Effects of insulin on food intake and plasma glucose level in fat-fed diabetic rats

Effects of insulin on food intake and plasma glucose level in fat-fed diabetic rats

Physiology & Behavior, Vol. 24, pp. 319-325. PergamonPress and BrainResearch Publ., 1980. Printedin the U.S.A. Effects of Insulin on Food Intake and ...

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Physiology & Behavior, Vol. 24, pp. 319-325. PergamonPress and BrainResearch Publ., 1980. Printedin the U.S.A.

Effects of Insulin on Food Intake and Plasma Glucose Level in Fat-Fed Diabetic Rats M A R K I. F R I E D M A N , z A L L I C E L. E M M E R I C H A N D K A R E N M. G I L

Biopsychology Program, University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 7 J u l y 1979 FRIEDMAN, M. I., A. L. EMMERICH AND K. M. GIL. Effects of insulin on food intake and plasma glucose level in fat-fed diabetic rats. PHYSIOL. BEHAV. 24(2) 31%325, 1980.--Schedules of insulin treatment which reliably increased eating in fat-fed diabetic rats were studied for their effect on plasma glucose concentrations. An inverse correlation between intake and plasma glucose was observed in fat-fed diabetics given long-term treatment with protamine-zinc insulin (PZI); however changes in glucose did not account for the differential effect of insulin on food intakes in normal controls or normal and diabetic rats fed a low-fat food. A single injection of 1 U PZI which increased eating in fat-fed diabetics but not normal controls 17-23 hr later did not reduce glucose concentrations from hyperglycemic levels in diabetics during the same time period. Injections of regular insulin increased eating in fat-fed diabetic and normal rats in a comparable fashion, but did not reduce plasma glucose in diabetics as low as in normal animals. The results show that the effect of exogenously administered insulin on food intake in fat-fed diabetics is largely unrelated to changes in circulating glucose levels and suggest that metabolic consequences of insulin treatment other than hypoglycemia may underlie the effect of the hormone on feeding in these animals. Insulin-induced eating

Food intake

Plasma glucose

DIABETIC rats that are maintained on a low-fat diet become hyperphagic, but reduce food intake towards normal levels when they are given daily injections of long-acting insulin [1, 4, 5, 8, 9]. In contrast, diabetic rats fed a diet rich in fat eat normal amounts of food and, when given insulin, become hyperphagic [4]. This overeating occurs with doses of insulin that otherwise serve as a hormone replacement for diabetics eating a low-fat diet. In addition, compared to normal rats, fat-fed diabetic animals increase eating in response to lower doses of insulin and to a greater extent. Insulin-induced eating is believed to result from a decrease in glucose utilization that ensues during hypoglycemia [10, 11, 15, 16]. Therefore, in the present experiments we sought to determine whether various schedules of insulin treatment, which reliably increase food intake of fat-fed diabetic rats, also make them hypoglycemic, and whether the more pronounced effect of insulin on food intake of fatfed diabetics is due to a greater degree of hypoglycemia. In order to avoid the confounding effect of food ingestion on blood glucose levels (e.g. [16]), we examined, the effects of different schedules of insulin administration on plasma glucose concentration when rats were deprived of food. The results show that the effects of insulin on food intake in diabetic rats fed a high-fat diet are largely unrelated to changes in blood glucose levels and thereby suggest that metabolic actions of exogenously administered insulin other than those on blood glucose levels may underlie the effect of the hormone on food intake in these animals.

Diabetes

Glucoreceptors

G E N E R A L METHOD

Subjects In the first three experiments female Sprague-Dawley rats (Camm Research or Charles River), weighing 250-350 g were used. In the fourth experiment, male Sprague-Dawley rats (Charles River) weighing 250-350 g were used. Rats were housed in hanging wire mesh cages and maintained on Purina Laboratory Chow and tap water ad lib except when noted. Lights in the colony room were on from 7:00 a.m.-7:00 p.m. New diets were introduced 2-4 weeks after induction of diabetes and were readily accepted by all animals. Due to the consistency of the experimental diets, spillage was negligible, but when it occurred, crumbs were collected and added to the remaining food before weighing.

