Glycogen Metabolism in Muscle: Its Circadian and Seasonal Variations Maria
‘*
Teresa Pessacq and Juan Josi Gagliardino rhythm. When the animals were submitted to a 29-hr fasting, the glycogen-24 hr-variation was still present, with similar
The glycogen content of mice diaphragm and “C-glucose incorporation into glycogen was studied around the clock at 4-hr intervals. It was found that glycogen content presents a circadian rhythm with a peak value around 4 hr; this rhythm was reproduced in four different months, but with changes both in the absolute values and in the range of oscillation around the 24-hr mean value. Although labeled glucose incorporation exhibited a circadian variation as well, its peak value was 180” out of phase compared with the glycogen
oscillation around the mean. However, the absolute glycogen values were one-third as high as the ones obtained in the control animals. Our results suggest that the rhythm of glycogen content is due, at least partly, to the circadian variation of its synthesis. Furthermore, food intake is not the main factor responsible for the above mentioned rhythm.
S
EVERAL PAPERS IN THE literature are concerned with the circadian rhythm of glycogen content in liver. An extensive survey on this field and a careful analysis of the data were performed some years ago by Sollberger.’ Conversely, not many publications deal with the behavior of glycogen in muscle.2*3 We have recently demonstrated that, in normal mice diaphragms, this parameter exhibits a clear circadian variation.4 But neither our experjments nor the available data studied the reproducibility and the constancy of this glycogen rhythm in muscle throughout the year. The underlying mechanism of this rhythm is also unknown. Therefore, we considered it interesting to study the glycogen content in mice diaphragm at regular intervals during the day in different seasons, as well as the effect of fasting on the glycogen circadian rhythm. The “C-glucose incorporation into glycogen around the clock was also studied in the present experimental design. The results showed that the 24-hr variation of glycogen content in muscle exhibits a definite seasonal influence. Fasting was unable to suppress the above mentioned rhythm. On the other hand, the labeled glucose incorporation into glycogen described a clear-cut circadian variation. MATERIAL AND
METHODS
Female mice of the C3H-S strain, aged 6 wk, from the Instituto de Biologia, Embriologia e Histologia de la Facultad de Ciencias Mtdicas, were used. They were housed eight per cage in a room at a temperature of 25’C with water and food ad libitum and illumination (fluorescent light 40W) from 6 (6 a.m.) to I8 (6 p.m.) hr. alternating with 12 hr darkness. The animals used were maintained under the specified conditions for at least 3 wk before being sacrificed. These
From the Cdtedra
de Fisiologia
con Biofisica.
Institute
de Fisiologia.
Facultad
M&dicas. Universidad Nacionai de La Plata. Caiie 60 y 120. La Piata. Argentina. Receivedfor publication December 18. 1974. Supported by grants from the Conicet and Comisi6n de Investigaciones Provincia de Buenos Aires. ~1975 by Grune & Stratton, Inc. Metabolism, Vol. 24, No. 6 (June), 1975
de
Cientificas
Cienria.y
de
la
737
738
PESSACQ
animals engaged in exercise the gastric content.’
and food intake
mainly
during
the darkness
AND GAGLIARDINO
period,
as indicated
by
Determination of Glycogen Content in Muscle Diaphragm For this type of experiment, six lots of eight animals each were killed by cervical dislocation and decapitation at 4, 8, 12, 16, 20, and 24 hr. The average weight in each lot was carefully kept around 20 g. Diaphragm was quickly removed from each animal, washed in cold physiologic saline, blotted between filter paper, and weighed. After this procedure, which took about 2 min, glycogen was extracted from the muscle and determined according to Seiffer’s procedure.6 The glycogen employed as standard in this determination was extracted from mouse livers according to the method of Somogyi.’ This experimental design was reproduced in four different months.
iTtJect of Fasting on Glycogen Content in Diaphragm For this purpose, During this period
food was removed from the cages 29 hr before the animals were the animals remained in the same cage, having free access to
sacrificed. water. A
control group, submitted to the dietary regimen previously described, was sacrificed together with these fasted animals at 4, 8, 12, 16, 20, and 24 hr. This experiment was done in September. The tissues from both groups of animals were simultaneously handled as stated above, with the only exception that glycogen was determined according to the method of Krisman.8 In our hands, this method always gives lower values than the anthrone one.
