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Sympathoadrenal activity and hypoglycemia in the hibernating garden dormouse
Physiology& Behavior, Vol. 48, pp. 783-787. ©PergamonPress plc, 1990.Printedin the U.S.A.
0031-9384/90$3.00 + .(30
Sympathoadrenal Activity and Hypoglycemia in the Hibernating Garden Dormouse C. ATGIE, M. NIBBELINK AND L. AMBID 1
Laboratoire des R~gulations des M~tabolismes et Nutrition, Universit~ Paul Sabatier rue Franfois Magendie 31400 Toulouse C~dex, France
ATGIE, C., M. NIBBELINKAND L. AMBID. Sympathoadrenal activity and hypoglycemia in the hibernating garden dormouse. PHYSIOL BEHAV 48(6) 783-787, 1990.--The hibernatinggarden dormouse is spontaneouslyhypophagic during the prehibernating period at which time we found a low peripheral sympathetic activity (S.A.). The aim of this work was to investigatethe link between dietary intake and S.A. The S.A. was evaluatedby measurementof catecholaminesin both plasma and adrenal glands by I-IPLC. Food intake, body weight, energy expenditureand plasma glucose were measured during the reentry phase of the hibernating period. The followingresults were obtained:the energy intake in pretorpid animals (55 to 83 kJ/24 h/100 g body weight) was less than energy expenditurewhich was between 145 and 97 kJ/24 h/100 g. The energy deficit induces marked hypoglycemiaimmediately before the onset of hypothermia (117 mg/dl vs. 76 mg/dl) and leads to a drastic drop in the peripheral sympathetic system. This, in turn, reduced energy production, causing the hypothermia. Hibernation Food intake Sympathoadrenalactivity Garden dormouse (Eliomys quercinus L.)
Catecholamines
STUDIES from our laboratory have shown that fasting or restricted caloric intake drastically reduces the sympathetic nervous system activity which is always associated with a fall in body temperature in the garden dormice (2, 3, 19). Reduction of sympathetic nervous system activity when caloric intake is reduced implies a basic mechanism within the central nervous system that assesses nutritional status and regulates sympathetic outflow accordingly. Experiments in rats with hypoglycemia suggest that the availability of glucose may be involved in the relationship between dietary intake and sympathetic activity (2, 3, 16). Garden dormice (Eliomys quercinus L.) are hibernating rodents that undergo spontaneous annual variations in food intake and body weight. In these animals, the fall in body temperature during hibernation is accompanied by a reduction in glucose levels (1). The nature of the signals that couple changes in dietary intake with changes in sympathetic activity is, however, still uncertain in hibernating rodents. The present paper reports on the changes in the activity of the sympathetic adrenal system (S.A.S.), during entrance into hibernation in midwinter and after periods of continuous hypothermia in a cold room at 6°C with a photoperiod L:D =0030:2330 h. The S.A.S. activity was evaluated by measurement of catecbolamine levels and dopamine-[3-hydroxylase (DflH) activity in the plasma and adrenal medulla. We then evaluated concomitant changes in food consumption, plasma glucose
DI3H activity
and energy expenditure during the bouts of homeothermia, hypothermia and awakening in the hibernating cycle. METHOD Anima/s The experiments were carded out on 60 garden dormice
(Eliomys quercinus L.) captured in the Dombes area of France, and subsequently maintained in the laboratory. They were housed in individual cages containing a wooden nest box, and had access to food (pellets for rats and apples) ad lib. Before the experiment, the animals were placed in a fight:dark photoperiod of 0030:2330 h in a cold room at 6°C. At this temperature, they become torpid, and their body temperature drops to the ambient temperature. From time to time, they wake up spontaneously, their body temperature increases, and they enter into a period of homeotbermic activity lasting several hours. They then fall back into a state of hypothermic torpor. The bouts of homeotbermia, hypothermia and arousal are schematized in Fig. 1. Changes in food intake were studied on the days before (stages 1 and 2) and after the periods of hypothermia (stage 3). For the daily measurement of food intake, 6 animals were placed in metabolism cages. Using the respective energetic values for pel-
~Requests for reprints should be addressed to L. Ambid.
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TABLE 1 BODY WEIGHTAND ENERGYINTAKEIN WAKEFULGARDENDORMICE OVER 5 MONTHSIN A COLDROOM AT 6°C WITH A PHOTOPERIOD L:D = 0300:2330 h Body Weight (g)
Energy Intake kJ/100 g/24 h
Nov
118 _-. 1.4 (24)
168 +_ 10.9 (32)
Dec
110 -+ 1.3 (24)
175 _ 5.8 (32)
Jan
105 -+ 1.5 (24)
190 _ 10.5 (37)
Feb
102 _ 2.5 (24)
200 _ 13.6 (32)
Mar
101 _+ 1.3 (24)
242 _+ 8.6 (32)
HYPOTHERMIA FIG. 1. Schematized cycle of arousal and lethargy during hibernation. Stage 1: normal wakefulness; stage 2:24 hours before hibernation; stage 3: return to homeothermia after a bout of hibernation. The hypothermic bout is broken by periodic arousal lasting about 24 hours at 6°C. The animals then become progressively hypothermic once more and at 6°C will remain lethargic for about 10 days.
lets and apple (12.96 and 2.18 kJ/g), the daily energy intake was calculated in kJ/100 g/24 h. Food intake for stage 1 was measured on the two days preceding stage 2. The animals were weighed once a week over a period of 6 months. The locomotion activity was measured continuously by an actographic system and the energy expenditure indirectly by the determination of the oxygen consumption (VO2) in a closed circuit respirometer at thermal ambiance (6°C). Rates of metabolic heat production were calculated from steady-state determinations of VO 2 assuming 20.16 kJ of heat were produced for each liter of 0 2 consumed.
Measurement of Sympathetic Adrenal System (S.A.S.) Activity The S.A.S. activity was estimated from the measurement of catecholamine (CA) levels and dopamine-13-hydroxylase (DI3H) activity in both the adrenal gland and the plasma. DBH is involved in the transformation of dopamine into norepinephrine (NE) in the synaptic vesicles of sympathetic nerve terminals, and is liberated along with NE in the process of exocytosis. The concomitant release of neurotransmitter and DI3H means that the plasma DI3H activity is representative of plasma levels of NE, and is thus a good indicator of sympathetic activity (7).