Experimental Diabetes In Experiments 1-3, diabetes was induced with a SC injection of 5% alloxan monohydrate (Sigma; 200 mg/kg, dissolved in 0.15 M NaC1). Approximately 75% of the rats injected with aUoxan survived and were severely diabetic showing glycosuria (>2% as measured with Lilly Tes-tapes), polydipsia, polyuria, hyperphagia and weight loss. Control, nondiabetic rats received no injection. In Experiment 4, rats were made diabetic with an injection of streptozotocin (Upjohn; 60 mg/kg, IP; dissolved in citrate buffer, pH 4.8 Sigma). Only rats showing plasma glucose levels over 400

~This research was supported by NIH Grant AM-20022 to M.I.F. We thank P. Smallman for her excellent technical assistance throughout these studies and I. Ramirez for his critical reading of an earlier version of this manuscript and his help with the statistical analysis. 2Address reprint requests to: Dr. Mark Friedman, Department of Psychology, University of Massachusetts, Amherst, MA 01003.

C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/020319-07502.00/1

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Statistical Analyses Except when noted, statistical comparisons were made with a Student t-Test, paired comparisons being made when appropriate. EXPERIMENT I In this experiment we examined the effects of long-term PZI treatment in normal and diabetic rats fed high- or low-fat diets. Because our primary concern was to examine the relationship between changes in food intake and plasma glucose concentrations in fat-fed diabetics, we monitored glucose levels only after these animals had reliably and consistently increased food intakes in response to insulin. In addition, because PZI does not have a constant action, but rather has delayed, peak effects, we monitored feeding and glucose levels at 6 hr intervals around the clock.

Procedure Eight diabetic and 7 normal rats were fed a high-fat diet consisting of powdered Purina chow and vegetable shortening (2:1, by weight), while 6 diabetic and 8 normal rats were given a low-fat, wet mash diet of powdered chow and water (1:1, by weight) which was prepared fresh daily. Diabetic and normal animals in each diet group were matched by body weight. After 3 weeks acclimation to the new diets, rats received subcutaneous injections of long-acting, protaminezinc insulin (PZI; Lilly) twice daily at 6 a.m. and 6 p.m. The starting dose was 1.5 U/day for two days which was then increased to 2.0 U/day for three days. The daily dose was divided evenly between the two injections. Four days before the start of insulin treatment, food intakes (to the nearest 0.1 g) were measured every 6 hr for 24 hr starting at 6 a.m. During this baseline period, animals were injected with saline at 6 a.m. and 6 p.m. Food intakes

were then measured daily for the next three days and Ibr the first three days of insulin treatment by which time diabetics had increased eating reliably for several days. On the fourth day of PZI treatment, intakes were again measured starting at 6 a.m. every 6 hr for 24 hr. At the same time intervals blood samples taken from the tip of the tail were collected into microhematocrit tubes, centrifuged and plasma samples assayed for glucose with a Beckman Glucose Analyzer (glucose oxidase method). On the last day of insulin treatment, rats were deprived of food at 6 p.m. and blood glucose samples taken at 12 a.m. were assayed for glucose.

Results Before insulin treatment, diabetic rats fed the low-fat diet were grossly hyperphagic (p<0.001) compared to normal controls; food intakes of those fed the high-fat diet were slightly elevated (/?<0.05) compared to controls (Table 1). During insulin treatment, normal rats eating the low-fat diet increased caloric intake only on the second day of insulin injections; normal rats fed the high-fat diet increased intakes only on the first day of insulin administration. Diabetic animals fed the low-fat diet showed no reliable change in caloric consumption during the course of PZI injections. In contrast, diabetics eating the high-fat diet increased caloric intake starting on the second day of insulin, and maintained this increase for the next two days. Caloric intakes (expressed in terms of change in caloric intake from saline baseline) and plasma glucose levels which were measured every 6 hr on the fourth day of insulin treatment after fat-fed diabetics had reliably increased intakes, are shown in Fig. 1. Only diabetic rats fed the high-fat diet increased intakes significantly over the 24 hr period and increases in food intake tended to occur during periods in which plasma glucose levels declined. Plasma glucose levels fluctuated during the 24 hr period in all groups of rats, but most dramatically in diabetics. Glucose concentrations were consistently highest in diabetics fed the low-fat diet. These animals decreased caloric intakes significantly (p<0.05) in the first two 6 hr periods, but not reliably for the entire 24 hr.

