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Diurnal variation of liver glycogen and plasma free fatty acids in rats fed ad libitum or single daily meal
Diurnal Variation of Liver Glycogen and Plasma Free Fatty Acids in Rats Fed ad Libitum or Single Daily Meal By RAY
W. FULLER
ANDEROLD R. DILLER
In rats allowed to eat ad libitum, liver glycogen and plasma free fatty acids varied rhythmically during a twenty-four hour period. There was an inverse relationship between their levels, i.e., at 8 p.m., glycogen was lowest and free fatty acids were highest, whereas at 5-8 a.m., the converse was true. Feeding a single daily meal (from 8 a.m. to noon) caused a shift in phase of about twelve hours in
both rhythms, with the inverse relationship between glycogen and free falty acid levels again apparent. The maguitude of both rhythms was accentuated by mealfeeding SO that the average levels of both glycogen and free fatty acids over the twenty-four hour period were slightly higher than in rats fed ad libitum. (Metabolism 19: No. 3, March, 226229, 1970)
OF MANY BIOLOGICAL CONSTITUENTS (metabolites and enzymes alike) do not remain constant but vary in a rhythmic manner during the 24-hour day. In some cases, the variations are due to the rhythmic ingestion of food. Among the metabolites that vary rhythmically are liver glycogenl and plasma free fatty acids.’ The effect of feeding rats a single daily meal instead of allowing them free access to food has been examined in respect to many biological parameters, including liver glycogen” and plasma free fatty acids.4 However, data on the diurnal rhythms in meal-fed animals compared to animals allowed free access to food are more limited. We report here the diurnal rhythmic changes of liver glycogen and of plasma free fatty acids in rats fed ad libitum or fed only a single daily meal.
T
HE AMOUNTS
MATERIALSAND METHODS Male albino rats from the Wistar strain, obtained from a local supplier, were used. The rats weighed approximately 150 g each. All rats were given water ad libitum and were fed Purina Lab Chow. One group of rats was fed ad libitum. The other group was given food only from 8 a.m. until noon for the two weeks preceding the experiment. The rats were killed by decapitation. Liver glycogen was isolated and hydrolyzed,5 then determined as glucose by a calorimetric method with glucose oxidase. Plasma free fatty acids were determined calorimetrically by a procedures modified for an auto-analyzer. There were five rats per group. Mean values, with standard errors of the means, are reported. RESULTS
In Fig. 1, the liver glycogen content and the plasma free fatty acid levels in From the Department of Metabolic Research, Lilly Research Laboratories, Indianapolis, lnd. Received for publication August 15. 1969. RAY W. FULLER, PH.D.: Head, Department of Metabolic Research, The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Ind. EROLD R. DILLER, MS.: Research Scientist, Department of Metabolic Research, The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Ind. 226
METABOLISM, VOL. 19, No. 3 (MARCH),
1970
LIVER
GLYCOGEN
AND PLASMA
227
FFA
rats fed ad libitum are shown. Throughout the light period and into the early part of the dark period, glycogen stores declined to a minimum at 8 p.m. Plasma free fatty acid levels rose steadily during that time to reach a maximum at 8 p.m. As glycogen stores then increased (8 p.m. to 5 a.m.), plasma free fatty acid levels fell. In terms of his overall metabolic economy, i.e. the substrates serving as sources of energy, the rat at 8 a.m. would seem to be substantially different from the rat at 8 p.m. The data in this figure are in general agreement with those published by others.l,” Figure 2 shows the diurnal rhythms of liver glycogen and plasma free fatty acids in rats fed a single daily meal. The phase of the glycogen rhythm was shifted by 12 hours in meal-fed rats compared to rats fed ad libitum. The lowest
l.-Liver Fig. glycogen and plasma free fatty acids in rats fed ad libitum.
‘8 I am
11 I
2I pm
5I
8I
11 I
2I
5I
8I ,
am
Fig. 2.-L i v e r glycogen and plasma free fatty acids in rats fed single daily meal (8 a.m. to noon).
