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Pancreatic glucagon administration, feeding, glycemia, and liver glycogen in Rats
Integration of Cenfral und Peripheral Receptors in Hunger und Energy Metrrholism Brain Research Bulletin, Vol. 5, Suppl. 4, pp. 17-21. Printed in the U.S.A.
Pancreatic Glucagon Administration, Feeding, Glycemia, and Liver Glycogen in Rats’ DENNIS
A. VANDERWEELE2
Division of Behuvioral Neurology,
AND EDWARD
HARACZKIEWICZ
New York University, School of Medicine,
New York, NY 10016
AND MICHAEL
Department
VANDERWEELE,
of Psychology,
D. A., E. HARACZKIEWICZ
A. DICONTI
Occidental College, Los Angeles,
AND M. A. DICONTI.
Pancreatic
g/wagon
CA 90041
adminisfration,
feeding,
g/yccJmicl.and liver ,q/~~c.ogc’n in rats. BRAIN RES. BULL. 5: Suppl. 4, 17-21, 1980.-Rats
were trained to bar press for Noyes pellets. Once trained, subjects were food deprived at 0800, injected with pancreatic glucagon or isotonic saline (0.35 mgikg BW, intraperitoneally in a volume of 0.1 cc per 300 g BW) at 1100, and then placed in the operant chamber at 1115 or 1415 for a duration of 3 hr. Animals placed immediately in the chamber showed a suppression of food intake accomplished by a lengthening of the interval following the first meal rather than a reduction in the size of that meal. Animals tested 3 hr post-injection showed an enhancement of feeding by increasing the size of the first meal compared to saline-injected subjects, ~~0.025. The behavioral effects of glucagon upon feeding depend on the point of the sequence of responses to the hormone during which the animal is studied. Early after glucagon administration, hyperglycemia and increased glucose utilization predominate and feeding was reduced; subsequently, blood glucose may be normal while the liver remains somewhat glycogen depleted and feeding is enhanced. Blood glucose levels Food intake Pancreatic glucagon Rat Short-term food intake regulation
A HIGH molar ratio of insulin: glucagon favors the storage or utilization of ingested nutrients while a very low ratio favors gluconeogenesis and metabolic fuel production [8]. It seems that alterations of this hormonal ratio might also alter feeding behavior, and studies are now appearing which attempt to assess the effects of manipulation of absolute titres of the hormones on ingestion and weight gain (see [4] and [lo] for summaries of these studies). Alternatively to the insu1in:glucagon ratio hypothesis, a proposal that the insu1in:growth hormone ratio might be influential upon feeding behavior has been forwarded [lo]. The important point stressed by each theory, however, is that nutrient flux should influence food ingestion and the present study attempts to assess this point. Recently, two studies demonstrated that a rise in glucagon levels which lowered the insulin:glucagon ratio and altered nutrient flux, also affected food ingestion [2,4]. The initial study reported that the administration of 0.2 mg glucagon to rats during an intermeal interval (IMI) produced a delay in the onset of the next meal with no effect upon the
Liver glycogen levels
Food deprivation
size of that meal. The increased IMI was attributed to an elevation of blood glucose, however, blood glucose levels were not reported. Insulin, on the other hand, was shown to hasten the onset of a subsequent meal when administered during the IMI. One methodological problem with this study was that the animals with the shortest baseline IMIs were assigned to the glucagon-injected group which may have predisposed this group to longer IMIs when injected. The second study administered glucagon over three consecutive days in rats and found that the hormone’s sole effect upon food intake was a reduction in meal size (MS). In this second study [4], 0.75 mg of zinc glucagon was administered over a 24 hr period via 3 subcutaneous injections and this procedure resulted in a 40% reduction in average MS and a depression of body weight gain. The authors suggest that less was eaten because of glucagon’s enhancement of glucose availability. As the zinc form of glucagon has a prolonged activity relative to simple glucagon [l], it seems quite likely that the rats were in a continuous hyperglycemic state and, therefore, reduced feeding. Blood
IThis work was supported by Grant AM-17259 (DAVW) and a Richter Foundation award to Occidental College (DAVW and MAD). We are greatly indebted to Drs. Theodore B. Van Itallie for ideas and support during this research and editorial assistance and F. Xavier Pi-Sunyer and his staff at the Obesity Research Center, St. Luke’s Hospital, Columbia University for the biochemical determinations of blood glucose and insulin levels. ‘Present address and address for reprints: Dr. Dennis A. VanderWeele, Psychology Department, Occidental College, Los Angeles, CA 90041.
