Physiology and Behavior, Vol. 9, pp. 741-746. Brain Research Publications Inc., 1972. Printed in U.S.A.
Analysis of Diabetic Hyperphagia and Polydipsia L. HERNANDEZ AND E. BRIESE 1
Department of Physiology, Universidad de Los Andes, Mbrida, Venezuela (Received 20 February 1972)
HERNANDEZ, L. AND E. BRIESE. Analysis of diabetic hyperphagia and polydipsia. PHYSIOL. BEHAV. 9(5)741-746, 1972.-Diabetic hyperphagia is similar to experimental hyperphagia obtained by ventromedial hypothalamic lesions in that it is associated with motivational deficit and finickiness. On the other hand it is different in that diabetic hyperphagia is asymptotieal while in the ventromedial hypothalamic lesioned animals hyperphagia subsides once obesity is attained. Also, diabetic hyperphagia is always accompanied by a polydipsia that is out of proportion to the increased food intake. This suggests that diabetes meUitus produces a biochemical lesion on an insulin dependent system probably more diffuse but more homogenous than electrolytic or radiofrequency hypothalamic lesions which result in hyperphagia. It is probable that this satiety insulin dependent system exerts a stronger inhibition on water than on food intake. Ventromedial hypothalamus Hyperphagia Insulin dependent system Satiety center
THE HYPERPHAGIA with hyperglycemia in diabetes mellitus was considered a paradox by Mayer . This indicates that the level of glucose alone is not the activator of the ventromedial chemoreceptors which should be rather sensitive to their own rate of glucose utilization . The paradox is apparent only if variations of glycemia are considered apart from other concurrent humoral variations and because extracellular environment was focused upon rather than the nutritional state of the cell. Concentration of extracellular glucose or even its rate of utilization are only factors which might influence or indicate the metabolic state of the cell but not the nutritional cell state in itself. It becomes increasingly evident t h a t for the nutritional state of the cell variations of insulinemia are more important than glycemia . As Kennedy observed "....an insulin-sensitive center would have considerable advantage over a purely glucose-sensitive one, since insulin levels fell after fasting, rose after feeding and rose still higher in obesity" . The work of Debons and his associates [6, 7, 8] and our own work [3, 4, 131 shown that insulin and probably other hormones such as somatotrophin , modulate ventromedial hypothalamic tone. Other r~ports [1, 2] indicate the same possibility. Consequently, Mayer's paradox vanishes. Diabetic hyperphagia is logical since lack of insulin yields a depression of hypothalamic ventromedial satiety center. Lack of insulin produces a functional or biochemical lesion homologous to physical lesions, electrolytic or by radiofrequency, and diabetic hyperphagia ensues. If this is true, diabetic hyperphagia should be similar to hypothalamic hyperphagia and this paper reports experiments aimed to investigate
this. Since the work of Miller, Bailey and Stevenson , Kennedy , and Teitelbaum , it is known that hypothalamic hyperphagia, obtained by bilateral lesions of ventromedial nuclei (VMH) is characteristically accompanied by a lessening of motivation for food and by finickiness. The motivational deficit manifests itself by a diminution of the readiness to work for food or to overcome obstacles in order to obtain food . Also, hypothalamic hyperphagic animals are more sensitive to the taste than to the caloric value of food . Consequently, we investigated some aspects of the feeding and drinking behavior of diabetic rats in order to see whether or not they are similar to those of rats with destroyed VMH.
METHOD AND RESULTS
General Methods Diabetes meUitus was induced by streptozotocin kindly donated by Dr. W. E. Dulin of Upjohn Company. Twentyseven male Sprague-Dawley rats with initial body weight between 272 and 464 g were used. During 1 4 - 1 9 days before the streptozotocin injection and 2 6 - 3 1 days after it, food and water intake, glucosuria and body weight were measured daily. Streptozotocin in citrate buffer pH 4.5 solution was injected into the jugular vein under superficial ether anesthesia or into the tail vein without anesthesia. To induce various degrees of diabetes, the rats were divided into 6 groups receiving different doses of streptozotocin as indicated in Table 1.
