Physiological and behavioral responses to glucoprivation in the golden hamster

Physiological and behavioral responses to glucoprivation in the golden hamster

Physiology & Behavior, Vol. 30, pp. 743-747. Pergamon Press Ltd., 1983. Printed in the U.S.A. Physiological and Behavioral Responses to Glucoprivatio...

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Physiology & Behavior, Vol. 30, pp. 743-747. Pergamon Press Ltd., 1983. Printed in the U.S.A.

Physiological and Behavioral Responses to Glucoprivation in the Golden Hamster NEIL ROWLAND

D e p a r t m e n t o f Psychology, University o f Florida, Gainesville, F L 3261I R e c e i v e d 22 D e c e m b e r 1982 ROWLAND, N. Physiological and behavioral responses to glucoprivation in the golden hamster. PHYSIOL BEHAV 30(5) 743-747, 1983.--Golden hamsters failed to increase their food intake following food deprivation alone or in combination with insulin or 2-deoxy-D-glucose (2DG) treatment. 2DG also failed to induce feeding in hamsters tested at night. In this latter experiment, there was no effect of 2DG on wheel running or general alertness. Insulin administration significantly decreased plasma levels of glucose and free fatty acids (FFA). 2DG treatment produced a dose-related hyperglycemia associated with increased ketone levels. These data are discussed in terms of cerebral energy status and its relation to food intake and physiological responses. Golden hamster Glucoprivation 2-deoxy-D-glucose Glucose Ketones Free fatty acids Ischymetry

Insulin

Fasting

Feeding

Activity

METHOD G O L D E N hamsters (Mesocricetus auratus) differ substantially from the much-studied laboratory rat in several aspects of their feeding behavior. They fail to increase meal size or food intake following food deprivation [1, 13, 16]. They also fail to increase food intake in response to acute glucoprivation induced by 2-deoxy-D-glucose (2DG) [10, 12, 14, 15] or 5-thioglucose (5TG) [3], both glucose antimetabolites which provoke sympathoadrenal activation and daytime feeding in rats [5, 6, 11, 17]. While 2DG does provoke hyperglycemia in hamsters [10,12], the physiological responses have been poorly studied. The purpose of the present experiments is to determine whether 2DG elicits feeding i n hamsters tested when food deprived (empty stomach), or at night when effects on activity can also be assessed. These data are complemented by plasma measures of glucose, ketones and free fatty acids ( F F A ) following 2DG treatment and, for comparison, insulin or deprivation conditions. The data suggest that systemic injections of 2DG may not in fact produce a cerebral metabolic emergency, and this could explain the lack of effect on food intake or general activity. E X P E R I M E N T I: E F F E C T O F PREVIOUS F O O D D E P R I V A T I O N ON F O O D I N T A K E A F T E R 2DG OR INSULIN It has been noted that the upper gastrointestinal tract of hamsters contains substantial amounts of undigested food at all times during ad lib feeding. In particular, the forestomach seems to fill and empty with about the periodicity of spontaneous meals, while the fundus is always full [13]. The forestomach may, then, contribute inhibitory signals to a feeding control mechanism. The present experiment examines whether a feeding response to glucoprivation might not be evident after food deprivation. A deprivation of 18 hr was used to ensure emptying of both forestomach and fundus.,

Animals and Housing Adult male and female golden hamsters (Mesocricetus auratus) were purchased from Charles River Laboratories, Lakeview, New Jersey, or were bred in this laboratory. They were housed individually in hanging wire cages with a plastic dish (for a bed) and chow pellets (Purina No. 5001) inside the cage. Tap water was also available ad lib.

Procedure Three males and four females were assigned to each of three weight-matched (mean=78 g) experimental groups. (In these, and many other experiments we have found no sex or age differences in hamster feeding measures.) Food was removed in the afternoon, 18 hr before the experiment. Next morning, the three groups received injections o f saline, 2DG (Sigma; 500 mg/kg in 100 mg/ml saline, IP) or regular insulin (Lilly, 100 U/kg at 1 ml/kg insulin SC) and were immediately returned to their home cage with a weighed amount of food. Intakes were recorded 30, 60, 120 and 240 min later, corrected for any spillage.

