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Suppression of food intake in diabetic rats by voluntary consumption and intrahypothalamic injection of glucose
Physiology & Behavior, Vol. 16, pp. 7 6 3 - 7 7 0 . Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Suppression of Food Intake in Diabetic Rats by Voluntary Consumption and Intrahypothalamic Injection of Glucose I JAAK PANKSEPP AND RICK MEEKER
Department o f Psychology, Bowling Green State University, Bowling Green, Ohio 43403 (Received 8 December 1974) PANKSEPP, J. AND R. MEEKER. Suppression of food intake in diabetic rats by voluntary consumption and intrahypothalamic injection of glucose. PHYSIOL. BEHAV. 16(6) 763-770, 1976. - - When given a choice between 2 concentrations of glucose (5 and 35%) normal rats prefer to consume most of their glucose from the concentrated source while diabetics prefer the dilute source. Although both consume approximately the same total amount of glucose, on a calorie basis the glucose depressed chow intake more in diabetics than in normal rats, indicating that diabetics can experience the satiating effects of glucose. Also, since the diabetics excreted more glucose in urine during the glucose access, the overall retention of calories appeared to be less than of controls, indicating the diabetics are effectively hypophagic as compared to controls when they ingested large quantities of glucose. Direct injection of glucose into the ventromedial hypothalamus suppressed feeding as effectively in diabetics as in controls, indicating that normal titers of insulin are probably not necessary for suppression of feeding by ingested glucose in diabetic animals. Diabetes
Feeding
Glucose
Satiety
Ventromedial hypothalamus
Energy balance
mannoheptulose, intragastric loads of glucose still produce normal inhibition of subsequent feeding [13]. To the contrary, it has been reported that small intraperitoneal and intragastric loads of glucose do not suppress feeding effectively in rats made diabetic with streptozotosin [2], but that repeated small daily intraperitoneal injections of glucose in hypophysectomized alloxan diabetic rats do reduce food intake [9]. In paraUel with this contradictory evidence, 2-deoxy-D-glucose is still capable of eliciting feeding in acutely diabetic animals [13], while in chronic alloxan diabetics, feeding has been reported to be completely intact by Kanner [7] and completely abolished by Nance and Gorski [ 10]. The present research was designed to further assess the presence or absence of glucose induced satiety in chronic diabetic (alloxan) animals. Because of previous contradictions in experiments using relatively small forced gastric and intraperitoneal loads of glucose, in the present experiment, daily stability of energy intake was monitored in animals allowed to voluntarily consume glucose solutions. Furthermore, to determine whether feeding behavior of diabetic hyperphagics could be explained in terms of glucose calories lost in the urine, levels of glucosuria were monitored. To determine any sweetness preference differences in normal and diabetic animals, free access was permitted to 2 glucose solutions of different concentrations (5 and 35%). The dilute solution was mixed as 5% because in unpublished work we have determined that diabetic rats subsist well with this solution as the only source of water,
ANIMALS with impaired insulin production (experimental diabetes) exhibit marked overeating, amounting at times to a doubling of daily food intake [8]. Despite this high energy intake, diabetic animals remain lean since nutrients cannot be effectively deposited as fat in the absence of insulin. It is unlikely that lack of insulin per se causes the hyperphagia, since the overeating requires several days to develop after treatment with rapidly acting diabetogenic drugs such as aUoxan and streptozotosin [3,8]. Also, acute diabetes produced either with mannoheptulose or anti-insulin serum does not produce any overeating [ 15]. Hence, the hyperphagia is probably a compensatory response to metabolic changes secondary to the diabetes. Presently there are 3 main possible causes of diabetic overeating: 1) Feeding regulatory ceils which normally depend on insulin for transmembrane transport of nutrients may be depleted of metabolic substrates more rapidly, simulating a metabolic state of starvation. 2) Feeding behavior may be energized secondarily as a consequence of the depletion of body nutrient stores. 3) Overeating may merely be a compensatory response for nutrients, primarily glucose, being lost in the urine. Since the major metabolic function of insulin is to permit blood glucose access to intracellular metabolic processes, the analysis of feeding behavior in diabetics has been primarily involved with determining whether glucose induced satiety can still occur in the absence of systemic insulin. The results from a number of studies have been contradictory. In rats made acutely diabetic with
1This research was supported by NSF Grant GB-40150, PHS Grant 1 RO1 AM17157-01, and Research Scientist Development Award 1-K2-MH-O086-01 to J. P. 763
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PANKSEPP AND MEEKER
and also because when given access to both water and a dilute glucose solution, diabetic animals consistently prefer to consume their water from the dilute glucose source [6]. The higher concentration was selected because of the clear preference which normal animals exhibit for this solution in 2-bottle preference tests. EXPERIMENT 1 METHOD
Animals Eighteen mature male Long-Evans hooded rats were used in the present study. Ten normal rats with body weights ranging from 396 to 546 g were used as controls and 8 diabetic rats with body weights ranging from 266 to 370 g as experimental animals. Prior to the induction of diabetes, body weights were approximately the same for both groups with normals ranging from 330 to 409 g and future diabetics ranging from 315 to 414 g. Diabetes was produced 5 weeks prior to the present experiment by an intraperitoneal injection of Alloxan Monohydrate (125 mg/kg) dissolved in 0.9% saline (50 mg/cc). All diabetic animals were verified to have greater than 500 mg% glucose in the urine with Clinistix. All of the diabetic rats and 3 of the control rats were housed in stainless steel wire mesh metabolism cages. Water and powdered food (Purina Lab Chow) were available ad lib with the food contained in spill-proof feeding jars. Lighting was on a 1 2 - 1 2 hr light-dark cycle with lights coming on at 8:00 a.m. Room temperature was maintained at 76 + 2°F.
Procedure Body weights (to 1.0 g), food intake (to 0.1 g), water or glucose intake (to 1.0 ml), urine output (to 1.0 ml), and urinary glucose levels (+SD = 184 mg%, as determined in reliability tests) were measured daily for 7 consecutive days. During the first 3 and the last 2 days, only water was available. On Days 4 and 5, solutions of 5% and 35% glucose (weight/volume) were available but no water. The 35% glucose solution was made by dissolving 700 g of d-glucose in tap water to make 2 liters of solution at least 24 hr prior to use. This solution was kept refrigerated and used as a stock solution for making up the 5% glucose solution. Cages were thoroughly cleaned on Days 1, 3, 4 and 5. Cotton was placed under the floor in the front portion of the cage to prevent spillage of glucose from the solutions into the urine collection bottles.
