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Food intake of rats administered with glycerol
Physiology &Behavior,Vol. 25, pp. 621-626. Pergamon Press and Brain Research Publ., 1980. Primed in the U.S.A.
Food Intake of Rats Administered with Glycerol ZVI GLICK 1
Department o f Nutrition, Hebrew University-Hadassah, Medical School, Jerusalem, Israel R e c e i v e d 21 M a r c h 1980 GLICK. Z. Food intake of rats administered with glycerol. PHYSIOL. BEHAV. 25(5) 621-626, 1980.--The effect of glycerol on rats food intake was determined when it was administered either via bolus gastric intubation, or by a continuous 24 hr infusion into the aorta. Both of these treatments resulted in a suppression of the 24 hr food consumption to an extent which was greater than that accounted for by the caloric value of the administered metabolite. Gastric loading with urea solutions equiosmotic to glycerol or with glucose solutions equicaloric to glycerol were less effective than glycerol in reducing the 24 hr food intake. The time course effect on food intake varied between gastric loading with glycerol and with equicaloric glucose solutions, with the former usually exerting a more delayed and longer lasting effect. Continuous intraaortal infusions of glycerol were more effective than glucose solutions in suppressing 24 hr food intake even though the latter had twice the caloric value of the former. Our data suggest that the action of glycerol on food intake is not mediated through its conversion to glucose. The possibility that glycerol may participate in a lipostatic control mechanism of food intake is discussed. Glycerol
Food intake
Energy balance
using this compound as an organic solvent c a r d e r for an anorectic drug tested in rats (Glick 1975, unpublished data). In this experiment, it was observed that the glycerol "control" was in itself a food intake suppressor. Therefore, the present study was undertaken to quantify the effect of glycerol on food intake of rats in relation to the administered dose, the diurnal phase of the alimentary cycle, and the route of its administration.
T H E hypothesis that body weight of mammals is regulated via the control of fat mass (lipostatic theory) proposed by Kennedy in 1953 [7] is still widely accepted. However, the physiologic monitoring system of adiposity and its relation to the mechanism which controls food intake is very little understood. Recent data from experiments with lipectomized rats suggest that the regulated factor in the lipostatic control of food intake is fat cell size rather than total body fat [4,8]. As a positive correlation has been found to exist between adipose cell size and the rate of glycerol release [3,13], glycerol might play a role in a monitoring system of adipose tissue size, and the control mechanism of food intake. Teleological reasoning for such a role for glycerol was made by Bray and Campfield [2]. More recently, Wirtschafter and Davis [21] presented evidence which strongly supports this suggestion. They have shown that subcutaneous injections into rats of small quantities of glycerol resulted in a suppression of daily food intake and in the maintenance of energy balance at a lower level of body weight in comparison with the injection of an equivalent amount of glucose. Termination of the glycerol treatment was followed by a return of body weight to normal level. Earlier data also showed that relatively small loads of glycerol (< 1 gm) administered subcutaneously or intraperitoneally into rats [ 16] or intraportally into rabbits [17] suppressed the daily food intake beyond its energy contribution. Intragastric loads of a similar order of magnitude were on the other hand without such an effect [1]. Our interest in glycerol effect on food intake began when
METHOD
Animals, Diet and Facilities A total of 81 female (virgin) rats of a Wistar derived Hebrew University strain, weighing 170 to 200 g were tested in this study. The rats were caged individually in rooms maintained at 24__2°C. The diet was standard chow pellets (Amba Ltd., Hedera, Israel) containing 3.6 kcal metabolizable energy per gram. Food intake corrected for spillage was weighed to the nearest 0.1 g. E X P E R I M E N T 1: E F F E C T O F I N T R A G A S T R I C A L L Y A D M I N I S T E R E D G L Y C E R O L ON F O O D I N T A K E Since in the rat the responsiveness of food intake to the same metabolic stimuli varies between day and night [9] the effect of glycerol on food intake was measured separately following its intubation before the dark (reversed cycle) and before the light (normal cycle) phases of the diurnal alimentary cycle.
1Present address: Dr. Zvi Glick, Division of Endocrinology, Harbor General Hospital-UCLA Campus, 1000 W. Carson Street, Torrance, CA 90509.
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FIG. 1. Food intake of rats maintained on a reversed lighting cycle by test solution and by time following gastric intubation (UE=urea solution equiosmotic to glycerol; GY=glycerol; GL=glucose; UR=urea solution equiosmotic to glucose; Low dose: 2.5 ml of a 40% glucose or glycerol or their respective equiosmotic solutions. High dose: 5 ml of these same solutions. *p<0.05, **p<0.01; ***p<0.001; one tail t-test.