Catecholamine Assays Plasma and adrenal catecholamines (NE and epinephrine E) were assayed by HPLC using an ODS ultrasphere column (25 cm) followed by an electrochemical detector (BAS-IAC). A detection potential of 0.60 volt was selected. The mobile phase was acetate-citrate buffer pH 5.2 (15). The accuracy of the method was checked by injecting known quantities of catecholamines. The overall error was 5%. Over 10 injections we found an 8% variation within a trial, and a 5% variation between trials. The overall sensitivity was 5 pg/I.d. The animals were killed by decapitation, and blood was collected on heparin, EGTA and glutathione. The blood samples were centrifuged, and the plasma decanted and kept at - 2 5 ° C until assay. After sacrifice, the left adrenal gland was rapidly removed and homogenized in a Potter apparatus in 6 ml of medium containing perchloric acid (0.1 M), EDTA (2.7 mM), glutathione (5 mM), and sodium metabisulfite (0.2 mM). The homogenate was centrifuged (10,000 × g, 4°C) and the supernatant was frozen at - 80°C
The mean and S.E.M, are shown. The total number of measurements is indicated in parentheses. Six animals were used.
before CA extraction. Plasma and adrenal supernatant CA were extracted on activated alumina (4) as previously described (9) before injection into the HPLC system.
Df3H Activity Assays DI3H activity was determined using the method described by Nagatsu and Undenfriend (20) based on the conversion of tyramine to octopamine. The results are expressed as units of activity (U = t~mole of octopamine, formed per hour at 37°C). After sacrifice of the garden dormouse, the right adrenal gland was immediately removed and homogenized in a Potter apparatus in 8 ml of medium containing Tris-HC1 (5 raM), NaC1 (150 mM), Triton X100 (0.2%), bovine serum albumin (0.2%) pH 7.2. The homogenate was then centrifuged (80,000 x g, 4°C) and the supernatant was frozen at - 80°C until DI3H activity determination.
Plasma Glucose Assays Plasma glucose was assayed by spectrophotometry with a Biotrol enzymatic kit A-02460. The results are presented as mean values and standard errors of the mean (SEM). The mean values were compared using Student's t-test for paired and unpaired samples as appropriate. RESULTS In garden dormice, during the period of hibernation (November to March) the mean body weight fell by 14%, and the energy intake during the periods of euthermia (rectal temperature t°r = 37°(2) ranged from 168 kJ/100 g/day in November to 242 in March (Table 1). Towards the spring arousal there was a progressive increase in food intake. In reality, the information given by these data in insufficient, because during the winter the food intake of the garden dormouse is variable and follows the cycles of lethargy and arousal. It is for this reason that an effort was made to defme the variations of the food intake more clearly in relation to the time, during the bouts of wakefulness and hibernation. Thus, changes in energy intake were studied on the days before and after the periods of hypothermia (Fig. 1). The results are shown in
SYMPATHOADRENAL ACTIVITY AND HYPOGLYCEMIA
785
TABLE 2
Energy Expenditure
ENERGY INTAKE IN THE GARDEN DORMOUSE 48 h (STAGE l) AND 24 h (STAGE 2) BEFORE THE START OF HIBERNATION AT 6°(: AND 24 h POSTAROUSAL (STAGE 3) AT 6°(2
J= ,q,
Stage 2
/r**
125 0 0
Energy Intake (kJ/100 g/24 h) Stage 1
150-
100 75
Stage 3
~ / r / t / r
50
Nov
168 - 10.9" (32)
67 -+ 13.5 (18)
168 - 11.8" (18)
Dec
175 -+ 5.8* (32)
74 -+ 7.6 (13)
160 _+ 9.0 (14)
Jan
190 _ 10.5" (32)
55 -+ 14.7 (11)
170 --+ 12.1' (18)
Feb
200 _ 13.6" (32)
76 -+ 13.5 (5)
201 -
242 - 8.6* (32)
83 - 15.4 (5)
240 _ 13.4" (8)
Mar
25
IIIIIIIIIIIII1
IIIIIllVIIIIIIIllrllllllllllYJ
36 °
17 °
34 °
J
6.5 °
9.6* (6)
(6°C) in a few hours. Before the drop in body temperature occurred in the pretorpid animals, energy intake (55 to 83 kJ/100 g/ 24 h, Table 2) fell lower than energy expenditure (145 to 97 kJ/ 100 g/24 h, Fig. 2). Thus, we observed alterations in the energy balance and loss of homeothermia. Among the systems participating in the regulation of energy metabolism in hibernating animals, the sympathoadrenal system is thought to play a major role.
Table 2. It can be seen that over the 24 h preceding hypothermia (stage 2), energy intake fell by an average of 62% with respect to the value observed 24 h beforehand (stage 1). In the 24-h period following arousal (stage 3), energy intake was much higher, and was close to that recorded during stage 1, 48 h prior to induction of the state of hibernation. Thus, hibernating animals exposed to cold ambient temperature (6°C) with ad lib access to food become spontaneously hypophagic during the late 24-h bout of homeothermia before entering into hypothermia. On the other hand, food intake is increased during the first 24 h after awakening. Changes in energy expenditure were also studied during the bouts of homeothermia, hypothermia and awakening in the hibernating cycle of garden dormice. The results are shown in Fig. 2. In the pretorpid animals, the energy expenditure ranged from 145 to 97 kJ/100 g/24 h. As soon as the dormice became drowsy, the energy expenditure values progressively decreased and their body temperature dropped. It fell rapidly to reach ambient temperature
Peripheral Sympathetic Activity During the period of homeothermic activity, plasma DI3H activity was at a maximum and was accompanied by high levels of NE and E (Fig. 3). Although plasma catecholamine levels in garden dormouse are higher than those reported in other species, no investigators have previously reported periodic variations in these compounds during the hibernation cycle in this species. Even though stress (e.g., handling) is known to increase plasma norepinephrine (10) all animals were subjected to the same treatment. In prelethargic animals, during the transition from homeothermia to hypothermia (from t°r = 37°C to 340(2) there was a fall of about 42% in plasma D[3H activity, associated with a fall in levels of NE (3-fold) and E (2-fold). In the adrenal gland of hypothermic animals, the activity of Dt3H was reduced by 90% with
Plasma Catecholamines
Plasma DBH Activity
10
r120
f
°°t! B I"" 4
0
37 °
~
FIG. 2. Energy expenditure before and during the induction of hibernation at 6°C in the garden dormouse. Seven animals were used for every stage of rectal temperature. ***p<0.001 compared with the values for the preceding rectal temperature.