Copyright
0 1980 ANKHO
International
Inc.-0361-9230/80/010017-05$01.00/O
IX
VANDERWEEII
glucose levels were, however, not reported. Both studies agree that glucagon reduced feeding, however, increased feeding should have resulted from the hormonal ratio favoring fuel production. In previous work, we had found that the hepatic-portal administration of pancreatic glucagon also had suppressive effects upon food intake in undeprived rabbits [9]. We had hypothesized that glucagon might be released upon feeding or contact of the gastrointestinal mucosa with significant amounts of carbohydrates (perhaps, specifically glucose) and, thus produce glycogenolysis as a significant portion of the rapid rise in blood glucose following a meal. This rapid rise in glycemia could participate in the induction of satiety. Our work showed that glucagon’s suppressive effect upon feeding occurs only when glycogen levels in the liver are replete. Thus, glucagon does not appear to act as a satiety signal itself, as its detection by receptors would be expected to produce a suppression in feeding independent of liver glycogen stores. It is more likely that the suppression is due to increased hepatic glucose efflux, an increase in blood insulin and glucose levels or consequent effects of this combination of events, as glucagon is glycogenolytic, insulinogenic, and gluconeogenic. The current experiment attempts to further investigate the relationship between glucagon, liver glycogen levels, and their effect upon feeding behavior. The first study was an extension of previous experiments, with animals receiving glucagon injections following 3-hr food deprivations. It was hypothesized that, relative to saline-injected controls, glucagon would suppress feeding. In the second study, glucagon was assessed for possible enhancement of feeding. This is a relevant direction for investigation, as glucagon commonly is released in highest concentration during increased fasting and decreased glycemia, a state marked by increased feeding when food becomes available. To induce this state, rats were fasted for 3 hr, injected with glucagon, and fasted an additional 3 hr to allow glycemic levels to decline. It was hypothesized that these manipulations would induce a more severe depletion of liver glycogen stores than similarly deprived, saline-injected animals, resulting in increased food ingestion. METHOD
Eighty adult male Holtzman rats of the Sprague-Dawley strain weighing between 393 and 589 g at the outset of the experiment were housed in a room lighted from 0600 to 1800 daily.
Acquisition
Each rat was placed in an operant chamber following 4 hr of food deprivation and magazine trained with l&20 Noyes food pellets (regular rodent formula). Each animal was left in the chamber over night and conditioned to press a lever for food. Once an animal had learned to spontaneously press the lever, it was given 3-daily 30-min sessions of CRF (one press-one pellet) in the operant chamber. This was the only food made available for those 3 days. By the end of the third session, all the animals were. pressing the lever at an average rate of 452 times per hour. Animals were then maintained on ad lib food (Purina chow) and water until initial body weights were recovered. at which time testing procedures were begun.
FY ‘41.
The animals were food deprived at 0800, and at 1100were injected intraperitoneally with either saline (0.9%) or 0.35 mg/kg BW pancreatic glucagon (Calbiochem, La Jolla, CA) dissolved in isotonic saline. Volumes injected remained equal across groups. Both saline and glucagon injected animals were assigned to either a short or long-term deprived condition. In the short-term condition, an animal was placed in the operant chamber 15 min following injection, whereas in the long-term condition, an animal was placed in the chamber 3 hr and I5 min after injection. Thus, including the 3-hr pre-injection deprivation period, animals in the short-term condition were deprived of food for a total of 3 hr and I5 min and, in the long-term condition, a total of 6 hr and IS min. Once in the operant chamber, the feeding behavior of animals in both conditions was recorded for 3 hr. Feeding behaviors recorded were initial latency to eat. MS and IMI for all meals occurring during the 3 hr, and total 3 hr ingestion. The initial latency was defined as the interval of time between the introduction of the animal into the operant chamber and the initial lever press of the first meal. MS definition was IO lever presses and the minimum IMI was IO min. The total number of lever presses during a given meal was recorded as MS, and the time between the last level press of a meal and the first lever press of the following meal was recorded as the IMI. Total intake was the total numher of presses during the 3-hr period. After the testing procedure. Ihe animals were immediately administered sodium pentobarbital (Nembutat), and blood samples were drawn by cardiac puncture for assay of glucose and insulin levels. RESULTS
The administration of exogenous pancreatic glucagon did not significantly alter the total 3-hr food intake when injected following a 3-hr fast. However, glucagon administration was found to significantly suppress food intake occurring after the first meal (compared to saline administration, ~~0.05). To account for this, 60% of the animals injected with glucagon failed to consume a second meal as compared to only 15% of the saline controls. Chi square performed on this difference was also significant (x2=8.64. p
Depri~wtiorl
The administration of exogenous glucagon in the middle of a 6-hr food deprivation increased, but not signiticantly. total 3-hr food intake following the fast and injections (compared to saline administration, p
In addition
to all of the animals
which were tested
behav-
GLUCAGON
EFFECTS
19
ON BLOOD, LIVER, AND FEEDING iorally
g
200
2 180 ;
160
3
140
ii 120 r 100 n
G
S
G
S
6hr Dep.
3hr Dep.