REVERSEDCYCLE(DARKPHASE) Method
Rats were kept on a reversed lighting cycle for two weeks prior to the experiment (10 a.m. to 10 p.m. dark; 10 p.m. to 10 a.m. light). Sham intubations were started 5 days before the experiment and consisted of single dally intubations without injections for 3 days, followed by two injections of 2.5 ml water for two additional days. Various test solutions were similarly administered in the days which followed. For this purpose, the rats were divided into 4 groups and each was given one of the following solutions: 40% glycerol (GY; n=12), 40% glucose (GL; n=12), also 26.4% urea (UE; n=12) and 13.2% urea (UR; n=6), the latter two were equiosmotic to the glycerol and the glucose test solutions respectively. These solutions were administered in a dally dose of 2.5 ml (low dose) for three days, followed by 5 ml (high dose) for three additional days. The rats were then untreated for two days, during which time food intake was not measured and then received 5 ml (high dose) daily for 3 additional days. Gastric intubations were done with the aid o f a 5 mi syringe and a 2 inch 18 gauge blunt needle on which end a soft plastic tubing was mounted, protruding by about 2
to 3 mm, in order to soften the passage of the needle towards the stomach. All intubations were made within the last 30 minutes before the beginning of the dark cycle (9:30 to 10:00 a.m.) and the order of intubation among the groups was changed each day. Preweighed food was offered at 10 a.m. Food intake was weighed every hour during the following 7 hours and also nearly 24 hours thereafter. Statistical analysis was done with the aid of student's t-test. Results
The effect on food intake of the various test solutions intubated before the dark phase are shown in Fig. 1. Two hour food intake following intubations with the low dose test solutions was minimal in the animals given giueose, and significantly smaller than intubation with the equiosmotic urea (UR). No difference in food intake was observed between rats given glycerol and equiosmotic urea (UE), Food intake at seven and at 24 hours following the low dose intubations were minimal in the animals given glycerol, and considerably smaller than in those given equiosmotic urea (UE). At 24 hours the difference in food intake between glycerol (GY) and equiosmotic urea (UE) was significant (p<0.05 or p<0.01) on each of the 3 days. The response to glucose
GLYCEROL AND FOOD INTAKE
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FIG. 2. Food intake of rats maintained on a normal lighting cycle. Symbols and headings as in Fig. 1.
unlike that to glycerol was not consistent and only on the first day was food intake at 24 hours significantly smaller than its equiosmotic urea (UR). After administration of the high dose food intake was considerably smaller in animals given glycerol, glucose and UE compared to that when the low dose of these solutions was given, and in the case of glycerol and glucose this difference at 7 and 24 hours post intubation was larger than due to the caloric content of the injected loads. The small dose consisted of about 4 kcal and the high of about 8 kcal per intubation equivalent to about 1 and 2 g of food respectively. Food consumption two hours after intubation was smallest in rats given glucose, but after 7 and 24 hours it was usually smallest following intubation with glycerol. The difference in food intake between rats given glucose and those given its equiosmotic control (UR) was always significant, but intake after glycerol intubation was significantly smaller than after its equiosmotic control (UE) only in the last day at 7 hours and in the last two days at 24 hours after intubation. Food intake following glycerol intubations was significantly smaller than following glucose only at 24 hours post intubation on the last day of treatment. NORMALCYCLE(LIGHTPHASE) Method The same general protocol was followed as in the re-
versed cycle experiment. However, the animals (previously untreated) were maintained on a normal lighting cycle of 9 a.m. to 9 p.m. light and 9 p.m. to 9 a.m. dark and 6 rats were used in each group. Intubations began at 9:30 a.m. and preweighed food was given at 10 a.m. Results The effect of the various test solutions intubated at the beginning of the light phase are shown in Fig. 2. Food consumption at 2 hours following intubations with glucose is again considerably smaller than its ¢quiosmotic control (UR), but during this time glycerol does not appear to suppress intake beyond its osmotic control (UE). At seven hours, however, there is a trend for glycerol to reduce food intake beyond its osmotic effect following the high dose, and in 2 out of 3 days beyond the effect of equicaloric glucose. At 24 hours following the high dose treatment these latter two trends observed at 7 hours are more marked, and statistically significant in the first two days when glycerol is compared to its equiosmotic control (UE), and in all three days when it is compared to glucose. It is evident that increasing glycerol load from "low" to "high" dose resulted in a considerably greater suppression of the 24 hour food intake in comparison with analogous increases of the other three test solutions. There were no signs of distress or debility following the administration of the test solutions during the dark and the light phases of the diurnal cycle.
624
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VOLUNTARY
INTAKE
of the test solutions changed between the animals. Each rat served as its own control and its 24 hr food intake during infusion was compared to food intake in the control day immediately preceding the infusion. Estrus cycles were determined in the morning of each day and no infusions were made during days of estrus, and during the day following estrus. Data were analyzed by a paired t test. Results
Both glycerol and glucose infusions resulted in a decrease of food intake (Fig. 3). In the case of glucose, the reduction in food intake was accounted for mostly by the energy equivalent of the infused glucose, while in the case of glycerol the total energy consumption (including that provided by the infusion) was significantly less than that of control days. A large individual variability was observed in the response of animals to any one of the test solutions. DISCUSSION Food Intake Following Gastric lntubations
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GL
FIG. 3. Mean 24 hr food intake before (c), and during continuous infusion of glycerol (GY) and of glucose (GL). Ca and Cz=one day before infusion of glycerol and glucose respectively. GY=during infusion of 9-10 ml of 10% glycerol. GL=during infusion of 9--10 ml of 20% glucose. The mean-SEM of the difference in voluntary food intake between control and respective test solution was 4.4_+1.4 for glycerol, and 2.9_+1.3 for glucose.