Urinary glucose levels were measured by a modification of the Raabo and Terkildsen procedure (Sigma Technical Bulletin 510). Samples of 0.1 mls of urine were pipetted into 100 mls of distilled water and 0.5 mls of the resulting solution incubated for 30 min at 37°C with glucose oxidase, peroxidase, and o-dianisidine hydrochloride. Absorbance readings were taken at 450 nm with a Turner Model 330 spectrophotometer and compared with those obtained for known glucose standards. Energy intakes and losses are expressed in terms of kilocalories (KCal). The utilizable energy content of the lab chow was calculated to be 3.61 KCal/g and of glucose, 3.75 KCal/g. RESULTS Glucose intake from dilute and concentrated sources and the percentage of glucose consumed as dilute solution are summarized in Table 1. Although diabetics and controls consume approximately the same total quantity of glucose, the control animals prefer to take it from the 35% solution (88% of daily glucose intake) while diabetics prefer the 5% solution (76% of daily intake). Urine volumes and urinary glucose concentrations for the 3 days prior to glucose, the 2 days of glucose access, and the 2 days following glucose for all diabetics are summarized in Fig. 1. During access to glucose the urine volume increased significantly by 60.5% over the average volume during the preglucose period (t = 3.24, df = 7, p<0.01, t-test for paired comparisons). Urine volumes dropped significantly upon termination of glucose (t = 4.92, d f = 7, p<0.01) to a level 13.0% lower than the average pre-glucose level (t = 6.73, df = 7, p<0.01). Urine volumes of control animals increased from 19 to 34 mls. Urinary glucose concentration increased slightly but nonsignificantly from the pre-glucose to the glucose period. However, following the glucose period, the urinary glucose concentrations showed a slight but statistically significant drop from both the glucose period (t = 3.46, df = 7, p<0.01) and the average of the pre-glucose period (t = 3.75, dr= 7, p<0.01). Daily caloric intakes for both groups as well as intakes corrected for calories lost as glucose in urine for diabetics are summarized in Fig. 2. In terms of total food consumed, the diabetic animals showed a much higher caloric intake than controls during all 3 phases of the experiment. During the period of glucose availability, there was a significant increase in the total caloric intake for the controls (t ---
TABLE 1 MEAN DAILY GLUCOSE INTAKES FOR ANIMALS HAVING FREE ACCESS TO 5 AND 35% d-GLUCOSE FOR 48 HR
5% Glucose Intake (mls) Controls (n = 10) Diabetics (n = 8)
35% Glucose Intake ( m l s )
Total Glucose (g)
% Daily Glucose Taken as Dilute (by weight)
45.0--_7.1
48.0___3.6
19.0_ + 1.3
12.0---2.2
302.1_+34.1"
12.5_+2.4"
19.5_+2.3
76.1 _+3.5*
Values are mean _+ SE. *p<0.01 (comparison of diabetics vs. controls).
DIABETES AND GLUCOSE INTAKE
765
350
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DAYS FIG. 1. Urine volumes (ntis) and urine glucose levels (mg%) for aUoxan diabetic animals having free access to food for 3 baseline days, 2 days when 5 and 35% glucose solutions were continuously available, and 2 post-glucose days.
200
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FIG. 2. Total and corrected caloric intakes for normal and diabetic animals having free access to food for 3 baseline days, 2 days when glucose solutions were continuously available, and 2 post-glucose days. The effective caloric intake of diabetics was computed by subtracting glucose calories lost in urine from the total daily caloric intake.
766
PANKSEPP AND MEEKER
6.57, df = 9, p<0.01) indicating deficient compensation for glucose intake. The smaller increase seen in diabetics was not significant (t = 1.35, df = 7, p>0.05). Comparison of the glucosuria corrected caloric intake of diabetics with the intake of controls (who exhibited no glucosuria) indicates that during the initial pre-glucose baseline period, the effective intake of diabetics did not differ significantly from controls (t = 0.12, df = 16, p>0.20). However, when glucose was made available, the corrected caloric intake of diabetics was actually reliably lower than of controls (t = 3.81, d f = 16, p<0.01). This failure to increase effective energy intake by diabetics is due both to the greater excretion of glucose (i.e. urine volume increased with no change in glucose concentration) as well as the greater compensatory reduction of chow intake. Control animals showed a 9.7 gram reduction in chow intake whereas diabetics reduced chow intake by 14.6 grams during the access to glucose. In terms of compensating for the glucose intake, these values represent a 49.0% compensation for controls and a 71.2% compensation for diabetics. Finally, on the first day after glucose, the control animals reduced their caloric intake to a level below the pre-glucose level (t = 2.15, df = 9, p < 0 . 0 5 ) while diabetics increased their corrected caloric intake to a value higher than the pre-glucose level (t = 4.08, dr= 7, p<0.01). Body weights were also affected differentially during the glucose period. On the first day of glucose access, the controls showed an average gain of 7.8 g versus an average daily gain of 2.7 g during the pre-glucose period (t = 2.54, df = 9, p<0.05). Conversely, on the first day of glucose, diabetics lost an average of 11.5 g versus an average daily loss of 3.5 g in the pre-glucose period, but due to a very high variance this difference was not statistically reliable by parametric techniques (t = 1.36, df = 7, p<0.10), but was so by nonparametric ones (Wilcoxon T = O, p<0.05).
intakes. It could be argued that had energy balance been calculated as a function of body weight, the diabetic animals could still have been considered hyperphagic under all conditions. This would be true, but it still could not account for the greater reduction in the food intake of diabetics. Furthermore, one has to consider that diabetics cannot deposit fat, thus their low weight is not natural, but obligatory, and absolute data comparisons should still be more appropriate than relative ones. EXPERIMENT 2 As an alternative to a caloric compensatory mechanism, it is possible that food intake reduction during glucose access in diabetics is due to a further increase in hyperosmolar blood glucose levels, and hence a secondary consequence of thirst. With only 5% and 35% glucose available, and no insulin to permit glucose entry into cells, thirst inducing extracellular hyperosmolarity may be forced upon the animal. Although this possibility is somewhat unlikely from the perspective that 5% glucose is hypoosmolar with respect to body fluids, it is conceivable that the slight tonicity was still insufficient to dilute the osmotic potential of the dry lab chow which was eaten. Accordingly, in the following experiment we measured food intake changes when diabetic and normal animals were given free access to a 35% glucose solution and water. With water available, the diabetic animals could readily correct any abnormal hyperosmolarity of blood above and beyond normally encountered in diabetes. Thus any food intake reduction to voluntary glucose intake would more strongly indicate the decrease in feeding to be a calorically mediated compensatory mechanism. METHOD
Animals DISCUSSION Although diabetic hyperphagia under certain conditions (chow only) can mathematically be explained as due to loss of glucose energy in the urine, this appears to be an oversimplification. When diabetic rats are given free access to glucose solutions, their effective energy intake is below normal. During glucose access, normal rats reduced their food intake by half that required to maintain stable energy intake. Diabetic animals showed a 71% compensation. Adding to this the 70% increase in glucosuria precipitated by the glucose intake, the diabetics were retaining reliably less energy in their bodies than controls. In fact their body weight loss was hastened by the glucose intake. This must mean that under physiological condition where insulin secretion is known to be less than 2% of normal, the behavioral expression of energy balance regulation can be at least as precise as in intact rats. In fact if there were an absolute necessity for insulin in glucose mediated control of feeding, it would have been expected that most of the ingested glucose would have passed through the body with no behavioral effect at all. Of course, the fact that the behavioral effect was larger than normal does not necessitate the conclusion that insulin does not participate in the regulation of hormonally intact animals. The hyperglycemia of diabetic animals may be sufficient to sustain adequate passive diffusion of nutrients into normally insulin sensitive cells. The above interpretations are based on absolute food
The same animals from Experiment 1 were used in Experiment 2. Housing conditions were as described in Experiment 1.