FIG. 1. Average meal size and total food intake over 3 hr in rats injected with glucagon (G) or saline (S). Each lever press produced a 0.045 g pellet of food and error bars represent the SEM. The only significant difference obtained was in 6-hr food-deprived subjects; glucagon-injected animals consumed significantly larger first meals than saline-injected animals @<0.05).
for food
intake
and then
sacrificed
for biochemical
analyses, an additional 50 animals were not tested behaviorally but assessed for glycemic and liver glycogen responses and insulin levels in response to food deprivations and drug injections. All animals were tested on exactly the same time schedule as animals assessed behaviorally and food was administered or withheld according to that regimen. Different groups of subjects were sampled for blood at introduction to the chambers (1115 for short-deprived and 1415 for long-deprived subjects), at 18 min later (half of the median initial IMI for short-deprived, saline-injected animals), and at the conclusion of behavioral testing. In addition, all subjects that were sacrificed at the normal time for the initiation of feeding (1115 for short-deprived and 1415 for long-deprived subjects) were decapitated and liver samples were removed for analysis of liver glycogen. Liver glycogen was assessed after KOH hydrolysis and blood glucose and insulin levels were assessed using the glucose oxidase enzymatic method in a Beckman glucose analyzer for glucose and a single-antibody, charcoal-separation method utilizing a rat-insulin standard for insulin. Glucagon produced a significant rise in glycemia associated with eating in short-deprived subjects but receding before testing in long-deprived subjects (see Table 1). At the initiation of behavioral testing, liver glycogen was decreased in both sets of glucagon-injected subjects, though more significantly in the short-deprived subjects. Surprisingly, glucose levels were not significantly elevated before feeding in short-deprived, glucagon-injected subjects. Insulin levels were never significantly different between saline and
TABLE
1
BIOCHEMICAL ANALYSES FOR ALL ANIMALS SUBJECTED TO FOOD DEPRIVATIONS AND GLUCAGON OR CONTROL SALINE INJECTIONS. ALL VALUES REPORTED ARE MEANS k SEM AND STATISTICAL COMPARISONS WERE MADE BETWEEN CONTROL AND DRUG CONDITIONS ONLY
Time:
1115
1133
1415
1715
Blood glucose (BG-mg%) and insulin levels (I-~U/ml) Short-term food deprivation
Saline injected
105.4 22.3 BG
158.1 24.6 BG
154.8 +5.9 BG
1115
1415
Liver glycogen (mg/g tissue)
-
9.7 20.9
-
-
8.0* -co.3
-
88.6 2 15.3 I Glucagon injected
105.1 ? 1.3 BG
173.0* 28.2 BG
160.4 k8.3 BG 76.8 29.8 I
Long-term food deprivation
Saline injected
118.9 +3.9 BG
92.1 k4.2 BG
182.0 k10.3 BG
-
5.3 21.0
70.1 513.1 I Glucagon injected
-
177.0* +6.0 BG
104.7 +3.1
164.3 +8.1 BG
-
3.61 kO.8
75.6 k9.5 I All animals were food deprived at 0800, short-deprived subjects were refed at 1115 and long-deprived at 1415 Glucagon or saline was injected for all subjects at 1100 (35 mg/kg BW). An * indicates different from saline at pCO.05 while t indicates different from saline at p
VANDEKWEELR
3,G.MP
t IMI
+
3.G.3.
tMS
MP
FIG. 2. Schematic representation of the methods and results of the present study where numbers indicate the hours of food deprivation, g indicates glucagon administration, mp indicates meal patterning (the dependent variable), and IMI (intermeal interval) and MS (meal size) the results. Stipping represents the glycemic stores; glycogen in the liver and glucose in the blood stream. In summary, glucagon’s immediate effects were to raise glycemic levels through glycogenolysis prolonging the period of non-feeding following a meal while with the addition of a second 3-hr deprivation period following the administration of glucagon, a significant increase in meal size was zeen.
glucagon-injected groups regardless of the amount of food deprivation, however, insulin levels were only assessed in animals which had fed for 3 hr and this may have attenuated the reputed insulin stimulation following glucagon administration. DISCUSSION This study has shown that exogenous glucagon can be used to both limit and enhance food intake. Effects upon food ingestion following glucagon is dependent upon nutritional reserves and glycemic levels. When liver glycogen stores are replete (after food deprivations of less than 8 hr in the rat), glucagon can affect feeding. This is consistent with our earlier findings [9], however, the glucagon enhancement of feeding represents a new phenomenon, not previously reported. While Woods PI trl. [IO] argue that lipolysis and gluconeogenesis should correlate with food deprivation and, hence, increased ingestion, most studies with glucagon have reported decreased food intake [2,4.7,9]. This is most likely attributable to the methods used as animals are assessed for feeding immediately following injections of glucagon or while under the influence of long-acting zinc glucagon. Glucagon’s immediate hyperglycemic effects through liver glycogenolysis might well explain this decreased ingestion; however, if animals continue to undergo food deprivation, glycemic levels should decline. These long-deprived animals with glucagon injections should have normal glycemic levels but depleted liver glycogen stores and, hence, should show increased feeding if this signal plays a role in the regulation of ingestion. This was exactly the effects produced in the
b:7 Al.
