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Carbohydrate metabolism and food intake in food restricted rats: Effects of an unexpected meal
Physiology & Behavior, Vol. 29, pp. 931-937. Pergamon Press, 1982. Printed in the U.S.A.
Carbohydrate Metabolism and Food Intake in Food Restricted Rats: Effects of an Unexpected Meal F. B. L I M A , N. S. H E L L ) C. T I M O - I A R I A M. S. D O L N I K O F F A N D A. A. P U P O
Institute o f Biomedical Sciences, University o f S i o Paulo Department o f Physiology & Pharmacology, 05008 Silo Paulo, SP-Brazil R e c e i v e d 4 J a n u a r y 1982 LIMA, F. B., N. S. HELL, C. TIMO-IARIA, M. S. DOLNIKOFF AND A. A. PUPO. Carbohydratemetabolism andfood intake in food restricted rats: Effects of an unexpected meal. PHYSIOL. BEHAV. 29(5) 931-937, 1982.mRats subjected to a daily single-meal feeding schedule were presented with an unexpected meal at three different times of the day and the effects on the carbohydrate metabolic patterns were determined. The results indicate that it is the actual metabolic moment of the organism, rather than the duration of the inter-meal period and the degree of gastric emptying, that determines the amount of food ingested. Food restriction
Carbohydrate metabolism
Food ingestion
FOOD restriction is known to induce an increase in intestinal absorption [1, 2, 4, 13], liver glycogen content [10,14], fat accumulation [ 11,12], lipogenesis in liver and adipose tissue [25], and a higher resistance to glycogen depletion during insulin hypoglycemia [6]. These changes may make more available energetic stuff for the emission of suddenly necessary, highly energy consuming behaviors [6]. Previous work [15] indicates that food restriction also causes a lesser insulin secretion, higher gastric capacitance and delayed gastric emptying, which suggests that food restricted animals better use circulating nutrients and pour energetic compounds into the blood at a slower rate. In rats fed at night, there appeared an increase in blood sugar concentration during the long inter-meal periods that was more pronounced than in those fed in day time. Liver glycogen decreased at 14 hours after the meal in both feeding groups, but greater decreases were observed in the rats fed during the day. Food ingestion was higher in rats fed at night despite similar inter-meal periods (22 hours) and gastric emptying for both groups. Glycemia and insulinemia at the time of the expected meal were also similar in both groups but hepatic glycogen content by then was significantly lower in the rats fed at night, which ingested an amount of food that was significantly larger than that ingested by the diurnal group. Food restriction is a useful experimental model to assess some factors involved in food intake regulation because conditions associated with feeding, such as the inter-meal period, the degree of gastric emptying, the quantity of food
available to the animals as well as the amount that is ingested, can be easily determined. Moreover, once the pattern of some metabolic parameters has been established other variables can be introduced in the experimental model at certain "metabolic moments" to determine those metabolic factors that regulate food ingestion. In the present study an unexpected meal was presented to rats adapted to a daffy single-meal feeding schedule, either diurnal or nocturnal. By assessing the metabolic changes caused by this surprise meal it was possible to obtain additional information regarding the role of metabolic factors in the regulation of food intake. METHOD
Male Wistar albino rats weighing about 200 g were kept in individual cages. During one week the animals received only one meal a day, either from 8:00 to I0:00 a.m. or from 8:00 to I0:00 p.m. As reported in a previous study [15] the experiments were performed throughout the year, repeating each experimental combination at different times of the year, aiming at assessing the influence of seasonal factors. Actually, so that the rats could be studied in conditions as natural as possible, such distribution of the experiments was adopted on purpose. Despite the different temperatures (between 8°C in winter and 36°C in deep summer), no differences were found to occur in the studied parameters that could be attributed to either variable. The animals were kept under nat-
Whis research was supported by the Sao Paulo State Research Foundation (FAPESP), the National Research Council (CNPq) and FINEP. fro whom requests for reprints should be addressed.
Copyright © 1982 P e r g a m o n Press--0031-9384/82/l10931-07503.00/0
932
I~IMA E7 ~A I .
g/1009 b.w. 6~
g/lOOg b.w. u
43-
I
8-
I
D+Ss
D+$14
N+$14
2~
O FIG. 1. Food intake during the unexpected meal, evaluated in g/100 g of body weight (me.an±standard error of the mean, s.e.m.) The meal was given to group D+S6 (n=56) from 4:00 to 6:00 p.m., to group D+$14 (n=56) from 12:00to 2:00 a.m., and to group N+S~4 (n=56) from 12:00to 2:00 p.m. 0
ural illumination to avoid artifical shifting of the naturally established locking of feeding behavior and metabolic patterns to the time of the day, as reflected by light and dark. Moreover, at the latitude of the laboratory (about 23° south) the drift of the sunset and of the sunrise is relatively narrow (less than two hours). Food restriction was also imposed at different times so that the entire 24 hour length of the day was covered. On the day that each experiment was to be completed (8th day) an unexpected meal was presented to the rats at a certain interval after the last usual meal according to the following schedule: (a) 56 animals trained to eat from 8:00 to I0:00 a.m. were given the surprise meal 6 hours later, i,e. from 4:00 to 6:00 p,m. (Group D+Se); (b) another 56, also routinely fed in day time (8:00 to 10:00 a.m.) had the extra meal 14 hours later, i.e. from 12:00 p.m, to 2:00a.m, (group D+S~4); (c) the 56 rats of the nocturnal group(8:00 to 10:00 p,m.) had the unexpected meal 14 hours later, i.e. from 12:00 to 2:00 p.m, (group N+$14). All the animals were permitted free access to water during the experiment. The day selected for the measurements the amount of food ingested during the routine and the unexpected meal was determined and then the rats were intraperitoneally anesthetized with sodium pentobarbiturate (40 mg/kg body weight,) The abdomen was rapidly opened and blood samples and the stomach plus its content were removed, and a few samples of liver were taken, as previously described [15]. The weight of the stomach and gastric contents were normalized according to body weight. This allowed corrections for differences in the sizes of the animals and was considered as an= index of gastric filling or emptying. The collection of these materials was performed in smaller groups of rats (n=8) every two hours be~nning at the onset of the extra meal and extended for a period of 12 hours. Blood glucose was determined by the ortho-toluidine method [3] and blood insulin by radioimmunoassay [16]. Liver glycogen was assayed by means of ~ e digestion, separation of the carbohydrate in ethanol [13], The extract obtained was hydrolised by heat and the resulting glucose concentration was determined by the ortho-toluidine method. The amount of food ingested was determined as the
t
r 4
I--1----7 8
1 12hr
FIG. 2. Time course of the relative weight of the stomach during 12 hours after the unexpected meal, expressed as g/lll~ g body weight as a function of time in hours after the meal, Groups defined as in Fig. 1. Continuous line: group D+$14. Dashed line: group N+S,4. Dotted line: D+S~. Mean_+s.e.m.
difference between the amount of food offered and that remaining at the conclusion of the two-hour period. The results were expressed in grams/100 grams body weight. The data were statistically processed at 95% of significance. Variation of the amount of food ingested among the three groups during the last routine-meal was analysed by Duncan's test. The same was done for t o m , s o n of f o ~ ingested during the surprise-meal presented to the three groups. For comparison of quantity of food Consumed by an animal at the last habitual meal and during surprise-meal, paired Student's t-test was used. Values of giycemia, h~atic glycogen, insulin and stomach fresh weight from all animals were analysed by non-paired Student's t-test. RESULTS Food Intake
During the last routine meal the amount of food ingested by the rats was 5.3_+0.2, 5.7_+0.2 and 7.4_+0.2 g/100 g body weight for groups D+S6, D÷Sz4 and N+$14, respectively. During the unexpected meal the animals ate 3.7__.0.2, 5.2-+0.1 and 4.5_+0.1 g/100 g body weight, respectively, as shown in Fig. 1. The metabolic moments at which these meals were given are marked in Figs. 3 and 4. For the three groups a significant difference was found between the routine meal and the extra meal. Analysis of the data revealed also that there was a significant difference among the three test groups, D+S~4 ingesting more food than N+S,4, and the latter more than D+ $6. W e i g h t o f the S t o m a c h .