Procedure Three days after the termination of Experiment 1 (i.e. 5 days after glucose termination), all animals were given free access to 35% glucose and water. A choice of the 2 solutions was allowed for 3 days, after which, all animals were given access to just water for a period of 2 days. Food was available ad lib throughout and food intakes measured daily. RESULTS Caloric intakes of normal and diabetic rats given access to water or water and 35% glucose are summarized in Table 2. Total caloric intake as glucose was equivalent for both normals and diabetics and amounted to slightly less than two thirds of the calories consumed as glucose when both 5% and 35% solutions were available in the previous experiment. A reduction in chow intake was again seen when glucose was consumed. Food intake of normal animals was reduced by 7.0 g (25.1 KCal) and of diabetics by 12.3 g (44.6 KCal). This reduction represents a compensation of 52.6% for controls and 100.3% for diabetics as compared to the pre-glucose caloric intake - values similar to those of 49.0%
DIABETES AND GLUCOSE INTAKE
767 TABLE 2
CALORIC INTAKE OF NORMAL AND DIABETIC ANIMALS GIVEN FREE ACCESS TO WATER OR WATER AND 35% GLUCOSE SOLUTION Preglucose Normals Diabetics Mean Chow Intake (KCal) Mean Glucose Intake (KCal) Mean Total Food Intake (KCal) Mean Water Intake (mls)
Glucose Normals Diabetics
Postglucose Normals Diabetics
93.4
160.4
68.3
115.8
91.2
145.7
--
--
47.7
44.4
--
--
93.4
160.4
116.1
160.2
91.2
145.7
43
192
237
33
182
and 71.2% obtained for controls and diabetics respectively in Experiment 1. Also, it should be noted that during glucose access diabetic animals increased water consumption (t = 2.72, d f = 7, p<0.05) while normal animals drank less (t = 5.77, d f = 9, p<0.01). Apparently, consumption of 35% glucose did increase the osmotic imbalance somewhat in diabetic animals. EXPERIMENT 3 From the first 2 experiments it is still not clear whether the presence of a hyperosmolar glucose solution is necessary in order to see a compensatory reduction in chow intake. In the following experiment, ad lib food intake was monitored with and without access to a 5% glucose solution and water. Further, to assess the possible contribution on non-glucose energy loss in the urine of diabetics, both glucosuria and ketosis were monitored. METHOD Animals
Five additional severely diabetic rats (Long-Evans, hooded) and five control animals (Long-Evans) were used in the present experiment. Animals were tested more than 4 weeks following production of diabetes. Procedure
A baseline of food intake, water intake, body weight, urine output and urinary glucose output was taken over a period of 3 days, whereupon a solution of 5% (w/v) d-glucose was made available in addition to the water for a period of 3 days, followed by 3 days of post-glucose baseline measurements. Each day, the ketone body content of the urine was checked with Ketostix® (Ames Co.) which will detect as little as 5 - 1 0 mg% of acetoacetate in urine. The reagent is less sensitive to acetone and does not react with 3hydroxybutyrate. Daily average urinary glucose output was measured potentiometrically with Yellow Springs Instruments Glucose Analyzer (YSI Model 23). RESULTS The mean chow intake, body weights, glucose intake and
5
glucose excretion of the diabetic and control rats for the various phases of the experiment are presented in Table 3 and these results were consistent from animal to animal. When a 5% solution of glucose was made available, both control and diabetic rats consumed large quantities in preference to the water. In the control animals, the average body weight increased 16 g for the 3 day period of glucose access. Urine volume increased approximately fourfold. Diabetic body weights dropped an average of 8 g during the access to glucose, urine volume almost doubled and urinary glucose concentrations increased 17%. With the exception of 1 diabetic and 1 control animal on 2 days of the pre-glucose baseline, fresh samples of both control and diabetic urine were consistently free of acetoacetate or acetone during all phases of the experiment. With only a dilute source of glucose available, the total daffy glucose consumed by diabetics was over 3 times that of controls. The compensatory reduction in chow intake for the added glucose calories and the effective diabetic energy intake are summarized in Fig. 3, and essentially replicate the pattern seen in Experiment 1. During glucose, total energy intake of controls increased by 19% while in diabetics the effective intake was reduced 26% (from 88.1 KCal to 65.2 KCal). Since this was done in the absence of any increase in water intake (in fact water intake dropped from 183 ml/day to 14 ml/day), the reduction of food intake is unlikely to be due to extra thirst induced by the glucose intake. It is noteworthy that diabetics again lost weight rapidly on the glucose regimen. EXPERIMENT 4 The ability of diabetic rats to compensate as well as controls for voluntarily ingested glucose suggests that the mechanisms which modulate the effects of glucose on energy intake are capable of functioning in diabetic rats. These receptors may lie within the ventromedial hypothalamus (VMH) since glucose has been shown to be effective in suppressing long term food intake when applied directly to the VMH [14]. Although a great deal of evidence indicates that the medial hypothalamic glucosesensitive system should require insulin for normal functioning [4,17], it is possible that extreme hyperglycemia may promote sufficient passage of nutrients to sustain near normal functioning in these cells in the stabilized diabetic. To test whether the hypothalamic response to glucose is
768
PANKSEPP AND MEEKER
TABLE 3 INTAKE AND EXCRETION PARAMETERS OF NORMAL AND DIABETIC RATS GIVEN FREE ACCESS TO A 5% GLUCOSE SOLUTION
Body Weight (g) Chow Intake (KCal.) Glucose Intake (KCal.) Water Intake (mls) Urine Vol. (mls.) Glucosuria* (mg%) Total Urinary Glucose (KCal.) Intake (KCal.) minus Glucose Loss (KCal.)
Pre
Controls Glucose
Post
Pre
Diabetics Glucose
Post
516 86.1 -39 19 77 0.1
532 82.4 20.0 2 76 39 0.1
531 90.1 -38 21 99 0.1
236 128.7 -183 154 7067 40.6
228 102.4 62.4 14 273 8385 99.6
234 128.8 -174 147 7556 42.0
86.0
102.3
90.0
88.1
65.2
86.8
*The relatively high levels of glucose in the urine of normal animals reflects the contribution of some unknown factor which yielded substantial readings on the YSI Glucose Analyzer. This factor was inversely related to the volume of urine in any given animal. Independent verification of the glucose levels spectrophotometrically confirmed that the urinary glucose of the normal 'animals was virtually zero.
o
o CONTROLS I
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25
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2
3
4
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6
7
8
9
Days FIG. 3. Total and corrected caloric intakes for normal and diabetic animals of Experiment 3.
DIABETES AND GLUCOSE INTAKE
769
still present in the presence of hyperglycemia and low endogenous levels of insulin, in the following experiment, glucose was injected directly into the VMH of diabetic and non-diabetic rats. METHOD
Animals and Surgery Eighteen female, Long-Evans hooded rats were used in the present experiment. Eight rats which had been made diabetic by intraperitoneal injections of 125 mg/kg of alloxan at least 3 weeks prior to surgery and 10 nondiabetic rats were anaesthetized with sodium pentobarbital (40 mg/kg) and implanted with bilateral stainless steel cannulae aimed for the VMH (0.5 mm posterior to bregma, 0.75 mm lateral to the midline, 9.5 mm below the surface of the skull, mouthbar 5 mm above intraural zero). Injection needles were cut to extend 1.0 mm past the tip of the cannulae. Cannulae were constructed of 23 ga stainless steel needle tubing with 29 ga inserts.
