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Diurnal rhythms of liver enzymes in the chicken, Gallus domesticus
Int. 3. Biochm.,
197 I,
DIURNAL
2,
581-584.
[Scientechnica
RHYTHMS CHICKEN,
K. A. CHANDRABOSE,
(Publishers) Ltd.]
581
OF SLIVER ENZYMES IN THE GALLUS ~~~EST~C~S
A. BENSADOUN,
AND C. K. CLIFFORD
Department of Poultry Science, Cornell University, Ithaca, New York, U.S.A.
ABSTRACT I. NADP-linked ma&c enzyme (ME) and glycerokinase (GR) show diurnal rhythms in the liver of cockerels. 2. Although the activities of both enzymes are markedly influenced by hypophysectomy and hormone replacement therapy, hypophysectomyabolishes the periodicity of ME, but not that of GK. 3. The rhy~mic~ty of ME dots not see.mto be related to malate concentration in the l&r or to the pattern of food intake. THE detection of quantitatively significant diurnal rhythms in the activities of enzymes has been reported in liver and other tissues (Goodwin, 1965 ; Potter, Gilbert, and Pitot, I 966). Several investigators have attempted to correlate diurnal variations in enzyme activities with corresponding rhythmicities in hormones known to be inducers of these enzymes (Civen, Ulrich, Trimmer, and Brown, 1967). We wish to report the results of studies on the variation of the activities of various liver and adipose tissue enzymes in intact and hypophysectomized roosters.
MATERIALS AND METHODS White Leghorn cockereb raised on a commercial diet were employed in this experiment. Hype_ physeetomy was performed when the birds were IQ-I I weeks old, by a transluccal approach (Rothchild, 1948). They were kept for 3 months after surgery before the experiment was performed. The animals were housed in individual cages in an isolated room with controlled temperature (2I' C.) andillumination (6.00 a.mA.oon.m.). Three. to 5 pairs of control and hypophysect&nized birds we& sacrificed at intervals of 4 hours. The followinn liver enzymes were analykd according to pro& dures given in a previous publication (Chandrabose and Bensadoun, I 97 I a) : citrate-cleavage enzyme
(E.G. 4. I. 3.8), NADP-linked ma& enzyme (E.C. I. I. I. 40), glucose-&phosphate dehydro-
genase (E.C. I. I. I. 4g), glycerokinase (EC. 2. 7. I. 30)~ and lipoprotein Iipase (glycerol ester hydrolase, E.C. 3. I. x. 3). Liver samples, quick-frozen in liquid nitrogen, were analysed for L-malate by the enzymatic procedure of Hohorst ( x963),
Statistical differences in enzyme activities at various times of the day were ascertained by Duncan’s multiple range test (Steel and Torrie, 1960). RESULTS
AND DISCUSSION
The results of enzymatic analyses are summarized in Table I and plotted in Fig. I. Only 2 enzymes, NADP-linked malic enzyme (ME) and glycerokinase (GK) , showed significant rhythmicity. The activity of ME was approximately threefold higher at I zoo noon than at any other time of the day (P. Both groups showed very similar food intake patterns, consuming most of their dietary intake between 12 noon and 4.00 p.m. When food intake was expressed per unit of metabolic size there were no significant differences in fbod consumption as a resuft of hy~physectomy. In the control birds the peak in enzyme activity preceded the peak of food consumption and occurred when they had eaten only 30 per cent of their daily food intake.
CHANDRABOSE
ET AL.
ht.
J.
Bimhem.