Statistics In this and subsequent experiments, the data were analyzed by A N O V A using the SAS release 79.6 programs. Significant main effects were examined using Duncan post hoc tests. In this case, a repeated measures A N O V A procedure was used. RESULTS The results are shown in Table 1 for 30 and 240 rain (the intermediate times gave data almost exactly as predicted by linear interpolation). All of the hamsters ate as soon as the food was presented, but there were no differences in the total

Copyright © 1983 Pergamon Press Ltd.--0031-9384/83/050743-05503.00

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TABLE 1 EFFECTS OF INSULIN AND 2DG ON FOOD INTAKE AFTER 18 HOURS FOOD DEPRIVATION IN HAMSTERS 30 min Deprived + Saline Deprived + Insulin (100 U/kg, SC) Deprived + 2DG (500 mg/kg, IP)

TABLE 2 THREE HOUR FOOD INTAKE OF SEDENTARY AND WHEEl_, EXERCISING HAMSTERS FOLLOWINGSALINE OR 2DG INJECTION AND CORRESPONDING WHEEL REVOLUTIONS IN THAT TIME

240 min

0.74 ± 0.08* 0.64 ± 0.09

1.96 ± 0.23 1.76 ± 0.14

0.67 ± 0.11

2.07 ± 0.26

*M ± SE food intake (g) for groups of 7.

Sedentary Food Intake (g) Exercising Food Intake (g) Activity (revs.)

Saline

2I)G ~1000 mg/kg. IP)

1.73 ± 0.19"

0.88 :: 0.20:i:

0.60 ± 0.314 5593 ± 318

0.94 ~ 0.46 5024 z 229

*M ± SE for groups of 6. gp<0.01 vs. sedentary group. Sp<0.01 vs. saline condition. amount consumed (Groups F=1.66, F=0.25, n.s.).

n.s.; G r o u p x T i m e

E X P E R I M E N T 2: E F F E C T S O F 2DG ON N O C T U R N A L F E E D I N G A N D ACTIVITY In the previous experiment we showed that glucoprivation did not induce additional food intake in deprived hamsters. These data thus complement previous observations in ad lib fed hamsters showing no hyperphagia in response to 2DG (sometimes a suppression), and a marginal and late increase in feeding after insulin [3, 10, 12]. However, all of these experiments have been performed during the daytime and, in other work in this laboratory, we have identified this as the lipogenic phase of the hamster's metabolic cycle. The rat, which is in a lipolytic phase during the day, shows good 2DG-induced eating at this time but poor stimulation of feeding during the nocturnal lipogenic phase [6]. One interpretation of these data is that 2DG is only effective during lipolytic periods. We therefore injected hamsters with 2DG at the start of their night and observed the subsequent feeding. An additional group of hamsters was studied in activity wheels in order to document the effects of 2DG on general activity (rats become lethargic with high doses of 2DG, and it is evident that some of the doses used in hamsters are enormous by rat standards). METHOD

Animals and Housing Twelve experimentally naive female hamsters were housed in a vivarium with lights off 2200-0800 hr. Six of the animals were placed in running wheels (Wahmann; 1 m running circumference) with revolution counters and side cages, two weeks prior to the experiment. During this time they all developed vigorous running patterns (9--21 thousand rev/night). The other six animals were housed individually in standard hanging cages. All had free access to Purina No. 5001 powdered chow (in glass jars) and tap water.