E X P E R I M E N T 2: E F F E C T OF I N T R A V A S C U L A R L Y A D M I N I S T E R E D G L Y C E R O L ON FOOD I N T A K E Having established that glycerol administered intragastrically could suppress food intake, it was the purpose of the second experiment to determine whether it affected food intake under more "physiologic" conditions, namely when continuously infused into the systemic circulation. In this experiment glycerol was compared to an equiosmotic glucose (about twice the caloric value) as affecting the 24 hr food intake. Method
Fifteen rats were used in this experiment. They were kept in constant lighting for 2 weeks before being implanted with intraaortal catheters as described by Scharrer et al. [19]. Several days after recovery from the operation, when food intake had returned to normal, the rats were infused continuously for 24 hours with 9 to 10 ml of either 10% glycerol or an equiosmotic (20%) glucose, both previously sterilized by autoclaving. Infusions were carded out with the aid of a syringe pump and during the infusion the rats could move freely in their cages. Each infusion was separated by intervals of at least 3 days during which no infusion took place,and the order of infusion
Being nocturnal animals the rats maintained on a reversed cycle were eating most of their 24 hr intakes within the 12 hours following intubation, while on the normal cycle only 12 hours thereafter. Thus, comparing the effect of glycerol to its equiosmotic control (UE), as affecting the 24 hr food intake, our observations can be explained as follows: In the reversed cycle (Fig. 1) when rats are intubated with the low dose test solutions, the osmotic effect of the load is apparently not great enough to override the specific effect of glycerol. Indeed, food consumption after UE is in the same range (slightly larger) as after UR, though the latter has half the osmotic concentration of the former. Under this dose, mean food intake following glycerol is significantly smaller than following its osmotic control (UE) at 24 hour postintubation. However, when the high dose is intubated just before the dark phase, during which time the rats are usually hyperphagic, a major determinant of the 24 hr food intake appears to be the osmotic effect. This is evident from the finding that intake after UE is considerably smaller than after UR. The osmotic effect is clearly apparent at the 2 and at the 7 hr food intakes, and appears to be carried over and result in a marked suppression of the 24 hr food intake. Nevertheless, in two out of three days the effect of glycerol is statistically distinguishable from its osmotic control (UE) at 24 hours post intubation. Moreover, during the third day when a possible adjustment to the UE may have occurred, causing a relative increase in food intake following its intubation, a very highly significant effect of glycerol (p<0.001) is observed. The absolute intake during the third day of glycerol administration does not differ much from intakes during the preceding two days. In rats which are accustomed to consuming most of their daily intakes only from about 12 hours after intubation (normal cycle), the osmotic effect does not seem to play an important role, as there is no consistent differences in the 24 hr food intake of animals intubated with either UE o r UR (Fig. 2). Here, too the more specific effect of glycerol on food intake can be evaluated. Even with the low dose, food consumption tended to be lower following intubation with glycerol as compared to its osmotic control solution (UE). This effect of glycerol becomes significant when the high dose is administered.
GLYCEROL AND F O O D I N T A K E The effect of glycerol on food intake appears to have a different pattern than glucose (Figs. 1 and 2). Two hours following the intubations, glucose suppresses intake more than glycerol. However, after 7 hours there is a trend for the glycerol to become as effective and at times even to exceed the effect of glucose in suppressing food intake. The mean 24 hr food intake, almost without exception is more reduced following the glycerol than the glucose intubations. The effect of glucose loading on the 24 hr food intake appears to be persistently evident only when the high dose was administered into rats maintained on the reversed cycle. Under these conditions the effect is clearly independent of the osmotic load (UR) at all three time periods in each of the three days (Fig. 1). A significant reduction in 24 hr food intake was previously observed following intraperitoneal administration of solutions containing about 1 to 1.5 g glucose [ 16]. This reduction extended beyond the osmotic or the caloric value of the administered glucose. However, Booth [1] reported no suppression of the 24 hr food intake beyond the caloric value of glucose loads of 2 g/day or more. The similarity between our results and those of Racotta et al. [ 16] is interesting in that in both experiments a glucose load was administered just before an active feeding period created in our experiment by the commencement of the dark phase and in theirs by 24 hr food deprivation. It appears that timing of glucose administration in relation to eating activity is critical for its effect on the 24 hr food intake, as glucose intubation before the light phase of the diurnal cycle had no apparent effect on the 24 hr food consumption. Our observation that intragastrically administered glucose suppressed food intake to an extent which was greater than due to its osmotic or energy value provides additional support for a specific role for this metabolite in controlling food intake. Glucoreceptors participating in this mechanism have been suggested to exist in the brain [6,10]; liver [18], and duodenum [13]. Food Intake During Intravascular Infusion Glycerol caused a significant reduction in the 24 hr food intake when infused continuously into the vascular system, at a dose equivalent to the " l o w " dose in the previous experiment. Again the effect of glycerol was distinguisable from that of glucose in that the latter, though containing twice the energy load of glycerol did not suppress food intake beyond its contribution in energy. The cause for the large individual variability in daily food intake in response to the administration of either one of the test solutions is not clear. It may indicate that the metabolic background against which these stimuli are added may be critical for their effect on food intake. In the case of glucose for instance it is well established that it is the rate of its utilization but not its blood concentration that will affect eating [10]. The effect of glycerol may be likewise modulated by endocrine factors, target tissue sensitivity, or other determinants. GENERAL DISCUSSION The main findings of our study are that glycerol suppressed daily food intake to an extent beyond that which could be attributed to its osmotic effect or to its contribution in energy, whether induced in a single bolus via gastric intubation, or infused continuously intravascularly. Also that the effect on food intake is distinctly different from that of glucose both in intensity of suppression and in the time course of its activity (Figs. 1, 2, 3). Glucose is clearly more effective during the first 3 to 4 hours post intubation, while glycerol