The number of measurements is shown in parentheses. *p<0.001 with respect to stage 2.
36 °
22 °
""
34 °
22 °
6.5o
36 ° 37 ° 340 22 ° 6.5 °
HG. 3. Plasma catecholamine concentration (ng/ml) and DI3H activity (U/l) during the start of hibernation in the garden dormouse. The number of animals at rectal temperatures 36°(2, 37°C, 34°C, 22°C and 6.5°C were, respectively, 7, 14, 7, 12 and 16. **p<0.01, ***p<0.001 compared with the values for the preceding body temperature. Rectal temperatures below 36-37°C represent an animal entering into hypothermia. Full hypothermia is reached at 6.5°C.
786
ATGIE, NIBBELINK AND AMBID
Adrenal Epinephrine m 30
Adrenal DSH Activity
rT-=
3 tD
m
~25
,
'k'k
i
20 15 10 5 0
•
37 o
Fin .
25 °
,
6.5 °
37 °
25 °
6.5 °
FIG. 4. Epinephrine concentration and DI3H activity in the adrenal gland during the start of hibernation in the garden dormouse. The number of animals at rectal temperatures 37°C, 25°C and 6.5°C were, respectively, 8, 5, and 7. **p<0.01, ***p<0.001 compared with the values for the preceding rectal temperature.
respect to euthermic controls (Fig. 4). As soon as the garden dormice became torpid adrenal DI3H activity and E level values progressively decreased. Thus, during the transition to hibernation there is a fall in adrenal activity with a marked drop in plasma epinephrine. We therefore evaluated the link between the spontaneous changes in food intake and the activity of the peripheral sympathetic nervous system. Among the available energetic substrates, glucose might be the link between dietary intake and sympathetic activity. The evolution of plasma glucose levels in dormice entering hypothermia at 6°C is shown in Fig. 5. The highest glycemia values were found in homeothermic dormice (t°r = 37°C). A drop in glycemia and a reduction of sympathetic activity slightly preceded the fall of body temperature in pretorpid garden dormice. During the body temperature transition from 37°C to 34°(2 plasma glucose levels were reduced by 35% and fell progressively during the following hypothermia at the ambient temperature of 6°(2 (Fig. 5). DISCUSSION These experiments demonstrate temporal relationships between food intake and sympathoadrenal system activity within the short cycle of hibernation in the garden dormouse (Eliomys quercinus L). During the bouts of homeothermia, hypothermia and awakening, we observed a fall in energy intake associated with spontaPlasma Glucose 120 ~100 O)
E 80 60 4O 20 0 36 ° 37 ° 34 ° 22 ° 6.5 °
FIG. 5. Plasma glucose of garden dormice entering hypothermia at 6°C. The mean and S.E.M. are shown. The number of animals at rectal temperature 36~C, 37°C, 34°C, 22°C and 6.5°(2 were, respectively, 7, 11, 7, 12, 8. ***p<0.001 compared with the values for the preceding rectal temperature.
neous alterations in energy balance during the beginning of this cycle. In the garden dormouse exposed to cold ambient temperature (6°C) with ad lib access to food, the fall in food intake is accompanied by a reduction in glucose level, inhibition of sympathetic tone and loss of homeothermia. It seems reasonable, in agreement with many authors (5, 11-14, 21, 23), to postulate that each hibernation bout is initiated and driven by the inhibition of sympathetic tone. Decreased norepinephrine activities may facilitate heat loss and suppress heat production for the entry into and maintenance of hibernation. The mechanisms culminating in hypothermia are not known. However, the results showing that the plasma glucose falls and that there is a reduction of the sympathoadrenal activity during the hours preceding the phases of torpor could possibly suggest a chain of cause and effect. This would imply that glucose deficiency leads to the inhibition of the sympathetic tonus. This suggestion is in complete agreement with the works of Young and Landsberg (23), and Landsberg et al. (16) who detected a reduction in sympathetic activity in hypoglycemic fasted gestating rats, and in fasted rats treated with phlorizin. Moreover, Avakian and Horvath (6) reported that norepinephrine turnover in rats fell with fasting and that a fall in plasma glucose level is associated with sympathetic nervous system activity reduction. By analogy with these results in the rat, which cannot be repeated in the dormouse since handling the animals causes a reversal of the tendency to enter fully into hibernation, we can note a comparable situation in the garden dormouse whose plasma glucose level was reduced by 30% on feeding with a protein-free diet. During this phase, DI3H activity and catecholamine levels fell markedly in the plasma and adrenal medulla. The fall in their levels led to a reduction in thermogenesis as demonstrated by loss of body temperature (3). Finally, the present experiments suggest that the fall of circulating glucose during the prehibernation period might reduce brain glucose availability within the central neurones involved in the regulation of sympathetic activity. So diminished glucose metabolism within the central nervous system could be an important link between dietary intake and sympathetic activity. This suggestion is in complete agreement with the hypotheses of Chafetz et al. (8) and of Leibowitz (17) who proposed a mechanism by which circulating glucose acts on the CNS via paraventficular nucleus neurones. Furthermore, studies of Rowe et al. (22) and Melnyk and Martin (18) suggest a role for insulin-mediated glucose metabolism within the CNS in the link between dietary intake and sympathetic activity. This hypothesis needs experimental
SYMPATHOADRENAL ACTIVITY AND HYPOGLYCEMIA
confn'mation in the hibernating garden dormouse. Our work, being limited to the quantitative aspect of the diet, raises the problem of the spontaneous onset of natural hibernation. During the days preceding hibernation, the animals have little appetite and stop eating. The immediate consequence of hypophagia is a disturbance of sugar metabolism since glycemia values progressively decrease. Hypoglycemia accompanied the fall in sympathetic activity and the depletion of adrenal catecholamine stores. The fall in plasma catecholamine levels leads to a reduction in energy expenditure. As we demonstrated, these fac-
787
tors combine to induce the loss of homeothermia. Therefore, our results indicate the important influence of nutritional factors on hibernation. The experiments were restricted to the garden dormouse but the same mechanism could well exist in other hibernators; this merits further investigation. ACKNOWLEDGEMENT This work was supported by Institut National de la Recherche Agronomique, grant No. 4434.