Procedure Approximately 4 weeks after surgery, half the animals were injected bilaterally with 1.0 ul of 10% (w/v) d-glucose and half with 10% (w/v) 2-deoxy-D-glucose (2-DG) dissolved in sterile physiological saline. Three injections were given per day separated by 7 hr. Three days after the first injection, the same procedure was followed except the injected solutions were reversed. Food intakes and body weight were measured daily. RESULTS Daily food intake on the day prior to testing and on the 2 injection days are summarized in Table 4. Glucose delivered to the VMH suppressed 24 hr food intakes in both non-diabetic (t = 2.60, d f = 9, p<0.05) and diabetic rats (t = 2.48, d f = 7, p < 0 . 0 5 ) relative to the control injections (2-DG). The control injection tended to increase food intake in control animals whereas in diabetic animals, it reliably suppressed feeding. Although we can put forward no cogent reason why 2-DG should have suppressed the food intake of the diabetic animals, it should be noted that peripheral injections of 2-DG in diabetics has also been found to suppress food intake [10]. These results indicate that glucose administered into the medial hypothalamus of diabetic rats can inhibit feeding as effectively as in normal animals. TABLE4 MEAN TWENTY-FOURHOUR FOOD INTAKES IN NON-DIABETIC AND DIABETIC RATS FOLLOWING INJECTIONS OF d-GLUCOSE INTO THE VENTROMEDIALHYPOTHALAMUS Pre Controls (n=10) Diabetics (n=10)
2-DG
d-Glucose
18.8---4.6
22.6---5.5
15.6-+6.5 *
27.4-+4.6
21.6-+4.4"~*
15.2+-8.5*
Values are mean --+ SD. *p<0.05 (correlated t-test; 2-DG minus d-Glucose). tp<0.05 (correlated t-test; 2-DG minus Pre).
GENERAL DISCUSSION Diabetic rats allowed free access to glucose solutions voluntarily consume large quantities, preferably from a very dilute source, and they reduce their normal food intake as much as normal animals to compensate for the calories ingested as glucose. In fact, the diabetics' effective energy intake (food intake corrected for glucose lost in urine) during glucose access is reliably lower than the caloric intake of control animals. This difference is due to both a greater percent reduction of food intake by diabetics as well as to an increased excretion of glucose in urine. These results support previous work indicating that a low level of endogenous insulin does not impair the capacity of glucose to inhibit feeding [9,15], but they do not necessarily negate studies which have observed impaired glucose sensitive control of feeding in diabetic animals [2,10]. These latter studies have employed small doses of glucose, and it may be that glucose can only modulate feeding normally in diabetics when a certain minimum amount of glucose has been consumed. Further, though these results clearly indicate that glucose can modulate feeding in the absence of normal insulin levels [7,14], the data cannot be interpreted to mean that diabetics still possess all the physiological mechanisms which normally participate in glucose induced suppression of feeding. Chronic diabetics may only exhibit intact adjustments of feeding in response to glucose intake because of compensatory physiological changes which have resulted from the diabetes. For instance, diabetic hyperglycemia may be so high that additional increases resulting from glucose consumption can lead to a sufficiently high transmembrane gradient of glucose to allow passive diffusion to cells which restores normal rates of intracellular glucose metabolism. Even though the data from Experiment 4 are consistent with such a mechanism, other explanations for the normal glucose inhibition of feeding in diabetics are possible. For example, in normal animals both insulin dependent and insulin independent glucose sensitive systems may operate at the hypothalamic level to control food intake [4, 15, 17]. Thus, the diabetic animal's feeding may be controlled primarily by an insulin independent mechanism in the lateral hypothalamus [17], while the intact animal also utilizes medial hypothalamic insulin sensitive mechanisms [4]. Since the ventromedial system appears to organize long-term regulation of energy balance while the lateral system organizes shorter-term controls [ 1, 11, 13], the diabetic animal may possess starved longer-term regulatory elements in the medial hypothalamus and oversatiated insulin-independent sensors in the lateral hypothalamus. Thereby, the diabetic animal could be an unusual preparation where an active long-term signal of body nutrient depletion normally coexists with a strong insulin independent short-term glucostatic signal of satiety. This might explain why ventromedial hypothalamic lesions can attenuate diabetic overeating [5,14]. The lesion would reduce the effect of the body nutrient depletion signal on feeding. This might also explain the reduction in chow intake we observed in diabetic animals voluntarily consuming glucose. An increase in passage of glucose into normally insulin sensitive receptors may have tended to replete the cells. Certainly the capacity of glucose injections into the VMH are compatible with such a proposal. The fact that 2-DG also reduced intake remains problematic,
770
PANKSEPP AND MEEKER
though it is heartening that the effect was not as large as produced by the d-glucose. Although the present data does give considerable support for the simple idea that diabetic hyperphagia could largely be the direct consequence of glucose energy lost in the urine, the situation is clearly more complex than that. It cannot explain the reduction of effective caloric intake during voluntary glucose consumption. Thus, during a time when excretion of glucose was reliably increased, food intake was not. Moreover, other features of diabetic feeding, such as increased meal sizes [12, 14, 16] are also difficult to explain simply by the energy losses in urine. If there were nothing but a rapidly acting causal link between energy loss in the urine and hyperphagia, then one would most probably predict that the frequency of feeding would be the major parameter to increase. Thus, it is more probable that the loss of energy in the urine establishes a chronic central state which is similar to starvation. It should be emphasized that the present work cannot be taken as a complete energy balance work-up on diabetic animals. We have primarily focused on how glucose loss
might relate to the regulatory pattern of diabetics. Although our data from Experiment 3 rule out the possibility that excretion of certain types of ketone bodies is pertinent to an energy balance analysis in severe chronic diabetics, there is an outside chance that other ketones such as/~-hydroxybutyrate were secreted in amounts that should be considered. Also, of the thousand or so other metabolites that are excreted in urine, there may be others which require attention. Also, a full energy balance analysis would require the measurement of differential energies expended in motor activity and non-behavioral thermogenesis. Certainly if it were found that glucose ingestion in the diabetic rat severely reduces these energy losses, our conclusion that diabetics actually consume less energy than controls could be brought into question. However, we consider these possibilities very unlikely, if but for the simple reason that during glucose access diabetic animals consistently lost weight. This indicates that their decrease in energy intake was at the expense of endogenous sources, and speaks strongly for the validity of our interpretations.
REFERENCES 1. Bernstein, I. L. Post-prandial EEG synchronization in normal hypothalamically lesioned rats. Physiol. Behav. 12: 535-545, 1974. 2. Booth, D. A. Feeding inhibition by glucose loads, compared between normal and diabetic rats. Physiol. Behav. 8: 801-805, 1972. 3. Booth, D. A. Some characteristics of feeding during streptozotocin-induced diabetes in the rat. J. comp. physiol. Psychol. 80: 238-249, 1972. 4. Debons, A. G., I. Krimsky and A. From. A direct action of insulin on the hypothalamic satiety center. Am. J. Physiol. 219: 938-943, 1970. 5. Goldman, J. K., J. D. Schnatz, L. L. Bernardis and L. A. Frohman. Effects of ventromedial hypothalamic destruction in rats with preexisting streptozotocin-induced diabetes. Metabolism 21: 132-136, 1972. 6. Kakolewski, J. W. and E. S. Valenstein. Glucose and saccharin preference in alloxan diabetic rats. J. comp. physiol. Psychol. 68: 31-37, 1969. 7. Kanner, M. Neurochemistry of feeding the diabetic rat. Paper presented at Symposium of Food and Fluid Intake, Satellite Meeting of XXVI International Congress o f Physiological Sciences, Jerusalem, October, 1974. 8. Kumaresan, P. and C. W. Turner. Effect of alloxan on feed consumption in the rat. Proc. Soc. exp. Biol. Med. 119: 400-402, 1965.