625 exhibits a more delayed effect. Our data suggest that the effect of glycerol on food intake is not mediated via its conversion to glucose as the glycerol infusate containing only half the energy value of glucose is more effective than glucose in suppressing the 24 hr food intake (Fig. 3). Finally, we demonstrate that glycerol administration results in the rats maintaining lower levels of daily energy intakes, with no clear evidence for catch-up increases in food intake throughout the six consecutive days of the glycerol treatment (Figs. 1, 2). Our data thus support the findings of Wirtshafter and Davis [21] on the long term nature of the effect of glycerol on food intake and energy balance. Our findings are also compatible with other previous reports showing that bolus administration of glycerol intraperitoneally into 24 hr food deprived rats [ 16], or intraportally into free feeding rabbits [ 17] suppressed their subsequent 24 hr food intakes. Booth [1] however reported that rats intubated intragastrically with glycerol loads similar to our "low dose" (about 1 g/day) before the dark phase, maintained an apparent 24 hr energy balance. However his rats were about twice as heavy as ours and this could possibly account for the variance in results. Our finding that a slow rate of intraaortal administration of glycerol, mimicking the route of glycerol release from adipose tissue, suppressed the 24 hr food intake, supports the hypothesis put forward by Wirtshafter and Davis [20] that glycerol might play a role in the lipostatic control of body weight. However, that this is so is difficult to prove. Clearly, a serum level of glycerol alone is not sufficient a cue for eliciting satiety, since it reaches maximal concentrations during food deprivation [12] namely in a state of hunger. Wirtshafter and Davis [21] suggested that the CNS may be responsive to glycerol only when it is accompanied by a pattern of other humoral signals, but the elements of such a pattern are not known. The origin of glycerol released from adipose tissue varies between the fed and the food deprived state. In the fed state adipose tissue lipoprotein lipase (LPL) activity is high resulting in a release of glycerol originating from lipolysis of VLDL and chlyomicra triglycerides at the surface of the capillary endothelial cells [5]. In the food deprived state glycerol is released from intracellular triglycerides by the action of the hormone sensitive lipase system. It is tempting to speculate that the CNS is able to differentiate between the two sources of serum glycerol, perhaps by some elements of the metabolic pattern accompanying each. For instance, activity of LPL is enhanced by insulin and inhibited by lipolytic hormones [11, 14, 15] and that of hormone sensitive lipase inhibited by insulin, and stimulated by lipolytic hormones. A possible role for glycerol in the control of food intake is compatible with findings that its serum level is increased during overfeeding of human subjects, but at the same time free fatty acid levels remain unchanged or decreased [20]. It is also compatible with the finding that lipectomy in mature rats does not result in a compensatory enlargement of remaining body fat [4,8], but in the animals maintaining a lower level of body energy, which is accompanied by an unchanged serum level of glycerol in the fed state [8]. However, such a role for glycerol is not easily reconciled with observations that obesity is usually accompanied by an increased activity of adipose tissue LPL [15] and a higher rate of glycerol release from adipose tissue [12]. It was previously suggested that CNS mechanism responsible for detecting glycerol may have reduced sensitivity to the glycerol signal in obesity [21]. This suggestion awaits experimental proof.
626
~;t ACK ACKNOWI.EDGEMENTS The authors thank Ms. S. Zarini for her able technical assistance, Mr. I. Einot for his important contribution in the statistical analysis, and to Dr. N. A. Kaufmann for his useful suggestions in the writing of the manuscripl.
REFERENCES 1. Booth, D. A. Postabsorptive induced suppression of appetite and the energostatic control of feeding. Physiol. Behav. 9: 199202, 1972. 2. Bray, G. A. and L. A. Campfield. Metabolic factors in the control of energy stores. Metabolism 24:99-117, 1975. 3. Bray, G. A., J. A. Glennon, L. B. Salans, E. S. Horton, E. Danforth and E. A. H. Sims. Spontaneous and experimental human obesity: Effects of diet and adipose cell size on lipolysis and lipogenesis. Metabolism 26: 739-747, 1977. 4. Faust, I. M., P. R. Johnson and J. Hirsch. Noncompensation of adipose mass in partially lipectomized mice and rats. Am. J. Physiol. 231: 538-544, 1976. 5. Fielding, C. J. and R. J. Havel. Lipoprotein lipase. Archs Path. Lab. Med. 101: 225-229, 1977. 6. Glick, Z. and J. Mayer. Hyperphagia caused by cerebral ventricular infusion with phlorizin. Nature 219: 1374, 1968. 7. Kennedy, G. L. The role of depot fat in the hypothalamic control of food intake in the rat. Proc. R. Soc. 140: 578-592, 1953. 8. Kral, J. G. Surgical reduction of adipose tissue in the male Sprague-Dawley rat. Am. J. Physiol. 231: 1090-1096, 1976. 9. LeMagnen, J. and M. Devos. Metabolic correlates of the meal onset in the free food intake of rats. Physiol. Behav. 5: 805-814, 1970. 10. Mayer, J. and D. W. Thomas. Regulation of food intake and obesity. Science 156: 328-337, 1967. 11. Nestel, P. J. and W. Austin. Relationship between adipose tissue lipoprotein lipase activity and compounds which affect intracellular lipolysis. LiJe Sci. 8: 157-164, 1969. 12. Nishizawa, Y. and G. A. Bray. Ventromedial hypothalamic lesions and the mobilization of fatty acids. J. olin. Invest. 61: 714-721, 1978.