REFERENCES 1. Agid, R.; Ambid, L. Effects of corporeal temperature on glucose metabolism in a homeotherm, the rat, and a hibernator, the garden dormouse. In: Musacchia, J.; Saunders, J. F., eds. Depressed metabolism. New York: American Elsevier; 1969:119-157. 2. Agid, R.; Ambid, L.; Sable-Amplis, R.; Sicart, R. Aspect of metabolic and endocrine changes in hibernation. In: Wang, L. C.; Hudson, J. W., eds. Strategies in cold natural torpidity and thermogenesis. New York: Academic Press; 1978:499-540. 3. Ambid, L.; Cazaneuve, C. Niveau prottique de la ration alimentaire, mttabolisme 6nergttique et activit~ du syst~me nerveux sympathique p~riphtrique chez le Ltrot (Eliomys quercinus L.). Bull. Soc. Ecophysiol. 9:209-223; 1984. 4. Anton, A.; Sayre, A. A study of the factor affections the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther. 138:360--375; 1962. 5. Atgie, C.; Nibbelink, M.; Ambid, L. Activit6 adrtnergique p~riph6rique et mtdullo-surrtnalienne au cours de l'induction de l'hibernation chez le Ltrot (Eliomys quercinus L.). Ann. Endocrinol. 41:255; 1987. 6. Avakian, E. V.; Horvath, S. M. Starvation suppresses sympathoadrenal medullary response to cold exposure in rat. Am. J. Physiol. 241:E316-E320; 1981. 7. Bacq, M. Les contrfiles de la libtration des m&tiateurs aux terminaisons des nerfs adrtnergiques. J. Physiol. (Paris) 72:371-542; 1976. 8. Chafetz, M. D.; Parko, K.; Diaz, S.; Leibowitz, S. F. Relationships between medial hypothalamic ot2-receptor binding, norepinephrine, and circulating glucose. Brain Res. 384:404--408; 1986. 9. Davies, C. L.; Molineux, S. G. Routine determination of plasma catecholamines using reverse-phase, ion-pair high-performance chromatography with electrochemical detection. J. Chromatogr. 231:4151; 1982. 10. Depocas, F.; Behrens, W. A. Effects of handling, decapitation, anesthesia and surgery on plasma norepinephrine levels in white rats. Can. J. Physiol. Pharmacol. 55:212-219; 1977. 11. Draskoczy, P. R.; Lyrnan, C. P. Turnover of catecholamines in active and hibernating ground squirrels. J. Pharmacol. Exp. Ther. 155: I01-111; 1967. 12. Feist, D. D.; Galster, W. A. Changes in hypothaiamic catecholamines and serotonin during hibernation and arousal in the arctic
ground squirrel. Comp. Biochem. Physiol. 48A:653-662; 1974. 13. Florant, G. L.; Weitzman, E. D.; Jayant, A.; Cote, L. J. Plasma catecholamines levels during cold acclimatation and hibernation in woodchucks (Marmota monax). J. Therm. Biol. 7:143-146; 1982. 14. Helle, K. B.; Bolstad, G.; Knudsen, R. Catecholamines, ATP and dopamine-13-hydroxylase in the adrenal medulla of the hedgehog in the prehibernating state and during hibernation. Cryobiology 17:7492; 1980. 15. Keller, R.; Oke, A.; Mefford, I.; Adams, R. N. Liquid chromatography analysis of catecholamine routine assay for regional brain mapping. Life Sci. 19:995-1004; 1976. 16. Landsberg, L.; Greff, L.; Gunn, S.; Young, J. B. Adrenergic mechanism in the metabolic adaptation to fasting and feeding: effects of phlorizin on diet-induced changes in sympathoadrenal activity in the rat. Metabolism 29:1128-1137; 1980. 17. Leibowitz, S. F. Hypothalamic paraventricular nucleus interaction between ct2-noradrenergic system and circulating hormones and nutrients in relation to energy balance. Neurosci. Biobehav. Rev. 12: 101-109; 1988. 18. Melnyk, R. B.; Martin, J. M. Insulin and central regulation of spontaneous fattening and weight loss. Am. J. Physiol. 249:R203-R208; 1985. 19. Montoya, R.; Ambid, L.; Agid, L. Torpor induced at any season by suppression of food proteins in a hibernator, the garden dormouse (Eliomys quercinus L.) Comp. Bioehem. Physiol. 62A:371-376; 1979. 20. Nagatsu, T.; Undenfriend, S. Photometric essay of dopamine-13-hydroxylase in human blood. Clin. Chem. 18:9813-983; 1972. 21. Petrovic, V. M.; Janic-Sibalic, V.; Roffi, J. Adrenal tyrosine hydroxylase activity in the ground squirrel--effect of cold and arousal from hibernation. Comp. Biocbem. Physiol. 61C:99-101; 1978. 22. Rowe, J. W.; Young, J. B.; Minaker, K. L. Effect of insulin and glucose infusion on sympathetic nervous system activity in normal man. Diabetes 30:219-225; 1981. 23. Sanerbier, I.; Lemmer, B. Seasonal variation in the turnover of noradrenalin of active and hibernating hedgehogs. Comp. Bioebem. Physiol. 57C:61-63; 1977. 24. Young, J. B.; Landsberg, L. Sympathoadrenal activity in fasting pregnant rats: dissociation of adrenal medullary and sympathetic nervous system responses. J. Clin. Invest. 64:109-116; 1979.