The possibility was also considered that the ME rhythmicity in control birds reflected a rhythm in the hepatic concentrations of Lmalate. There were no significant differences in L-malate concentrations at various times of the day (Table III). The above experiments strongly suggest that an intact anterior pituitary is necessary for the ME daily rhythm. It has been shown previously that ph~iologi~al levels of thyroxine (Chandrabose and Bensadoun, rg7r b) will induce ME when injected in hypophysectomized roosters. It is conceivabIe that thyroxine might act as a major physiological inducer of malic enzyme, Iodohormone plasma levels, measured at various times of the day in another group of roosters, showed a significant diurnal rhythmicity, with a peak iodohormone activity at 4.00 p.m. (Sadovsky and Bensadoun, I g7 I). It is premature to attempt to relate the peak ME activity with the peak iodohormone levels until both rne~u~en~ have been made in the same animals. The short half-life of 3.23 hours, reported by Singh, Reinke, and Ringer (x967) for thyroxine and triiodothyroxine, is consistent with the existence of a diurnal rhythmicity for these hormones. Glycerokinase (GK) showed diurnal rhythmicity in both control and hypophysectomized birds. GK activity at 12.00 noon was twofold higher than at any other time of the day in both control and hypophysectomized birds (P
‘971,
2
DIURNAL
RHYTHMS
abolishes the periodicity of ME, it does not affectthat ofGi(. Theseiwoexamples illustrate that although an enzyme is inducible or repressible in a short time by exogenous hormone injections, these hormones are not
OF LIVER
583
ENZYMES
necessarily the major physiological factors responsible for its diurnal rhythms. The other enzyme studied, citrate-cleavage enzyme, gluco~-~phosphate dehydrogenase, and lipoprotein hpase, did not show any
60 MALIC
GLYCEROL
ENZYME
KINASE
50 .e E . .s
40
“c ii g30 . -: i! 520 3 E
I 800
2000 I600 TIME OF DAY
,
I
5
1200
IO
2400
400
800
1200
1600 TIME
2000 OF DAY
1
I
1200
1600 TIME
2000 OF DAY
2400
2400
4
.G
$ .c 0)
15
6 CITRATE
CLEAVAGE
ENZYME
P
2000 TIME OF DAY
FIG.I.-Diurnal
2400
400
800
TIME OF DAY
400
rhythms of liver malic enzyme, glycerokinase, citrate-cleavage enzyme, glucose-6., phosphate dehydmgenase, and adipose tissue lipoprotein lipase, in control and hypophysectomized cockerels. (800, 1200, 1600, 2000, 2400, and 400 standfor8.00a.m.,12.00 noon, 4.00 p.m., 8.00 p.m., I 2.00 midnight, and 4.00 a.m. respectively.)
CHANDRABOSE
584
ET
AL.
Table II.-VOLUNTARY FOOD CONSUMPIION OF CONTROLAND HYPOPHYSECTOMIZEDCOCKERELS (g. per kg. body-weight)+”
TIME BIRDS 8.&ym.
Control Hypophysectomized
1.5fo.8 I. I *a*
I
8.oo12.00 a.m.
IZoo4.00 p.m.
4.oo8.00 p.m.
8.oo12.00 p.m.
IZ.oo4.00 a.m.
2.6ko.6
19.1 k2.2
0.6fo.5
o-4*0.2
0.7 io.2
o-7 io.5
16.3f1.8
0.4Co.2
0.8fo.3
0.7io.3
I
I
I
Mean body-weight of 4 control cockerels=2061 g. Mean body-weight of 4 hypophysectomized cockerels=n~zo g. The values represent means fS.E.M. of 4 observations. Table III-L-MALATE CONCENTRATIONS (umoles L-malate per g. protein) IN THE LIVERY OF CONTROL AND HYPOPHYSECTOWZED COCKERELSAT VARIOUS TIMES OF THE DAY
TIMEOF DAY TREATMENT 8.00 a.m.
12.00 noon
4.00 p.m.
12.00 midnight
8.00 p.m.
4.00 a.m.
Control Hypophysectomized Number of -chickens
1
6
1
6
1
8
1
8
1
6
1
8
-
* Standard error of the mean. diurnal
variations when cockerels were fed under the conditions described in the Methods section. ad libitum
REFERENCES CHANDRABOSE, K. A., and BENSADOUN, A. (I g7 Ia), ‘ Effects of hypophysectomy on some enzymes involved in lipid metabolism of the domestic chicken (Callus domesticus) ‘, Comp. Biochem. Physiol., 39, 45-54.