“C-glucose incorporation Into Muscle Glycogen The procedure described by Clausen’ was employed in this type of experiment. Mice were injected with “C-glucose (2uCi/mouse, intraperitoneally) 1 hr before sacrifice. Groups of eight animals each were submitted to this treatment and were sacrificed at 4, 8, 12, 16, 20, and 24 hr. The isolated diaphragms obtained were treated for glycogen determination as previously described. A suitable aliquot of the glycogen solution was employed to determine the 14Cglucose incorporation into glycogen. Sufficient counts were recorded in order to avoid errors higher than 5%. This experiment was performed in March. The paired
t test was employed
for the statistical
analysis
of results.
RESULTS
Table 1 shows the glycogen content of muscle diaphragm expressed in micrograms in four different months in two consecutive years. It can be observed that the mean value (average from the six time points) corresponding to December and October fall within the same range. The same parallelism can be drawn between June and August values. The latter are about three times as great as the Table 1. Glycogen Content (pg) and Concentration (pg/mg
wet weight)
of Normal Mice Diaphragm During the Day at Different Months of the Year”
Time 04
141 * 21.0
1.7 z+z0.25
191 f 44.5
2.6 l 0.65
54 f 15.8
1.0 f 0.29
50 zt 6.1
08
164 zt 24.2
1.8 f 0.21
139 zt 24.4
1.7 f 0.28
55zt16.0"
l.0i0.24
90 zt 11.2"
1.5 11~0.16
12
169 + 10.3"
1.9 zk 0.13
172 + 25.2"
2.1 I 0.33
34 zk 4.0
0.6 f 0.08
45 +
0.9 I 0.08
1.2 ho.23
4.0
0.8 zt 0.09
110 f 10.2
1.4 * 0.15
10 f
0.4'
0.2 f 0.06
14 +
1.6' 0.2 zt 0.02
20
147 + 28.7
1.6 * 0.26
96 * 12.1
1.4 z!=0.18
25 f
2.8
0.4 f 0.03
27 +
3.8
24
135 zt 16.4
1.6 f 0.21
95 zt 9.2'
1.3 + 0.14
48 + 10.8
0.9 f 0.20
63 r+ 8.6
16
Number
81 + 12.9'
(4)
(9)
(10)
‘*
Teresa Pessacq and Juan Josi Gagliardino rhythm. When the animals were submitted to a 29-hr fasting, the glycogen-24 hr-variation was still present, with similar
The glycogen content of mice diaphragm and “C-glucose incorporation into glycogen was studied around the clock at 4-hr intervals. It was found that glycogen content presents a circadian rhythm with a peak value around 4 hr; this rhythm was reproduced in four different months, but with changes both in the absolute values and in the range of oscillation around the 24-hr mean value. Although labeled glucose incorporation exhibited a circadian variation as well, its peak value was 180” out of phase compared with the glycogen
oscillation around the mean. However, the absolute glycogen values were one-third as high as the ones obtained in the control animals. Our results suggest that the rhythm of glycogen content is due, at least partly, to the circadian variation of its synthesis. Furthermore, food intake is not the main factor responsible for the above mentioned rhythm.
S
EVERAL PAPERS IN THE literature are concerned with the circadian rhythm of glycogen content in liver. An extensive survey on this field and a careful analysis of the data were performed some years ago by Sollberger.’ Conversely, not many publications deal with the behavior of glycogen in muscle.2*3 We have recently demonstrated that, in normal mice diaphragms, this parameter exhibits a clear circadian variation.4 But neither our experjments nor the available data studied the reproducibility and the constancy of this glycogen rhythm in muscle throughout the year. The underlying mechanism of this rhythm is also unknown. Therefore, we considered it interesting to study the glycogen content in mice diaphragm at regular intervals during the day in different seasons, as well as the effect of fasting on the glycogen circadian rhythm. The “C-glucose incorporation into glycogen around the clock was also studied in the present experimental design. The results showed that the 24-hr variation of glycogen content in muscle exhibits a definite seasonal influence. Fasting was unable to suppress the above mentioned rhythm. On the other hand, the labeled glucose incorporation into glycogen described a clear-cut circadian variation. MATERIAL AND
METHODS
Female mice of the C3H-S strain, aged 6 wk, from the Instituto de Biologia, Embriologia e Histologia de la Facultad de Ciencias Mtdicas, were used. They were housed eight per cage in a room at a temperature of 25’C with water and food ad libitum and illumination (fluorescent light 40W) from 6 (6 a.m.) to I8 (6 p.m.) hr. alternating with 12 hr darkness. The animals used were maintained under the specified conditions for at least 3 wk before being sacrificed. These
From the Cdtedra
de Fisiologia
con Biofisica.