0031-9384/90$3.00 + .(30
Sympathoadrenal Activity and Hypoglycemia in the Hibernating Garden Dormouse C. ATGIE, M. NIBBELINK AND L. AMBID 1
Laboratoire des R~gulations des M~tabolismes et Nutrition, Universit~ Paul Sabatier rue Franfois Magendie 31400 Toulouse C~dex, France
ATGIE, C., M. NIBBELINKAND L. AMBID. Sympathoadrenal activity and hypoglycemia in the hibernating garden dormouse. PHYSIOL BEHAV 48(6) 783-787, 1990.--The hibernatinggarden dormouse is spontaneouslyhypophagic during the prehibernating period at which time we found a low peripheral sympathetic activity (S.A.). The aim of this work was to investigatethe link between dietary intake and S.A. The S.A. was evaluatedby measurementof catecholaminesin both plasma and adrenal glands by I-IPLC. Food intake, body weight, energy expenditureand plasma glucose were measured during the reentry phase of the hibernating period. The followingresults were obtained:the energy intake in pretorpid animals (55 to 83 kJ/24 h/100 g body weight) was less than energy expenditurewhich was between 145 and 97 kJ/24 h/100 g. The energy deficit induces marked hypoglycemiaimmediately before the onset of hypothermia (117 mg/dl vs. 76 mg/dl) and leads to a drastic drop in the peripheral sympathetic system. This, in turn, reduced energy production, causing the hypothermia. Hibernation Food intake Sympathoadrenalactivity Garden dormouse (Eliomys quercinus L.)
Catecholamines
STUDIES from our laboratory have shown that fasting or restricted caloric intake drastically reduces the sympathetic nervous system activity which is always associated with a fall in body temperature in the garden dormice (2, 3, 19). Reduction of sympathetic nervous system activity when caloric intake is reduced implies a basic mechanism within the central nervous system that assesses nutritional status and regulates sympathetic outflow accordingly. Experiments in rats with hypoglycemia suggest that the availability of glucose may be involved in the relationship between dietary intake and sympathetic activity (2, 3, 16). Garden dormice (Eliomys quercinus L.) are hibernating rodents that undergo spontaneous annual variations in food intake and body weight. In these animals, the fall in body temperature during hibernation is accompanied by a reduction in glucose levels (1). The nature of the signals that couple changes in dietary intake with changes in sympathetic activity is, however, still uncertain in hibernating rodents. The present paper reports on the changes in the activity of the sympathetic adrenal system (S.A.S.), during entrance into hibernation in midwinter and after periods of continuous hypothermia in a cold room at 6°C with a photoperiod L:D =0030:2330 h. The S.A.S. activity was evaluated by measurement of catecbolamine levels and dopamine-[3-hydroxylase (DflH) activity in the plasma and adrenal medulla. We then evaluated concomitant changes in food consumption, plasma glucose
DI3H activity
and energy expenditure during the bouts of homeothermia, hypothermia and awakening in the hibernating cycle. METHOD Anima/s The experiments were carded out on 60 garden dormice
(Eliomys quercinus L.) captured in the Dombes area of France, and subsequently maintained in the laboratory. They were housed in individual cages containing a wooden nest box, and had access to food (pellets for rats and apples) ad lib. Before the experiment, the animals were placed in a fight:dark photoperiod of 0030:2330 h in a cold room at 6°C. At this temperature, they become torpid, and their body temperature drops to the ambient temperature. From time to time, they wake up spontaneously, their body temperature increases, and they enter into a period of homeotbermic activity lasting several hours. They then fall back into a state of hypothermic torpor. The bouts of homeotbermia, hypothermia and arousal are schematized in Fig. 1. Changes in food intake were studied on the days before (stages 1 and 2) and after the periods of hypothermia (stage 3). For the daily measurement of food intake, 6 animals were placed in metabolism cages. Using the respective energetic values for pel-
~Requests for reprints should be addressed to L. Ambid.
783
784
I I
ATGIE, NIBBELINK AND AMBID
STAGE 1
I I
STAGE2
HOMEOTHERMIA
STAGE 3
I
!
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|
124 hours|
|
I I | |
I I I
I I | |
TABLE 1 BODY WEIGHTAND ENERGYINTAKEIN WAKEFULGARDENDORMICE OVER 5 MONTHSIN A COLDROOM AT 6°C WITH A PHOTOPERIOD L:D = 0300:2330 h Body Weight (g)
Energy Intake kJ/100 g/24 h
Nov
118 _-. 1.4 (24)
168 +_ 10.9 (32)
Dec
110 -+ 1.3 (24)
175 _ 5.8 (32)
Jan
105 -+ 1.5 (24)
190 _ 10.5 (37)
Feb
102 _ 2.5 (24)
200 _ 13.6 (32)
Mar
101 _+ 1.3 (24)
242 _+ 8.6 (32)
HYPOTHERMIA FIG. 1. Schematized cycle of arousal and lethargy during hibernation. Stage 1: normal wakefulness; stage 2:24 hours before hibernation; stage 3: return to homeothermia after a bout of hibernation. The hypothermic bout is broken by periodic arousal lasting about 24 hours at 6°C. The animals then become progressively hypothermic once more and at 6°C will remain lethargic for about 10 days.
lets and apple (12.96 and 2.18 kJ/g), the daily energy intake was calculated in kJ/100 g/24 h. Food intake for stage 1 was measured on the two days preceding stage 2. The animals were weighed once a week over a period of 6 months. The locomotion activity was measured continuously by an actographic system and the energy expenditure indirectly by the determination of the oxygen consumption (VO2) in a closed circuit respirometer at thermal ambiance (6°C). Rates of metabolic heat production were calculated from steady-state determinations of VO 2 assuming 20.16 kJ of heat were produced for each liter of 0 2 consumed.
Measurement of Sympathetic Adrenal System (S.A.S.) Activity The S.A.S. activity was estimated from the measurement of catecholamine (CA) levels and dopamine-13-hydroxylase (DI3H) activity in both the adrenal gland and the plasma. DBH is involved in the transformation of dopamine into norepinephrine (NE) in the synaptic vesicles of sympathetic nerve terminals, and is liberated along with NE in the process of exocytosis. The concomitant release of neurotransmitter and DI3H means that the plasma DI3H activity is representative of plasma levels of NE, and is thus a good indicator of sympathetic activity (7).