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cally for 24 hr in fat-fed diabetic and normal rats undergoing long-term PZI treatment when they were deprived of food. In addition, larger doses of PZI were used in an attempt to reliably induce hyperphagia in normal rats as well. Procedure

At no time did plasma glucose levels of fat-fed diabetics fall below those of normal controls. Plasma glucose concentrations measured on the fifth day of insulin treatment after 6 hr food deprivation were equally low (about 50 mg%) in all groups. In fat-fed diabetic rats, there was a significant negative correlation (r= -0.81, p<0.02) between plasma glucose concentration at the end of 6 hr periods and changes in caloric intake from baseline during the preceding intervals. Note that this correlation represents an estimate of the common population correlation and was derived from averaging weighed r's of individual animals ([3], pp. 83-85). For purposes of illustration, individual values.(4 for each of 8 animals) are displayed in the scatter plot in Fig. 2. No reliable correlation of this sort was found in the other groups of rats, and, in fat-fed diabetics, there was no significant relationship between glucose levels at the beginning of a period and changes in caloric intake in the subsequent interval. EXPERIMENT 2 In the last experiment, plasma glucose determinations were made, except for one time point, when animals had access to food. Because only fat-fed diabetics increased food intakes on the day blood was sampled, it is possible that this hyperphagia may have confounded the glucose measures and obscured the impact of PZI treatment in these animals. Therefore, in this experiment, in order to .control for differences in food intake, we measured plasma glucose periodi-

Eight diabetic and 8 normal rats were given the high-fat diet described above in place of Purina chow. Ten days later, insulin treatment began. Rats were given PZI at 9:30 a.m. and 9:30 p.m. The starting dose was 1.5 U/day for two days, which was then increased to 2.0 U/day for two days, and finally to 3.0 U/day where it was maintained for four days. The daily dose was divided evenly between injections except for the 3.0 U/day dose for which 2.0 U was given in the morning and 1.0 U given in the evening. Food intakes were measured daily starting the day before and during the eight days of insulin treatment. On the ninth day, rats were deprived of food at 9:30 a.m. Every 6 hr, blood was collected from the tail and plasma glucose measured as above. RESULTS

Before insulin treatment, mean daily food intakes of diabetic and normal rats were approximately the same (Fig. 3). During insulin treatment, diabetic rats increased food intake above pretreatment baseline starting on the fourth day at a dose of 2 U/day and continued to increase intakes until the end of treatment. In contrast, normal rats increased food intake starting on the sixth day of insulin treatment at a dose of 3 U/day, however this increase was reliably maintained for only one additional day so that on the last day of treatment food consumption was not significantly different from baseline. Analysis of food intakes over the course of insulin treatment showed that diabetic rats increased food intakes more than normal controls, F(1,14)=33.2; p<0.01.

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FIG. 3. Effects of protamine-zinc insulin treatment on daily food intakes of normal and alloxan-diabetic rats maintained on a high-fat diet. Asterisks denote points significantly different from baseline (*. p<0.02: **, p<0.00l). On the ninth day of insulin treatment rats were deprived of food. Plasma glucose levels fluctuated over this 24 hr period in both groups of rats with diabetic rats showing the most dramatic changes (Fig. 4). At no time point studied did the plasma glucose levels of diabetics fall significantly below those of normal rats. In this experiment, as well as in the last, normal rats were hyperglycemic at various points during the 24 hr observation period. This elevation of plasma glucose does not appear to occur following a single injection of PZI ([10], see Experiment 3 below), but has been observed following long-term PZI treatment (Friedman, unpublished observations). EXPERIMENT 3 In the last two experiments, plasma glucose was monitored at 6 hr intervals. In order to examine more closely the effects of PZI treatment on glucose levels in fat-fed diabetic rats, in the following experiment we sampled blood more frequently during a discrete 6 hr period. As shown above (Table 1 and Fig. 3) and previously [4], fat-fed diabetic rats increase eating in response to as little as 2 U/day of PZI given in two injections of 1U each. Therefore, in order to study the effects in fat-fed diabetics of a lower effective dose of insulin, we gave rats only one injection of 1 U PZI. In addition, because the peak actions of PZI are delayed, and because pilot studies in other animals show that the effects on food intakes in diabetics of a single injection of PZI were manifest starting approximately 18 hr later, we measured plasma glucose and food intake starting 17 hr after PZI treatment at a time when these rats normally ate very little.