CHANDRABOSE, K. A., and BENSADOUN, A. ( Ig7 Ib) , ‘ Effect of hypophysectomy on liver enzymes in the chicken, Callus domesticus. Replacement therapy with growth hormone and thyroxine ‘, Corn@Biochem. Physiol., 39, 55-59. CNEN, M., ULRICH, R., TRIMMER,B. M., and BROWN, C. B. (Ig67), ‘ Circadian rhythms of liver enzymes and their relationship to enzyme induction ‘, S&rue, N. Y., 157, I 563-1564. GOODWIN,B. C. (I g65), ‘ Oscillatory behavior in enzyme control processes ‘, in Advances in Enzyme Z2egulation (ed. WEBER,G.), vol. 3, pp. 425-438. HOHORST,H. J. (Ig65), ’ L( -)Malate determination with malic dehydrogenase and DPN ‘, in
of Enzymatic Analysis (ed. BERGMEYER, H. V.), pp. 328-332. New York: Academic Press. POTTER,V. R., GILBERT,R. A., and PITOT,H. C. ( 1g66), ‘ Enzyme levels in rats adapted to 36hour fasting ‘, in Advances in Enzyme Regulation (ed. WEBER,G.), vol. 4, pp. 247-265. Oxford: Pergamon. ROTHCHILD. I. ( 16~81. ‘ A simDlified techniaue for hypophy&ctdm;’ of the domestic fowl ‘,’ EndoMetho&
~.nology, 4% 293-297.
R., and BENSADOUN, A. (Ig71), ‘ Thyroid iodohormones in the plasma of the rooster (Gallus domesticus) ‘, Gen. camp. Endocr., in the press. SINGH, A., REINKE, E. P., and RINGER, R. E. (Ig67), ’ Thyroxine and tri-iodothyronine turnover in the chicken, the Bobwhite and the Japanese quail ‘, Gen. camp. Endocr., g, 353-381. STEEL, R. G. D., and TORRIE, J. H. ( Ig6o), Principles and Procedures of Statistics. New York: McGraw-Hill. SADOVSKY,
Key Word Index: Diurnal rhythm, NADP-linked malic enzyme, lipid metabolism.
197 I,
DIURNAL
2,
581-584.
[Scientechnica
RHYTHMS CHICKEN,
K. A. CHANDRABOSE,
(Publishers) Ltd.]
581
OF SLIVER ENZYMES IN THE GALLUS ~~~EST~C~S
A. BENSADOUN,
AND C. K. CLIFFORD
Department of Poultry Science, Cornell University, Ithaca, New York, U.S.A.
ABSTRACT I. NADP-linked ma&c enzyme (ME) and glycerokinase (GR) show diurnal rhythms in the liver of cockerels. 2. Although the activities of both enzymes are markedly influenced by hypophysectomy and hormone replacement therapy, hypophysectomyabolishes the periodicity of ME, but not that of GK. 3. The rhy~mic~ty of ME dots not see.mto be related to malate concentration in the l&r or to the pattern of food intake. THE detection of quantitatively significant diurnal rhythms in the activities of enzymes has been reported in liver and other tissues (Goodwin, 1965 ; Potter, Gilbert, and Pitot, I 966). Several investigators have attempted to correlate diurnal variations in enzyme activities with corresponding rhythmicities in hormones known to be inducers of these enzymes (Civen, Ulrich, Trimmer, and Brown, 1967). We wish to report the results of studies on the variation of the activities of various liver and adipose tissue enzymes in intact and hypophysectomized roosters.