The relative weight of the stomach just before the unexpected meal in each group was 5.0_+0.4 (D+S0, 1.2_+0.1 (D+S14)---lower than in group D+S~---and 1.8_+0.1 g/100 g
CARBOHYDRATE METABOLISM AND FOOD INTAKE
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FIG. 3. Time course of glycogen (continuous fine), glycemia (dotted fine) and insufinemia (dashed line) in rats fed on a single-meal schedule given every day from 8:00 to 10:00 a.m. The vertical shaded areas indicate the periods at which the unexpected meals were administered: to group D one meal was offered 6 hours after the regular meal (group D+Se) and 14 hours after the meal (group D+$14). In ordinates: glycemia expressed as rag/100 ral of blood, hepatic glycogen concentration as mg/100 mg of liver tissue, and insulinemia as ~U/ml of serum. In abscissae, time in hours.
2°°t+
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.u/ml ............
'O
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FIG. 4. Time course of fiver glycogen concentration (continuous fine), glycemia (dotted fine) and insulin (dashed fine) in rats of the nocturnal group, fed on a singlemeal given every day from 8:00 to 10:00 p.m. The vertical shaded area indicates the period at which the unexpected meal was offered. Units expressed as in Fig. 3.
body weight (N+Sl4)---also lower than in group D+Se. The corresponding weights after the extra meal are shown in Fig. 2, which reveals that the time course o f gastric emptying was much about the same for both diurnal groups (D+S6 and D+SI+) whereas in the nocturnal group (N+SI4) the process was faster and leveled off sooner than in the rats fed habitually in day time.
Glycemia The variation o f glycemia as a function o f time in the interval between two regular, expected meals given to the rats o f the diurnal groups is depicted in Fig. 3. To identify the metabolic moments at which the rats received the unexpected meals two bars were added to the figure, one for
D+S6 and another for D+$14. Two hours after the regular meal glycemia went up and at the sixth hour it decreased to 119.6_+4.1 rag/100 ml. F r o m this value it increased almost linearly, attaining 142.2_+4.8 rag/100 ml at the 14th hour. At this time it declined, reducing glucose concentration to 103.9-+3.5 rag/100 ml in 6 hours. The extra meal was given to one group (D+Se) at the first lower peak and to the other (D+$14) at the highest peak. Variations of glycemia in the nocturnal group as a function o f time between the two regular meals is shown in Fig. 4. Glycemia decreased from 117.4_+5.8 to 98.8_+3.9 mg/100 ml in the two hours following the meal and then increased slowly to 176.7_+8.6 along the subsequent 18 hours, after which a steep decline to the pre-meal levels occurred. The
934
I.IMA ET AL
LJ/ml
mg/lOOm I mg/lOOmg 200 4 ........
mg/lOOml mg/lOOmg
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FIG. 5. Hepatic glycogen (continuous line), glycemia (dotted line) and insulin (dashed line) during and all along the 12 hours ensuing the unexpected meal in group D+S6. The feeding period is marked by the shaded area. Units as in Fig. 3, expressed as means_+s.e.m.
FIG. 6. Hepatic glycogen (continous line), glycemia (dotted line) and insufinemia (dashed fine) during and all along the t2 hours ensuing the unexpected meal in group D+SI,, The feeding period is marked by the shaded area. Units as in Fig. 3, expressed as mean±s,e.m.
unexpected meal was offered, as indicated in Fig. 4, 14 hours after the regular meal. Just before the extra meal glycemia was 119.6_+4.1 in group D+Se, 142.2_+4.8 in group D+$14, and 148.7_+3.4 mg/100 ml in group N+S~4. For the former two groups, i.e., when the unexpected meal was given 14 hours after the regular feeding period, glycemia was statistically similar but significantly higher than that for D+S6. After the surprise meal giycemia decreased in both D+S~4 and N+S~4 (Figs. 6 and 7), staying at levels lower than those attained before the meal all along the 12 following hours. Yet, a hyperglycemic propensity in group N+S~4 can be detected in Fig. 7. In group D+ Ss there was a brief increase in glycemia during the meal period and after a steady decline lasting 8 hours another mild increase restored the pre-meal level (Fig. 5).
m~OOml rag/lOOm9 4-i .......
-80
, 150-
iiiiiii!i!i 3-i!iiii
-60
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Hepatic Glycogen Hepatic glycogen concentration in the diurnal groups increased steadily from the beginning of the meal to reach its peak 6 hours later, when a slow decline brought it from 3.27_+0.21 to 2.76_+0.13 rag/100 nag in 8 hours. From then on a sudden fall to 1.1_+0.06 rag/100 mg required only two hours to occur. In the nocturnal group the time course of glycogen variation followed the same trend, with maintenance of high level till 14 hours of fasting elapsed (2.6_+0.08 rag/100 nag). A steep fall started in both diurnal and nocturnal groups 14 hours after the regular meal. As shown in Figs, 3 and 4, the decrease of glycogen in the nocturnal group was much slower than in the diurnal groups, although it went down to an even lower level just before the f o l l o ~ meal, Hepatic glycogen concentrations at the moment an unexpected meal was given to the rats were high, namely 3.27_+0.21 for D+Ss, 2.76_+0.13 for D+S~4 and 2,6_+0,08 rag/100 mg for group N+S~4 (Figs. 5, 6 and 7, respectively, and Fig. 8). The values for D+S6 were s i g n i f i c ~ y more elevated than those for D+S~4 and N+SI, but in all cases they were considerably high. Immediately after the unexpected meal a small increase occurred in group D+S8 (Fig. 5). After a slow oscillation a
laU/ml
200-
~
-20
b
'
~I '
,8 i ll2hr
-
0
FIG. 7. Glycogen (continuous line), glycemia (dotted line) and insulinemia (dashed fine) during and along the 12 hours f o l l o ~ : t h e unexpected meal in group N+$14. The feeding period is marked by the shaded area. Units as in Fig. 3, expressed as means_+s.e.m.
definite decrease started 6 hours past the meal. In sharp contrast, in group D+S14 the extra meal was followed by a rapid fall in glycogen and then an increase. Six hours after the meal it fell again to the same level attained at the end of the extra meal period (nearly 2 rag/100 rag), paralleling the final portion of the curve for group D+Se. However, the average hepatic glycogen in group D+S~4 (Fig. 6) was generally lower than in the D+Se group. In groupN+S~4 glycogen did not change significantly during t h e l 2 hours following the extra meal (Fig. 7). Insulinemia In the diurnal group insulinemia oscillated about 30 /~U/ml during 16 hours after the regular meal, then decreasing to 22_+1.5 ~U/ml up to the next meal. In the nocturnal group, however, insulinemia decreased from 42_+2.0/~U/ml
CARBOHYDRATE METABOLISM AND FOOD INTAKE
~/l~g
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.-~ 150 ~
°
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~U/ml 40~
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hr
FIG. 8. Liver glycogen, glycemia and insulinemia as a function of time during the extra meal period (shaded mark at the bottom of each graph) and the ensuing 12 hours in the three groups. Homologous curves are grouped for comparison. The corresponding s.e.m. which are found in the other figures, were deleted to enhance the trends of each variable.
after the meal to 21-+2.0/zU/ml 4 hours later and then went up to a peak 56_+6.0/zU/ml at the tenth hour after the meal. Another intense decrease to 23_+2.0 ?zU/ml occurred at the sixteenth hour, and was followed by a slight increase that reached 28-+2.0/~U/ml by the time of the next meal. Thus, a 12-hour cycle seemed to modulate the blood insulin concentration in the nocturnal group. When the unexpected meal was given to the rats of the diurnal group 6 hours after the regular meal (D+Se) a drastic increase from 25-+ 1.0/~U/ml to 37_+4.0/zU/ml was found. A slow decrease then ensued that reduced insulinemia to about 50% of the initial concentration prevailing when the extra meal started (Fig. 5). In D+S14 and N'kS14 insulinemia did not change much all along the period ensuing the unexpected meal. A comparison of hepatic glycogen level, glycemia and insulinemia from the three groups after the extra meal is presented in Fig. 8.