9. 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. 10. Nance, D. M. and R. A. Gorski. Effects of chronic diabetes on a 2-Deoxy-D-glucose induced feeding and drinking. Pharmac. Biochem. Behav. 1: 483-485, 1973. 11. Panksepp, J. A re-examination of the rule of the ventromedial hypothalamus in feeding behavior. Physiol Behav. 7: 385-394, 1971. 12. Panksepp, J. A reanalysis of feeding patterns in the rat. J. comp. physiol. Psychol. 82: 78-94, 1973. 13. Panksepp, J. Hypothalamic regulation of energy balance and feeding behavior. Fedn Proc. 33: 1150-1165, 1974. 14. Panksepp, J. and D. M. Nance. Insulin, glucose and hypothaiamic regulation of feeding. Physiol. Behav. 9:447-451, 1972. 15. Panksepp, J., D. Tonge and K. Oatley. Insulin and glucostatic control of feeding. J. eomp. physiol. Psychol. 78: 226-232, 1972. 16. Panksepp, J. and M. Ritter. Mathematical analysis of energy regulatory patterns of normal and diabetic rats. J. comp. physiol. Psychol. 1975, in press. 17. Smith, C. J. V. Hypothalamic glucoreptors - the influence of gold thioglucose implants in the ventromedial and lateral hypothaiamic areas of normal and diabetic rats. Physiol. Behav. 9: 391-396, 1972.
Suppression of Food Intake in Diabetic Rats by Voluntary Consumption and Intrahypothalamic Injection of Glucose I JAAK PANKSEPP AND RICK MEEKER
Department o f Psychology, Bowling Green State University, Bowling Green, Ohio 43403 (Received 8 December 1974) PANKSEPP, J. AND R. MEEKER. Suppression of food intake in diabetic rats by voluntary consumption and intrahypothalamic injection of glucose. PHYSIOL. BEHAV. 16(6) 763-770, 1976. - - When given a choice between 2 concentrations of glucose (5 and 35%) normal rats prefer to consume most of their glucose from the concentrated source while diabetics prefer the dilute source. Although both consume approximately the same total amount of glucose, on a calorie basis the glucose depressed chow intake more in diabetics than in normal rats, indicating that diabetics can experience the satiating effects of glucose. Also, since the diabetics excreted more glucose in urine during the glucose access, the overall retention of calories appeared to be less than of controls, indicating the diabetics are effectively hypophagic as compared to controls when they ingested large quantities of glucose. Direct injection of glucose into the ventromedial hypothalamus suppressed feeding as effectively in diabetics as in controls, indicating that normal titers of insulin are probably not necessary for suppression of feeding by ingested glucose in diabetic animals. Diabetes
Feeding
Glucose
Satiety
Ventromedial hypothalamus
Energy balance
mannoheptulose, intragastric loads of glucose still produce normal inhibition of subsequent feeding [13]. To the contrary, it has been reported that small intraperitoneal and intragastric loads of glucose do not suppress feeding effectively in rats made diabetic with streptozotosin [2], but that repeated small daily intraperitoneal injections of glucose in hypophysectomized alloxan diabetic rats do reduce food intake [9]. In paraUel with this contradictory evidence, 2-deoxy-D-glucose is still capable of eliciting feeding in acutely diabetic animals [13], while in chronic alloxan diabetics, feeding has been reported to be completely intact by Kanner [7] and completely abolished by Nance and Gorski [ 10]. The present research was designed to further assess the presence or absence of glucose induced satiety in chronic diabetic (alloxan) animals. Because of previous contradictions in experiments using relatively small forced gastric and intraperitoneal loads of glucose, in the present experiment, daily stability of energy intake was monitored in animals allowed to voluntarily consume glucose solutions. Furthermore, to determine whether feeding behavior of diabetic hyperphagics could be explained in terms of glucose calories lost in the urine, levels of glucosuria were monitored. To determine any sweetness preference differences in normal and diabetic animals, free access was permitted to 2 glucose solutions of different concentrations (5 and 35%). The dilute solution was mixed as 5% because in unpublished work we have determined that diabetic rats subsist well with this solution as the only source of water,
ANIMALS with impaired insulin production (experimental diabetes) exhibit marked overeating, amounting at times to a doubling of daily food intake [8]. Despite this high energy intake, diabetic animals remain lean since nutrients cannot be effectively deposited as fat in the absence of insulin. It is unlikely that lack of insulin per se causes the hyperphagia, since the overeating requires several days to develop after treatment with rapidly acting diabetogenic drugs such as aUoxan and streptozotosin [3,8]. Also, acute diabetes produced either with mannoheptulose or anti-insulin serum does not produce any overeating [ 15]. Hence, the hyperphagia is probably a compensatory response to metabolic changes secondary to the diabetes. Presently there are 3 main possible causes of diabetic overeating: 1) Feeding regulatory ceils which normally depend on insulin for transmembrane transport of nutrients may be depleted of metabolic substrates more rapidly, simulating a metabolic state of starvation. 2) Feeding behavior may be energized secondarily as a consequence of the depletion of body nutrient stores. 3) Overeating may merely be a compensatory response for nutrients, primarily glucose, being lost in the urine. Since the major metabolic function of insulin is to permit blood glucose access to intracellular metabolic processes, the analysis of feeding behavior in diabetics has been primarily involved with determining whether glucose induced satiety can still occur in the absence of systemic insulin. The results from a number of studies have been contradictory. In rats made acutely diabetic with
1This research was supported by NSF Grant GB-40150, PHS Grant 1 RO1 AM17157-01, and Research Scientist Development Award 1-K2-MH-O086-01 to J. P. 763
764
PANKSEPP AND MEEKER
and also because when given access to both water and a dilute glucose solution, diabetic animals consistently prefer to consume their water from the dilute glucose source [6]. The higher concentration was selected because of the clear preference which normal animals exhibit for this solution in 2-bottle preference tests. EXPERIMENT 1 METHOD
Animals Eighteen mature male Long-Evans hooded rats were used in the present study. Ten normal rats with body weights ranging from 396 to 546 g were used as controls and 8 diabetic rats with body weights ranging from 266 to 370 g as experimental animals. Prior to the induction of diabetes, body weights were approximately the same for both groups with normals ranging from 330 to 409 g and future diabetics ranging from 315 to 414 g. Diabetes was produced 5 weeks prior to the present experiment by an intraperitoneal injection of Alloxan Monohydrate (125 mg/kg) dissolved in 0.9% saline (50 mg/cc). All diabetic animals were verified to have greater than 500 mg% glucose in the urine with Clinistix. All of the diabetic rats and 3 of the control rats were housed in stainless steel wire mesh metabolism cages. Water and powdered food (Purina Lab Chow) were available ad lib with the food contained in spill-proof feeding jars. Lighting was on a 1 2 - 1 2 hr light-dark cycle with lights coming on at 8:00 a.m. Room temperature was maintained at 76 + 2°F.
Procedure Body weights (to 1.0 g), food intake (to 0.1 g), water or glucose intake (to 1.0 ml), urine output (to 1.0 ml), and urinary glucose levels (+SD = 184 mg%, as determined in reliability tests) were measured daily for 7 consecutive days. During the first 3 and the last 2 days, only water was available. On Days 4 and 5, solutions of 5% and 35% glucose (weight/volume) were available but no water. The 35% glucose solution was made by dissolving 700 g of d-glucose in tap water to make 2 liters of solution at least 24 hr prior to use. This solution was kept refrigerated and used as a stock solution for making up the 5% glucose solution. Cages were thoroughly cleaned on Days 1, 3, 4 and 5. Cotton was placed under the floor in the front portion of the cage to prevent spillage of glucose from the solutions into the urine collection bottles.
