968), the pig (Swiatek et al, 1970), and in the kidney of the fasted sheep (Taylor et al, 1971). In contrast, hepatic PC remained unchanged in the fasted rabbit (Garber and Hanson, 1971; Ray, 1976). Corresponding changes in the hepatic and renal PEPCK activities are shown in Figure 2. The hepatic mitochondrial or the cytosolic enzyme was virtually unaffected by fasting; refeeding after a 4-day fast significantly decreased the mitochondrial activity. The renal mitochondrial enzyme activity increased slightly only after a 4-day fast, but the cytosolic activity more than doubled after a 2-day fast; refeeding decreased both activities to slightly lower levels than those of the controls. The increased cytosolic PEPCK activity in the kidney was not due to the leakage of the enzyme from the mitochondria into the cytosol. As seen in Figure 3, while the renal PEPCK activity in the cytosol increased from 35% in the fed chickens to 56% in the 2-day fasted chickens, the activity of the mitochondrial marker enzyme, GDH, remained unchanged in the cytosol. In agreement with an earlier report, both hepatic and renal mitochondrial PEPCK activities in the chicken did not fluctuate markedly during fasting (Brady et al, 1978b).
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distilled water and used for the assay of PC, mitochondrial PEPCK, and G6Pase. Glutamate dehydrogenase (GDH) was used as a mitochondrial marker enzyme; its activity was assayed in both fractions. Enzyme Assays. PC and PEPCK were assayed by the radiochemical carboxylation assay as described by Deodhar and Mistry (1969) and Ballard and Hanson (1967), respectively. In both assays the incorporation of 14 C-bicarbonate into oxalacetate was measured as citrate and malate, respectively. After termination of the reaction and centrifugation, the residual 1 C-bicarbonate was removed from the proteinfree supernates by repeated additions of small pieces of dry ice over a period of 1 hr. Aliquots were then counted in a Packard liquid scintillation counter after adding 15 ml of Aquasol. G6Pase was assayed according to Baginski et al. (1974); FDPase was determined spectrophotometrically in a coupled reaction system as described by Latzko and Gibbs (1974). GDH was determined spectrophotometrically as described by Schmidt (1974). PC, PEPCK, and G6Pase were determined at 37 C, whereas F6Pase and GDH were measured at 30 C. Enzyme activities were expressed as Mtnoles of substrate consumed or product formed per minute per gram of wet tissue. The appropriate dilution which gave a linear relationship of enzymatic activity with respect to time and protein concentration was determined beforehand for each enzyme assay.
891
892
SHEN AND MISTRY TABLE 1. Effect of fasting and fasting and refeeding on body weight, liver weight, and kidney weight of growing chickens*b
Group A Fed Body weight, g
629
± 13
1.71 ±
.08
Kidney weight, g/lOOgbody wt
.88 ±
.06
Group C Fasted for 4 days
Group D Fasted for 4 days and refed for 4 days
530 ± 18 P<.01 1.49 ± .06 P<.025
435 ±5 P<.005 1.47 ± .10 P<.05
701 ± 29 P<.05 2.28 ± .13 P-C.025
.76 ± P<.05
.77 ± .04 P<.05
.04
.81 ± NS
.04
Each value represents the mean ± SEM of four chickens. Significance of difference (P) from the t-test was calculated by testing the mean of the fasted and fasted and refed chickens against the control (Group A). "NS" denotes not significant, i.e., P>.05.
T h a t fasting increased the cytosolic b u t n o t t h e m i t o c h o n d r i a l hepatic PEPCK activity has b e e n s h o w n for t h e rat (Filsell et al., 1 9 6 9 ; Hanson and Garber, 1 9 7 2 ; Shrago et al., 1 9 6 3 ) ; t h e guinea pig ( L a r d y et al, 1 9 6 4 ; Soling et al, 1970), and t h e rabbit (Johnson et al, 1 9 7 0 ; Ray, 1976). In sheep (Taylor et al, 1971) and pig (Swiatek et al, 1 9 7 0 ) liver, fasting m a r k e d ly increased b o t h t h e m i t o c h o n d r i a l and t h e cytosolic e n z y m e activities b u t t h e increase in
t h e cytosolic activity was in part due t o t h e leakage of t h e e n z y m e from t h e m i t o c h o n d r i a since t h e y were m o r e fragile in t h e fasting state and, hence, m o r e susceptible t o breakage during h o m o g e n i z a t i o n and subcellular fractionation (Taylor et al, 1 9 7 1 ) . This was also t h e case for
H e p a t i c PEPCK cytosolic
H e p a t i c PEPCK mitochondrial 20-
ft
m o o
rh
P<0
\
rh
_
<0.05
-
l
15-
5 -
a.