Procedure Baseline measures of food intake and running were recorded on the first day of the experiment. The animals were injected with 0.9% saline (0.5 ml) IP between 2140 and 2155 hr. The running animals were typically in their side cages at this time, and were captured by closing the door to the wheel. (Difficulty in non-stressful capture o f these animals while they were running dictated our protocol of injection

just before lights out, rather than in the middle of the night.) The food jars and wheel revolutions were recorded l, 2 and 3 hr after lights off. The following day, the same procedure was followed except that the injection was 1000 mg/kg 2DG IP, given in 5 ml/kg 0.9% NaC1 vehicle. This hypertonic solution caused only transient signs of distress. The mean body weights of both exercising and sedentary groups was 112 g at the time of injections, and the 24 hr intakes for 2 days prior to the experiment were 12.6 and 12.8 g for exercising and sedentary groups, respectively. RESULTS The results are shown in Table 2. The baseline intakes were different between the two groups (p<0.01; t-test); only the 3 hr data are shown, but in each case the hourly subtotals were almost exactly 33% of the 3 hr total. The wheel running animals ate less than the sedentary controls (which were, nonetheless, active within their home cage for much of the time). Treatment with 2DG suppressed the food intake of the sedentary animals, but had no effect on either the intake or the wheel running of the exercising hamsters. Interestingly, the sedentary hamsters appeared to be more active after 2DG treatment, making more attempts to escape while the food was being measured relative to the baseline day. They also were warm to the touch at this time. DISCUSSION Hamsters showed no feeding response to 2DG at night suggesting that their daytime failures are not simply a reflection of the stage of their metabolic circadian cycle. They also did not show impairments in activity or general alertness after this high dose of 2DG. Indeed, the decreased intake in the sedentary animals which might have been attributed to expected hypoactivity during glucoprivation, may instead be an effect of increased home cage activity. E X P E R I M E N T 3: M E T A B O L I C C H A N G E S A S S O C I A T E D W I T H G L U C O P R I V A T I O N IN HAMSTERS It has been shown that hamsters, like rats, exhibit a hyperglycemic response to 2DG [10,12]. Their failure to feed after 2DG might, however, be the result of adequate utiliza-

2DG IN H A M S T E R S

745 TABLE 3 PLASMA CONCENTRATIONS (MEANS - SE) OF GLUCOSE, TOTAL KETONES, AND FREE FATTY ACIDS (FFA) OF HAMSTERS AND RATS 1 HOUR FOLLOWING2DG OR INSULIN TREATMENT Glucose (mg/dl) Experiment 3A (Hamsters) Ad Lib (6) Deprived 24 hr (4) Insulin (100 U/kg) (5) 2DG (500 mg/kg) (5) Experiment 3B (Hamsters) Ad Lib (6) 2DG (500 mg/kg) (6) 2DG (1000 mg/kg) (6) Experiment 3B (Rats) Ad Lib (5) 2DG (300 mg/kg) (5) Number *p<0.05 tp<0.05 $p<0.05

120 ± 123 ± 46 ± 134 ±

5 12 7* 5

Ketones (mM)

1.04 2.22 0.46 0.97

± 0.58 _+ 0.40* ± 0.05 + 0.19

FFA (mM)

0.85 1.03 0.58 0.88

± ±

0.09 0.07 0.03* 0.05

145 ___ 7 218 _+ 24t 332 ± 13:~

1.16 +_ 0.22 1.81 ±_ 0.68 3.00 ± 0.48t

0.48 ± 0.04 0.48 ± 0.08 0.55 ± 0.04

159 ± 2 322 ± 9t

0.35 ± 0.16 0.51 ± 0.12

0.23 ± 0.08 0.52 ± 0.06~

of animals in parentheses. different from all other groups (Exp. 3A). different from corresponding control (ad lib) group (Exp. 3B). different from both ad lib and 2DG (500) groups.

tion of alternative metabolic fuels by energy sensitive neurons. We have reported that 2DG feeding in rats is abolished by infusions of ketones, a substrate which is well-utilized by brain [17]. It is possible that hamsters produce large amounts of ketones after 2DG, and any brain receptors might not then be in a metabolic state of emergency. In the first part of this experiment we have compared the glucose, F F A , and ketone levels in blood of fed, fasted, insulin or 2DG-treated hamsters. In the second part of the experiment we present data from two doses of 2DG in hamsters, and comparative data from rats. METHOD