MATERIALS AND METHODS White Leghorn cockereb raised on a commercial diet were employed in this experiment. Hype_ physeetomy was performed when the birds were IQ-I I weeks old, by a transluccal approach (Rothchild, 1948). They were kept for 3 months after surgery before the experiment was performed. The animals were housed in individual cages in an isolated room with controlled temperature (2I' C.) andillumination (6.00 a.mA.oon.m.). Three. to 5 pairs of control and hypophysect&nized birds we& sacrificed at intervals of 4 hours. The followinn liver enzymes were analykd according to pro& dures given in a previous publication (Chandrabose and Bensadoun, I 97 I a) : citrate-cleavage enzyme
(E.G. 4. I. 3.8), NADP-linked ma& enzyme (E.C. I. I. I. 40), glucose-&phosphate dehydro-
genase (E.C. I. I. I. 4g), glycerokinase (EC. 2. 7. I. 30)~ and lipoprotein Iipase (glycerol ester hydrolase, E.C. 3. I. x. 3). Liver samples, quick-frozen in liquid nitrogen, were analysed for L-malate by the enzymatic procedure of Hohorst ( x963),
Statistical differences in enzyme activities at various times of the day were ascertained by Duncan’s multiple range test (Steel and Torrie, 1960). RESULTS
AND DISCUSSION
The results of enzymatic analyses are summarized in Table I and plotted in Fig. I. Only 2 enzymes, NADP-linked malic enzyme (ME) and glycerokinase (GK) , showed significant rhythmicity. The activity of ME was approximately threefold higher at I zoo noon than at any other time of the day (P
CHANDRABOSE
ET AL.
ht.
J.
Bimhem.
The possibility was also considered that the ME rhythmicity in control birds reflected a rhythm in the hepatic concentrations of Lmalate. There were no significant differences in L-malate concentrations at various times of the day (Table III). The above experiments strongly suggest that an intact anterior pituitary is necessary for the ME daily rhythm. It has been shown previously that ph~iologi~al levels of thyroxine (Chandrabose and Bensadoun, rg7r b) will induce ME when injected in hypophysectomized roosters. It is conceivabIe that thyroxine might act as a major physiological inducer of malic enzyme, Iodohormone plasma levels, measured at various times of the day in another group of roosters, showed a significant diurnal rhythmicity, with a peak iodohormone activity at 4.00 p.m. (Sadovsky and Bensadoun, I g7 I). It is premature to attempt to relate the peak ME activity with the peak iodohormone levels until both rne~u~en~ have been made in the same animals. The short half-life of 3.23 hours, reported by Singh, Reinke, and Ringer (x967) for thyroxine and triiodothyroxine, is consistent with the existence of a diurnal rhythmicity for these hormones. Glycerokinase (GK) showed diurnal rhythmicity in both control and hypophysectomized birds. GK activity at 12.00 noon was twofold higher than at any other time of the day in both control and hypophysectomized birds (P
‘971,
2
DIURNAL
RHYTHMS
abolishes the periodicity of ME, it does not affectthat ofGi(. Theseiwoexamples illustrate that although an enzyme is inducible or repressible in a short time by exogenous hormone injections, these hormones are not
OF LIVER
583
ENZYMES
necessarily the major physiological factors responsible for its diurnal rhythms. The other enzyme studied, citrate-cleavage enzyme, gluco~-~phosphate dehydrogenase, and lipoprotein hpase, did not show any
60 MALIC
GLYCEROL
ENZYME
KINASE
50 .e E . .s
40
“c ii g30 . -: i! 520 3 E
I 800
2000 I600 TIME OF DAY
,
I
5
1200
IO
2400
400
800
1200
1600 TIME
2000 OF DAY
1
I
1200
1600 TIME
2000 OF DAY
2400
2400
4
.G
$ .c 0)
15
6 CITRATE
CLEAVAGE
ENZYME
P
2000 TIME OF DAY
FIG.I.-Diurnal
2400
400
800
TIME OF DAY
400
rhythms of liver malic enzyme, glycerokinase, citrate-cleavage enzyme, glucose-6., phosphate dehydmgenase, and adipose tissue lipoprotein lipase, in control and hypophysectomized cockerels. (800, 1200, 1600, 2000, 2400, and 400 standfor8.00a.m.,12.00 noon, 4.00 p.m., 8.00 p.m., I 2.00 midnight, and 4.00 a.m. respectively.)