DISCUSSION One of the main trends in the hypotheses intended to exPlain triggering and regulation of energetic food ingestion is that both depend upon some intracellular metabolic cue. Because of its role in metabolism and of the wide evidence of
935 hepatic chemoreceptors signaling changes in local glucose concentration, the liver is likely to play a key role in such processes [5, 17, 25]. Nevertheless, powerful mechanisms arising in chemoreceptors sensitive to changes in intracellular glucose which are located in the area of the medial forebrain bundle of the hypothalamus and in the nuclei of the solitary tract are probably involved also [7,8]. It has been demonstrated [28] that food ingestion activated by insulin or 2-deoxy-D-glucose is related to the hour of the day in which either substance is administered. Such differences are bound to the hepatic glycogen content, so much so that when glycogen is high the amount of food ingested is low and when glycogen is low, more food is ingested. In the present work such a relationship was confirmed. The reactivity of the carbohydrate metabolic system was tested by the unexpected meals, which were given when liver glycogen was very high (around 3 mg/100 mg of liver tissue). The unexpected meal was ingested more intensely by the group that received the extra meal 14 hours after the regular meal, more by D+S14 than by N+$14, and least by the group that had the extra meal 6 hours after the regular one (D+Ss). The reason why D+Se ate less than N+$14 and Dq-S14 might be the incomplete emptying of the stomach only 6 hours after the previous meal. In fact, 6 hours after the meal the relative weight of the D+S6 group stomach plus its content was 5_+0.5 g/100 g body weight, much more than the 1.2+0.1 and 1.9_+0.1 g/100 g body weight, respectively for Dq-St4 and N-kS14. However, this difference cannot account for the higher ingestion of food in N + S ~ and D+$14, since in free fed rats the stomach plus its content weighed 5 g/100 g body weight, yet they ate as much as when the stomach weighed 1.5+0.07 [15]. On the other hand, examination of the curves that describe the time course of hepatic glycogen concentration (Figs. 3 and 4) show that it did not change significantly in group D+Ss during the period in which the extra meal was presented whereas it decreased moderately (but significantly) in group N+S~4 and declined quite steeply in group D+S~4. The rats of groups D+So, N+Sx4 and D+S~4 ate progressively more in that order. Glycogen concentration in the liver decreased at the rates of 0.05 rag/100 mg per hour in D+Se, 0.27 mg/100 mg in N+S~4 and 0.83 rag/100 mg per hour in D+$14. It thus seems that there is a relationship between the depletion of glycogen in the liver and how much food is ingested. Such a relationship will be quantified by expanding the resolution, within the two-hour period in which the extra meal is given, to intervals as short as 15 minutes. Attention should be drawn to the fact that as related to group D+S6 (which ate the least), group N+$14 ate 0.4 g/100 g body weight per hour more than D+Se and group D+SI4 ate 0.75 g/100 g body weight per hour above D+So. To stress further the relevance of the metabolic factors in regulation of food intake it should be pointed out that group D+S~4 ingested more food than N+$14, despite the time elapsed after the previous regular meal being the same (14 hours) and the relative weight of the stomach attaining similar values. That indicates a secondary role played by the intermeal interval, in disagreement with which has been claimed [9], and the degree of gastric emptying as main factors in determining the food intake. As to this statement, however, the fact that the animals in our experiments were subjected to a chronic food restriction and, therefore, adapted to a metabolic cycle locked to a fLxed pattern feeding schedule, should be taken into account. When a relationship between glycemia and food intake is sought in our data it seems that this parameter cannot play a
936
lAMA L'J A t .
direct role in regulating the amount of food ingested, only an indirect one. Actually, at the moment the unexpected meal was presented glycemia was the highest in rats of the group D+S~4, less in group N+S~4 and the least in D+S0. However, the rats ate progesssively more of the food in that same order. If glycemia just preceding the meal was directly involved, an entirely reverse order should be expected. After the extra meal the time course of the glycemic curve evolved very similarly in the three groups, the shifting of one curve as to the others being very small soon after the feeding had stopped. However, if basal glycemia is not directly involved in regulating the amount of food to be ingested, its evolution during the meal period should be an important signal. In fact, D+S6 group received the extra meal during an early phase of fasting hyperglycemia and N+$14 was refed during a clear hyperglycemic period. On the contrary, the period corresponding to an extra meal of D+$14 corresponded to a clear-cut decrease in blood glucose. Adding to that is the striking fact that in group N+$14 glycemia was raising towards a very high level (about 177 mg/100 ml), reached 20 hours after the regular meal, and to a not so high level (about 140 mg/100 ml) in group D+S~4 14 hours after the regular meals whereas it was to decrease slightly above 100 mg/100 ml 20 hours following the meal. It appears that after the unexpected meal glycemia was rapidly brought to a stable concentration that changed very slowly as a function of time in succeeding hours, until a new cycle would start over again. Glycogen was very high 6 hours after the regular meal in groups habitually fed at day time and tended to decrease at a low rate along the ensuing 8 hours (Fig. 3). When the unexpected meal was ingested starting at the sixth hour after the regular meal (Fig. 5) glycogen concentration increased similarly to the highest usual fasting level and remained so for the next 6 hours following the extra meal. In contrast, when the extra meal was given 14 hours after the regular meal liver glycogen was suddenly reduced to about half its concentration and then went up again before resuming the regular descent to attain, 12 hours after such meal, a value similar to those reached in the other two groups (Fig. 8). In the nocturnal group glycogen did not change significantly all along the 10 hours following the extra meal, converging during the latest two hours to meet the D+$14 and D+S6 corresponding curves. It may be reasoned that glycogen sharp decrease that was about to occur at the time the meals $14 and N~4 were given was prevented by the extra supply of food. At $6 glycogen was very high and tended to be even higher because of the unexpected supply but glycemia, as discussed above, was stabilized. The excess in glycogen must have played a key role in determining the lesser intake of food by the D+S6 group in relation to D+$14 and N+S~4. Gastric emptying was similar in both D + S~4and D+ $6 groups after the extra meal ingestion stopped and thus regulation of the glucose input to the liver was not due to a controlled gastric output of food to the intestines. A delay in intestinal absorption might be involved as a mechanism to impede a glycogen overload of the liver but it is more likely that high glycogen
concentration probably saturated the glycogenetic mechanism. These data support the hypothesis that metabolic factors are important in the mechanisms that regulate food intake. They supply additional evidence that glycogen metabolism is directly related to such mechanisms. This relationship may be relevant as a way of measuring the relative urge to restore, by feeding, energetic stores that have been used up. Several other evidences are in line with such views, since it has been shown [20, 21, 22, 23] that regulation of glycogen metabolism is modulated by neural structures located in the ventromedial and lateral hypothalamic areas and exerting, respectively, a control of liver phosphorylases and glycogen synthetase. In addition, neurons of the lateral hypothalamus are sensitive to portal infusion of glucose [18,19] and hyperglycemic reflexes are elicited by functional cytogtucopenia in the liver [7] and in the nuclei of solitary tract [27]. Such hyperglycemic reflexes, which are triggered by cytoglucopenia in the liver, have been considered [7] metabolic early adjustments that keep glycemia within a narrow homeostatic range. According to such hypothesis the hyperglycemic reflexes prevent a deep hypoglycemia due to an increasing mobilization of glycogenetic mechanisms (including both glycogenolysis and gluconeogenesis), until a signal arising from some metabolic step triggers a full feeding behavior. The present work disclosed a connection between the. rate of glycogenolysis and the amount of food ingested. Such a connection may well reflect the activation of the above suggested mechanism and then the hyperglycemic reflexes may be at one end of a series of mechanisms aiming at keeping normoglycemia, at the other end of which is feeding behavior. Our data did not show a clear-cut relationship between serum insulin and the quantity of food ingested. The three groups studied in our experimental model received an extra meal when insulinemia was similar in all three. This extra meal, on the other hand, was unable to modify the habitual variation of this hormone during the post-absorptive period. The present work provides evidence that the length of the inter-meal period is not primarily related to the amount of food ingested and that the main factor is related to the metabolic pattern. The regulating mechanisms acquired by experience and due to the natural or artifically (ecologically, experimentally and socially) imposed availability of food is likely to be closer to the experimental frame adopted for the rats that were subjected to the unexpected meal. However, several other complex factors are involved, including the relative amount of carbohydrate, fat and protein in food. Another possible factor is the photoperiod, whose significance for feeding is still unsettled. In our experiments, group D+S ~4 was given the unexpected meal in day time. On the other hand, group N+$14 had the extra meal in day time after having received the regular meal at night. Yet, the amount of food both groups ate was strikingly different in contrast to what has been reported by other authors [9], who found that neither permanent lighting nor continuous dark influence food intake.