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HERNANDEZ AND BRIESE
Food intake was measured every 24 hr by weighing a spill-proof feeder  containing a known amount of pulverized standard rat food. Water was given in 250 ml inverted bottles with stainless steel drinking spouts and intakes measured by weighing the bottles on a scale. Preliminary tests were done to determine spillage in both normal and diabetic rats. For food measurements maximum spillage under any circumstances was less than 1% of the quantities measured and for water measurements less than 2%. Glucosuria was tested with Gluco-Cinta M75 Ely Lilly & Co. Hyperphagia and polydipsia were determined comparing the intake during the 10 days preceding the streptozotocin injection with the intake obtained in 10 consecutive days starting after the seventh postinjection day. An animal was considered hyperphagic and polydipsic when differences between the two periods were statistically significant according to an analysis of variance test. In our series of rats there was no hyperphagia nor polydipsia without glucosuria and without body weight decrease. According to these four criteria 22 animals became diabetics after streptozotocin as is shown in Table 1.
FIG. 1. Food intake in rat made diabetic by 55 mg/kg of streptozotocin,
TABLE I DOSES OF STREPTOZOTOCIN INJECTED BY VEIN USED TO PRODUCE DIABETES
Rats Streptozotocin (mg/kg)
65 55 50 45 35 30 Total
3 3 3 8 5 5
2 0 0 0 0 0
1 3 3 6 4 5
E 2.5 ¢D
,,=t I-Z 0 0 0
*Rats with glucosuria, hyperphagia, polydipsia and body weight decrease.
Hyperphagia Analysis Since after streptozotocin food intake increased asymptotically as shown in Fig. 1, a regression analysis was made for each dose group. Daily mean increments were calculated for each dose group, and the regression coefficient was calculated for each group. A linear relationship between log of food intake increments and time was found for each dose group with probability for the null hypothesis p< 0.01 to <0.001. Figure 2 shows this relationship for the 55 mg/kg dose group. It was also found that a linear relationship existed between the log of regression coefficients and doses as showed in Fig. 3. This indicates that there was a dose response relationship between streptozotocin and hyperphagia.
FIG. 2. Lineal relationship between the log food intake increment means and time for 3 rats made diabetic by 55 mg/kg of streptozotocin.
Polydipsia Analysis Daily increments of water intake were calculated in the same way as food intake increments, A linear relationship between water intake log and time was found (p<0.01) for each dose group. However no such relationship was found between the regression coefficient log of water intake increment and doses which indicates that with the present data dose r e s p o n s e r e l a t i o n s h i p between streptozotocin and polydipsia can not be established.
MOTIVATIONAL DEFICIT IN DIABETIC HYPERPHAGIA
0 ..J IZ bJ b. LL LU O (..)
03 03 bJ [E ~9 L~ n-
743 significant with a probability for the null hypothesis p< 0.001. With regard to the hunger motivated instrumental response, all animals, thoroughly trained, were first tested in a Skinner box on continuous reinforcement and on variable ratio (VR) 5 and VR 10 schedules of reinforcement, after 8 hr of fasting. The food reinforcement given was 45 mg standard food pellets (Noyes Company) delivered by pellet dispensers (Gerbrands Co.). On all these tests diabetic rats worked more than normal ones (middle diagram of Fig. 5). Then, a special pre-feeding schedule, similar to that indicated by Miller, Bailey and Stevenson , was tested, as follows: eleven hr of ad lib feeding in the home cage, 12 hr without food, half hour ad lib feeding on mash (powdered rat food and water 50%) and finally one half hr in the Skinner box on a VR 10 schedule of reinforcement. On this schedule, as can be seen in the extreme right diagram of Fig. 5 the diabetic rats, in order to obtain food, pressed the lever much less frequently than the normal animals. This difference was highly significant according to an analysis of variance test (p< 0.001 ).