am
Pm
am
228
FULLER AND DILLER
glycogen level in the meal-fed rats was at 8 a.m. By 11 a.m., glycogen stores had increased slightly, and by 2 p.m., they had risen to the plateau level that was maintained for the next twelve hours. That level was somewhat higher than in rats fed ad libitum. Meal-feeding also resulted in a shift in the plasma free fatty acid rhythm. In meal-fed rats, highest free fatty acid levels again coincided with lowest glycogen levels and occurred at 8 a.m., 12 hours apart from the peak in rats fed ad libitum. DISCUSSION
The results of feeding a single meal on free fatty acid levels in rats are similar to those reported by Bortz et al. for humans.’ In rats fed a single meal, plasmafree fatty acid levels were higher (.35 to .72 mEq./L.) and reached a sharper peak than in rats fed ad libitum (where the range was .22 to .44 mEq./L. ) . Although there was a tendency toward higher overall glycogen and free fatty acid levels in rats fed the single daily meal, results of comparison of the actual level of each constituent in meal-fed rats and rats fed ad libitum depended entirely upon the time of day. For instance, if one compared glycogen levels in meal-fed rats without regard for the daily rhythm, he could conclude that mealfeeding elevated glycogen (if he measured levels only at 8 p.m.), reduced glycogen (if he measured only at 8 a.m.) or did not significantly affect glycogen (if he measured at a time when the lines crossed, at 2-5 a.m. or about noon). It was probably not meal-feeding per se that caused shifts in the glycogen and free fatty acid rhythms; instead the shifts occurred because the meal was fed during the day rather than at night. Although the laboratory rat with food available continuously consumes food in a more diffuse pattern than rats given a single meal, most data show that the great proportion of the food is consumed during the dark period.* Thus, what some workers call ( 1) “nibblers” (rats fed ad libitum) and (2) “meal-fed’ rats could probably also be considered (1) “rats whose food intake is predominantly at night” and (2) “rats whose food intake is entirely during a short period of the day,” respectively. Based on the results in Figs. 1 and 2, it seems that a comparison of metabolite levels at only one time of day in rats fed a single meal to those in rats fed ad libitum is meaningless if that metabolite varies in a daily rhythm. What controls the rhythms in glycogen and free fatty acids and how are those rhythms related? Both rhythms appear to be linked to food intake, although the connection between food intake and the rhythms cannot be clearly stated. Possibly some specific metabolites arising from ingested food have regulatory roles. Hormonal control may be inportant. Hormones known to affect glycogen and free fatty acid metabolism and whose secretion may be controlled partly by food intake, e.g. growth hormone,s insulin,l” and glucagon,ll may vary in a diurnal rhythm. Finally, evidence exists that the autonomic nervous system may exert control over both glycogenl” and plasma free fatty acid levels,l”-l5 and a neural component in the daily rhythms must be considered. REFERENCES 1. Sollberger, A.: The control of circadian glycogen rhythms. Ann. N.Y. Acad. Sci.
117:519, 1964. 2. Barrett, A. M.:
Adventitious
factors
LIVER GLYCOGEN AND PLASMA FFA
affecting the concentration of free fatty acids in the plasma of rata. Brit. J. Pharmacol. 22:577, 1964. 3. Hollifield, G., and Parson, W.: Metabolic adaptations to a “stuff and starve” feeding program. I. Studies of adipose tissue and liver glycogen in rats limited to a short daily feeding period. J. Clin. Invest. 41:245, 1962. 4. Leveille, G. A., and Chakrabarty, K.: Diurnal variations in tissue glycogen and liver weight of meal-fed rats. J. Nutr. 93: 546, 1967. 5. Good, C. A., Kramer, H., and Somogyi, M.: The determination of glycogen, J. Biol. Chem. 100:485, 1933. 6. Laurell, S., and Tibbling, G.: Colorimetric micro-determination of free fatty acids in plasma. Clin. Chim. Acta 16:57, 1967. 7. Bortz, W. M., Howat, P., and Holmes, W. L.: The effect of feeding frequency on diurnal plasma free fatty acids and glucose levels. Metabolism 18: 120, 1969. 8. Suttie, J. W.: Effect of dietary fluoride on the pattern of food intake in the rat and the development of a programmed pellet dispenser. J. Nutr. 96:529, 1968. 9. Morris, H. G., and Jorgensen, J. R.: Circadian pattern of plasma GH concentra-
229 tion in children. Clin. Res. 16:148, 1968. 10. Freinkel, N., Mager, M., and Vinnick, L.: Cyclicity in the interrelationships between plasma insulin and glucose during starvation in normal young men. J. Lab. Clin. Med. 71:171, 1968. 11. Hellman, B., and Hellerstrom, C.: Diurnal changes in the function of the pancreatic islets of rats as indicated by nuclear size in the islet cells. Acta Endocrin. 31: 267, 1959. 12. Shimazu, T., and Amakawa, A.: Regulation of glycogen metabolism in liver by the autonomic nervous system. II. Neural control of glycogenolytic enzymes. Biochim. Biophys. Acta 165:335, 1968. 13. Havel, R. I., and Goldfien, A.: The role of the sympathetic nervous system in the metabolism of free fatty acids. J. Lipid Res. 1:102, 1959. 14. Rosell, S.: Release of free fatty acids from subcutaneous adipose tissue in dogs following sympathetic nerve stimulation. Acta Physiol. Stand. 67:343, 1966. 15. Conway, M. J., Goodner, C. J., and Gale, C. C.: The effect of glucose on CNS control of lipolysis in the baboon. Clin. Res. 16:148, 1968.