Urinary glucose levels were measured by a modification of the Raabo and Terkildsen procedure (Sigma Technical Bulletin 510). Samples of 0.1 mls of urine were pipetted into 100 mls of distilled water and 0.5 mls of the resulting solution incubated for 30 min at 37°C with glucose oxidase, peroxidase, and o-dianisidine hydrochloride. Absorbance readings were taken at 450 nm with a Turner Model 330 spectrophotometer and compared with those obtained for known glucose standards. Energy intakes and losses are expressed in terms of kilocalories (KCal). The utilizable energy content of the lab chow was calculated to be 3.61 KCal/g and of glucose, 3.75 KCal/g. RESULTS Glucose intake from dilute and concentrated sources and the percentage of glucose consumed as dilute solution are summarized in Table 1. Although diabetics and controls consume approximately the same total quantity of glucose, the control animals prefer to take it from the 35% solution (88% of daily glucose intake) while diabetics prefer the 5% solution (76% of daily intake). Urine volumes and urinary glucose concentrations for the 3 days prior to glucose, the 2 days of glucose access, and the 2 days following glucose for all diabetics are summarized in Fig. 1. During access to glucose the urine volume increased significantly by 60.5% over the average volume during the preglucose period (t = 3.24, df = 7, p<0.01, t-test for paired comparisons). Urine volumes dropped significantly upon termination of glucose (t = 4.92, d f = 7, p<0.01) to a level 13.0% lower than the average pre-glucose level (t = 6.73, df = 7, p<0.01). Urine volumes of control animals increased from 19 to 34 mls. Urinary glucose concentration increased slightly but nonsignificantly from the pre-glucose to the glucose period. However, following the glucose period, the urinary glucose concentrations showed a slight but statistically significant drop from both the glucose period (t = 3.46, df = 7, p<0.01) and the average of the pre-glucose period (t = 3.75, dr= 7, p<0.01). Daily caloric intakes for both groups as well as intakes corrected for calories lost as glucose in urine for diabetics are summarized in Fig. 2. In terms of total food consumed, the diabetic animals showed a much higher caloric intake than controls during all 3 phases of the experiment. During the period of glucose availability, there was a significant increase in the total caloric intake for the controls (t ---
TABLE 1 MEAN DAILY GLUCOSE INTAKES FOR ANIMALS HAVING FREE ACCESS TO 5 AND 35% d-GLUCOSE FOR 48 HR
5% Glucose Intake (mls) Controls (n = 10) Diabetics (n = 8)
35% Glucose Intake ( m l s )
Total Glucose (g)
% Daily Glucose Taken as Dilute (by weight)
45.0--_7.1
48.0___3.6
19.0_ + 1.3
12.0---2.2
302.1_+34.1"
12.5_+2.4"
19.5_+2.3
76.1 _+3.5*
Values are mean _+ SE. *p<0.01 (comparison of diabetics vs. controls).
DIABETES AND GLUCOSE INTAKE
765
350
IO000
51 AND35% GLUCOSEAVAILABLE
T
I000 6
300
#/0.
A °
"
/
/
.
.
.
.
8
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200
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150
100
I
I
I
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I
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I
1
2
3
4
5
6
1
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DAYS FIG. 1. Urine volumes (ntis) and urine glucose levels (mg%) for aUoxan diabetic animals having free access to food for 3 baseline days, 2 days when 5 and 35% glucose solutions were continuously available, and 2 post-glucose days.
200
5% AND 35% O
O
0-----0 A-----el
GLUCOSE AVAILABLE
"'~'" I /,.e- ............ " "
GLUCOSURIA
175
~
CONTROLS
DIABETICS, TOTALFOOD DIABETICS, FOODMINUS
•#js"•s
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,,
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.=,.
-- 125 I.I-
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75
i
2
3
4
5
6
7
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FIG. 2. Total and corrected caloric intakes for normal and diabetic animals having free access to food for 3 baseline days, 2 days when glucose solutions were continuously available, and 2 post-glucose days. The effective caloric intake of diabetics was computed by subtracting glucose calories lost in urine from the total daily caloric intake.
766
PANKSEPP AND MEEKER
6.57, df = 9, p<0.01) indicating deficient compensation for glucose intake. The smaller increase seen in diabetics was not significant (t = 1.35, df = 7, p>0.05). Comparison of the glucosuria corrected caloric intake of diabetics with the intake of controls (who exhibited no glucosuria) indicates that during the initial pre-glucose baseline period, the effective intake of diabetics did not differ significantly from controls (t = 0.12, df = 16, p>0.20). However, when glucose was made available, the corrected caloric intake of diabetics was actually reliably lower than of controls (t = 3.81, d f = 16, p<0.01). This failure to increase effective energy intake by diabetics is due both to the greater excretion of glucose (i.e. urine volume increased with no change in glucose concentration) as well as the greater compensatory reduction of chow intake. Control animals showed a 9.7 gram reduction in chow intake whereas diabetics reduced chow intake by 14.6 grams during the access to glucose. In terms of compensating for the glucose intake, these values represent a 49.0% compensation for controls and a 71.2% compensation for diabetics. Finally, on the first day after glucose, the control animals reduced their caloric intake to a level below the pre-glucose level (t = 2.15, df = 9, p < 0 . 0 5 ) while diabetics increased their corrected caloric intake to a value higher than the pre-glucose level (t = 4.08, dr= 7, p<0.01). Body weights were also affected differentially during the glucose period. On the first day of glucose access, the controls showed an average gain of 7.8 g versus an average daily gain of 2.7 g during the pre-glucose period (t = 2.54, df = 9, p<0.05). Conversely, on the first day of glucose, diabetics lost an average of 11.5 g versus an average daily loss of 3.5 g in the pre-glucose period, but due to a very high variance this difference was not statistically reliable by parametric techniques (t = 1.36, df = 7, p<0.10), but was so by nonparametric ones (Wilcoxon T = O, p<0.05).
intakes. It could be argued that had energy balance been calculated as a function of body weight, the diabetic animals could still have been considered hyperphagic under all conditions. This would be true, but it still could not account for the greater reduction in the food intake of diabetics. Furthermore, one has to consider that diabetics cannot deposit fat, thus their low weight is not natural, but obligatory, and absolute data comparisons should still be more appropriate than relative ones. EXPERIMENT 2 As an alternative to a caloric compensatory mechanism, it is possible that food intake reduction during glucose access in diabetics is due to a further increase in hyperosmolar blood glucose levels, and hence a secondary consequence of thirst. With only 5% and 35% glucose available, and no insulin to permit glucose entry into cells, thirst inducing extracellular hyperosmolarity may be forced upon the animal. Although this possibility is somewhat unlikely from the perspective that 5% glucose is hypoosmolar with respect to body fluids, it is conceivable that the slight tonicity was still insufficient to dilute the osmotic potential of the dry lab chow which was eaten. Accordingly, in the following experiment we measured food intake changes when diabetic and normal animals were given free access to a 35% glucose solution and water. With water available, the diabetic animals could readily correct any abnormal hyperosmolarity of blood above and beyond normally encountered in diabetes. Thus any food intake reduction to voluntary glucose intake would more strongly indicate the decrease in feeding to be a calorically mediated compensatory mechanism. METHOD
Animals DISCUSSION Although diabetic hyperphagia under certain conditions (chow only) can mathematically be explained as due to loss of glucose energy in the urine, this appears to be an oversimplification. When diabetic rats are given free access to glucose solutions, their effective energy intake is below normal. During glucose access, normal rats reduced their food intake by half that required to maintain stable energy intake. Diabetic animals showed a 71% compensation. Adding to this the 70% increase in glucosuria precipitated by the glucose intake, the diabetics were retaining reliably less energy in their bodies than controls. In fact their body weight loss was hastened by the glucose intake. This must mean that under physiological condition where insulin secretion is known to be less than 2% of normal, the behavioral expression of energy balance regulation can be at least as precise as in intact rats. In fact if there were an absolute necessity for insulin in glucose mediated control of feeding, it would have been expected that most of the ingested glucose would have passed through the body with no behavioral effect at all. Of course, the fact that the behavioral effect was larger than normal does not necessitate the conclusion that insulin does not participate in the regulation of hormonally intact animals. The hyperglycemia of diabetic animals may be sufficient to sustain adequate passive diffusion of nutrients into normally insulin sensitive cells. The above interpretations are based on absolute food
The same animals from Experiment 1 were used in Experiment 2. Housing conditions were as described in Experiment 1.