REFERENCES
1. Cohn, C., D. Joseph and E. Srago. Effect of diet on body composition. I. The production of increased body fat without overweight (non obese obesity) by force feeding the normal rat. Metabolism 6: 381-387, 1957.
2. Cohn, C. and D. Joseph. Changes in body compositionattendant on forced feeding, Am, J. Physiol. 196: 965--968, 1959. 3. Dubowski, K. H. An o-toluidine method for body fluid glucose determination. Clin. Chem. 8: 215-235, 1962.
CARBOHYDRATE METABOLISM A N D FOOD I N T A K E 4. Ffibry, P. Metabolic consequences of the pattern of food intake. In: Handbook of Physiology, Section 6: Alimentary Canal, vol. 1: Food and Water Intake, edited by C. F. Code. Washington, DC: American Physiological Society, 1967, pp. 31-49. 5. Friedman, M. I. and E, M. Stricker. The physiological psychology of hunger: a physiological perspective. Psychol. Rev. 83: 409-431, 1975. 6. Hell, N. S., L. B. Costa de Oliveira, M. S. Dolnikoff, R. Scivoletto and C. Timo-Iaria. Changes of carbohydrate metabolism caused by food restriction, as detected by insulin administration. Physiol. Behav. 24: 473-478, 1980. 7. Kadekaro, M., C. Timo-Iaria and M. L. M. Vicentini. Control of gastric secretion by the central nervous system. In: Nerves and the Gut, edited by F. P. Brooks and P. W. Evers. Philadelphia: Charles B. Slack Inc., 1977, pp. 377-429. 8. Kadekaro, M., C. Timo-Iaria and M. L. M. Vicentini. Gastric secretion provoked by functional cytoglucopenia in the nuclei of the solitary tract in the cat. J. Physiol., Lond. 299: 397-407, 1980. 9. Le Magnen, J., M. Devos and C. Larue Achagiotis. Food deprivation induced parallel changes in blood glucose, plasma free fatty acids and feeding during the two parts of the diurnal cycle in rats. Neurosci. Biobehav. Rev. 4: Suppl. 1: 17-23, 1980. 10. Leveille, G. A. Glycogen metabolism in meal-fed rats and chicks and the time sequence of lipogenic and enzymatic adaptative changes. J. Nutr. 90: 449-460, 1966. 11. Leveille, G. A. Adipose tissue metabolism: influence of periodicity of eating and diet composition. Fedn Proc. 29: 1294-1301, 1970. 12. Leveille, G. A. and R. W. Hanson. Influence of periodicity of eating on adipose tissue metabolism in the cat. Can. J. Physiol. Pharmac. 43: 857-865, 1965. 13. Leveille, G. A. and K. Chakrabarty. Diurnal variations in tissue glycogen and liver weight of meal-fed rats. J. Nutr. 93: 546--554, 1967. 14. Leveille, G. A. and K. Chakrabarty. Absorption and utilization of glucose by meal-fed and nibbling rats. J. Nutr. 96: 69-75, 1968. 15. Lima, F. B., N. S. Hell, C. Timo-Iaria, R. Scivoletto, M. S. Dolnikoff and A. A. Pupo. Metabolic consequences of food restriction in rats. Physiol. Behav. 27: 115--123, 1981.
937 16. Morgan, C. R. and A. Lazarow. Immunoassay of insulin. Two antibody system. Plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 12: 115-126, 1962. 17. Russek, M. Algunas consideraciones sobre la regulaci6n de la ingesti6n de alimento. Cieneia 25: 73-90, 1967. 18. Schraitt, M. Circadian rhythmicity in response of cells in the lateral hypothalamus. Am. J. Physiol. 225: 1096-1101, 1973. 19. Schmitt, M. Influences of hepatic portal receptors on hypothalamic feeding and satiety centers. Am. J. Physiol. 225: 10891095, 1973. 20. Shimazu, T. Glycogen synthetase activity in liver: regulation by the autonomic nerves. Science 156: 1256-1257, 1967. 21. Shimazu, T. and A. Fukuda. Increased activities of glycogenolytic enzyme in liver after splanchnic nerve stimulation. Science 150: 1607-1608, 1965. 22. Shimazu, T. and A. Amakawa. Regulation of glycogen metabolism in liver by autonomic nervous system. II. Neural control of glycogenolytic enzymes. Biochem. biophys, acta 175: 335--348, 1968. 23. Shimazu, T. and A. Amakawa. Regulation of glycogen metabolism in liver by the autonomic nervous system. III. Differential effects of sympathetic nerve stimulation and of catecholamines on liver phosphorylase. Biochim. biophys, acta 165: 349-356, 1968. 24. Shimazu, T., H. Matsushita and K. Ishikawa. Hypothalamic control of liver glycogen metabolism in adult and aged rats. Brain Res. 144: 243-252. 25. Sawchenko, P. E. and M. I. Friedman. Sensory functions of the liver: a review. Am. J. Physiol. Regul. Integr. comp. Physiol. 5: RS-R20, 1979. 26. Tepperman, ]. and H. M. Tepperman. Effect of antecedent food intake pattern on hepatic lipogenesis. Am. J. Physiol. 193: 55-64, 1958. 27. Timo-Iaria, C., M. Kadekaro and M. L. M. Vicentini. Rhomboencephalic reflex loops involved in vegetative response to cytoglucopenia. Symposium on Food and Water Intake, Gifsur-Yvette, France, 1977. 28. Vanderweel, D. A., D. Novin, M. Rezek and J. D. Sanderson. Duodenal or hepatic portal glucose-perfusion: evidence for duodenally-based satiety. Physiol. Behav. 12: 467-473, 1974.