Procedure: Experiment 3A The subjects from Experiment 1 were allowed 3 days to recover, then were randomly assigned to four groups. One group was fasted for 24 hr. The other three groups had food freely available until injection of saline, insulin (100 U/kg, SC) or 2DG (500 mg/kg, IP). Food was then removed and 1 hr later the animals were sacrificed by decapitation. Trunk blood was collected in chilled tubes containing heparin and NaF, and the plasma was saved for subsequent determination of glucose (Yellow Springs glucose analyzer 23A), F F A (using a colorimetric micro-method developed by Ramii'ez) (cited in [4]), and total ketones [19].

Procedure: Experiment 3B Eighteen experimentally naive female hamsters (mean body weight 120 g), from the same vendors batch as used in Experiment 2, received IP injections of saline, or 2DG (500 or 1000 mg/kg). F o o d was removed and, 1 hr later, they were anesthetized with ether and 0.7 ml blood was taken by cardiac puncture. The plasma was assayed as above. Ten naive female Sprague-Dawley (Zivic Miller) rats (mean body weight 198 g) were similarly injected with saline

or 2DG (300 mg/kg), and blood was taken 1 hr later by cardiac puncture. RESULTS The results of the plasma determinations are shown in Table 3.

Experiment 3A The A N O V A tests showed significant group main effects for all three plasma metabolites (F glucose=29.3, p<0.0001; F ketone=5.0, p<0.05; F F F A = 5 . 6 , p<0.01). Deprivation produced a significant elevation of ketone bodies and good maintenance of glucose levels in agreement with our previous observations [13]. Insulin treatment produced substantial hypoglycemia, and suppression of both F F A and ketones although this latter was not statistically significant. 2DG (500 mg/kg), surprisingly, had no effect whatsoever on any of the plasma metabolites. It is, therefore, maybe not so strange that hamsters in this group did not eat to this dose of 2DG (Experiment 1).

Experiment 3B The A N O V A tests showed significant group main effects for glucose (F=26.5, p<0.001), but not for ketones (F=2.8, p<0.10) or F F A (F=0.3, p > 0 . 5 ) in hamsters. The glucose values were different between all three groups. All of the hamsters given 1000 mg/kg 2DG showed blood glucose levels over 270 mg/dl; in contrast, at the 500 mg/kg dose, 2DG provoked marked hyperglycemia (>200 mg/dl) in only 3 of the 6 animals. In this respect, this dose seems to be close to a threshold or ED50. Despite the non-significant (0.1 >p>0.05) overall F value, the ketone level in the 1000 mg/kg group was substantially elevated (p<0.05, Duncan) but F F A values were not affected. There was a significant correlation between plasma glucose and ketone concentrations,

r(18)=+0.57, p<0.01, and between ketones and F F A levels (r= +0.75, p<0,01 ). In rats, the much lower dose of 300 mg/kg 2DG produced a comparably large hyperglycemia. F F A levels were also elevated significantly, but the change in ketones was not significant. Notice that the ketone and F F A values are considerably lower in the rat groups than in corresponding hamster groups. DISCUSSION Treatment of hamsters with insulin, or food deprivation, produced physiological effects which are largely comparable to those that have been widely documented in rats. The major results concern the effects of 2DG in hamsters. In Experiment 3A, 2DG had no effect at a dose of 500 mg/kg, but in Experiment 3B this dose was close to an ED50. The only obvious difference between the experiments is the prior deprivation experience of the hamsters in 3A. A high enough dose of 2DG produced large elevations in blood glucose and corresponding rapid increases in ketone levels without change in F F A . This observation suggests that the biochemical trigger of ketogenesis may differ between hamsters and rats during 2DG glucoprivation. These results are at slight variance with those of Ritter and Balch [1(}] who reported a hyperglycemia of 200 mg/dl above control l e v e l s - comparable to the physiological effect in our high dose g r o u p - - b u t they administered only 500 mg/kg 2DG. Differences in the initial physiological status of the animals and/or the availability of food might have produced an apparent shift downwards in the dose-effect curve. Their procedure of successive blood sampling by retro-orbital puncture (anesthesia was not mentioned) might also affect the data. In experiments with hamsters bearing indwelling jugular vein catheters I have found that serial blood sampling interferes with the clearance from plasma of a glucose load. In the Ritter and Balch experiment, therefore, a small 2DG-induced hyperglycemia might have been greatly exaggerated by their procedure. The data from rats confirm the well-known hyperglycemia and increased F F A levels in animals studied during the daytime [2,18]. We also show a trend toward increased ketone levels, and further work is needed to see whether the increase here is greater at longer intervals after 2DG injection and/or higher doses. These data are at variance with a report [6] that 2DG suppresses F F A levels during the daytime in fed or fasted rats; those investigators found a nocturnal mobilization of F F A by 2DG and suggested such a (sympathetically-mediated?) lipomobilization might prevent nocturnal feeding to 2DG. GENERAL DISCUSSION The failure of hamsters to eat at 2DG is a robust phenomenon. In addition to occurring during the daytime to a variety of doses and chow [10, 12, 15] or sunflower seed [ 14] diet, it now has been observed after food deprivation, by night, and in exercising animals which have a lower baseline intake.