Catecholamine Assays Plasma and adrenal catecholamines (NE and epinephrine E) were assayed by HPLC using an ODS ultrasphere column (25 cm) followed by an electrochemical detector (BAS-IAC). A detection potential of 0.60 volt was selected. The mobile phase was acetate-citrate buffer pH 5.2 (15). The accuracy of the method was checked by injecting known quantities of catecholamines. The overall error was 5%. Over 10 injections we found an 8% variation within a trial, and a 5% variation between trials. The overall sensitivity was 5 pg/I.d. The animals were killed by decapitation, and blood was collected on heparin, EGTA and glutathione. The blood samples were centrifuged, and the plasma decanted and kept at - 2 5 ° C until assay. After sacrifice, the left adrenal gland was rapidly removed and homogenized in a Potter apparatus in 6 ml of medium containing perchloric acid (0.1 M), EDTA (2.7 mM), glutathione (5 mM), and sodium metabisulfite (0.2 mM). The homogenate was centrifuged (10,000 × g, 4°C) and the supernatant was frozen at - 80°C
The mean and S.E.M, are shown. The total number of measurements is indicated in parentheses. Six animals were used.
before CA extraction. Plasma and adrenal supernatant CA were extracted on activated alumina (4) as previously described (9) before injection into the HPLC system.
Df3H Activity Assays DI3H activity was determined using the method described by Nagatsu and Undenfriend (20) based on the conversion of tyramine to octopamine. The results are expressed as units of activity (U = t~mole of octopamine, formed per hour at 37°C). After sacrifice of the garden dormouse, the right adrenal gland was immediately removed and homogenized in a Potter apparatus in 8 ml of medium containing Tris-HC1 (5 raM), NaC1 (150 mM), Triton X100 (0.2%), bovine serum albumin (0.2%) pH 7.2. The homogenate was then centrifuged (80,000 x g, 4°C) and the supernatant was frozen at - 80°C until DI3H activity determination.
Plasma Glucose Assays Plasma glucose was assayed by spectrophotometry with a Biotrol enzymatic kit A-02460. The results are presented as mean values and standard errors of the mean (SEM). The mean values were compared using Student's t-test for paired and unpaired samples as appropriate. RESULTS In garden dormice, during the period of hibernation (November to March) the mean body weight fell by 14%, and the energy intake during the periods of euthermia (rectal temperature t°r = 37°(2) ranged from 168 kJ/100 g/day in November to 242 in March (Table 1). Towards the spring arousal there was a progressive increase in food intake. In reality, the information given by these data in insufficient, because during the winter the food intake of the garden dormouse is variable and follows the cycles of lethargy and arousal. It is for this reason that an effort was made to defme the variations of the food intake more clearly in relation to the time, during the bouts of wakefulness and hibernation. Thus, changes in energy intake were studied on the days before and after the periods of hypothermia (Fig. 1). The results are shown in
SYMPATHOADRENAL ACTIVITY AND HYPOGLYCEMIA
785
TABLE 2
Energy Expenditure
ENERGY INTAKE IN THE GARDEN DORMOUSE 48 h (STAGE l) AND 24 h (STAGE 2) BEFORE THE START OF HIBERNATION AT 6°(: AND 24 h POSTAROUSAL (STAGE 3) AT 6°(2
J= ,q,
Stage 2
/r**
125 0 0
Energy Intake (kJ/100 g/24 h) Stage 1
150-
100 75
Stage 3
~ / r / t / r
50
Nov
168 - 10.9" (32)
67 -+ 13.5 (18)
168 - 11.8" (18)
Dec
175 -+ 5.8* (32)
74 -+ 7.6 (13)
160 _+ 9.0 (14)
Jan
190 _ 10.5" (32)
55 -+ 14.7 (11)
170 --+ 12.1' (18)
Feb
200 _ 13.6" (32)
76 -+ 13.5 (5)
201 -
242 - 8.6* (32)
83 - 15.4 (5)
240 _ 13.4" (8)
Mar
25
IIIIIIIIIIIII1
IIIIIllVIIIIIIIllrllllllllllYJ
36 °
17 °
34 °
J
6.5 °
9.6* (6)
(6°C) in a few hours. Before the drop in body temperature occurred in the pretorpid animals, energy intake (55 to 83 kJ/100 g/ 24 h, Table 2) fell lower than energy expenditure (145 to 97 kJ/ 100 g/24 h, Fig. 2). Thus, we observed alterations in the energy balance and loss of homeothermia. Among the systems participating in the regulation of energy metabolism in hibernating animals, the sympathoadrenal system is thought to play a major role.
Table 2. It can be seen that over the 24 h preceding hypothermia (stage 2), energy intake fell by an average of 62% with respect to the value observed 24 h beforehand (stage 1). In the 24-h period following arousal (stage 3), energy intake was much higher, and was close to that recorded during stage 1, 48 h prior to induction of the state of hibernation. Thus, hibernating animals exposed to cold ambient temperature (6°C) with ad lib access to food become spontaneously hypophagic during the late 24-h bout of homeothermia before entering into hypothermia. On the other hand, food intake is increased during the first 24 h after awakening. Changes in energy expenditure were also studied during the bouts of homeothermia, hypothermia and awakening in the hibernating cycle of garden dormice. The results are shown in Fig. 2. In the pretorpid animals, the energy expenditure ranged from 145 to 97 kJ/100 g/24 h. As soon as the dormice became drowsy, the energy expenditure values progressively decreased and their body temperature dropped. It fell rapidly to reach ambient temperature
Peripheral Sympathetic Activity During the period of homeothermic activity, plasma DI3H activity was at a maximum and was accompanied by high levels of NE and E (Fig. 3). Although plasma catecholamine levels in garden dormouse are higher than those reported in other species, no investigators have previously reported periodic variations in these compounds during the hibernation cycle in this species. Even though stress (e.g., handling) is known to increase plasma norepinephrine (10) all animals were subjected to the same treatment. In prelethargic animals, during the transition from homeothermia to hypothermia (from t°r = 37°C to 340(2) there was a fall of about 42% in plasma D[3H activity, associated with a fall in levels of NE (3-fold) and E (2-fold). In the adrenal gland of hypothermic animals, the activity of Dt3H was reduced by 90% with
Plasma Catecholamines
Plasma DBH Activity
10
r120
f
°°t! B I"" 4
0
37 °
~
FIG. 2. Energy expenditure before and during the induction of hibernation at 6°C in the garden dormouse. Seven animals were used for every stage of rectal temperature. ***p<0.001 compared with the values for the preceding rectal temperature.