n
1 o _ Renal S 15
PEPCK mitochondrial
_ Renal PEPCK cytosolic
£ N
FIG. 1. Effect of fasting and fasting and refeeding on pyruvate carboxylase of the chicken. Abbreviations: A, fed control; B, 2-day-fasted; C, 4-day-fasted; and D, 4-day-fasted and 4-day refed chickens. Data are expressed as the mean of four animals ± SEM (vertical lines). Significance of the difference (P) from the t-test was calculated by testing the mean values of the fasted and fasted and refed chickens against the control.
10
r h
,.i
m Q ov a. r-S-
0 B
C
m o q o a.
n n
S g v ?, §v
£°
rh
V 0.
a.
B
£
q
n D
FIG. 2. Effect of fasting and fasting and refeeding on phosphoenolpyruvate carboxykinase of the chicken. Abbreviations and other details are the same as in the legend to Figure 1.
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Liver weight, g/lOOgbody wt
Group B Fasted for 2 days
GLUCONEOGENIC ENZYMES IN THE CHICKEN
Hepatic PEPCK
Hepatic GDH
Hepatic
1 00
10 in
o o d v a.
80
^FDPase o o d v a.
o d v a.
40
o o 6 v a.
o o d v
a. a a E3
4 PTl E3Renal GDH
Particulate Cytosol
ih £
>> 0
B
Q_
60
^ 40
D
FIG. 4. Effect of fasting and fasting and refeeding on fructose-l,6-diphosphatase of the chicken. Abbreviations and other details are the same as in the legend to Figure 1.
20
FIG. 3. Comparison of the subcellular distribution of phosphoenolpyruvate carboxykinase and glutamate dehydrogenase of the chicken. The 100,000 X g sediment and supernate are described as particulate and cytosol fractions, respectively. Abbreviations are the same as in the legend to Figure 1.
t h e hepatic m i t o c h o n d r i a from fasted (Walter, 1 9 7 6 ) and diabetic (Hall et al, I 9 6 0 ; Matsub u r a and T o c h i n o , 1969) rats. Fasting m a r k e d l y increased t h e hepatic and renal FDPase activities in the chicken (Figure 4 ) ; t h e activities increased by a b o u t 8 0 and 90%, respectively, after a 4-day fast. U p o n refeeding, t h e activities r e t u r n e d t o near control levels. Starvation was also reported t o increase t h e e n z y m e activity in t h e liver and t h e kidney of t h e sheep (Filsell et al, 1 9 6 9 ) . In contrast, fasting had n o effect on hepatic FDPase in t h e rat (Filsell et al, 1 9 6 9 ; Soling etal, 1 9 7 0 ) , t h e rabbit (Ray, 1976), and t h e guinea pig (Soling etal, 1970). Both hepatic and renal G6Pase activities increased b y 15 and 30% respectively in t h e 4 day fasted chickens and r e t u r n e d t o near c o n t r o l values u p o n refeeding (Figure 5). Increase in G6Pase activity during starvation has been observed in t h e liver and t h e k i d n e y of t h e rat (Nordlie etal, 1 9 6 8 ; Soling et al, 1970) and t h e sheep (Filsell et al, 1 9 6 9 ) and in t h e liver of t h e pig (Swiatek et al, 1 9 7 0 ) and
t h e guinea pig (Soling et al, 1970). T h e increased G6Pase activity in fasted animals was in p a r t due t o an increase in glycogenolysis (Nordlie and Jorgenson, 1975). ACKNOWLEDGMENTS This study was s u p p o r t e d in p a r t b y United States Public Health Service, National Institutes of Health research grant A M - 0 8 3 7 3 . We t h a n k A. Frick for taking care of t h e animals.
A
B
C
D
A
B
C
D
FIG. 5. Effect of fasting and fasting and refeeding on glucose-6-phosphatase of the chicken. Abbreviations and other details are the same as in the legend to Figure 1.