Four diabetic and 4 normal rats were maintained on the high-fat diet described above for 2 weeks. On different days, rats were injected with either 0.15 M NaCI or 1 U PZI at 2 p.m. and either food intake or plasma glucose measured starting at 7 a.m. the next morning. Food intakes were measured every 2 hr for 6 hr. For the glucose determinations, rats were deprived of food at 7 a.m. and blood samples were taken at that time and then every 2 hr for 6 hr. Each test was repeated twice and no sooner than 3 days apart. RESULTS

The results of the third experiment are shown in Fig. 5. Normal rats showed no change in food intake or plasma glucose concentrations 17-23 hr after a single injection (1 U) of long acting insulin. On the other hand, diabetic rats increased food intakes in response to insulin so that by the end of the observation period these rats had eaten more than when given saline (p<0.01) and more than normal controls given insulin (/)<0.02). Without insulin treatment, plasma glucose levels in diabetic rats were greatly elevated compared to normal animals. When diabetics were given insulin, plasma glucose concentrations showed a small, but reliable, increase from these already hyperglycemic levels during the observation period. EXPERIMENT 4 In the preceding experiments we studied the effects of long-acting PZI in fat-fed, female rats with alloxan diabetes. In this last experiment we attempted to generalize our findings by examining the effects of short-acting, regular insulin on food intake and plasma glucose in male, streptozotocindiabetic rats that were fed another high-fat diet that has been shown previously to normalize food intake in these animals [51.

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FIG. 5. Food intakes and plasma glucose concentrations in fat-fed normal and alloxan-diabetic rats 17-23 hr after a single injection of saline (open circles) or I U protamine-zinc insulin (closed circles). Each point represents mean for eight determinations, two for each animal; bars show SEM. For plasma glucose determinations, rats were deprived of food during the test period. Numbers of rats are shown in parentheses.

Procedure Five streptozotocin-diabetic and 6 normal r a t s were placed on a high-fat diet comprised of 25% vegetable shortening (Crisco), 50% cornstarch, 20% casein, 4% salt mix, and 1% viatmin mix (all dry ingredients from ICN Pharmaceuticals). •Two-three weeks later, testing began. Rats were injected SC with either 3 U or 5 U/kg of short-acting, regular insulin (Lilly) or 0.15 M NaCI at 9 a.m. and food intake or plasma glucose measured for the next 6 hr. Food intakes were determined every 2 hr. F o r the glucose determinations, rats were deprived of food and blood samples collected as above at 9 a.m. and then every 2 hr. Tests were conducted in a partially counterbalanced order and were separated by 4-7 days, except when food intakes were measured in which case feeding tests with insulin were preceded by two consecutive days of feeding tests with saline. RESULTS Injections of regular, short-acting insulin (5 U/kg) increased food intakes in both normal and diabetic rats (p<0.02 and p<0.01, respectively; Fig. 6). Injection of 3 U/kg had no reliable effect on food intake in either group of animals. In food-deprived normal rats given 5 U/kg of insulin, plasma glucose fell to an average of 48 mg% (range=3074 rag%) two hr after injection and then gradually returned to saline control levels by six hr. Plasma glucose of fooddeprived diabetic rats given 5 U/kg of insulin fell to an average of 203 mg% (range=81-496 mg%) and also gradually returned toward saline control levels. Injections of 3 U/kg produced a drop in plasma glucose by 2 hr that was comparable to that observed after injection of 5 U/kg. There was some