13. Novin, D., J. D. Sanderson and D. A. Vanderweele. The effect of isotonic glucose on eating as a function of feeding condition and infusion site. Physiol. Behav. 13: 3-7, 1974. 14. Patten, R. L. The reciprocal regulation of lipoprotein lipase activity and hormone-sensitive lipase activity in rat adipocytes. J. biol. Chem. 245: 5577-5584, 1970. 15. Pykalisto, O. J., P. H. Smith and J. D. Brunzeli. Determinants of human adipose tissue lipoprotein lipase. Effects of diabetes and obesity on basal and diet induced activity. J. Him Invest. 56: 1108-1117, 1975. 16. Racotta, R. and M. Russek. Food and water intake of rats 'after intraperitoneal and subcutaneous administration of glucose, glycerol and sodium lactate. Physiol. Behav. 18: 267-273, 1977. 17. Rezek, M. and D. Novin. Hepatic-portal nutrient infusion: Effect on feeding in intact and vagotomized rabbits. Am. J. Physiol. 232: EI19-E130, 1977. 18. Russek, M. Participation of hepatic glucoreceptors in the control of intake of food. Nature 197: 79--80, 1963. 19. Scharrer, E., D. W. Thomas and J. Mayer. Absence of effect of intraaortal glucose infusions upon spontaneous meals of rats. Pfliigers Arch. ges. Physiol. 351: 315--322, 1974. 20. Sims, E. A. H., E. Danforth, E. S. Horton, G. A. Bray, J. A. Glennon and L. B. Salans. Endocrine and metabolic effects of experimental obesity in man. Rec. Prog. tlorm. Res. 29: 457496, 1973. 21. Wirtshafter, D. and J. D. Davis. Body weight: Reduction by long-term glycerol treatment. Science 198: 1271-1274, 1977. 22. York, D. A. and G. A. Bray. Genetic obesity in rats. ii. The effect of food restriction on the metabolism of adipose tissue. Metabolism 22: 443-454, 1973.
Food Intake of Rats Administered with Glycerol ZVI GLICK 1
Department o f Nutrition, Hebrew University-Hadassah, Medical School, Jerusalem, Israel R e c e i v e d 21 M a r c h 1980 GLICK. Z. Food intake of rats administered with glycerol. PHYSIOL. BEHAV. 25(5) 621-626, 1980.--The effect of glycerol on rats food intake was determined when it was administered either via bolus gastric intubation, or by a continuous 24 hr infusion into the aorta. Both of these treatments resulted in a suppression of the 24 hr food consumption to an extent which was greater than that accounted for by the caloric value of the administered metabolite. Gastric loading with urea solutions equiosmotic to glycerol or with glucose solutions equicaloric to glycerol were less effective than glycerol in reducing the 24 hr food intake. The time course effect on food intake varied between gastric loading with glycerol and with equicaloric glucose solutions, with the former usually exerting a more delayed and longer lasting effect. Continuous intraaortal infusions of glycerol were more effective than glucose solutions in suppressing 24 hr food intake even though the latter had twice the caloric value of the former. Our data suggest that the action of glycerol on food intake is not mediated through its conversion to glucose. The possibility that glycerol may participate in a lipostatic control mechanism of food intake is discussed. Glycerol
Food intake
Energy balance
using this compound as an organic solvent c a r d e r for an anorectic drug tested in rats (Glick 1975, unpublished data). In this experiment, it was observed that the glycerol "control" was in itself a food intake suppressor. Therefore, the present study was undertaken to quantify the effect of glycerol on food intake of rats in relation to the administered dose, the diurnal phase of the alimentary cycle, and the route of its administration.
T H E hypothesis that body weight of mammals is regulated via the control of fat mass (lipostatic theory) proposed by Kennedy in 1953 [7] is still widely accepted. However, the physiologic monitoring system of adiposity and its relation to the mechanism which controls food intake is very little understood. Recent data from experiments with lipectomized rats suggest that the regulated factor in the lipostatic control of food intake is fat cell size rather than total body fat [4,8]. As a positive correlation has been found to exist between adipose cell size and the rate of glycerol release [3,13], glycerol might play a role in a monitoring system of adipose tissue size, and the control mechanism of food intake. Teleological reasoning for such a role for glycerol was made by Bray and Campfield [2]. More recently, Wirtschafter and Davis [21] presented evidence which strongly supports this suggestion. They have shown that subcutaneous injections into rats of small quantities of glycerol resulted in a suppression of daily food intake and in the maintenance of energy balance at a lower level of body weight in comparison with the injection of an equivalent amount of glucose. Termination of the glycerol treatment was followed by a return of body weight to normal level. Earlier data also showed that relatively small loads of glycerol (< 1 gm) administered subcutaneously or intraperitoneally into rats [ 16] or intraportally into rabbits [17] suppressed the daily food intake beyond its energy contribution. Intragastric loads of a similar order of magnitude were on the other hand without such an effect [1]. Our interest in glycerol effect on food intake began when
METHOD
Animals, Diet and Facilities A total of 81 female (virgin) rats of a Wistar derived Hebrew University strain, weighing 170 to 200 g were tested in this study. The rats were caged individually in rooms maintained at 24__2°C. The diet was standard chow pellets (Amba Ltd., Hedera, Israel) containing 3.6 kcal metabolizable energy per gram. Food intake corrected for spillage was weighed to the nearest 0.1 g. E X P E R I M E N T 1: E F F E C T O F I N T R A G A S T R I C A L L Y A D M I N I S T E R E D G L Y C E R O L ON F O O D I N T A K E Since in the rat the responsiveness of food intake to the same metabolic stimuli varies between day and night [9] the effect of glycerol on food intake was measured separately following its intubation before the dark (reversed cycle) and before the light (normal cycle) phases of the diurnal alimentary cycle.
1Present address: Dr. Zvi Glick, Division of Endocrinology, Harbor General Hospital-UCLA Campus, 1000 W. Carson Street, Torrance, CA 90509.
621
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FIG. 1. Food intake of rats maintained on a reversed lighting cycle by test solution and by time following gastric intubation (UE=urea solution equiosmotic to glycerol; GY=glycerol; GL=glucose; UR=urea solution equiosmotic to glucose; Low dose: 2.5 ml of a 40% glucose or glycerol or their respective equiosmotic solutions. High dose: 5 ml of these same solutions. *p<0.05, **p<0.01; ***p<0.001; one tail t-test.