Carbohydrate Metabolism and Food Intake in Food Restricted Rats: Effects of an Unexpected Meal F. B. L I M A , N. S. H E L L ) C. T I M O - I A R I A M. S. D O L N I K O F F A N D A. A. P U P O
Institute o f Biomedical Sciences, University o f S i o Paulo Department o f Physiology & Pharmacology, 05008 Silo Paulo, SP-Brazil R e c e i v e d 4 J a n u a r y 1982 LIMA, F. B., N. S. HELL, C. TIMO-IARIA, M. S. DOLNIKOFF AND A. A. PUPO. Carbohydratemetabolism andfood intake in food restricted rats: Effects of an unexpected meal. PHYSIOL. BEHAV. 29(5) 931-937, 1982.mRats subjected to a daily single-meal feeding schedule were presented with an unexpected meal at three different times of the day and the effects on the carbohydrate metabolic patterns were determined. The results indicate that it is the actual metabolic moment of the organism, rather than the duration of the inter-meal period and the degree of gastric emptying, that determines the amount of food ingested. Food restriction
Carbohydrate metabolism
Food ingestion
FOOD restriction is known to induce an increase in intestinal absorption [1, 2, 4, 13], liver glycogen content [10,14], fat accumulation [ 11,12], lipogenesis in liver and adipose tissue [25], and a higher resistance to glycogen depletion during insulin hypoglycemia [6]. These changes may make more available energetic stuff for the emission of suddenly necessary, highly energy consuming behaviors [6]. Previous work [15] indicates that food restriction also causes a lesser insulin secretion, higher gastric capacitance and delayed gastric emptying, which suggests that food restricted animals better use circulating nutrients and pour energetic compounds into the blood at a slower rate. In rats fed at night, there appeared an increase in blood sugar concentration during the long inter-meal periods that was more pronounced than in those fed in day time. Liver glycogen decreased at 14 hours after the meal in both feeding groups, but greater decreases were observed in the rats fed during the day. Food ingestion was higher in rats fed at night despite similar inter-meal periods (22 hours) and gastric emptying for both groups. Glycemia and insulinemia at the time of the expected meal were also similar in both groups but hepatic glycogen content by then was significantly lower in the rats fed at night, which ingested an amount of food that was significantly larger than that ingested by the diurnal group. Food restriction is a useful experimental model to assess some factors involved in food intake regulation because conditions associated with feeding, such as the inter-meal period, the degree of gastric emptying, the quantity of food
available to the animals as well as the amount that is ingested, can be easily determined. Moreover, once the pattern of some metabolic parameters has been established other variables can be introduced in the experimental model at certain "metabolic moments" to determine those metabolic factors that regulate food ingestion. In the present study an unexpected meal was presented to rats adapted to a daffy single-meal feeding schedule, either diurnal or nocturnal. By assessing the metabolic changes caused by this surprise meal it was possible to obtain additional information regarding the role of metabolic factors in the regulation of food intake. METHOD
Male Wistar albino rats weighing about 200 g were kept in individual cages. During one week the animals received only one meal a day, either from 8:00 to I0:00 a.m. or from 8:00 to I0:00 p.m. As reported in a previous study [15] the experiments were performed throughout the year, repeating each experimental combination at different times of the year, aiming at assessing the influence of seasonal factors. Actually, so that the rats could be studied in conditions as natural as possible, such distribution of the experiments was adopted on purpose. Despite the different temperatures (between 8°C in winter and 36°C in deep summer), no differences were found to occur in the studied parameters that could be attributed to either variable. The animals were kept under nat-
Whis research was supported by the Sao Paulo State Research Foundation (FAPESP), the National Research Council (CNPq) and FINEP. fro whom requests for reprints should be addressed.
Copyright © 1982 P e r g a m o n Press--0031-9384/82/l10931-07503.00/0
932
I~IMA E7 ~A I .
g/1009 b.w. 6~
g/lOOg b.w. u
43-
I
8-
I
D+Ss
D+$14
N+$14
2~
O FIG. 1. Food intake during the unexpected meal, evaluated in g/100 g of body weight (me.an±standard error of the mean, s.e.m.) The meal was given to group D+S6 (n=56) from 4:00 to 6:00 p.m., to group D+$14 (n=56) from 12:00to 2:00 a.m., and to group N+S~4 (n=56) from 12:00to 2:00 p.m. 0
ural illumination to avoid artifical shifting of the naturally established locking of feeding behavior and metabolic patterns to the time of the day, as reflected by light and dark. Moreover, at the latitude of the laboratory (about 23° south) the drift of the sunset and of the sunrise is relatively narrow (less than two hours). Food restriction was also imposed at different times so that the entire 24 hour length of the day was covered. On the day that each experiment was to be completed (8th day) an unexpected meal was presented to the rats at a certain interval after the last usual meal according to the following schedule: (a) 56 animals trained to eat from 8:00 to I0:00 a.m. were given the surprise meal 6 hours later, i,e. from 4:00 to 6:00 p,m. (Group D+Se); (b) another 56, also routinely fed in day time (8:00 to 10:00 a.m.) had the extra meal 14 hours later, i.e. from 12:00 p.m, to 2:00a.m, (group D+S~4); (c) the 56 rats of the nocturnal group(8:00 to 10:00 p,m.) had the unexpected meal 14 hours later, i.e. from 12:00 to 2:00 p.m, (group N+$14). All the animals were permitted free access to water during the experiment. The day selected for the measurements the amount of food ingested during the routine and the unexpected meal was determined and then the rats were intraperitoneally anesthetized with sodium pentobarbiturate (40 mg/kg body weight,) The abdomen was rapidly opened and blood samples and the stomach plus its content were removed, and a few samples of liver were taken, as previously described [15]. The weight of the stomach and gastric contents were normalized according to body weight. This allowed corrections for differences in the sizes of the animals and was considered as an= index of gastric filling or emptying. The collection of these materials was performed in smaller groups of rats (n=8) every two hours be~nning at the onset of the extra meal and extended for a period of 12 hours. Blood glucose was determined by the ortho-toluidine method [3] and blood insulin by radioimmunoassay [16]. Liver glycogen was assayed by means of ~ e digestion, separation of the carbohydrate in ethanol [13], The extract obtained was hydrolised by heat and the resulting glucose concentration was determined by the ortho-toluidine method. The amount of food ingested was determined as the
t
r 4
I--1----7 8
1 12hr
FIG. 2. Time course of the relative weight of the stomach during 12 hours after the unexpected meal, expressed as g/lll~ g body weight as a function of time in hours after the meal, Groups defined as in Fig. 1. Continuous line: group D+$14. Dashed line: group N+S,4. Dotted line: D+S~. Mean_+s.e.m.
difference between the amount of food offered and that remaining at the conclusion of the two-hour period. The results were expressed in grams/100 grams body weight. The data were statistically processed at 95% of significance. Variation of the amount of food ingested among the three groups during the last routine-meal was analysed by Duncan's test. The same was done for t o m , s o n of f o ~ ingested during the surprise-meal presented to the three groups. For comparison of quantity of food Consumed by an animal at the last habitual meal and during surprise-meal, paired Student's t-test was used. Values of giycemia, h~atic glycogen, insulin and stomach fresh weight from all animals were analysed by non-paired Student's t-test. RESULTS Food Intake
During the last routine meal the amount of food ingested by the rats was 5.3_+0.2, 5.7_+0.2 and 7.4_+0.2 g/100 g body weight for groups D+S6, D÷Sz4 and N+$14, respectively. During the unexpected meal the animals ate 3.7__.0.2, 5.2-+0.1 and 4.5_+0.1 g/100 g body weight, respectively, as shown in Fig. 1. The metabolic moments at which these meals were given are marked in Figs. 3 and 4. For the three groups a significant difference was found between the routine meal and the extra meal. Analysis of the data revealed also that there was a significant difference among the three test groups, D+S~4 ingesting more food than N+S,4, and the latter more than D+ $6. W e i g h t o f the S t o m a c h .