W. FULLER
ANDEROLD R. DILLER
In rats allowed to eat ad libitum, liver glycogen and plasma free fatty acids varied rhythmically during a twenty-four hour period. There was an inverse relationship between their levels, i.e., at 8 p.m., glycogen was lowest and free fatty acids were highest, whereas at 5-8 a.m., the converse was true. Feeding a single daily meal (from 8 a.m. to noon) caused a shift in phase of about twelve hours in
both rhythms, with the inverse relationship between glycogen and free falty acid levels again apparent. The maguitude of both rhythms was accentuated by mealfeeding SO that the average levels of both glycogen and free fatty acids over the twenty-four hour period were slightly higher than in rats fed ad libitum. (Metabolism 19: No. 3, March, 226229, 1970)
OF MANY BIOLOGICAL CONSTITUENTS (metabolites and enzymes alike) do not remain constant but vary in a rhythmic manner during the 24-hour day. In some cases, the variations are due to the rhythmic ingestion of food. Among the metabolites that vary rhythmically are liver glycogenl and plasma free fatty acids.’ The effect of feeding rats a single daily meal instead of allowing them free access to food has been examined in respect to many biological parameters, including liver glycogen” and plasma free fatty acids.4 However, data on the diurnal rhythms in meal-fed animals compared to animals allowed free access to food are more limited. We report here the diurnal rhythmic changes of liver glycogen and of plasma free fatty acids in rats fed ad libitum or fed only a single daily meal.
T
HE AMOUNTS
MATERIALSAND METHODS Male albino rats from the Wistar strain, obtained from a local supplier, were used. The rats weighed approximately 150 g each. All rats were given water ad libitum and were fed Purina Lab Chow. One group of rats was fed ad libitum. The other group was given food only from 8 a.m. until noon for the two weeks preceding the experiment. The rats were killed by decapitation. Liver glycogen was isolated and hydrolyzed,5 then determined as glucose by a calorimetric method with glucose oxidase. Plasma free fatty acids were determined calorimetrically by a procedures modified for an auto-analyzer. There were five rats per group. Mean values, with standard errors of the means, are reported. RESULTS
In Fig. 1, the liver glycogen content and the plasma free fatty acid levels in From the Department of Metabolic Research, Lilly Research Laboratories, Indianapolis, lnd. Received for publication August 15. 1969. RAY W. FULLER, PH.D.: Head, Department of Metabolic Research, The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Ind. EROLD R. DILLER, MS.: Research Scientist, Department of Metabolic Research, The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Ind. 226
METABOLISM, VOL. 19, No. 3 (MARCH),
1970
LIVER
GLYCOGEN
AND PLASMA
227
FFA
rats fed ad libitum are shown. Throughout the light period and into the early part of the dark period, glycogen stores declined to a minimum at 8 p.m. Plasma free fatty acid levels rose steadily during that time to reach a maximum at 8 p.m. As glycogen stores then increased (8 p.m. to 5 a.m.), plasma free fatty acid levels fell. In terms of his overall metabolic economy, i.e. the substrates serving as sources of energy, the rat at 8 a.m. would seem to be substantially different from the rat at 8 p.m. The data in this figure are in general agreement with those published by others.l,” Figure 2 shows the diurnal rhythms of liver glycogen and plasma free fatty acids in rats fed a single daily meal. The phase of the glycogen rhythm was shifted by 12 hours in meal-fed rats compared to rats fed ad libitum. The lowest
l.-Liver Fig. glycogen and plasma free fatty acids in rats fed ad libitum.
‘8 I am
11 I
2I pm
5I
8I
11 I
2I
5I
8I ,
am
Fig. 2.-L i v e r glycogen and plasma free fatty acids in rats fed single daily meal (8 a.m. to noon).