REVERSEDCYCLE(DARKPHASE) Method
Rats were kept on a reversed lighting cycle for two weeks prior to the experiment (10 a.m. to 10 p.m. dark; 10 p.m. to 10 a.m. light). Sham intubations were started 5 days before the experiment and consisted of single dally intubations without injections for 3 days, followed by two injections of 2.5 ml water for two additional days. Various test solutions were similarly administered in the days which followed. For this purpose, the rats were divided into 4 groups and each was given one of the following solutions: 40% glycerol (GY; n=12), 40% glucose (GL; n=12), also 26.4% urea (UE; n=12) and 13.2% urea (UR; n=6), the latter two were equiosmotic to the glycerol and the glucose test solutions respectively. These solutions were administered in a dally dose of 2.5 ml (low dose) for three days, followed by 5 ml (high dose) for three additional days. The rats were then untreated for two days, during which time food intake was not measured and then received 5 ml (high dose) daily for 3 additional days. Gastric intubations were done with the aid o f a 5 mi syringe and a 2 inch 18 gauge blunt needle on which end a soft plastic tubing was mounted, protruding by about 2
to 3 mm, in order to soften the passage of the needle towards the stomach. All intubations were made within the last 30 minutes before the beginning of the dark cycle (9:30 to 10:00 a.m.) and the order of intubation among the groups was changed each day. Preweighed food was offered at 10 a.m. Food intake was weighed every hour during the following 7 hours and also nearly 24 hours thereafter. Statistical analysis was done with the aid of student's t-test. Results
The effect on food intake of the various test solutions intubated before the dark phase are shown in Fig. 1. Two hour food intake following intubations with the low dose test solutions was minimal in the animals given giueose, and significantly smaller than intubation with the equiosmotic urea (UR). No difference in food intake was observed between rats given glycerol and equiosmotic urea (UE), Food intake at seven and at 24 hours following the low dose intubations were minimal in the animals given glycerol, and considerably smaller than in those given equiosmotic urea (UE). At 24 hours the difference in food intake between glycerol (GY) and equiosmotic urea (UE) was significant (p<0.05 or p<0.01) on each of the 3 days. The response to glucose
GLYCEROL AND FOOD INTAKE
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LOW DOSE
CYCLE HIGH
DOSE
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2 DAY
3
FIG. 2. Food intake of rats maintained on a normal lighting cycle. Symbols and headings as in Fig. 1.
unlike that to glycerol was not consistent and only on the first day was food intake at 24 hours significantly smaller than its equiosmotic urea (UR). After administration of the high dose food intake was considerably smaller in animals given glycerol, glucose and UE compared to that when the low dose of these solutions was given, and in the case of glycerol and glucose this difference at 7 and 24 hours post intubation was larger than due to the caloric content of the injected loads. The small dose consisted of about 4 kcal and the high of about 8 kcal per intubation equivalent to about 1 and 2 g of food respectively. Food consumption two hours after intubation was smallest in rats given glucose, but after 7 and 24 hours it was usually smallest following intubation with glycerol. The difference in food intake between rats given glucose and those given its equiosmotic control (UR) was always significant, but intake after glycerol intubation was significantly smaller than after its equiosmotic control (UE) only in the last day at 7 hours and in the last two days at 24 hours after intubation. Food intake following glycerol intubations was significantly smaller than following glucose only at 24 hours post intubation on the last day of treatment. NORMALCYCLE(LIGHTPHASE) Method The same general protocol was followed as in the re-
versed cycle experiment. However, the animals (previously untreated) were maintained on a normal lighting cycle of 9 a.m. to 9 p.m. light and 9 p.m. to 9 a.m. dark and 6 rats were used in each group. Intubations began at 9:30 a.m. and preweighed food was given at 10 a.m. Results The effect of the various test solutions intubated at the beginning of the light phase are shown in Fig. 2. Food consumption at 2 hours following intubations with glucose is again considerably smaller than its ¢quiosmotic control (UR), but during this time glycerol does not appear to suppress intake beyond its osmotic control (UE). At seven hours, however, there is a trend for glycerol to reduce food intake beyond its osmotic effect following the high dose, and in 2 out of 3 days beyond the effect of equicaloric glucose. At 24 hours following the high dose treatment these latter two trends observed at 7 hours are more marked, and statistically significant in the first two days when glycerol is compared to its equiosmotic control (UE), and in all three days when it is compared to glucose. It is evident that increasing glycerol load from "low" to "high" dose resulted in a considerably greater suppression of the 24 hour food intake in comparison with analogous increases of the other three test solutions. There were no signs of distress or debility following the administration of the test solutions during the dark and the light phases of the diurnal cycle.
624
(; 1 1CK
VOLUNTARY
INTAKE
of the test solutions changed between the animals. Each rat served as its own control and its 24 hr food intake during infusion was compared to food intake in the control day immediately preceding the infusion. Estrus cycles were determined in the morning of each day and no infusions were made during days of estrus, and during the day following estrus. Data were analyzed by a paired t test. Results
Both glycerol and glucose infusions resulted in a decrease of food intake (Fig. 3). In the case of glucose, the reduction in food intake was accounted for mostly by the energy equivalent of the infused glucose, while in the case of glycerol the total energy consumption (including that provided by the infusion) was significantly less than that of control days. A large individual variability was observed in the response of animals to any one of the test solutions. DISCUSSION Food Intake Following Gastric lntubations
z o[ -
CI
G¥
¢2
GL
FIG. 3. Mean 24 hr food intake before (c), and during continuous infusion of glycerol (GY) and of glucose (GL). Ca and Cz=one day before infusion of glycerol and glucose respectively. GY=during infusion of 9-10 ml of 10% glycerol. GL=during infusion of 9--10 ml of 20% glucose. The mean-SEM of the difference in voluntary food intake between control and respective test solution was 4.4_+1.4 for glycerol, and 2.9_+1.3 for glucose.