present study. When rats were mjecterl with glucagon and immediately tested for feeding, a suppression of food intake appeared but when subjects were fasted for an additional period of time to allow normalization of glycemia with continued glycogen depletion, an enhancement of eating was witnessed. The immediate effects of exogenous glucagon appeared to be a prolonging of the IMI following the initial postinjection meal (consistent with the report of Balagura c’/ t/l. 121). Glucagon appears to prolong satiety by making the same amount of ingestion last longer. Meal size was not significantly different between saline and glucagon-injected animals, however. fewer glucagon-injected subjects consumed a second meal within the 3-hr period following the injections, if tested immediately. When glucagon injections were given during the middle of a longer food deprivation period (6-hr group), an increase in the initial MS alone was seen. These results suggest that in the liver glycogendepleted animal with declining glycemic levels. food may hc less satiating. Glucagon’s sole effect appears indirect upon food intake proceeding from its glycogenolytic :md gluconeogenic capacity. By heightening glucose levels from glycogenolysis, glucagon may have a short-term suppressive effect, enhancing the satiety produced from ingesta (similar to Brobeck’s hypothesis 131). The cpecific effect of decreasing liver glycogen through glucagon injections. however. appears to be an increase in the amount of food consumed during the meal following the injection (see schematic summary in Fig. 2). It seems clear that this hormonal system does influence food intake in a manner consistent with the glucostatic theories of feeding regulation. The paradox apparent in the literature (glucagon produces satiety while insulin has been used to promote food intake: (2. 1. 5. 61) is not really a paradox. The hormones can correlate behaviorally with the physiological state inducing their secretion. if it is recognized that they are part of a sequence of signals regulating food ingestion. Glucagon has been studied when food is made available and animals are likely to cat while insulin has been administered in large dosages or in long-acting forms such that it will continue to be available when ingestion has been reduced. Our present results seem most consistent with those ot Balagura Ed N/. [2] rather than those of decastro PI ctl. 141. We noted a lengthening of the IMI immediately following glucagon administration without an effect upon MS. At this time. blood glucose levels were elevated in glucagon-injected animals as compared to saline-treated subjects ( 173.0 +- 8.2 mg% to 158. I 7 4.6 mg% ( respectively). as predicted but not reported by both groups. MS was. however. markedly increased if the hyperglycemic action of glucagon was ailowcd to abate with liver glycogen remaining in a depleted state. When liver glycogen levels averaged 3.6% wet tissue weight as compared to 5.3% for saline-injected controls. MS WRS increased by over 60%. It is interesting to speculate that glucagon administered portally can suppress food intake in free-feeding animals because glucagon raises glycemia iI\ well as stimulates insulin secretion. The present report. however, did not show any significant differences in insulin levels between saline and glucagon-injected animals: insulin levels were normal free-feeding titres in both injection conditions. It appears that glucagon can be used both to encourage oi limit short-term food intake. While the administration of glucagon alters short-term food ingestion, caloric balance is largely maintained as total 3-hr food intakes following both
GLUCAGON
EFFECTS
ON BLOOD, LIVER, AND FEEDING
food deprivation conditions are not significantly different from that following saline injection. Hence, shifts in nutrient
I. Assan, R. and J. Delaunay. Comparative biological activity of standard glucagon and various long acting preparations. Path. Biol. Paris 20: 979-984, 1972. 2. Balagura, S., M. Kanner and L. E. Harrell. Modification of feeding patterns by glucodynamic hormones. Behav. Bid/. 13: 457-465, 1975. 3. Brobeck, J. R. Nature of satiety signals. Am. 1. din. Nutr. 28: 806-807, 1975. 4. decastro, J., S. Paullin and G. DeLugas. Insuhn and glucagon as determinants of body weight set point and microregulation in rats. .I. comg. physiol. Psycho/. 92: 571-579, 1978. 5. Hoebel, B. G. and P. Teitelbaum. Weight regulation in normal and hypothalamic hyperphagic rats. J. camp. physiol. Psycho/. 61: 189-193, 1966.
21
stores alters short-term feeding but if no excess or decrement in energy intake occurs, long-term regulation is maintained.
6. MacKay, E. M., J. W. Callaway and R. H. Barnes. Hyperalimentation in normal animals produced by protamine insulin. J. Nutr. 20: 5966, 1940. 7. Martin, J. R., D. Novin and D. A. VanderWeele. Loss of glucagon suppression of feeding after vagotomy in rats. Am. J. Physioi. 234: E314-E318, 1978. 8. Unger, R. H. and P. J. Lefebvre. Glucagon: ~uiecu~ur Physiology, Ctinicnl und Therapeutic Irnp~~~~lti~~s.New York: Pergamon, 1972. 9. VanderWeele, D. A., P. J. Geiselman and D. Novin. Pancreatic glucagon, food deprivation and feeding in intact and vagotomized rabbits. Physiol. Behav. 23: 155-158, 1979. 10. Woods, S. C., E. Decke and J. R. Vasselli. Metabolic hormones and regulation of body weight. P.F)‘c.~~J/.Rev. 81: 2-3.1974.