The relative weight of the stomach just before the unexpected meal in each group was 5.0_+0.4 (D+S0, 1.2_+0.1 (D+S14)---lower than in group D+S~---and 1.8_+0.1 g/100 g
CARBOHYDRATE METABOLISM AND FOOD INTAKE
rn0Oral g/1mg~0Omg
D+S6
4-
o .... 150
933
~M/ml
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-8O
....
3).
i+i+i
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.......
-20 -= 0
0
' ~4 I
18 ~
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112 116 ~ 2bhl
FIG. 3. Time course of glycogen (continuous fine), glycemia (dotted fine) and insufinemia (dashed line) in rats fed on a single-meal schedule given every day from 8:00 to 10:00 a.m. The vertical shaded areas indicate the periods at which the unexpected meals were administered: to group D one meal was offered 6 hours after the regular meal (group D+Se) and 14 hours after the meal (group D+$14). In ordinates: glycemia expressed as rag/100 ral of blood, hepatic glycogen concentration as mg/100 mg of liver tissue, and insulinemia as ~U/ml of serum. In abscissae, time in hours.
2°°t+
rng/ oo /l°°n 150
.u/ml ............
'O
3-
...... ~,, " 0 ......
T~
~-80
N+$14
'
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0 ~
"~" ~
~10
',
~!~i:~~i:~:il 'e'
1',
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-40
i-EI20 ~" o
FIG. 4. Time course of fiver glycogen concentration (continuous fine), glycemia (dotted fine) and insulin (dashed fine) in rats of the nocturnal group, fed on a singlemeal given every day from 8:00 to 10:00 p.m. The vertical shaded area indicates the period at which the unexpected meal was offered. Units expressed as in Fig. 3.
body weight (N+Sl4)---also lower than in group D+Se. The corresponding weights after the extra meal are shown in Fig. 2, which reveals that the time course o f gastric emptying was much about the same for both diurnal groups (D+S6 and D+SI+) whereas in the nocturnal group (N+SI4) the process was faster and leveled off sooner than in the rats fed habitually in day time.
Glycemia The variation o f glycemia as a function o f time in the interval between two regular, expected meals given to the rats o f the diurnal groups is depicted in Fig. 3. To identify the metabolic moments at which the rats received the unexpected meals two bars were added to the figure, one for
D+S6 and another for D+$14. Two hours after the regular meal glycemia went up and at the sixth hour it decreased to 119.6_+4.1 rag/100 ml. F r o m this value it increased almost linearly, attaining 142.2_+4.8 rag/100 ml at the 14th hour. At this time it declined, reducing glucose concentration to 103.9-+3.5 rag/100 ml in 6 hours. The extra meal was given to one group (D+Se) at the first lower peak and to the other (D+$14) at the highest peak. Variations of glycemia in the nocturnal group as a function o f time between the two regular meals is shown in Fig. 4. Glycemia decreased from 117.4_+5.8 to 98.8_+3.9 mg/100 ml in the two hours following the meal and then increased slowly to 176.7_+8.6 along the subsequent 18 hours, after which a steep decline to the pre-meal levels occurred. The
934
I.IMA ET AL
LJ/ml
mg/lOOm I mg/lOOmg 200 4 ........
mg/lOOml mg/lOOmg
~U/ml -80 L_
-60
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-60
.... ........
........
_
..
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-~iiii~iii!::~i",,l ~ :::::::::::::::::: .............. /..,...,,.. ,.../•..
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40
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0
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b
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4
'
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lt2hr
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i
t8 I l'2hr
FIG. 5. Hepatic glycogen (continuous line), glycemia (dotted line) and insulin (dashed line) during and all along the 12 hours ensuing the unexpected meal in group D+S6. The feeding period is marked by the shaded area. Units as in Fig. 3, expressed as means_+s.e.m.
FIG. 6. Hepatic glycogen (continous line), glycemia (dotted line) and insufinemia (dashed fine) during and all along the t2 hours ensuing the unexpected meal in group D+SI,, The feeding period is marked by the shaded area. Units as in Fig. 3, expressed as mean±s,e.m.
unexpected meal was offered, as indicated in Fig. 4, 14 hours after the regular meal. Just before the extra meal glycemia was 119.6_+4.1 in group D+Se, 142.2_+4.8 in group D+$14, and 148.7_+3.4 mg/100 ml in group N+S~4. For the former two groups, i.e., when the unexpected meal was given 14 hours after the regular feeding period, glycemia was statistically similar but significantly higher than that for D+S6. After the surprise meal giycemia decreased in both D+S~4 and N+S~4 (Figs. 6 and 7), staying at levels lower than those attained before the meal all along the 12 following hours. Yet, a hyperglycemic propensity in group N+S~4 can be detected in Fig. 7. In group D+ Ss there was a brief increase in glycemia during the meal period and after a steady decline lasting 8 hours another mild increase restored the pre-meal level (Fig. 5).
m~OOml rag/lOOm9 4-i .......
-80
, 150-
iiiiiii!i!i 3-i!iiii
-60
.~ 100--]
2- .:.:i::i!i!
-40
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0
Hepatic Glycogen Hepatic glycogen concentration in the diurnal groups increased steadily from the beginning of the meal to reach its peak 6 hours later, when a slow decline brought it from 3.27_+0.21 to 2.76_+0.13 rag/100 nag in 8 hours. From then on a sudden fall to 1.1_+0.06 rag/100 mg required only two hours to occur. In the nocturnal group the time course of glycogen variation followed the same trend, with maintenance of high level till 14 hours of fasting elapsed (2.6_+0.08 rag/100 nag). A steep fall started in both diurnal and nocturnal groups 14 hours after the regular meal. As shown in Figs, 3 and 4, the decrease of glycogen in the nocturnal group was much slower than in the diurnal groups, although it went down to an even lower level just before the f o l l o ~ meal, Hepatic glycogen concentrations at the moment an unexpected meal was given to the rats were high, namely 3.27_+0.21 for D+Ss, 2.76_+0.13 for D+S~4 and 2,6_+0,08 rag/100 mg for group N+S~4 (Figs. 5, 6 and 7, respectively, and Fig. 8). The values for D+S6 were s i g n i f i c ~ y more elevated than those for D+S~4 and N+SI, but in all cases they were considerably high. Immediately after the unexpected meal a small increase occurred in group D+S8 (Fig. 5). After a slow oscillation a
laU/ml
200-
~
-20
b
'
~I '
,8 i ll2hr
-
0
FIG. 7. Glycogen (continuous line), glycemia (dotted line) and insulinemia (dashed fine) during and along the 12 hours f o l l o ~ : t h e unexpected meal in group N+$14. The feeding period is marked by the shaded area. Units as in Fig. 3, expressed as means_+s.e.m.
definite decrease started 6 hours past the meal. In sharp contrast, in group D+S14 the extra meal was followed by a rapid fall in glycogen and then an increase. Six hours after the meal it fell again to the same level attained at the end of the extra meal period (nearly 2 rag/100 rag), paralleling the final portion of the curve for group D+Se. However, the average hepatic glycogen in group D+S~4 (Fig. 6) was generally lower than in the D+Se group. In groupN+S~4 glycogen did not change significantly during t h e l 2 hours following the extra meal (Fig. 7). Insulinemia In the diurnal group insulinemia oscillated about 30 /~U/ml during 16 hours after the regular meal, then decreasing to 22_+1.5 ~U/ml up to the next meal. In the nocturnal group, however, insulinemia decreased from 42_+2.0/~U/ml
CARBOHYDRATE METABOLISM AND FOOD INTAKE
~/l~g
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2 ::i:i:i:i:~:b-
~so.-.. o
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.-~ 150 ~
°
,.°
~U/ml 40~
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:i:i:i:~:~:i:i: ~
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hr
FIG. 8. Liver glycogen, glycemia and insulinemia as a function of time during the extra meal period (shaded mark at the bottom of each graph) and the ensuing 12 hours in the three groups. Homologous curves are grouped for comparison. The corresponding s.e.m. which are found in the other figures, were deleted to enhance the trends of each variable.
after the meal to 21-+2.0/zU/ml 4 hours later and then went up to a peak 56_+6.0/zU/ml at the tenth hour after the meal. Another intense decrease to 23_+2.0 ?zU/ml occurred at the sixteenth hour, and was followed by a slight increase that reached 28-+2.0/~U/ml by the time of the next meal. Thus, a 12-hour cycle seemed to modulate the blood insulin concentration in the nocturnal group. When the unexpected meal was given to the rats of the diurnal group 6 hours after the regular meal (D+Se) a drastic increase from 25-+ 1.0/~U/ml to 37_+4.0/zU/ml was found. A slow decrease then ensued that reduced insulinemia to about 50% of the initial concentration prevailing when the extra meal started (Fig. 5). In D+S14 and N'kS14 insulinemia did not change much all along the period ensuing the unexpected meal. A comparison of hepatic glycogen level, glycemia and insulinemia from the three groups after the extra meal is presented in Fig. 8.