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FIG. 6. Cumulative food intakes and plasma glucose concentrations in fat-fed normal and streptozotocin-diabetic rats following injections of saline or regular insulin. For plasma glucose determinations, rats were deprived of food during the test period. tendency for blood glucose levels to rise more quickly after injection of the lower dose. In normal rats, plasma glucose concentrations were significantly higher (p<0.05) 4 hr after injections of 3 U/kg than they were after injection of the 5 U/kg dose. In diabetic rats, plasma glucose returned to preinjection levels by the end of the 6 hr test following injection of the lower but not higher dose, although this effect was not statistically reliable. DISCUSSION The results described above confirm and extend earlier observations on the effects of insulin on food intake of fatfed diabetic rats [4]. Unlike diabetics eating a low-fat diet, those fed a food rich in fat increased eating in response to long-term treatment with PZI (Table 1 and Fig. 1). In addition, fat-fed diabetics increased food intake more reliably, to a greater extent, and, typically, in response to lower doses of PZI than normal controls (Table 1 and Figs. 1, 3, and 5). Finally, the results of Experiment 4 suggest that insulininduced eating in fat-fed diabetics is not necessarily dependent on the type of insulin, sex of the animal, composition of the high-fat diet, or the manner of inducing diabetes used previously. Because the experiments reported here were designed to examine the relationship between plasma glucose level and food intake in insulin-treated, fat-fed diabetics, conclusions regarding this relationship in normal and diabetic rats fed a low-fat diet or normal rats fed a high-fat food are limited. However, the findings obtained from these other kinds of animals do provide a basis for comparison for observations made with fat-fed diabetics and will be used in this discussion primarily for this purpose. In this regard, it should be noted that, with the exception of normal rats given 3 U/kg of regular insulin which produced a marked drop in plasma glucose but did not stimulate eating (Fig. 6), non-diabetic rats tended to increase food intake in response to insulin treat-

324 ment that made them hypoglycemic. This finding is consistent with previous observations on the relationship of blood glucose levels and food intake after insulin [10, I I, 15, 161. In the first experiment, we observed an inverse relationship between food intake during 6 hr periods and plasma glucose concentration at the end of those periods in fat-fed diabetic rats receiving injections of insulin (Fig. 2). Because there was no significant correlation between caloric intake and glucose level at the start of the period, the changes in eating probably were due to metabolic events occurring during, rather than before, the 6 hr intervals. This conclusion is supported by the findings that mean food intakes increased during periods when glucose levels fell and decreased as animals became hyperglycemic (Fig. 1). Because similar fluctuations in plasma glucose concentrations were observed in food-deprived diabetics that were treated with insulin in a similar manner (Fig. 4), and because it is unlikely that glucose levels would rise and fall as a result of, respectively, decreases and increases in food intake, the results may indicate thai changes in blood glucose were the cause of variations in food intake in the fat-fed diabetic rats in this experiment. Such an interpretation is consistent with the notion that decreased blood glucose levels provide the stimulus to eat after injections of insulin [10, 11, 15, 16]. On the other hand, since the observed fluctuations in plasma glucose probably reflect changes in the delayed release of protamine-bound insulin, they may be only an indication of insulin action; other metabolic effects of the exogenous insulin, of which there are many, that were not inonitored may have been responsible for the observed alterations in food intake. In several respects, the present findings strongly suggest that other consequences of insulin administration besides hypoglycemia may underlie the effect of the hormone on food intake in fat-fed diabetics. First, the effects of insulin treatment on plasma glucose levels do not account for the differential effect of the hormone on food intake of normal and diabetic rats. Fat-fed diabetics in the first two experiments described above increased eating more than normal rats in response to a schedule of insulin treatment which did not make them more hypoglycemic, in fact, despite food deprivation, mean plasma glucose levels of diabetics in Experiment 2 exceeded 200 mg% for three of the five time points studied (Fig. 4). In the third experiment, insulin treatment increased food intake only in diabetic rats, and did so at a time when blood glucose levels were grossly elevated above normal (Fig. 5). Finally, in Experiment 4, diabetic rats increased food intake in a manner comparable to normal rats after injection of short-acting insulin (5 U/kg), although this dose of insulin did not reduce plasma glucose concentrations as much in diabetics (Fig. 6). Collectively, these findings show that the more pronounced effect of insulin on food intake of fat-fed diabetic rats does not result because diabetics become more hypogtycemic, and suggest thai the hormone may provoke feeding behavior in normal and diabetic rats for different reasons. The present results which indicate that insulin may stimulate eating in fat-fed diabetic rats without decreasing blood glucose below normal also suggest that consequences of insulin administration other than hypoglycemia may elicit feeding behavior in these animals. In the third experiment, a regimen of insulin treatment which reliably increased food intake of fat-fed diabetic rats did not decrease plasma glucose levels in the same rats when they were deprived of food: in fact, insulin treated diabetic rats were