Pancreatic Glucagon Administration, Feeding, Glycemia, and Liver Glycogen in Rats’ DENNIS
A. VANDERWEELE2
Division of Behuvioral Neurology,
AND EDWARD
HARACZKIEWICZ
New York University, School of Medicine,
New York, NY 10016
AND MICHAEL
Department
VANDERWEELE,
of Psychology,
D. A., E. HARACZKIEWICZ
A. DICONTI
Occidental College, Los Angeles,
AND M. A. DICONTI.
Pancreatic
g/wagon
CA 90041
adminisfration,
feeding,
g/yccJmicl.and liver ,q/~~c.ogc’n in rats. BRAIN RES. BULL. 5: Suppl. 4, 17-21, 1980.-Rats
were trained to bar press for Noyes pellets. Once trained, subjects were food deprived at 0800, injected with pancreatic glucagon or isotonic saline (0.35 mgikg BW, intraperitoneally in a volume of 0.1 cc per 300 g BW) at 1100, and then placed in the operant chamber at 1115 or 1415 for a duration of 3 hr. Animals placed immediately in the chamber showed a suppression of food intake accomplished by a lengthening of the interval following the first meal rather than a reduction in the size of that meal. Animals tested 3 hr post-injection showed an enhancement of feeding by increasing the size of the first meal compared to saline-injected subjects, ~~0.025. The behavioral effects of glucagon upon feeding depend on the point of the sequence of responses to the hormone during which the animal is studied. Early after glucagon administration, hyperglycemia and increased glucose utilization predominate and feeding was reduced; subsequently, blood glucose may be normal while the liver remains somewhat glycogen depleted and feeding is enhanced. Blood glucose levels Food intake Pancreatic glucagon Rat Short-term food intake regulation
A HIGH molar ratio of insulin: glucagon favors the storage or utilization of ingested nutrients while a very low ratio favors gluconeogenesis and metabolic fuel production [8]. It seems that alterations of this hormonal ratio might also alter feeding behavior, and studies are now appearing which attempt to assess the effects of manipulation of absolute titres of the hormones on ingestion and weight gain (see [4] and [lo] for summaries of these studies). Alternatively to the insu1in:glucagon ratio hypothesis, a proposal that the insu1in:growth hormone ratio might be influential upon feeding behavior has been forwarded [lo]. The important point stressed by each theory, however, is that nutrient flux should influence food ingestion and the present study attempts to assess this point. Recently, two studies demonstrated that a rise in glucagon levels which lowered the insulin:glucagon ratio and altered nutrient flux, also affected food ingestion [2,4]. The initial study reported that the administration of 0.2 mg glucagon to rats during an intermeal interval (IMI) produced a delay in the onset of the next meal with no effect upon the
Liver glycogen levels
Food deprivation
size of that meal. The increased IMI was attributed to an elevation of blood glucose, however, blood glucose levels were not reported. Insulin, on the other hand, was shown to hasten the onset of a subsequent meal when administered during the IMI. One methodological problem with this study was that the animals with the shortest baseline IMIs were assigned to the glucagon-injected group which may have predisposed this group to longer IMIs when injected. The second study administered glucagon over three consecutive days in rats and found that the hormone’s sole effect upon food intake was a reduction in meal size (MS). In this second study [4], 0.75 mg of zinc glucagon was administered over a 24 hr period via 3 subcutaneous injections and this procedure resulted in a 40% reduction in average MS and a depression of body weight gain. The authors suggest that less was eaten because of glucagon’s enhancement of glucose availability. As the zinc form of glucagon has a prolonged activity relative to simple glucagon [l], it seems quite likely that the rats were in a continuous hyperglycemic state and, therefore, reduced feeding. Blood
IThis work was supported by Grant AM-17259 (DAVW) and a Richter Foundation award to Occidental College (DAVW and MAD). We are greatly indebted to Drs. Theodore B. Van Itallie for ideas and support during this research and editorial assistance and F. Xavier Pi-Sunyer and his staff at the Obesity Research Center, St. Luke’s Hospital, Columbia University for the biochemical determinations of blood glucose and insulin levels. ‘Present address and address for reprints: Dr. Dennis A. VanderWeele, Psychology Department, Occidental College, Los Angeles, CA 90041.