DISCUSSION One of the main trends in the hypotheses intended to exPlain triggering and regulation of energetic food ingestion is that both depend upon some intracellular metabolic cue. Because of its role in metabolism and of the wide evidence of
935 hepatic chemoreceptors signaling changes in local glucose concentration, the liver is likely to play a key role in such processes [5, 17, 25]. Nevertheless, powerful mechanisms arising in chemoreceptors sensitive to changes in intracellular glucose which are located in the area of the medial forebrain bundle of the hypothalamus and in the nuclei of the solitary tract are probably involved also [7,8]. It has been demonstrated [28] that food ingestion activated by insulin or 2-deoxy-D-glucose is related to the hour of the day in which either substance is administered. Such differences are bound to the hepatic glycogen content, so much so that when glycogen is high the amount of food ingested is low and when glycogen is low, more food is ingested. In the present work such a relationship was confirmed. The reactivity of the carbohydrate metabolic system was tested by the unexpected meals, which were given when liver glycogen was very high (around 3 mg/100 mg of liver tissue). The unexpected meal was ingested more intensely by the group that received the extra meal 14 hours after the regular meal, more by D+S14 than by N+$14, and least by the group that had the extra meal 6 hours after the regular one (D+Ss). The reason why D+Se ate less than N+$14 and Dq-S14 might be the incomplete emptying of the stomach only 6 hours after the previous meal. In fact, 6 hours after the meal the relative weight of the D+S6 group stomach plus its content was 5_+0.5 g/100 g body weight, much more than the 1.2+0.1 and 1.9_+0.1 g/100 g body weight, respectively for Dq-St4 and N-kS14. However, this difference cannot account for the higher ingestion of food in N + S ~ and D+$14, since in free fed rats the stomach plus its content weighed 5 g/100 g body weight, yet they ate as much as when the stomach weighed 1.5+0.07 [15]. On the other hand, examination of the curves that describe the time course of hepatic glycogen concentration (Figs. 3 and 4) show that it did not change significantly in group D+Ss during the period in which the extra meal was presented whereas it decreased moderately (but significantly) in group N+S~4 and declined quite steeply in group D+S~4. The rats of groups D+So, N+Sx4 and D+S~4 ate progressively more in that order. Glycogen concentration in the liver decreased at the rates of 0.05 rag/100 mg per hour in D+Se, 0.27 mg/100 mg in N+S~4 and 0.83 rag/100 mg per hour in D+$14. It thus seems that there is a relationship between the depletion of glycogen in the liver and how much food is ingested. Such a relationship will be quantified by expanding the resolution, within the two-hour period in which the extra meal is given, to intervals as short as 15 minutes. Attention should be drawn to the fact that as related to group D+S6 (which ate the least), group N+$14 ate 0.4 g/100 g body weight per hour more than D+Se and group D+SI4 ate 0.75 g/100 g body weight per hour above D+So. To stress further the relevance of the metabolic factors in regulation of food intake it should be pointed out that group D+S~4 ingested more food than N+$14, despite the time elapsed after the previous regular meal being the same (14 hours) and the relative weight of the stomach attaining similar values. That indicates a secondary role played by the intermeal interval, in disagreement with which has been claimed [9], and the degree of gastric emptying as main factors in determining the food intake. As to this statement, however, the fact that the animals in our experiments were subjected to a chronic food restriction and, therefore, adapted to a metabolic cycle locked to a fLxed pattern feeding schedule, should be taken into account. When a relationship between glycemia and food intake is sought in our data it seems that this parameter cannot play a
936
lAMA L'J A t .
direct role in regulating the amount of food ingested, only an indirect one. Actually, at the moment the unexpected meal was presented glycemia was the highest in rats of the group D+S~4, less in group N+S~4 and the least in D+S0. However, the rats ate progesssively more of the food in that same order. If glycemia just preceding the meal was directly involved, an entirely reverse order should be expected. After the extra meal the time course of the glycemic curve evolved very similarly in the three groups, the shifting of one curve as to the others being very small soon after the feeding had stopped. However, if basal glycemia is not directly involved in regulating the amount of food to be ingested, its evolution during the meal period should be an important signal. In fact, D+S6 group received the extra meal during an early phase of fasting hyperglycemia and N+$14 was refed during a clear hyperglycemic period. On the contrary, the period corresponding to an extra meal of D+$14 corresponded to a clear-cut decrease in blood glucose. Adding to that is the striking fact that in group N+$14 glycemia was raising towards a very high level (about 177 mg/100 ml), reached 20 hours after the regular meal, and to a not so high level (about 140 mg/100 ml) in group D+S~4 14 hours after the regular meals whereas it was to decrease slightly above 100 mg/100 ml 20 hours following the meal. It appears that after the unexpected meal glycemia was rapidly brought to a stable concentration that changed very slowly as a function of time in succeeding hours, until a new cycle would start over again. Glycogen was very high 6 hours after the regular meal in groups habitually fed at day time and tended to decrease at a low rate along the ensuing 8 hours (Fig. 3). When the unexpected meal was ingested starting at the sixth hour after the regular meal (Fig. 5) glycogen concentration increased similarly to the highest usual fasting level and remained so for the next 6 hours following the extra meal. In contrast, when the extra meal was given 14 hours after the regular meal liver glycogen was suddenly reduced to about half its concentration and then went up again before resuming the regular descent to attain, 12 hours after such meal, a value similar to those reached in the other two groups (Fig. 8). In the nocturnal group glycogen did not change significantly all along the 10 hours following the extra meal, converging during the latest two hours to meet the D+$14 and D+S6 corresponding curves. It may be reasoned that glycogen sharp decrease that was about to occur at the time the meals $14 and N~4 were given was prevented by the extra supply of food. At $6 glycogen was very high and tended to be even higher because of the unexpected supply but glycemia, as discussed above, was stabilized. The excess in glycogen must have played a key role in determining the lesser intake of food by the D+S6 group in relation to D+$14 and N+S~4. Gastric emptying was similar in both D + S~4and D+ $6 groups after the extra meal ingestion stopped and thus regulation of the glucose input to the liver was not due to a controlled gastric output of food to the intestines. A delay in intestinal absorption might be involved as a mechanism to impede a glycogen overload of the liver but it is more likely that high glycogen
concentration probably saturated the glycogenetic mechanism. These data support the hypothesis that metabolic factors are important in the mechanisms that regulate food intake. They supply additional evidence that glycogen metabolism is directly related to such mechanisms. This relationship may be relevant as a way of measuring the relative urge to restore, by feeding, energetic stores that have been used up. Several other evidences are in line with such views, since it has been shown [20, 21, 22, 23] that regulation of glycogen metabolism is modulated by neural structures located in the ventromedial and lateral hypothalamic areas and exerting, respectively, a control of liver phosphorylases and glycogen synthetase. In addition, neurons of the lateral hypothalamus are sensitive to portal infusion of glucose [18,19] and hyperglycemic reflexes are elicited by functional cytogtucopenia in the liver [7] and in the nuclei of solitary tract [27]. Such hyperglycemic reflexes, which are triggered by cytoglucopenia in the liver, have been considered [7] metabolic early adjustments that keep glycemia within a narrow homeostatic range. According to such hypothesis the hyperglycemic reflexes prevent a deep hypoglycemia due to an increasing mobilization of glycogenetic mechanisms (including both glycogenolysis and gluconeogenesis), until a signal arising from some metabolic step triggers a full feeding behavior. The present work disclosed a connection between the. rate of glycogenolysis and the amount of food ingested. Such a connection may well reflect the activation of the above suggested mechanism and then the hyperglycemic reflexes may be at one end of a series of mechanisms aiming at keeping normoglycemia, at the other end of which is feeding behavior. Our data did not show a clear-cut relationship between serum insulin and the quantity of food ingested. The three groups studied in our experimental model received an extra meal when insulinemia was similar in all three. This extra meal, on the other hand, was unable to modify the habitual variation of this hormone during the post-absorptive period. The present work provides evidence that the length of the inter-meal period is not primarily related to the amount of food ingested and that the main factor is related to the metabolic pattern. The regulating mechanisms acquired by experience and due to the natural or artifically (ecologically, experimentally and socially) imposed availability of food is likely to be closer to the experimental frame adopted for the rats that were subjected to the unexpected meal. However, several other complex factors are involved, including the relative amount of carbohydrate, fat and protein in food. Another possible factor is the photoperiod, whose significance for feeding is still unsettled. In our experiments, group D+S ~4 was given the unexpected meal in day time. On the other hand, group N+$14 had the extra meal in day time after having received the regular meal at night. Yet, the amount of food both groups ate was strikingly different in contrast to what has been reported by other authors [9], who found that neither permanent lighting nor continuous dark influence food intake.