DOSE (mg/kg) (3)
FIG. 3. Lineal relationship between the regression coefficient logs and streptozotocin doses. o
Relationship Between Hyperphagia and Polydipsia Three kinds of comparison were carried out: (1) water intake increments with food intake increments for each dose group by a linear regression analysis; (2) for each animal the ratio water intake/food intake (W/F), calculated for the 10 days preceding the streptozotocin injection and for the 10 days beginning with the seventh day after the injection (the ratios were compared by an analysis of variance); and (3) linear regression analysis between dose group means of W/F ratios and doses. We found that there was a linear relationship between the water intake increments and food intake increments. On the other hand the ratio W/F was significantly much higher in diabetic (p<0.001) animals than in the same animals before diabetes (Fig. 4). Finally a linear relationship was found (p<0.02) between W/F log and streptozotocin doses, with higher W/F increments for higher doses. The relationship between the doses of streptozotocin and W/F ratio is shown to be (W/F) d = (W/F)ieKlJ where (W/F) d is the ratio f o r a given s t r e p t o z o t o c i n dose, ( W / F ) i is the i n i t i a l ratio, K is the regression constant, e the natural log base and D the dose.
Behavioral Characteristics of Diabetic Hyperphagia F o u r rats with streptozotocin induced diabetes were compared with three normal rats of similar b o d y weight regarding their consumatory behavior and their hunger motivated instrumental behavior reinforced at variable ratio. The extreme left diagram of Fig. 5 illustrates the consumatory behavior of the four diabetic rats as compared with the three normal ones. All animals were trained, for at least 20 days, to get all their powdered food from the special feeders mentioned under general methods. An analysis o f variance test showed that the difference was
FIG. 4. Water/food intake ratios before (white columns) and after (striped columns) different doses of streptozotocin producing diabetes mellitus. Figures above columns indicate number of rats in each dose group. ¢
¢ w > w
03 o3 Lt~ ew 0_ ¢= LU > ..J
AFTER D E P R I V A T I ON
FIG. 5. Comparison of consumatory and instrumental behavior motivated by hunger and reinforced at VR l0 in 3 normal rats (striped columns) and 4 streptozotocin diabetic rats (white columns). Middle columns indicate the instrumental behavior after 8 hr of deprivation and extreme right columns illustrated the behavior after 12 hr of fasting followed by half hr of feeding on mash.
HERNANDEZ AND BRIESE
Taste Versus Calories
Since hyperphagic animals bearing hypothalamic lesions are more sensitive to taste than to caloric content of food, that is, they overemphasize the sensory and neglect the metabolic signals for food intake control , we tried to find out if diabetic hyperphagic animals behave alike. Consumption of the standard diet was compared to those of more pallatable or unpallatable diets. The pallatable diet consisted of powdered standard diet mixed with dextrose (D-Glucose No. 8343 E. Merck) 50% by weight . The reason dextrose was used rather than saccharin was to make our results comparable with those of Teitelbaum  and because other studies on glucose and saccharin preference in diabetic rats  were done with solutions to be drunk (not eaten) by the animals. Furthermore in these other studies the diabetic rats were compared with normal ones; consequently, there are no data on the behavior of hypothalamic hyperphagic rats. Our standard rat food, as stated by the commercial company which manufactures it (Protinal) yields 3.38 kcal per g; dextrose yields 4.1 kcal per g. The food intake of four normal rats in 24 hr was compared with that of four diabetic rats of similar body weight when they ate standard powdered rat food and when they ate the dextrose mixed diet. Before measuring glucose mixed diet intake, three days were allowed in order to accustom the animals to the new diet. As Fig. 6 shows the diabetic rats increased their food intake when given dextrose mixed diet while normal animals did not modify their intake. The differences were significant (p< 0.001) according to an analysis of variance test. Quinine was used as an addition to make the diet unpallatable. Instead of powdered standard food, each animal received on the floor of its home cage a block of food prepared according to the following formula: corn flour 500 g, gelatin 350 g, standard powdered rat food 250 g, powdered milk 250 g, vegetal