Copyright
0 1980 ANKHO
International
Inc.-0361-9230/80/010017-05$01.00/O
IX
VANDERWEEII
glucose levels were, however, not reported. Both studies agree that glucagon reduced feeding, however, increased feeding should have resulted from the hormonal ratio favoring fuel production. In previous work, we had found that the hepatic-portal administration of pancreatic glucagon also had suppressive effects upon food intake in undeprived rabbits [9]. We had hypothesized that glucagon might be released upon feeding or contact of the gastrointestinal mucosa with significant amounts of carbohydrates (perhaps, specifically glucose) and, thus produce glycogenolysis as a significant portion of the rapid rise in blood glucose following a meal. This rapid rise in glycemia could participate in the induction of satiety. Our work showed that glucagon’s suppressive effect upon feeding occurs only when glycogen levels in the liver are replete. Thus, glucagon does not appear to act as a satiety signal itself, as its detection by receptors would be expected to produce a suppression in feeding independent of liver glycogen stores. It is more likely that the suppression is due to increased hepatic glucose efflux, an increase in blood insulin and glucose levels or consequent effects of this combination of events, as glucagon is glycogenolytic, insulinogenic, and gluconeogenic. The current experiment attempts to further investigate the relationship between glucagon, liver glycogen levels, and their effect upon feeding behavior. The first study was an extension of previous experiments, with animals receiving glucagon injections following 3-hr food deprivations. It was hypothesized that, relative to saline-injected controls, glucagon would suppress feeding. In the second study, glucagon was assessed for possible enhancement of feeding. This is a relevant direction for investigation, as glucagon commonly is released in highest concentration during increased fasting and decreased glycemia, a state marked by increased feeding when food becomes available. To induce this state, rats were fasted for 3 hr, injected with glucagon, and fasted an additional 3 hr to allow glycemic levels to decline. It was hypothesized that these manipulations would induce a more severe depletion of liver glycogen stores than similarly deprived, saline-injected animals, resulting in increased food ingestion. METHOD
Eighty adult male Holtzman rats of the Sprague-Dawley strain weighing between 393 and 589 g at the outset of the experiment were housed in a room lighted from 0600 to 1800 daily.
Acquisition
Each rat was placed in an operant chamber following 4 hr of food deprivation and magazine trained with l&20 Noyes food pellets (regular rodent formula). Each animal was left in the chamber over night and conditioned to press a lever for food. Once an animal had learned to spontaneously press the lever, it was given 3-daily 30-min sessions of CRF (one press-one pellet) in the operant chamber. This was the only food made available for those 3 days. By the end of the third session, all the animals were. pressing the lever at an average rate of 452 times per hour. Animals were then maintained on ad lib food (Purina chow) and water until initial body weights were recovered. at which time testing procedures were begun.
FY ‘41.
The animals were food deprived at 0800, and at 1100were injected intraperitoneally with either saline (0.9%) or 0.35 mg/kg BW pancreatic glucagon (Calbiochem, La Jolla, CA) dissolved in isotonic saline. Volumes injected remained equal across groups. Both saline and glucagon injected animals were assigned to either a short or long-term deprived condition. In the short-term condition, an animal was placed in the operant chamber 15 min following injection, whereas in the long-term condition, an animal was placed in the chamber 3 hr and I5 min after injection. Thus, including the 3-hr pre-injection deprivation period, animals in the short-term condition were deprived of food for a total of 3 hr and I5 min and, in the long-term condition, a total of 6 hr and IS min. Once in the operant chamber, the feeding behavior of animals in both conditions was recorded for 3 hr. Feeding behaviors recorded were initial latency to eat. MS and IMI for all meals occurring during the 3 hr, and total 3 hr ingestion. The initial latency was defined as the interval of time between the introduction of the animal into the operant chamber and the initial lever press of the first meal. MS definition was IO lever presses and the minimum IMI was IO min. The total number of lever presses during a given meal was recorded as MS, and the time between the last level press of a meal and the first lever press of the following meal was recorded as the IMI. Total intake was the total numher of presses during the 3-hr period. After the testing procedure. Ihe animals were immediately administered sodium pentobarbital (Nembutat), and blood samples were drawn by cardiac puncture for assay of glucose and insulin levels. RESULTS
The administration of exogenous pancreatic glucagon did not significantly alter the total 3-hr food intake when injected following a 3-hr fast. However, glucagon administration was found to significantly suppress food intake occurring after the first meal (compared to saline administration, ~~0.05). To account for this, 60% of the animals injected with glucagon failed to consume a second meal as compared to only 15% of the saline controls. Chi square performed on this difference was also significant (x2=8.64. p
Depri~wtiorl
The administration of exogenous glucagon in the middle of a 6-hr food deprivation increased, but not signiticantly. total 3-hr food intake following the fast and injections (compared to saline administration, p
In addition
to all of the animals
which were tested
behav-
GLUCAGON
EFFECTS
19
ON BLOOD, LIVER, AND FEEDING iorally
g
200
2 180 ;
160
3
140
ii 120 r 100 n
G
S
G
S
6hr Dep.
3hr Dep.