The number of measurements is shown in parentheses. *p<0.001 with respect to stage 2.
36 °
22 °
""
34 °
22 °
6.5o
36 ° 37 ° 340 22 ° 6.5 °
HG. 3. Plasma catecholamine concentration (ng/ml) and DI3H activity (U/l) during the start of hibernation in the garden dormouse. The number of animals at rectal temperatures 36°(2, 37°C, 34°C, 22°C and 6.5°C were, respectively, 7, 14, 7, 12 and 16. **p<0.01, ***p<0.001 compared with the values for the preceding body temperature. Rectal temperatures below 36-37°C represent an animal entering into hypothermia. Full hypothermia is reached at 6.5°C.
786
ATGIE, NIBBELINK AND AMBID
Adrenal Epinephrine m 30
Adrenal DSH Activity
rT-=
3 tD
m
~25
,
'k'k
i
20 15 10 5 0
•
37 o
Fin .
25 °
,
6.5 °
37 °
25 °
6.5 °
FIG. 4. Epinephrine concentration and DI3H activity in the adrenal gland during the start of hibernation in the garden dormouse. The number of animals at rectal temperatures 37°C, 25°C and 6.5°C were, respectively, 8, 5, and 7. **p<0.01, ***p<0.001 compared with the values for the preceding rectal temperature.
respect to euthermic controls (Fig. 4). As soon as the garden dormice became torpid adrenal DI3H activity and E level values progressively decreased. Thus, during the transition to hibernation there is a fall in adrenal activity with a marked drop in plasma epinephrine. We therefore evaluated the link between the spontaneous changes in food intake and the activity of the peripheral sympathetic nervous system. Among the available energetic substrates, glucose might be the link between dietary intake and sympathetic activity. The evolution of plasma glucose levels in dormice entering hypothermia at 6°C is shown in Fig. 5. The highest glycemia values were found in homeothermic dormice (t°r = 37°C). A drop in glycemia and a reduction of sympathetic activity slightly preceded the fall of body temperature in pretorpid garden dormice. During the body temperature transition from 37°C to 34°(2 plasma glucose levels were reduced by 35% and fell progressively during the following hypothermia at the ambient temperature of 6°(2 (Fig. 5). DISCUSSION These experiments demonstrate temporal relationships between food intake and sympathoadrenal system activity within the short cycle of hibernation in the garden dormouse (Eliomys quercinus L). During the bouts of homeothermia, hypothermia and awakening, we observed a fall in energy intake associated with spontaPlasma Glucose 120 ~100 O)
E 80 60 4O 20 0 36 ° 37 ° 34 ° 22 ° 6.5 °
FIG. 5. Plasma glucose of garden dormice entering hypothermia at 6°C. The mean and S.E.M. are shown. The number of animals at rectal temperature 36~C, 37°C, 34°C, 22°C and 6.5°(2 were, respectively, 7, 11, 7, 12, 8. ***p<0.001 compared with the values for the preceding rectal temperature.
neous alterations in energy balance during the beginning of this cycle. In the garden dormouse exposed to cold ambient temperature (6°C) with ad lib access to food, the fall in food intake is accompanied by a reduction in glucose level, inhibition of sympathetic tone and loss of homeothermia. It seems reasonable, in agreement with many authors (5, 11-14, 21, 23), to postulate that each hibernation bout is initiated and driven by the inhibition of sympathetic tone. Decreased norepinephrine activities may facilitate heat loss and suppress heat production for the entry into and maintenance of hibernation. The mechanisms culminating in hypothermia are not known. However, the results showing that the plasma glucose falls and that there is a reduction of the sympathoadrenal activity during the hours preceding the phases of torpor could possibly suggest a chain of cause and effect. This would imply that glucose deficiency leads to the inhibition of the sympathetic tonus. This suggestion is in complete agreement with the works of Young and Landsberg (23), and Landsberg et al. (16) who detected a reduction in sympathetic activity in hypoglycemic fasted gestating rats, and in fasted rats treated with phlorizin. Moreover, Avakian and Horvath (6) reported that norepinephrine turnover in rats fell with fasting and that a fall in plasma glucose level is associated with sympathetic nervous system activity reduction. By analogy with these results in the rat, which cannot be repeated in the dormouse since handling the animals causes a reversal of the tendency to enter fully into hibernation, we can note a comparable situation in the garden dormouse whose plasma glucose level was reduced by 30% on feeding with a protein-free diet. During this phase, DI3H activity and catecholamine levels fell markedly in the plasma and adrenal medulla. The fall in their levels led to a reduction in thermogenesis as demonstrated by loss of body temperature (3). Finally, the present experiments suggest that the fall of circulating glucose during the prehibernation period might reduce brain glucose availability within the central neurones involved in the regulation of sympathetic activity. So diminished glucose metabolism within the central nervous system could be an important link between dietary intake and sympathetic activity. This suggestion is in complete agreement with the hypotheses of Chafetz et al. (8) and of Leibowitz (17) who proposed a mechanism by which circulating glucose acts on the CNS via paraventficular nucleus neurones. Furthermore, studies of Rowe et al. (22) and Melnyk and Martin (18) suggest a role for insulin-mediated glucose metabolism within the CNS in the link between dietary intake and sympathetic activity. This hypothesis needs experimental
SYMPATHOADRENAL ACTIVITY AND HYPOGLYCEMIA
confn'mation in the hibernating garden dormouse. Our work, being limited to the quantitative aspect of the diet, raises the problem of the spontaneous onset of natural hibernation. During the days preceding hibernation, the animals have little appetite and stop eating. The immediate consequence of hypophagia is a disturbance of sugar metabolism since glycemia values progressively decrease. Hypoglycemia accompanied the fall in sympathetic activity and the depletion of adrenal catecholamine stores. The fall in plasma catecholamine levels leads to a reduction in energy expenditure. As we demonstrated, these fac-
787
tors combine to induce the loss of homeothermia. Therefore, our results indicate the important influence of nutritional factors on hibernation. The experiments were restricted to the garden dormouse but the same mechanism could well exist in other hibernators; this merits further investigation. ACKNOWLEDGEMENT This work was supported by Institut National de la Recherche Agronomique, grant No. 4434.