REFERENCES
1. Cohn, C., D. Joseph and E. Srago. Effect of diet on body composition. I. The production of increased body fat without overweight (non obese obesity) by force feeding the normal rat. Metabolism 6: 381-387, 1957.
2. Cohn, C. and D. Joseph. Changes in body compositionattendant on forced feeding, Am, J. Physiol. 196: 965--968, 1959. 3. Dubowski, K. H. An o-toluidine method for body fluid glucose determination. Clin. Chem. 8: 215-235, 1962.
CARBOHYDRATE METABOLISM A N D FOOD I N T A K E 4. Ffibry, P. Metabolic consequences of the pattern of food intake. In: Handbook of Physiology, Section 6: Alimentary Canal, vol. 1: Food and Water Intake, edited by C. F. Code. Washington, DC: American Physiological Society, 1967, pp. 31-49. 5. Friedman, M. I. and E, M. Stricker. The physiological psychology of hunger: a physiological perspective. Psychol. Rev. 83: 409-431, 1975. 6. Hell, N. S., L. B. Costa de Oliveira, M. S. Dolnikoff, R. Scivoletto and C. Timo-Iaria. Changes of carbohydrate metabolism caused by food restriction, as detected by insulin administration. Physiol. Behav. 24: 473-478, 1980. 7. Kadekaro, M., C. Timo-Iaria and M. L. M. Vicentini. Control of gastric secretion by the central nervous system. In: Nerves and the Gut, edited by F. P. Brooks and P. W. Evers. Philadelphia: Charles B. Slack Inc., 1977, pp. 377-429. 8. Kadekaro, M., C. Timo-Iaria and M. L. M. Vicentini. Gastric secretion provoked by functional cytoglucopenia in the nuclei of the solitary tract in the cat. J. Physiol., Lond. 299: 397-407, 1980. 9. Le Magnen, J., M. Devos and C. Larue Achagiotis. Food deprivation induced parallel changes in blood glucose, plasma free fatty acids and feeding during the two parts of the diurnal cycle in rats. Neurosci. Biobehav. Rev. 4: Suppl. 1: 17-23, 1980. 10. Leveille, G. A. Glycogen metabolism in meal-fed rats and chicks and the time sequence of lipogenic and enzymatic adaptative changes. J. Nutr. 90: 449-460, 1966. 11. Leveille, G. A. Adipose tissue metabolism: influence of periodicity of eating and diet composition. Fedn Proc. 29: 1294-1301, 1970. 12. Leveille, G. A. and R. W. Hanson. Influence of periodicity of eating on adipose tissue metabolism in the cat. Can. J. Physiol. Pharmac. 43: 857-865, 1965. 13. Leveille, G. A. and K. Chakrabarty. Diurnal variations in tissue glycogen and liver weight of meal-fed rats. J. Nutr. 93: 546--554, 1967. 14. Leveille, G. A. and K. Chakrabarty. Absorption and utilization of glucose by meal-fed and nibbling rats. J. Nutr. 96: 69-75, 1968. 15. Lima, F. B., N. S. Hell, C. Timo-Iaria, R. Scivoletto, M. S. Dolnikoff and A. A. Pupo. Metabolic consequences of food restriction in rats. Physiol. Behav. 27: 115--123, 1981.
937 16. Morgan, C. R. and A. Lazarow. Immunoassay of insulin. Two antibody system. Plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 12: 115-126, 1962. 17. Russek, M. Algunas consideraciones sobre la regulaci6n de la ingesti6n de alimento. Cieneia 25: 73-90, 1967. 18. Schraitt, M. Circadian rhythmicity in response of cells in the lateral hypothalamus. Am. J. Physiol. 225: 1096-1101, 1973. 19. Schmitt, M. Influences of hepatic portal receptors on hypothalamic feeding and satiety centers. Am. J. Physiol. 225: 10891095, 1973. 20. Shimazu, T. Glycogen synthetase activity in liver: regulation by the autonomic nerves. Science 156: 1256-1257, 1967. 21. Shimazu, T. and A. Fukuda. Increased activities of glycogenolytic enzyme in liver after splanchnic nerve stimulation. Science 150: 1607-1608, 1965. 22. Shimazu, T. and A. Amakawa. Regulation of glycogen metabolism in liver by autonomic nervous system. II. Neural control of glycogenolytic enzymes. Biochem. biophys, acta 175: 335--348, 1968. 23. Shimazu, T. and A. Amakawa. Regulation of glycogen metabolism in liver by the autonomic nervous system. III. Differential effects of sympathetic nerve stimulation and of catecholamines on liver phosphorylase. Biochim. biophys, acta 165: 349-356, 1968. 24. Shimazu, T., H. Matsushita and K. Ishikawa. Hypothalamic control of liver glycogen metabolism in adult and aged rats. Brain Res. 144: 243-252. 25. Sawchenko, P. E. and M. I. Friedman. Sensory functions of the liver: a review. Am. J. Physiol. Regul. Integr. comp. Physiol. 5: RS-R20, 1979. 26. Tepperman, ]. and H. M. Tepperman. Effect of antecedent food intake pattern on hepatic lipogenesis. Am. J. Physiol. 193: 55-64, 1958. 27. Timo-Iaria, C., M. Kadekaro and M. L. M. Vicentini. Rhomboencephalic reflex loops involved in vegetative response to cytoglucopenia. Symposium on Food and Water Intake, Gifsur-Yvette, France, 1977. 28. Vanderweel, D. A., D. Novin, M. Rezek and J. D. Sanderson. Duodenal or hepatic portal glucose-perfusion: evidence for duodenally-based satiety. Physiol. Behav. 12: 467-473, 1974.