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80
in
ci-
Renal PEPCK
^ 100 "
Renal FDPase
in
60
20
893
894
SHEN AND MISTRY
REFERENCES
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Achuta Murthy, P. N., C. S. Shen, and S. P. Mistry, 1977. Effect of fasting and refeeding high-carbohydrate, high-fat and high-protein diets on the activities of some enzymes of carbohydrate metabolism in rat kidney. Federation Proc. 36:1172. (Abstr.) Allred, J. B., and K. L. Boehrig, 1970. Hepatic gluconeogenesis and glycolysis in chickens fed "carbohydrate free" diets. J. Nutr. 100:615-622. Baginski, E. S., P. P. Foa, and B. Zak, 1974. Page 876—880 in Methods of enzymatic analysis. Vol. 2. H. U. Bergmeyer, ed. Academic Press, New York and London. Ballard, F. J., and R. W. Hanson, 1967. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver. Biochem. J. 104:866—871. Ballard, F. J., R. W. Hanson, D. S. Kronfeld, and F. Raggi, 1968. Metabolic changes in liver associated with spontaneous ketosis and starvation in cows. J. Nutr. 94:160-172. Belo, P. S., D. R. Romsos, and G. A. Levielle, 1976. Blood metabolites and glucose metabolism in the fed and fasted chicken. J. Nutr. 106:1135-1143. Brady, L. J., D. R. Romsos, P. S. Brady, W. G. Bergen, and G. A. Leveille, 1978b. The effect of fasting on body composition, glucose turnover, enzymes and metabolites in the chick. J. Nutr. 108:648-657. Brady, L., D. R. Romsos, G. A. Leveille, and E. R. Miller, 1978a. Gluconeogenesis in isolated hepatocytes. Federation Proc. 37:677. (Abstr.) Deodhar, A. D., and S. P. Mistry, 1969. Gluconeogenesis in biotin deficiency: in vitro synthesis of pyruvate holocarboxylase in biotin-deficient rat liver. Biochem. Biophys. Res. Comm. 34:755-759. Exton, J. H., 1972. Gluconeogenesis. Metabolism 2 1 : 945-990. Feigenbaum, A. S., and H. Fisher, 1963. Changes in fatty acid composition in nutritional fatty degeneration of the liver. 1. Effect of starvation. Brit. J. Nutr. 17:31-37. Filsell, O. H., I. G. Jarrett, P. H. Taylor, and D. B. Keech, 1969. Effects of fasting, diabetes and glucocorticoids on gluconeogenic enzymes in the sheep. Biochim. Biophys. Acta 184:54—63. Garber, A. J., and R. W. Hanson, 1971. The control of phosphoenolpyruvate formation by rabbit liver mitochondria. J. Biol. Chem. 246:5555-5562. Hall, J. C , L. A. Sordahl, and P. L. Stefko, 1960. The effect of insulin on oxidative phosphorylation in normal and diabetic mitochondria. J. Biol. Chem. 235:1536-1539. Hanson, R. W., and A. J. Garber, 1972. Phosphoenolpyruvate carboxykinase I. Its role in gluconeogenesis. Amer. J. Clin. Nutr. 25:1010-1021. Johnson, D. C , K. A. Ebert, and P. D. Ray, 1970. Hepatic gluconeogenesis of the rabbit. Biochem. Biophys. Res. Comm. 39:750-756. Krebs, H. A., 1963. Renal gluconeogenesis. Adv. Enz. Reg. 1:385-400. Krebs, H. A., 1966. The regulation of the release of ketone bodies by the liver. Adv. Enz. Reg. 4:339— 353. Lardy, H. A., D. O. Foster, E. Shargo, and P. D. Ray, 1964. Metabolic and hormonal regulation of phosphopyruvate synthesis. Adv. in Enzyme Reg. 2: 39-47.
GLUCONEOGENIC ENZYMES IN THE CHICKEN Soling, H.-D., B. Willms, J. Kleineke, and M. Gehloff, 1970. Regulation of gluconeogenesis in guinea pig liver. Europ. J. Biochem. 16:289-302. Swiatek, K. R., K. L. Chao, H. L. Chao, M. Cornblath, and T. Tildon, 1970. Distribution of phosphoenolpyruvate carboxykinase in pig liver. Biochim. Biophys. Acta 2 0 6 : 3 1 6 - 3 2 1 . Taylor, P. H., J. C. Wallace, and D. B. Keech, 1971. Gluconeogenic enzymes in sheep liver. Intracellu-
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lar localization of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in normal, fasted and diabetic sheep. Biochim. Biophys. Acta 237: 179-191. Walter, P., 1976. Pyruvate carboxylase: Intracellular localization and regulation. Page 239—265 in Gluconeogenesis: Its regulation in mammalian species. R. W. Hanson and M. A. Mehlman, ed. John Wiley and Sons, New York and London.
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