E X P E R I M E N T 2: E F F E C T OF I N T R A V A S C U L A R L Y A D M I N I S T E R E D G L Y C E R O L ON FOOD I N T A K E Having established that glycerol administered intragastrically could suppress food intake, it was the purpose of the second experiment to determine whether it affected food intake under more "physiologic" conditions, namely when continuously infused into the systemic circulation. In this experiment glycerol was compared to an equiosmotic glucose (about twice the caloric value) as affecting the 24 hr food intake. Method
Fifteen rats were used in this experiment. They were kept in constant lighting for 2 weeks before being implanted with intraaortal catheters as described by Scharrer et al. [19]. Several days after recovery from the operation, when food intake had returned to normal, the rats were infused continuously for 24 hours with 9 to 10 ml of either 10% glycerol or an equiosmotic (20%) glucose, both previously sterilized by autoclaving. Infusions were carded out with the aid of a syringe pump and during the infusion the rats could move freely in their cages. Each infusion was separated by intervals of at least 3 days during which no infusion took place,and the order of infusion
Being nocturnal animals the rats maintained on a reversed cycle were eating most of their 24 hr intakes within the 12 hours following intubation, while on the normal cycle only 12 hours thereafter. Thus, comparing the effect of glycerol to its equiosmotic control (UE), as affecting the 24 hr food intake, our observations can be explained as follows: In the reversed cycle (Fig. 1) when rats are intubated with the low dose test solutions, the osmotic effect of the load is apparently not great enough to override the specific effect of glycerol. Indeed, food consumption after UE is in the same range (slightly larger) as after UR, though the latter has half the osmotic concentration of the former. Under this dose, mean food intake following glycerol is significantly smaller than following its osmotic control (UE) at 24 hour postintubation. However, when the high dose is intubated just before the dark phase, during which time the rats are usually hyperphagic, a major determinant of the 24 hr food intake appears to be the osmotic effect. This is evident from the finding that intake after UE is considerably smaller than after UR. The osmotic effect is clearly apparent at the 2 and at the 7 hr food intakes, and appears to be carried over and result in a marked suppression of the 24 hr food intake. Nevertheless, in two out of three days the effect of glycerol is statistically distinguishable from its osmotic control (UE) at 24 hours post intubation. Moreover, during the third day when a possible adjustment to the UE may have occurred, causing a relative increase in food intake following its intubation, a very highly significant effect of glycerol (p<0.001) is observed. The absolute intake during the third day of glycerol administration does not differ much from intakes during the preceding two days. In rats which are accustomed to consuming most of their daily intakes only from about 12 hours after intubation (normal cycle), the osmotic effect does not seem to play an important role, as there is no consistent differences in the 24 hr food intake of animals intubated with either UE o r UR (Fig. 2). Here, too the more specific effect of glycerol on food intake can be evaluated. Even with the low dose, food consumption tended to be lower following intubation with glycerol as compared to its osmotic control solution (UE). This effect of glycerol becomes significant when the high dose is administered.
GLYCEROL AND F O O D I N T A K E The effect of glycerol on food intake appears to have a different pattern than glucose (Figs. 1 and 2). Two hours following the intubations, glucose suppresses intake more than glycerol. However, after 7 hours there is a trend for the glycerol to become as effective and at times even to exceed the effect of glucose in suppressing food intake. The mean 24 hr food intake, almost without exception is more reduced following the glycerol than the glucose intubations. The effect of glucose loading on the 24 hr food intake appears to be persistently evident only when the high dose was administered into rats maintained on the reversed cycle. Under these conditions the effect is clearly independent of the osmotic load (UR) at all three time periods in each of the three days (Fig. 1). A significant reduction in 24 hr food intake was previously observed following intraperitoneal administration of solutions containing about 1 to 1.5 g glucose [ 16]. This reduction extended beyond the osmotic or the caloric value of the administered glucose. However, Booth [1] reported no suppression of the 24 hr food intake beyond the caloric value of glucose loads of 2 g/day or more. The similarity between our results and those of Racotta et al. [ 16] is interesting in that in both experiments a glucose load was administered just before an active feeding period created in our experiment by the commencement of the dark phase and in theirs by 24 hr food deprivation. It appears that timing of glucose administration in relation to eating activity is critical for its effect on the 24 hr food intake, as glucose intubation before the light phase of the diurnal cycle had no apparent effect on the 24 hr food consumption. Our observation that intragastrically administered glucose suppressed food intake to an extent which was greater than due to its osmotic or energy value provides additional support for a specific role for this metabolite in controlling food intake. Glucoreceptors participating in this mechanism have been suggested to exist in the brain [6,10]; liver [18], and duodenum [13]. Food Intake During Intravascular Infusion Glycerol caused a significant reduction in the 24 hr food intake when infused continuously into the vascular system, at a dose equivalent to the " l o w " dose in the previous experiment. Again the effect of glycerol was distinguisable from that of glucose in that the latter, though containing twice the energy load of glycerol did not suppress food intake beyond its contribution in energy. The cause for the large individual variability in daily food intake in response to the administration of either one of the test solutions is not clear. It may indicate that the metabolic background against which these stimuli are added may be critical for their effect on food intake. In the case of glucose for instance it is well established that it is the rate of its utilization but not its blood concentration that will affect eating [10]. The effect of glycerol may be likewise modulated by endocrine factors, target tissue sensitivity, or other determinants. GENERAL DISCUSSION The main findings of our study are that glycerol suppressed daily food intake to an extent beyond that which could be attributed to its osmotic effect or to its contribution in energy, whether induced in a single bolus via gastric intubation, or infused continuously intravascularly. Also that the effect on food intake is distinctly different from that of glucose both in intensity of suppression and in the time course of its activity (Figs. 1, 2, 3). Glucose is clearly more effective during the first 3 to 4 hours post intubation, while glycerol