The critical issue to be addressed is v~hcther 21)(J itscff produces glucoprivation and decreased cerebral metabolic rate. We have shown that the feeding response I~ 2I)(; m rats, like the sympathoadrenal response, Zs generated b~, a receptor in the CNS [ 17]. Since this receptor is responsive t~ alternate melabolic fuels such as ketones and the existence of such neurons has recently been demonstrated eiectrophysiologically [8], then its metabolic rate or ischymetJ,v would not be affected by 2DG if there were a large endogenous supply of ketones. This seems to be the case since, whenever hyperglycemia was observed (Experiment 3), hyperketonemia also occurred. Further verification of this speculation must await whole body and brain respiration experiments. The failure of hamsters to eat at 2DG might, therefore, be because the brain is never metabolically compromised. From the insulin experiments, when no such alternate fuel is available, it seems that hamster brain is particularly well-protected from glucose- related emergencies (hamsters with glucose levels as low as 20 mg/dl do not show neurological symptoms). However, a truly acute emergency induced by intracerebroventricular injection of 2DG (5 rag) does induce food intake with a latency of 15 rain in hamsters, but the amount eaten is about a normal meal I0.5-I g) !unpublished observations, November, 1982.i While this manuscript was in preparation, DiBattista [3] presented data showing that hamsters fail to eat in response to 5-thioglucose (5TG), a glucose antimetabolite which, in rats, causes feeding and physiological changes similar to 2DG [1 I]. DiBattista also showed that 5TG produced F F A mobilization in hamsters, a difference from the present 2DG data, but did not present ketone levels. However, it is possible that higher doses of 2DG and/or longer postinjection intervals might reveal significant increases in F F A since the lower dose 5TG treatments produced hyperglycemia without elevated F F A [3]. Our finding of a strong correlation between ketone and F F A levels supports this suggestion. Regardless of the F F A issue, however, it appears ketogenesis occurs after 2DG in hamsters but not in rats. It is at least possible that this ketogenesis in hamsters is sufficient to sustain the ischymetry, or metabolic rate, or essential CNS neurons [8]. In this case. the nature of ~he receptor responsible for triggering ketogenesis (and/or F F A mobilization) in hamsters becomes less clear. It would be intriguing if it were not located in the brain (see [17] for discussion). Finally, glucoprivation may engage feeding in rats by activation of various neurotransmitter systems including endorphin/enkephalins [7,9], In this regard, it is interesting that hamster feeding seems to be opiate-insensitive [7], indicating that quite different neural controls exist in this species. ACKNOWLEDGEMENT Supported by NIH grants AM 30660 and AM 30669. 1 thank Tim Bartness, Linda Bellush, Susan Cochran, Gerri Lennon and Gloria Smith for assistance.

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2DG IN HAMSTERS

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