Procedure Three days after the termination of Experiment 1 (i.e. 5 days after glucose termination), all animals were given free access to 35% glucose and water. A choice of the 2 solutions was allowed for 3 days, after which, all animals were given access to just water for a period of 2 days. Food was available ad lib throughout and food intakes measured daily. RESULTS Caloric intakes of normal and diabetic rats given access to water or water and 35% glucose are summarized in Table 2. Total caloric intake as glucose was equivalent for both normals and diabetics and amounted to slightly less than two thirds of the calories consumed as glucose when both 5% and 35% solutions were available in the previous experiment. A reduction in chow intake was again seen when glucose was consumed. Food intake of normal animals was reduced by 7.0 g (25.1 KCal) and of diabetics by 12.3 g (44.6 KCal). This reduction represents a compensation of 52.6% for controls and 100.3% for diabetics as compared to the pre-glucose caloric intake - values similar to those of 49.0%
DIABETES AND GLUCOSE INTAKE
767 TABLE 2
CALORIC INTAKE OF NORMAL AND DIABETIC ANIMALS GIVEN FREE ACCESS TO WATER OR WATER AND 35% GLUCOSE SOLUTION Preglucose Normals Diabetics Mean Chow Intake (KCal) Mean Glucose Intake (KCal) Mean Total Food Intake (KCal) Mean Water Intake (mls)
Glucose Normals Diabetics
Postglucose Normals Diabetics
93.4
160.4
68.3
115.8
91.2
145.7
--
--
47.7
44.4
--
--
93.4
160.4
116.1
160.2
91.2
145.7
43
192
237
33
182
and 71.2% obtained for controls and diabetics respectively in Experiment 1. Also, it should be noted that during glucose access diabetic animals increased water consumption (t = 2.72, d f = 7, p<0.05) while normal animals drank less (t = 5.77, d f = 9, p<0.01). Apparently, consumption of 35% glucose did increase the osmotic imbalance somewhat in diabetic animals. EXPERIMENT 3 From the first 2 experiments it is still not clear whether the presence of a hyperosmolar glucose solution is necessary in order to see a compensatory reduction in chow intake. In the following experiment, ad lib food intake was monitored with and without access to a 5% glucose solution and water. Further, to assess the possible contribution on non-glucose energy loss in the urine of diabetics, both glucosuria and ketosis were monitored. METHOD Animals
Five additional severely diabetic rats (Long-Evans, hooded) and five control animals (Long-Evans) were used in the present experiment. Animals were tested more than 4 weeks following production of diabetes. Procedure
A baseline of food intake, water intake, body weight, urine output and urinary glucose output was taken over a period of 3 days, whereupon a solution of 5% (w/v) d-glucose was made available in addition to the water for a period of 3 days, followed by 3 days of post-glucose baseline measurements. Each day, the ketone body content of the urine was checked with Ketostix® (Ames Co.) which will detect as little as 5 - 1 0 mg% of acetoacetate in urine. The reagent is less sensitive to acetone and does not react with 3hydroxybutyrate. Daily average urinary glucose output was measured potentiometrically with Yellow Springs Instruments Glucose Analyzer (YSI Model 23). RESULTS The mean chow intake, body weights, glucose intake and
5
glucose excretion of the diabetic and control rats for the various phases of the experiment are presented in Table 3 and these results were consistent from animal to animal. When a 5% solution of glucose was made available, both control and diabetic rats consumed large quantities in preference to the water. In the control animals, the average body weight increased 16 g for the 3 day period of glucose access. Urine volume increased approximately fourfold. Diabetic body weights dropped an average of 8 g during the access to glucose, urine volume almost doubled and urinary glucose concentrations increased 17%. With the exception of 1 diabetic and 1 control animal on 2 days of the pre-glucose baseline, fresh samples of both control and diabetic urine were consistently free of acetoacetate or acetone during all phases of the experiment. With only a dilute source of glucose available, the total daffy glucose consumed by diabetics was over 3 times that of controls. The compensatory reduction in chow intake for the added glucose calories and the effective diabetic energy intake are summarized in Fig. 3, and essentially replicate the pattern seen in Experiment 1. During glucose, total energy intake of controls increased by 19% while in diabetics the effective intake was reduced 26% (from 88.1 KCal to 65.2 KCal). Since this was done in the absence of any increase in water intake (in fact water intake dropped from 183 ml/day to 14 ml/day), the reduction of food intake is unlikely to be due to extra thirst induced by the glucose intake. It is noteworthy that diabetics again lost weight rapidly on the glucose regimen. EXPERIMENT 4 The ability of diabetic rats to compensate as well as controls for voluntarily ingested glucose suggests that the mechanisms which modulate the effects of glucose on energy intake are capable of functioning in diabetic rats. These receptors may lie within the ventromedial hypothalamus (VMH) since glucose has been shown to be effective in suppressing long term food intake when applied directly to the VMH [14]. Although a great deal of evidence indicates that the medial hypothalamic glucosesensitive system should require insulin for normal functioning [4,17], it is possible that extreme hyperglycemia may promote sufficient passage of nutrients to sustain near normal functioning in these cells in the stabilized diabetic. To test whether the hypothalamic response to glucose is
768
PANKSEPP AND MEEKER
TABLE 3 INTAKE AND EXCRETION PARAMETERS OF NORMAL AND DIABETIC RATS GIVEN FREE ACCESS TO A 5% GLUCOSE SOLUTION
Body Weight (g) Chow Intake (KCal.) Glucose Intake (KCal.) Water Intake (mls) Urine Vol. (mls.) Glucosuria* (mg%) Total Urinary Glucose (KCal.) Intake (KCal.) minus Glucose Loss (KCal.)
Pre
Controls Glucose
Post
Pre
Diabetics Glucose
Post
516 86.1 -39 19 77 0.1
532 82.4 20.0 2 76 39 0.1
531 90.1 -38 21 99 0.1
236 128.7 -183 154 7067 40.6
228 102.4 62.4 14 273 8385 99.6
234 128.8 -174 147 7556 42.0
86.0
102.3
90.0
88.1
65.2
86.8
*The relatively high levels of glucose in the urine of normal animals reflects the contribution of some unknown factor which yielded substantial readings on the YSI Glucose Analyzer. This factor was inversely related to the volume of urine in any given animal. Independent verification of the glucose levels spectrophotometrically confirmed that the urinary glucose of the normal 'animals was virtually zero.
o
o CONTROLS I
150
o---o
DIABETICS, TOTAL FOO
~---~
DIABETICS, FOOD
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MINUSGLucOSURIA/
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xs
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•
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I
% %
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%
#
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25
•
1
)
•
"
"
I
I
I
I
I
2
3
4
S
6
7
8
9
Days FIG. 3. Total and corrected caloric intakes for normal and diabetic animals of Experiment 3.