butter 250 g, water 1760 ml, boiled together and left to set in a refrigerator. The blocks had a rubber consistency and there was no spilling. This diet was given to six normal and six diabetic adult male rats of similar body weight. Two days were allowed to accustom animals to the new diet. During the next three days, food intake was measured at 4, 8, 12, 16 and 24 hr after giving a fresh food block each day at 8 a.m. The fourth day the rats were deprived of food for 24 hr and, finally, the next day at 8 a.m. received a block of food with 1.024% of quinine sulfate. Food intake was determined at the same intervals as during the three days when unadulterated food was available. It can be seen in Fig. 7 that normal rats compensate for caloric deficit imposed by food deprivation; they ate more than usual in spite of being offered quinine bittered food. In contrast, diabetic rats were less motivated since, in spite of the 24 hr deprivation, they ate less of quinine adulterated food than of the unadulterated food which they ate normally. Figure 7 shows also that after the first 16 hr of feeding on quinine adulterated food, normal rats behave like diabetic ones, that is, they eat less adulterated food than their normal food intake. That is, during this period they were less motivated since they had already compensate for the caloric deficit by overeating during the first 16 hr. DISCUSSION These results indicate that there are striking similarities
20L L I0
60 50 hi V
3O Q 0
FIG. 6. Food intake in 24 hr of 4 normal rats (top) and 4 diabetic rats (bottom) when they ate standard diet (white columns) or glucose mixed diet (black columns).Verticalbars indicate standard error of means.
FIG. 7. Intakes of quinine adulterated diet (black columns) after 24 hr deprivation compared with usual standard food intakes in 6 diabetic (upper) and 6 normal (bottom) rats. The extreme right columns represent cumulative values in 24 hr; other columns represent intakes during the discrete intervals as indicated in abseisa.
between diabetic hyperphagia and hyperphagia obtained by physical lesions of VMH. Although diabetic rats consume more food if it is freely available they are less motivated for food than normal animals when they have to obtain it by performing a learned instrumental task reinforced at variable ratio. Diabetic rats are also more sensitive than normal ones to taste qualities of food. This suggests that diabetic hyperphagia is based on a mechanism similar to hypothalamic hyperphagia, that is, a decrease of VMH inhibitory tone. This constitutes an argument favorable to the view that VMH elements involved in food and water control are insulin dependent and that absolute or relative
MOTIVATIONAL DEFICIT IN DIABETIC HYPERPHAGIA deficit of insulin, as in diabetes, produces a functional lesion of those elements. Classifically, hunger and polyphagia in diabetes mellitus are attributed to loss of glucose in urine, that is, deprivation of part of total calories required for maintenance . But this theory does not mention the mechanism by which the loss of calories produces hunger. It only records a fact without giving a satisfactory explanation. Furthermore, glucosuria is not always associated with increased appetite; for instance, in elderly patients with mild diabetes, polyphagia may precede glycosuria for many years  and in congenital renal glycosuria there is no polyuria, polydipsia nor polyphagia . In addition, Young and Liu  reported that insulin reduces hyperphagia in diabetic rats while having no effect on the hyperphagia of diabetic rats with VMH lesions. This indicates that insulin exerts its effect through the VMH and that satieting function of VMH is insulin dependent. On the other hand there are important differences between the two kinds of hyperphagia. First, the hyperphagia in diabetic rats is asymptotically perfect while the hyperphagia by VMH lesions subsides once a certain level of obesity is reached [9, 24]. This is favorable to the idea that VMH lesions, both physical or biochemical, induce a rise in the set point for body weight regulation but since this new point is never reached in diabetic rats, they remain hyperphagic. Since eventually food intake in diabetic animals reaches the asymptote in spite of chronic body weight reduction, this also suggests that the new level of food intake in diabetic animals is limited by other factors different from body weight (obesity). Second, all our diabetic rats were hyperphagic and their hyperphagia depended on the degree of diabetes since