FIG. 1. Average meal size and total food intake over 3 hr in rats injected with glucagon (G) or saline (S). Each lever press produced a 0.045 g pellet of food and error bars represent the SEM. The only significant difference obtained was in 6-hr food-deprived subjects; glucagon-injected animals consumed significantly larger first meals than saline-injected animals @<0.05).
for food
intake
and then
sacrificed
for biochemical
analyses, an additional 50 animals were not tested behaviorally but assessed for glycemic and liver glycogen responses and insulin levels in response to food deprivations and drug injections. All animals were tested on exactly the same time schedule as animals assessed behaviorally and food was administered or withheld according to that regimen. Different groups of subjects were sampled for blood at introduction to the chambers (1115 for short-deprived and 1415 for long-deprived subjects), at 18 min later (half of the median initial IMI for short-deprived, saline-injected animals), and at the conclusion of behavioral testing. In addition, all subjects that were sacrificed at the normal time for the initiation of feeding (1115 for short-deprived and 1415 for long-deprived subjects) were decapitated and liver samples were removed for analysis of liver glycogen. Liver glycogen was assessed after KOH hydrolysis and blood glucose and insulin levels were assessed using the glucose oxidase enzymatic method in a Beckman glucose analyzer for glucose and a single-antibody, charcoal-separation method utilizing a rat-insulin standard for insulin. Glucagon produced a significant rise in glycemia associated with eating in short-deprived subjects but receding before testing in long-deprived subjects (see Table 1). At the initiation of behavioral testing, liver glycogen was decreased in both sets of glucagon-injected subjects, though more significantly in the short-deprived subjects. Surprisingly, glucose levels were not significantly elevated before feeding in short-deprived, glucagon-injected subjects. Insulin levels were never significantly different between saline and
TABLE
1
BIOCHEMICAL ANALYSES FOR ALL ANIMALS SUBJECTED TO FOOD DEPRIVATIONS AND GLUCAGON OR CONTROL SALINE INJECTIONS. ALL VALUES REPORTED ARE MEANS k SEM AND STATISTICAL COMPARISONS WERE MADE BETWEEN CONTROL AND DRUG CONDITIONS ONLY
Time:
1115
1133
1415
1715
Blood glucose (BG-mg%) and insulin levels (I-~U/ml) Short-term food deprivation
Saline injected
105.4 22.3 BG
158.1 24.6 BG
154.8 +5.9 BG
1115
1415
Liver glycogen (mg/g tissue)
-
9.7 20.9
-
-
8.0* -co.3
-
88.6 2 15.3 I Glucagon injected
105.1 ? 1.3 BG
173.0* 28.2 BG
160.4 k8.3 BG 76.8 29.8 I
Long-term food deprivation
Saline injected
118.9 +3.9 BG
92.1 k4.2 BG
182.0 k10.3 BG
-
5.3 21.0
70.1 513.1 I Glucagon injected
-
177.0* +6.0 BG
104.7 +3.1
164.3 +8.1 BG
-
3.61 kO.8
75.6 k9.5 I All animals were food deprived at 0800, short-deprived subjects were refed at 1115 and long-deprived at 1415 Glucagon or saline was injected for all subjects at 1100 (35 mg/kg BW). An * indicates different from saline at pCO.05 while t indicates different from saline at p
VANDEKWEELR
3,G.MP
t IMI
+
3.G.3.
tMS
MP
FIG. 2. Schematic representation of the methods and results of the present study where numbers indicate the hours of food deprivation, g indicates glucagon administration, mp indicates meal patterning (the dependent variable), and IMI (intermeal interval) and MS (meal size) the results. Stipping represents the glycemic stores; glycogen in the liver and glucose in the blood stream. In summary, glucagon’s immediate effects were to raise glycemic levels through glycogenolysis prolonging the period of non-feeding following a meal while with the addition of a second 3-hr deprivation period following the administration of glucagon, a significant increase in meal size was zeen.
glucagon-injected groups regardless of the amount of food deprivation, however, insulin levels were only assessed in animals which had fed for 3 hr and this may have attenuated the reputed insulin stimulation following glucagon administration. DISCUSSION This study has shown that exogenous glucagon can be used to both limit and enhance food intake. Effects upon food ingestion following glucagon is dependent upon nutritional reserves and glycemic levels. When liver glycogen stores are replete (after food deprivations of less than 8 hr in the rat), glucagon can affect feeding. This is consistent with our earlier findings [9], however, the glucagon enhancement of feeding represents a new phenomenon, not previously reported. While Woods PI trl. [IO] argue that lipolysis and gluconeogenesis should correlate with food deprivation and, hence, increased ingestion, most studies with glucagon have reported decreased food intake [2,4.7,9]. This is most likely attributable to the methods used as animals are assessed for feeding immediately following injections of glucagon or while under the influence of long-acting zinc glucagon. Glucagon’s immediate hyperglycemic effects through liver glycogenolysis might well explain this decreased ingestion; however, if animals continue to undergo food deprivation, glycemic levels should decline. These long-deprived animals with glucagon injections should have normal glycemic levels but depleted liver glycogen stores and, hence, should show increased feeding if this signal plays a role in the regulation of ingestion. This was exactly the effects produced in the
b:7 Al.