FRIEDMAN, EMMERI(TH ~\Ni)(~i~ hyperglycemic at the start of the lest perioci and becainc. even more so by the end (Fig. 5). These effects o | insulin were reliably observed between repeated test,,: during lhc two test periods in which glucose was nlcasured, piasm~ glucose levels of the diabetics exceeded 340 ing(~ for 3(} ou..: of the 32 individual determinations and the lowest concerti ration observed was 126 mgg~ which is well whhm the normal, ted range for the rats we used (see Fig. 2i. Given tha~ diabetics were hyperglycemic at the start of the observation period, it seems unlikely that they became hypoglycemic between 2-hr determinations, especially since their glucose levels increased steadily over this time. Similarly, in Exper~ iment 4, fat-fed diabetic rats increased food retake when given insulin in a dose (5 U/kg) which on the average did not reduce plasma glucose below hyperglycemic levels. However, because regular insulin has a rapid action, it is possible that plasma glucose may have fallen lower than observed between 2-hr determinations. On the other hand, it should be noted that plasma glucose did not fall below 265 ing*:i m some of the diabetics and that because the effects of insulin on glucose levels were examined when rats were deprived of food, the observed values may be a conservative estimale of the effect of insulin on circulating glucose concentrations during feeding tests when food was freely available. Insulin-induced eating without hypoglycemia has been observed previously. Mayer and Bates [ 12] reported thai injections of NPH insulin increased food intake of normal rals without making them hypoglycemic. However, since lhe schedule of insulin injections and blood sampling was nol specified and because glucose levels fluctuate dramatically during treatment with long-acting insulin (Figs. I and 4), it is possible that .'ats in this earlier study ma$. have become hypoglycemic at a time that was not monilored. Avoiding this problem, Steffans [16] has measured feeding and blood glucose levels concurrently in fi'eely-feeding rals given short-acting insulin. He found that whereas normal rats begin eating after injections of insulin when whole blood glucose concentrations fall to about 50 mgC';, rats with lesions in the ventromedial hypothalamus start eating when blood glucose falls to about 90 m ~ , which is well within the normal range for whole blood. The lack of circulating glucose during hypoglycemia is thought to compromise glucose utilization in cerebral glucoreceptors which are activated by a decrease in their own glucose utilization and which, in turn. trigger feeding behavior [11, 15, 16]. To the extent that the present and earlier [12,16] findings show that injection of insulin may stimulate eating without decreasing blood glucose levels below those found in normal, fed rats, they suggest that eating induced by insulin is not necessarily under the control of receptors in the brain that are sensitive to a lack of glucose. Ritter and his colleagues have found that a hypoglycemic episode experienced by rats in the absence of food will stimulate food intake at a later time when blood glucose levels have returned to normal [14]. This observation led them to suggest that glucoprivic eating induced by insulin may occur without any sign of ongoing glucoprivation. The studies report here extend their observation by indicating that insulin administration may increase eating without the occurrence of glucoprivation and thereby raise doubts whether insulin's effect on food intake necessarily reflecl,~ a glucoprivic control of feeding behavior (see also [6,17]). Decreases in blood glucose per se also do not appear to account for the effects of insulin on food intake in normal or diabetic rats. Diabetics fed a low-fat diet in Experiment I