a dose response relationship between streptozotocin and hyperphagia was found, while, as it is well known, only a fraction of VMH lesioned animals usually become hyperphagics. This difference may be attributed to the fact that insulin deficiency yields a selective and inescapable biochemical lesion of all the VMH insulin dependent elements involved in the satiety system, while the physical lesions are limited to only some parts of that system. In fact, in an additional experiment we found that from 19 male adult rats with histological identical electrolytic lesions only 11 became hyperphagics. Concerning the W/F intake ratios our findings are the opposite of what Miller, Bailey and Stevenson described as hypothalamic hypodipsia . In our diabetic rats polydipsia was more important than hyperphagia, that is, the W/F intake ratios were higher than normal. Recently Ferguson and Keesey  reported in VMH lesioned female rats W/F intake ratios comparable with control animals and suggested that polydipsia is due to hyperphagia and not a consequence of the lesion itself. However, in a recent experiment, not to be reported in detail here, in 11 rats with hyperphagia by electrolytic VMH lesions we found that the W/F ratio increased in 6, remained the same in 3 and decreased in 2. So, polydipsia can not be simply a
745 response to increased food intake. Also, the data reported by Kakolewski et aL  indicate a substantial and persistent increase of W/F ratio after electrolytic VMH lesions. The authors interpreted this relative polydipsia as due to transient diabetes insipidus, but it is clear from their data that the increased W/F coefficient do not diminish with time but rather augment. One can speculate that small anatomical differences in the physical VMH lesions produce hyperphagia with distinct W/F ratios while the biochemical lesion due to diabetes, being homogenous, invariably yields a higher polydipsia than hyperphagia, that is, a higher W/F ratio. Were that true one could deduce that normally the VMH insulin dependent system exerts a stronger inhibition on drinking than on feeding. This, of course, contradicts the classical ideas on diabetic polydipsia which is believed to be secondary to the well-known osmotic polyuria, that is, unabsorbed glucose prevents tubular absorption of water by virtue of its osmotic pressure . However, since glycosuria per se does not always produce polyuria nor polydipsia,  the hypothetic existence of an additional hypothalamic primary factor in the diabetic polydipsia should not be dismissed. The idea that the VMH inhibition is normally stronger on drinking than on feeding is also suggested by the well known stages of recovery of food and water regulation following lateral hypothalarnic lesions . The recovery proceeds through four sequential stages: (1) aphagia and adipsia, (2) anorexia and adipsia, (3) adipsia; and, (4) partial recovery. Immature rats acquire normal adult regulatory behavior through the same sequential stages . The fact that feeding regulation recovers or develops first and drinking later suggests that VMH inhibition on drinking is normally stronger than on feeding. An analogous conclusion can be drawn from the fact that genetically obsese rats maintained on limited food intake have larger water intake than controls [301. Teitelbaum  reported that dynamic hyperphagic animals in contrast to obese hyperphagic ones showed no significant decrease in their intake of quinine adulterated diet. He reported that "....it is not the hypothalamic lesions, but rather the obesity, which reduces hunger motivation." In our diabetic rats however, there was a hunger motivation deficit without obesity. On the contrary, diabetic rats are always below the normal body weight. This is in accord with the work of Graff and Stellar  which has shown that obesity is not a necessary condition for finickiness and that maximal finickiness was seen in animals that were not obese. Since hyperphagia, obesity and finickiness can be found independent of each other, these a u t h o r s suggested separate but overlapping neural mechanisms for hyperphagia and finickiness. In conclusion the experiments reported indicate that hyperphagia of rats with streptozotocin induced diabetes was dose dependent and paired with a motivational deficit similar to the hyperphagia obtained by physical lesions of VMH. This suggests the existance of an insulin dependent system in the VMH region probably identical to the so-called satiety center.
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