625 exhibits a more delayed effect. Our data suggest that the effect of glycerol on food intake is not mediated via its conversion to glucose as the glycerol infusate containing only half the energy value of glucose is more effective than glucose in suppressing the 24 hr food intake (Fig. 3). Finally, we demonstrate that glycerol administration results in the rats maintaining lower levels of daily energy intakes, with no clear evidence for catch-up increases in food intake throughout the six consecutive days of the glycerol treatment (Figs. 1, 2). Our data thus support the findings of Wirtshafter and Davis [21] on the long term nature of the effect of glycerol on food intake and energy balance. Our findings are also compatible with other previous reports showing that bolus administration of glycerol intraperitoneally into 24 hr food deprived rats [ 16], or intraportally into free feeding rabbits [ 17] suppressed their subsequent 24 hr food intakes. Booth [1] however reported that rats intubated intragastrically with glycerol loads similar to our "low dose" (about 1 g/day) before the dark phase, maintained an apparent 24 hr energy balance. However his rats were about twice as heavy as ours and this could possibly account for the variance in results. Our finding that a slow rate of intraaortal administration of glycerol, mimicking the route of glycerol release from adipose tissue, suppressed the 24 hr food intake, supports the hypothesis put forward by Wirtshafter and Davis [20] that glycerol might play a role in the lipostatic control of body weight. However, that this is so is difficult to prove. Clearly, a serum level of glycerol alone is not sufficient a cue for eliciting satiety, since it reaches maximal concentrations during food deprivation [12] namely in a state of hunger. Wirtshafter and Davis [21] suggested that the CNS may be responsive to glycerol only when it is accompanied by a pattern of other humoral signals, but the elements of such a pattern are not known. The origin of glycerol released from adipose tissue varies between the fed and the food deprived state. In the fed state adipose tissue lipoprotein lipase (LPL) activity is high resulting in a release of glycerol originating from lipolysis of VLDL and chlyomicra triglycerides at the surface of the capillary endothelial cells [5]. In the food deprived state glycerol is released from intracellular triglycerides by the action of the hormone sensitive lipase system. It is tempting to speculate that the CNS is able to differentiate between the two sources of serum glycerol, perhaps by some elements of the metabolic pattern accompanying each. For instance, activity of LPL is enhanced by insulin and inhibited by lipolytic hormones [11, 14, 15] and that of hormone sensitive lipase inhibited by insulin, and stimulated by lipolytic hormones. A possible role for glycerol in the control of food intake is compatible with findings that its serum level is increased during overfeeding of human subjects, but at the same time free fatty acid levels remain unchanged or decreased [20]. It is also compatible with the finding that lipectomy in mature rats does not result in a compensatory enlargement of remaining body fat [4,8], but in the animals maintaining a lower level of body energy, which is accompanied by an unchanged serum level of glycerol in the fed state [8]. However, such a role for glycerol is not easily reconciled with observations that obesity is usually accompanied by an increased activity of adipose tissue LPL [15] and a higher rate of glycerol release from adipose tissue [12]. It was previously suggested that CNS mechanism responsible for detecting glycerol may have reduced sensitivity to the glycerol signal in obesity [21]. This suggestion awaits experimental proof.
626
~;t ACK ACKNOWI.EDGEMENTS The authors thank Ms. S. Zarini for her able technical assistance, Mr. I. Einot for his important contribution in the statistical analysis, and to Dr. N. A. Kaufmann for his useful suggestions in the writing of the manuscripl.
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13. Novin, D., J. D. Sanderson and D. A. Vanderweele. The effect of isotonic glucose on eating as a function of feeding condition and infusion site. Physiol. Behav. 13: 3-7, 1974. 14. Patten, R. L. The reciprocal regulation of lipoprotein lipase activity and hormone-sensitive lipase activity in rat adipocytes. J. biol. Chem. 245: 5577-5584, 1970. 15. Pykalisto, O. J., P. H. Smith and J. D. Brunzeli. Determinants of human adipose tissue lipoprotein lipase. Effects of diabetes and obesity on basal and diet induced activity. J. Him Invest. 56: 1108-1117, 1975. 16. Racotta, R. and M. Russek. Food and water intake of rats 'after intraperitoneal and subcutaneous administration of glucose, glycerol and sodium lactate. Physiol. Behav. 18: 267-273, 1977. 17. Rezek, M. and D. Novin. Hepatic-portal nutrient infusion: Effect on feeding in intact and vagotomized rabbits. Am. J. Physiol. 232: EI19-E130, 1977. 18. Russek, M. Participation of hepatic glucoreceptors in the control of intake of food. Nature 197: 79--80, 1963. 19. Scharrer, E., D. W. Thomas and J. Mayer. Absence of effect of intraaortal glucose infusions upon spontaneous meals of rats. Pfliigers Arch. ges. Physiol. 351: 315--322, 1974. 20. Sims, E. A. H., E. Danforth, E. S. Horton, G. A. Bray, J. A. Glennon and L. B. Salans. Endocrine and metabolic effects of experimental obesity in man. Rec. Prog. tlorm. Res. 29: 457496, 1973. 21. Wirtshafter, D. and J. D. Davis. Body weight: Reduction by long-term glycerol treatment. Science 198: 1271-1274, 1977. 22. York, D. A. and G. A. Bray. Genetic obesity in rats. ii. The effect of food restriction on the metabolism of adipose tissue. Metabolism 22: 443-454, 1973.