CHANDRABOSE
584
ET
AL.
Table II.-VOLUNTARY FOOD CONSUMPIION OF CONTROLAND HYPOPHYSECTOMIZEDCOCKERELS (g. per kg. body-weight)+”
TIME BIRDS 8.&ym.
Control Hypophysectomized
1.5fo.8 I. I *a*
I
8.oo12.00 a.m.
IZoo4.00 p.m.
4.oo8.00 p.m.
8.oo12.00 p.m.
IZ.oo4.00 a.m.
2.6ko.6
19.1 k2.2
0.6fo.5
o-4*0.2
0.7 io.2
o-7 io.5
16.3f1.8
0.4Co.2
0.8fo.3
0.7io.3
I
I
I
Mean body-weight of 4 control cockerels=2061 g. Mean body-weight of 4 hypophysectomized cockerels=n~zo g. The values represent means fS.E.M. of 4 observations. Table III-L-MALATE CONCENTRATIONS (umoles L-malate per g. protein) IN THE LIVERY OF CONTROL AND HYPOPHYSECTOWZED COCKERELSAT VARIOUS TIMES OF THE DAY
TIMEOF DAY TREATMENT 8.00 a.m.
12.00 noon
4.00 p.m.
12.00 midnight
8.00 p.m.
4.00 a.m.
Control Hypophysectomized Number of -chickens
1
6
1
6
1
8
1
8
1
6
1
8
-
* Standard error of the mean. diurnal
variations when cockerels were fed under the conditions described in the Methods section. ad libitum
REFERENCES CHANDRABOSE, K. A., and BENSADOUN, A. (I g7 Ia), ‘ Effects of hypophysectomy on some enzymes involved in lipid metabolism of the domestic chicken (Callus domesticus) ‘, Comp. Biochem. Physiol., 39, 45-54.
CHANDRABOSE, K. A., and BENSADOUN, A. ( Ig7 Ib) , ‘ Effect of hypophysectomy on liver enzymes in the chicken, Callus domesticus. Replacement therapy with growth hormone and thyroxine ‘, Corn@Biochem. Physiol., 39, 55-59. CNEN, M., ULRICH, R., TRIMMER,B. M., and BROWN, C. B. (Ig67), ‘ Circadian rhythms of liver enzymes and their relationship to enzyme induction ‘, S&rue, N. Y., 157, I 563-1564. GOODWIN,B. C. (I g65), ‘ Oscillatory behavior in enzyme control processes ‘, in Advances in Enzyme Z2egulation (ed. WEBER,G.), vol. 3, pp. 425-438. HOHORST,H. J. (Ig65), ’ L( -)Malate determination with malic dehydrogenase and DPN ‘, in
of Enzymatic Analysis (ed. BERGMEYER, H. V.), pp. 328-332. New York: Academic Press. POTTER,V. R., GILBERT,R. A., and PITOT,H. C. ( 1g66), ‘ Enzyme levels in rats adapted to 36hour fasting ‘, in Advances in Enzyme Regulation (ed. WEBER,G.), vol. 4, pp. 247-265. Oxford: Pergamon. ROTHCHILD. I. ( 16~81. ‘ A simDlified techniaue for hypophy&ctdm;’ of the domestic fowl ‘,’ EndoMetho&
~.nology, 4% 293-297.
R., and BENSADOUN, A. (Ig71), ‘ Thyroid iodohormones in the plasma of the rooster (Gallus domesticus) ‘, Gen. camp. Endocr., in the press. SINGH, A., REINKE, E. P., and RINGER, R. E. (Ig67), ’ Thyroxine and tri-iodothyronine turnover in the chicken, the Bobwhite and the Japanese quail ‘, Gen. camp. Endocr., g, 353-381. STEEL, R. G. D., and TORRIE, J. H. ( Ig6o), Principles and Procedures of Statistics. New York: McGraw-Hill. SADOVSKY,
Key Word Index: Diurnal rhythm, NADP-linked malic enzyme, lipid metabolism.