am
Pm
am
228
FULLER AND DILLER
glycogen level in the meal-fed rats was at 8 a.m. By 11 a.m., glycogen stores had increased slightly, and by 2 p.m., they had risen to the plateau level that was maintained for the next twelve hours. That level was somewhat higher than in rats fed ad libitum. Meal-feeding also resulted in a shift in the plasma free fatty acid rhythm. In meal-fed rats, highest free fatty acid levels again coincided with lowest glycogen levels and occurred at 8 a.m., 12 hours apart from the peak in rats fed ad libitum. DISCUSSION
The results of feeding a single meal on free fatty acid levels in rats are similar to those reported by Bortz et al. for humans.’ In rats fed a single meal, plasmafree fatty acid levels were higher (.35 to .72 mEq./L.) and reached a sharper peak than in rats fed ad libitum (where the range was .22 to .44 mEq./L. ) . Although there was a tendency toward higher overall glycogen and free fatty acid levels in rats fed the single daily meal, results of comparison of the actual level of each constituent in meal-fed rats and rats fed ad libitum depended entirely upon the time of day. For instance, if one compared glycogen levels in meal-fed rats without regard for the daily rhythm, he could conclude that mealfeeding elevated glycogen (if he measured levels only at 8 p.m.), reduced glycogen (if he measured only at 8 a.m.) or did not significantly affect glycogen (if he measured at a time when the lines crossed, at 2-5 a.m. or about noon). It was probably not meal-feeding per se that caused shifts in the glycogen and free fatty acid rhythms; instead the shifts occurred because the meal was fed during the day rather than at night. Although the laboratory rat with food available continuously consumes food in a more diffuse pattern than rats given a single meal, most data show that the great proportion of the food is consumed during the dark period.* Thus, what some workers call ( 1) “nibblers” (rats fed ad libitum) and (2) “meal-fed’ rats could probably also be considered (1) “rats whose food intake is predominantly at night” and (2) “rats whose food intake is entirely during a short period of the day,” respectively. Based on the results in Figs. 1 and 2, it seems that a comparison of metabolite levels at only one time of day in rats fed a single meal to those in rats fed ad libitum is meaningless if that metabolite varies in a daily rhythm. What controls the rhythms in glycogen and free fatty acids and how are those rhythms related? Both rhythms appear to be linked to food intake, although the connection between food intake and the rhythms cannot be clearly stated. Possibly some specific metabolites arising from ingested food have regulatory roles. Hormonal control may be inportant. Hormones known to affect glycogen and free fatty acid metabolism and whose secretion may be controlled partly by food intake, e.g. growth hormone,s insulin,l” and glucagon,ll may vary in a diurnal rhythm. Finally, evidence exists that the autonomic nervous system may exert control over both glycogenl” and plasma free fatty acid levels,l”-l5 and a neural component in the daily rhythms must be considered. REFERENCES 1. Sollberger, A.: The control of circadian glycogen rhythms. Ann. N.Y. Acad. Sci.
117:519, 1964. 2. Barrett, A. M.:
Adventitious
factors
LIVER GLYCOGEN AND PLASMA FFA
affecting the concentration of free fatty acids in the plasma of rata. Brit. J. Pharmacol. 22:577, 1964. 3. Hollifield, G., and Parson, W.: Metabolic adaptations to a “stuff and starve” feeding program. I. Studies of adipose tissue and liver glycogen in rats limited to a short daily feeding period. J. Clin. Invest. 41:245, 1962. 4. Leveille, G. A., and Chakrabarty, K.: Diurnal variations in tissue glycogen and liver weight of meal-fed rats. J. Nutr. 93: 546, 1967. 5. Good, C. A., Kramer, H., and Somogyi, M.: The determination of glycogen, J. Biol. Chem. 100:485, 1933. 6. Laurell, S., and Tibbling, G.: Colorimetric micro-determination of free fatty acids in plasma. Clin. Chim. Acta 16:57, 1967. 7. Bortz, W. M., Howat, P., and Holmes, W. L.: The effect of feeding frequency on diurnal plasma free fatty acids and glucose levels. Metabolism 18: 120, 1969. 8. Suttie, J. W.: Effect of dietary fluoride on the pattern of food intake in the rat and the development of a programmed pellet dispenser. J. Nutr. 96:529, 1968. 9. Morris, H. G., and Jorgensen, J. R.: Circadian pattern of plasma GH concentra-
229 tion in children. Clin. Res. 16:148, 1968. 10. Freinkel, N., Mager, M., and Vinnick, L.: Cyclicity in the interrelationships between plasma insulin and glucose during starvation in normal young men. J. Lab. Clin. Med. 71:171, 1968. 11. Hellman, B., and Hellerstrom, C.: Diurnal changes in the function of the pancreatic islets of rats as indicated by nuclear size in the islet cells. Acta Endocrin. 31: 267, 1959. 12. Shimazu, T., and Amakawa, A.: Regulation of glycogen metabolism in liver by the autonomic nervous system. II. Neural control of glycogenolytic enzymes. Biochim. Biophys. Acta 165:335, 1968. 13. Havel, R. I., and Goldfien, A.: The role of the sympathetic nervous system in the metabolism of free fatty acids. J. Lipid Res. 1:102, 1959. 14. Rosell, S.: Release of free fatty acids from subcutaneous adipose tissue in dogs following sympathetic nerve stimulation. Acta Physiol. Stand. 67:343, 1966. 15. Conway, M. J., Goodner, C. J., and Gale, C. C.: The effect of glucose on CNS control of lipolysis in the baboon. Clin. Res. 16:148, 1968.