DIABETES AND GLUCOSE INTAKE
769
still present in the presence of hyperglycemia and low endogenous levels of insulin, in the following experiment, glucose was injected directly into the VMH of diabetic and non-diabetic rats. METHOD
Animals and Surgery Eighteen female, Long-Evans hooded rats were used in the present experiment. Eight rats which had been made diabetic by intraperitoneal injections of 125 mg/kg of alloxan at least 3 weeks prior to surgery and 10 nondiabetic rats were anaesthetized with sodium pentobarbital (40 mg/kg) and implanted with bilateral stainless steel cannulae aimed for the VMH (0.5 mm posterior to bregma, 0.75 mm lateral to the midline, 9.5 mm below the surface of the skull, mouthbar 5 mm above intraural zero). Injection needles were cut to extend 1.0 mm past the tip of the cannulae. Cannulae were constructed of 23 ga stainless steel needle tubing with 29 ga inserts.
Procedure Approximately 4 weeks after surgery, half the animals were injected bilaterally with 1.0 ul of 10% (w/v) d-glucose and half with 10% (w/v) 2-deoxy-D-glucose (2-DG) dissolved in sterile physiological saline. Three injections were given per day separated by 7 hr. Three days after the first injection, the same procedure was followed except the injected solutions were reversed. Food intakes and body weight were measured daily. RESULTS Daily food intake on the day prior to testing and on the 2 injection days are summarized in Table 4. Glucose delivered to the VMH suppressed 24 hr food intakes in both non-diabetic (t = 2.60, d f = 9, p<0.05) and diabetic rats (t = 2.48, d f = 7, p < 0 . 0 5 ) relative to the control injections (2-DG). The control injection tended to increase food intake in control animals whereas in diabetic animals, it reliably suppressed feeding. Although we can put forward no cogent reason why 2-DG should have suppressed the food intake of the diabetic animals, it should be noted that peripheral injections of 2-DG in diabetics has also been found to suppress food intake [10]. These results indicate that glucose administered into the medial hypothalamus of diabetic rats can inhibit feeding as effectively as in normal animals. TABLE4 MEAN TWENTY-FOURHOUR FOOD INTAKES IN NON-DIABETIC AND DIABETIC RATS FOLLOWING INJECTIONS OF d-GLUCOSE INTO THE VENTROMEDIALHYPOTHALAMUS Pre Controls (n=10) Diabetics (n=10)
2-DG
d-Glucose
18.8---4.6
22.6---5.5
15.6-+6.5 *
27.4-+4.6
21.6-+4.4"~*
15.2+-8.5*
Values are mean --+ SD. *p<0.05 (correlated t-test; 2-DG minus d-Glucose). tp<0.05 (correlated t-test; 2-DG minus Pre).
GENERAL DISCUSSION Diabetic rats allowed free access to glucose solutions voluntarily consume large quantities, preferably from a very dilute source, and they reduce their normal food intake as much as normal animals to compensate for the calories ingested as glucose. In fact, the diabetics' effective energy intake (food intake corrected for glucose lost in urine) during glucose access is reliably lower than the caloric intake of control animals. This difference is due to both a greater percent reduction of food intake by diabetics as well as to an increased excretion of glucose in urine. These results support previous work indicating that a low level of endogenous insulin does not impair the capacity of glucose to inhibit feeding [9,15], but they do not necessarily negate studies which have observed impaired glucose sensitive control of feeding in diabetic animals [2,10]. These latter studies have employed small doses of glucose, and it may be that glucose can only modulate feeding normally in diabetics when a certain minimum amount of glucose has been consumed. Further, though these results clearly indicate that glucose can modulate feeding in the absence of normal insulin levels [7,14], the data cannot be interpreted to mean that diabetics still possess all the physiological mechanisms which normally participate in glucose induced suppression of feeding. Chronic diabetics may only exhibit intact adjustments of feeding in response to glucose intake because of compensatory physiological changes which have resulted from the diabetes. For instance, diabetic hyperglycemia may be so high that additional increases resulting from glucose consumption can lead to a sufficiently high transmembrane gradient of glucose to allow passive diffusion to cells which restores normal rates of intracellular glucose metabolism. Even though the data from Experiment 4 are consistent with such a mechanism, other explanations for the normal glucose inhibition of feeding in diabetics are possible. For example, in normal animals both insulin dependent and insulin independent glucose sensitive systems may operate at the hypothalamic level to control food intake [4, 15, 17]. Thus, the diabetic animal's feeding may be controlled primarily by an insulin independent mechanism in the lateral hypothalamus [17], while the intact animal also utilizes medial hypothalamic insulin sensitive mechanisms [4]. Since the ventromedial system appears to organize long-term regulation of energy balance while the lateral system organizes shorter-term controls [ 1, 11, 13], the diabetic animal may possess starved longer-term regulatory elements in the medial hypothalamus and oversatiated insulin-independent sensors in the lateral hypothalamus. Thereby, the diabetic animal could be an unusual preparation where an active long-term signal of body nutrient depletion normally coexists with a strong insulin independent short-term glucostatic signal of satiety. This might explain why ventromedial hypothalamic lesions can attenuate diabetic overeating [5,14]. The lesion would reduce the effect of the body nutrient depletion signal on feeding. This might also explain the reduction in chow intake we observed in diabetic animals voluntarily consuming glucose. An increase in passage of glucose into normally insulin sensitive receptors may have tended to replete the cells. Certainly the capacity of glucose injections into the VMH are compatible with such a proposal. The fact that 2-DG also reduced intake remains problematic,
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PANKSEPP AND MEEKER
though it is heartening that the effect was not as large as produced by the d-glucose. Although the present data does give considerable support for the simple idea that diabetic hyperphagia could largely be the direct consequence of glucose energy lost in the urine, the situation is clearly more complex than that. It cannot explain the reduction of effective caloric intake during voluntary glucose consumption. Thus, during a time when excretion of glucose was reliably increased, food intake was not. Moreover, other features of diabetic feeding, such as increased meal sizes [12, 14, 16] are also difficult to explain simply by the energy losses in urine. If there were nothing but a rapidly acting causal link between energy loss in the urine and hyperphagia, then one would most probably predict that the frequency of feeding would be the major parameter to increase. Thus, it is more probable that the loss of energy in the urine establishes a chronic central state which is similar to starvation. It should be emphasized that the present work cannot be taken as a complete energy balance work-up on diabetic animals. We have primarily focused on how glucose loss
might relate to the regulatory pattern of diabetics. Although our data from Experiment 3 rule out the possibility that excretion of certain types of ketone bodies is pertinent to an energy balance analysis in severe chronic diabetics, there is an outside chance that other ketones such as/~-hydroxybutyrate were secreted in amounts that should be considered. Also, of the thousand or so other metabolites that are excreted in urine, there may be others which require attention. Also, a full energy balance analysis would require the measurement of differential energies expended in motor activity and non-behavioral thermogenesis. Certainly if it were found that glucose ingestion in the diabetic rat severely reduces these energy losses, our conclusion that diabetics actually consume less energy than controls could be brought into question. However, we consider these possibilities very unlikely, if but for the simple reason that during glucose access diabetic animals consistently lost weight. This indicates that their decrease in energy intake was at the expense of endogenous sources, and speaks strongly for the validity of our interpretations.
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