Institute
de Fisiologia.
Facultad
M&dicas. Universidad Nacionai de La Plata. Caiie 60 y 120. La Piata. Argentina. Receivedfor publication December 18. 1974. Supported by grants from the Conicet and Comisi6n de Investigaciones Provincia de Buenos Aires. ~1975 by Grune & Stratton, Inc. Metabolism, Vol. 24, No. 6 (June), 1975
de
Cientificas
Cienria.y
de
la
737
738
PESSACQ
animals engaged in exercise the gastric content.’
and food intake
mainly
during
the darkness
AND GAGLIARDINO
period,
as indicated
by
Determination of Glycogen Content in Muscle Diaphragm For this type of experiment, six lots of eight animals each were killed by cervical dislocation and decapitation at 4, 8, 12, 16, 20, and 24 hr. The average weight in each lot was carefully kept around 20 g. Diaphragm was quickly removed from each animal, washed in cold physiologic saline, blotted between filter paper, and weighed. After this procedure, which took about 2 min, glycogen was extracted from the muscle and determined according to Seiffer’s procedure.6 The glycogen employed as standard in this determination was extracted from mouse livers according to the method of Somogyi.’ This experimental design was reproduced in four different months.
iTtJect of Fasting on Glycogen Content in Diaphragm For this purpose, During this period
food was removed from the cages 29 hr before the animals were the animals remained in the same cage, having free access to
sacrificed. water. A
control group, submitted to the dietary regimen previously described, was sacrificed together with these fasted animals at 4, 8, 12, 16, 20, and 24 hr. This experiment was done in September. The tissues from both groups of animals were simultaneously handled as stated above, with the only exception that glycogen was determined according to the method of Krisman.8 In our hands, this method always gives lower values than the anthrone one.
“C-glucose incorporation Into Muscle Glycogen The procedure described by Clausen’ was employed in this type of experiment. Mice were injected with “C-glucose (2uCi/mouse, intraperitoneally) 1 hr before sacrifice. Groups of eight animals each were submitted to this treatment and were sacrificed at 4, 8, 12, 16, 20, and 24 hr. The isolated diaphragms obtained were treated for glycogen determination as previously described. A suitable aliquot of the glycogen solution was employed to determine the 14Cglucose incorporation into glycogen. Sufficient counts were recorded in order to avoid errors higher than 5%. This experiment was performed in March. The paired
t test was employed
for the statistical
analysis
of results.
RESULTS
Table 1 shows the glycogen content of muscle diaphragm expressed in micrograms in four different months in two consecutive years. It can be observed that the mean value (average from the six time points) corresponding to December and October fall within the same range. The same parallelism can be drawn between June and August values. The latter are about three times as great as the Table 1. Glycogen Content (pg) and Concentration (pg/mg
wet weight)
of Normal Mice Diaphragm During the Day at Different Months of the Year”
Time 04
141 * 21.0
1.7 z+z0.25
191 f 44.5
2.6 l 0.65
54 f 15.8
1.0 f 0.29
50 zt 6.1
08
164 zt 24.2
1.8 f 0.21
139 zt 24.4
1.7 f 0.28
55zt16.0"
l.0i0.24
90 zt 11.2"
1.5 11~0.16
12
169 + 10.3"
1.9 zk 0.13
172 + 25.2"
2.1 I 0.33
34 zk 4.0
0.6 f 0.08
45 +
0.9 I 0.08
1.2 ho.23
4.0
0.8 zt 0.09
110 f 10.2
1.4 * 0.15
10 f
0.4'
0.2 f 0.06
14 +
1.6' 0.2 zt 0.02
20
147 + 28.7
1.6 * 0.26
96 * 12.1
1.4 z!=0.18
25 f
2.8
0.4 f 0.03
27 +
3.8
24
135 zt 16.4
1.6 f 0.21
95 zt 9.2'
1.3 + 0.14
48 + 10.8
0.9 f 0.20
63 r+ 8.6
16
Number
81 + 12.9'
(4)
(9)
(10)