present study. When rats were mjecterl with glucagon and immediately tested for feeding, a suppression of food intake appeared but when subjects were fasted for an additional period of time to allow normalization of glycemia with continued glycogen depletion, an enhancement of eating was witnessed. The immediate effects of exogenous glucagon appeared to be a prolonging of the IMI following the initial postinjection meal (consistent with the report of Balagura c’/ t/l. 121). Glucagon appears to prolong satiety by making the same amount of ingestion last longer. Meal size was not significantly different between saline and glucagon-injected animals, however. fewer glucagon-injected subjects consumed a second meal within the 3-hr period following the injections, if tested immediately. When glucagon injections were given during the middle of a longer food deprivation period (6-hr group), an increase in the initial MS alone was seen. These results suggest that in the liver glycogendepleted animal with declining glycemic levels. food may hc less satiating. Glucagon’s sole effect appears indirect upon food intake proceeding from its glycogenolytic :md gluconeogenic capacity. By heightening glucose levels from glycogenolysis, glucagon may have a short-term suppressive effect, enhancing the satiety produced from ingesta (similar to Brobeck’s hypothesis 131). The cpecific effect of decreasing liver glycogen through glucagon injections. however. appears to be an increase in the amount of food consumed during the meal following the injection (see schematic summary in Fig. 2). It seems clear that this hormonal system does influence food intake in a manner consistent with the glucostatic theories of feeding regulation. The paradox apparent in the literature (glucagon produces satiety while insulin has been used to promote food intake: (2. 1. 5. 61) is not really a paradox. The hormones can correlate behaviorally with the physiological state inducing their secretion. if it is recognized that they are part of a sequence of signals regulating food ingestion. Glucagon has been studied when food is made available and animals are likely to cat while insulin has been administered in large dosages or in long-acting forms such that it will continue to be available when ingestion has been reduced. Our present results seem most consistent with those ot Balagura Ed N/. [2] rather than those of decastro PI ctl. 141. We noted a lengthening of the IMI immediately following glucagon administration without an effect upon MS. At this time. blood glucose levels were elevated in glucagon-injected animals as compared to saline-treated subjects ( 173.0 +- 8.2 mg% to 158. I 7 4.6 mg% ( respectively). as predicted but not reported by both groups. MS was. however. markedly increased if the hyperglycemic action of glucagon was ailowcd to abate with liver glycogen remaining in a depleted state. When liver glycogen levels averaged 3.6% wet tissue weight as compared to 5.3% for saline-injected controls. MS WRS increased by over 60%. It is interesting to speculate that glucagon administered portally can suppress food intake in free-feeding animals because glucagon raises glycemia iI\ well as stimulates insulin secretion. The present report. however, did not show any significant differences in insulin levels between saline and glucagon-injected animals: insulin levels were normal free-feeding titres in both injection conditions. It appears that glucagon can be used both to encourage oi limit short-term food intake. While the administration of glucagon alters short-term food ingestion, caloric balance is largely maintained as total 3-hr food intakes following both
GLUCAGON
EFFECTS
ON BLOOD, LIVER, AND FEEDING
food deprivation conditions are not significantly different from that following saline injection. Hence, shifts in nutrient
I. Assan, R. and J. Delaunay. Comparative biological activity of standard glucagon and various long acting preparations. Path. Biol. Paris 20: 979-984, 1972. 2. Balagura, S., M. Kanner and L. E. Harrell. Modification of feeding patterns by glucodynamic hormones. Behav. Bid/. 13: 457-465, 1975. 3. Brobeck, J. R. Nature of satiety signals. Am. 1. din. Nutr. 28: 806-807, 1975. 4. decastro, J., S. Paullin and G. DeLugas. Insuhn and glucagon as determinants of body weight set point and microregulation in rats. .I. comg. physiol. Psycho/. 92: 571-579, 1978. 5. Hoebel, B. G. and P. Teitelbaum. Weight regulation in normal and hypothalamic hyperphagic rats. J. camp. physiol. Psycho/. 61: 189-193, 1966.
21
stores alters short-term feeding but if no excess or decrement in energy intake occurs, long-term regulation is maintained.
6. MacKay, E. M., J. W. Callaway and R. H. Barnes. Hyperalimentation in normal animals produced by protamine insulin. J. Nutr. 20: 5966, 1940. 7. Martin, J. R., D. Novin and D. A. VanderWeele. Loss of glucagon suppression of feeding after vagotomy in rats. Am. J. Physioi. 234: E314-E318, 1978. 8. Unger, R. H. and P. J. Lefebvre. Glucagon: ~uiecu~ur Physiology, Ctinicnl und Therapeutic Irnp~~~~lti~~s.New York: Pergamon, 1972. 9. VanderWeele, D. A., P. J. Geiselman and D. Novin. Pancreatic glucagon, food deprivation and feeding in intact and vagotomized rabbits. Physiol. Behav. 23: 155-158, 1979. 10. Woods, S. C., E. Decke and J. R. Vasselli. Metabolic hormones and regulation of body weight. P.F)‘c.~~J/.Rev. 81: 2-3.1974.