REFERENCES 1. Agid, R.; Ambid, L. Effects of corporeal temperature on glucose metabolism in a homeotherm, the rat, and a hibernator, the garden dormouse. In: Musacchia, J.; Saunders, J. F., eds. Depressed metabolism. New York: American Elsevier; 1969:119-157. 2. Agid, R.; Ambid, L.; Sable-Amplis, R.; Sicart, R. Aspect of metabolic and endocrine changes in hibernation. In: Wang, L. C.; Hudson, J. W., eds. Strategies in cold natural torpidity and thermogenesis. New York: Academic Press; 1978:499-540. 3. Ambid, L.; Cazaneuve, C. Niveau prottique de la ration alimentaire, mttabolisme 6nergttique et activit~ du syst~me nerveux sympathique p~riphtrique chez le Ltrot (Eliomys quercinus L.). Bull. Soc. Ecophysiol. 9:209-223; 1984. 4. Anton, A.; Sayre, A. A study of the factor affections the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther. 138:360--375; 1962. 5. Atgie, C.; Nibbelink, M.; Ambid, L. Activit6 adrtnergique p~riph6rique et mtdullo-surrtnalienne au cours de l'induction de l'hibernation chez le Ltrot (Eliomys quercinus L.). Ann. Endocrinol. 41:255; 1987. 6. Avakian, E. V.; Horvath, S. M. Starvation suppresses sympathoadrenal medullary response to cold exposure in rat. Am. J. Physiol. 241:E316-E320; 1981. 7. Bacq, M. Les contrfiles de la libtration des m&tiateurs aux terminaisons des nerfs adrtnergiques. J. Physiol. (Paris) 72:371-542; 1976. 8. Chafetz, M. D.; Parko, K.; Diaz, S.; Leibowitz, S. F. Relationships between medial hypothalamic ot2-receptor binding, norepinephrine, and circulating glucose. Brain Res. 384:404--408; 1986. 9. Davies, C. L.; Molineux, S. G. Routine determination of plasma catecholamines using reverse-phase, ion-pair high-performance chromatography with electrochemical detection. J. Chromatogr. 231:4151; 1982. 10. Depocas, F.; Behrens, W. A. Effects of handling, decapitation, anesthesia and surgery on plasma norepinephrine levels in white rats. Can. J. Physiol. Pharmacol. 55:212-219; 1977. 11. Draskoczy, P. R.; Lyrnan, C. P. Turnover of catecholamines in active and hibernating ground squirrels. J. Pharmacol. Exp. Ther. 155: I01-111; 1967. 12. Feist, D. D.; Galster, W. A. Changes in hypothaiamic catecholamines and serotonin during hibernation and arousal in the arctic
ground squirrel. Comp. Biochem. Physiol. 48A:653-662; 1974. 13. Florant, G. L.; Weitzman, E. D.; Jayant, A.; Cote, L. J. Plasma catecholamines levels during cold acclimatation and hibernation in woodchucks (Marmota monax). J. Therm. Biol. 7:143-146; 1982. 14. Helle, K. B.; Bolstad, G.; Knudsen, R. Catecholamines, ATP and dopamine-13-hydroxylase in the adrenal medulla of the hedgehog in the prehibernating state and during hibernation. Cryobiology 17:7492; 1980. 15. Keller, R.; Oke, A.; Mefford, I.; Adams, R. N. Liquid chromatography analysis of catecholamine routine assay for regional brain mapping. Life Sci. 19:995-1004; 1976. 16. Landsberg, L.; Greff, L.; Gunn, S.; Young, J. B. Adrenergic mechanism in the metabolic adaptation to fasting and feeding: effects of phlorizin on diet-induced changes in sympathoadrenal activity in the rat. Metabolism 29:1128-1137; 1980. 17. Leibowitz, S. F. Hypothalamic paraventricular nucleus interaction between ct2-noradrenergic system and circulating hormones and nutrients in relation to energy balance. Neurosci. Biobehav. Rev. 12: 101-109; 1988. 18. Melnyk, R. B.; Martin, J. M. Insulin and central regulation of spontaneous fattening and weight loss. Am. J. Physiol. 249:R203-R208; 1985. 19. Montoya, R.; Ambid, L.; Agid, L. Torpor induced at any season by suppression of food proteins in a hibernator, the garden dormouse (Eliomys quercinus L.) Comp. Bioehem. Physiol. 62A:371-376; 1979. 20. Nagatsu, T.; Undenfriend, S. Photometric essay of dopamine-13-hydroxylase in human blood. Clin. Chem. 18:9813-983; 1972. 21. Petrovic, V. M.; Janic-Sibalic, V.; Roffi, J. Adrenal tyrosine hydroxylase activity in the ground squirrel--effect of cold and arousal from hibernation. Comp. Biocbem. Physiol. 61C:99-101; 1978. 22. Rowe, J. W.; Young, J. B.; Minaker, K. L. Effect of insulin and glucose infusion on sympathetic nervous system activity in normal man. Diabetes 30:219-225; 1981. 23. Sanerbier, I.; Lemmer, B. Seasonal variation in the turnover of noradrenalin of active and hibernating hedgehogs. Comp. Bioebem. Physiol. 57C:61-63; 1977. 24. Young, J. B.; Landsberg, L. Sympathoadrenal activity in fasting pregnant rats: dissociation of adrenal medullary and sympathetic nervous system responses. J. Clin. Invest. 64:109-116; 1979.