I N S U L I N - I N D U C E D EATING AND PLASMA G L U C O S E showed dramatic decreases in blood glucOse levels during insulin therapy, yet these animals did not increase food intake. Injection of 3 U/kg of short-acting insulin appeared to produce a similar reduction of blood glucose in normal and diabetic rats as did injections of 5 U/kg; however, only the larger dose reliably increased food intake. Finally, as discussed above, insulin treatment which produced an increase in plasma glucose during a 6 hr observation period in Experiment 3 when fat-fed diabetics were food-deprived nevertheless induced them to eat during the same interval when they had access to food. According to the glucostatic hypothesis [11,12], it is the decreased utilization of glucose that occurs during hypoglycemia, rather than a reduction in blood glucose levels, that provides the stimulus to eat after insulin administration. Although one must be cautious when inferring changes in utilization of a metabolic substrate from alterations in its circulating level, the results of Experiments 1 and 4, in contrast to the glucostatic hypothesis, indicate that fat-fed diabetic rats may increase eating in response to insulin at a time when glucose utilization is increased; that is, when plasma glucose levels are falling from hyperglycemia, diabetic levels toward normal. On the other hand, it is possible that decreased utilization of glucose within a particular metabolic pathway may be critical and that insulin treatment may have increased eating in these experiments because the hormone directed glucose, say, away from oxidation toward fat synthesis [6]. However, again the results of Experiment 3 argue against the generality of such a mechanism since, in this case, insulin-induced eating appeared to be associated with an elevation of plasma glucose. Which metabolic effects of insulin other than hypoglycemia underlie the effect of the hormone on food intake in fat-fed diabetics is a matter for speculation. Because insulininduced feeding is observed in diabetics fed a high-fat diet but not in those eating a low-fat food ([[4,9], Fig. 1), overeating may result from some action of the hormone on lipid

325 metabolism. Doses of insulin which have no appreciable effect on blood glucose level will clear the circulation of lipid metabolites (e.g. [13,18]), and this reduced availability of circulating fat fuels may stimulate feeding in diabetics which rely on lipids as a major source of energy [5,6]. Such an interpretation would explain the failure of chronic diabetics fed a low-fat diet to overeat in response to insulin since those animals already have relatively low levels of circulating fats [5]. Similarly, it would explain the more pronounced hyperphagia of fat-fed diabetics in comparison to normal rats in that normal rats are not restricted to fats as their primary source of energy and, unlike diabetics, are able to utilize glucose as well for that purpose. Presumably, hypoglycemia is associated with insulin-induced feeding in normal rats because the lack of circulating glucose is an indication that other metabolic fuels are also less available [6,14]. It has been reported that normal rats begin to eat after injection of insulin when blood glucose stabilizes at a hypoglycemic level [2,16], a finding which may suggest that eating elicited by insulin administration occurs when the production of glucose from endogenous sources matches the removal of the hexose from the circulation [2]. In the third experiment, insulin treatment led to an increase in blood glucose levels of diabetic rats that were deprived of food which paralleled the increase in food intake observed when the same animals were given insulin and allowed to eat (Fig. 5). This finding is reminiscent of the observation that humans made hypoglycemic with insulin report hunger after blood glucose levels have already begun to return to normal [7], as well as the more recent finding that rats increase food intake after blood glucose levels have been restored in the wake of an insulin-induced hypoglycemic episode [4]. Inasmuch as such increases in plasma glucose levels after insulin reflect an enhanced production of glucose, it is possible that mobilization of endogenous reserves from the liver or gastrointestinal tract may provide a stimulus for insulin-induced eating.

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11. Mayer, J. Regulation of the energy intake and the body weight: The glucostatic theory and the lipostatic hypothesis. Ann. N. Y. Acad. Sci. 63: 15-42, 1955. 12. Mayer, J. and M. W. Bates. Blood glucose and food intake in normal and hypophysectomized, alloxan-treated rats. Am. J. Physiol. 168: 812-819, 1952. 13. Mirsky, 1. A. Relative effects of insulin, oxytocin and vasopressin on the free fatty acid concentration of the plasma of nondiabetic and diabetic dogs. Endocrinology 73: 613-618, 1963. 14. Ritter, R. C., M. Roelke and M. Neville. Glucoprivic feeding behavior in absence of other signs of glucoprivation. Am. J. Physiol. 234: E617-E621, 1978. 15. Smith, G. P. and A. N. Epstein. Increased feeding in response to decreased glucose utilization in the rat and monkey. Am J. Physiol. 217: 1083-1087, 1969. 16. Steffans, A. B. The influence of insulin injections and infusions on eating and blood glucose level in the rat, Physiol. Behav. 4: 823-828, 1969. 17. Stricker, E. M., N. Rowland, C. F. Saller and M. I. Friedman. Homeostasis during hypoglycemia: Central control of adrenal secretion and peripheral control of feeding. Science 196: 79-81, 1977. 18. Zierler, K. L. and D. Robinowitz. Effects of very small concentrations of insulin on forearm metabolism. Persistance of its effect on potassium and fatty acids without its effect on glucose. J. olin. Invest. 43: 950-962, 1964.