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Effects of Adrenaline on the Dynamics of Carbohydrate Metabolism in Rats Treated Chronically with Adrenaline1 SUZANNE WBUSSEAU-MIGNERON,~ JACQUESLEBEANC,~ A N D LOUISELAFRANCE Department of$hy.siology, School of Medicine, Lava1 b'nilversity, Qrreb~c,Que. GI V 4G2 Dm'va'sion($Ba'ological Sciences, National Researc-11Coztncil s f C a n ~ d uOtta~va, , Ccancada K I A OR6
Received June 26,1974 ~OUSSEAL~-MIGNERON, S . , EEBLANC,J., LAFRANCE, L., and DEPOCAS,F. 1975. Effects of adrenaline on the dynamics of carbohydrate metabolisna in rats treated chronically with adrenaline. Can. J . Physiol. Pharmacol. 53, 124-128. FoBlowing a subcutaneous injection of adrenaline (300 pglkg), blood-gliicose levels were lower in rats treated chronically with adrenaline (300 pglkg twice a day for 28 days) than in control rats during at least 2.5 h after the injection. To explain this difference of response, the turnover rate of glucose was measured in control and adrenaline-treated rats during adrenaline infusion (0.75 p g l k g l min-I), with [U-14C]lglucoseas tracer. It was found that the rate of appearance of glucose was greater in the control than in the adrenaline-treated group after a 120-nain infusion of adrenaline. The rate of disappearance of glucose in the treated rats increased during the first 60 min of infusion and stayed at this elevated level for a subsequent 2 h, whereas in the control rats. it remained unchanged at the beginning of adrenaline infusion and significantly increased only duidng the second and third hours of infusion. In addition, the metabolic-clearance rate of glucose was not modified by adrenaline in the treated group, belt in the controlgroup, the initial clearance rate was significantly less tHan in the treated group, and decreased during the first hour of adrenaline infusion even though blood glucose reached values of 244 mg1108 ml. From these data, it is suggested that rats adapt to a chronic exogenous supply of adrenaline by a reduced increase in glucose production in response to adrenaline infusion and a better glucose utilization, which possibly indicates a decrease in the inhibitory effect of adrenaline on insulin secretion. ROUSSEAU-MIGNERON, S., LEBLANC,J., LAFRANCE, L. et DEPOCAS,F. 1975. EfTects of adrenaline on the dynamics of carbohydrate metabolism in rats treated chronically with adrenaline. Can. J. Physiol. Pharrnacol. 53, 124-128. Nous avons mesure la concentration du glucose sanguin apres une injection sous-cutade d'adrenaline (300 pglkg) chez des rats tCmoins et des rats traites chroniquernent B l'adrenaline (300 pglkg deux fois par jour. pendant 28 jours); nous avons remarque au cours des 150 min d'observation que la concentration du glucose sanguin est beaucoup plus faible chez les rats trait& que chez les ternoins. Afin d'expliquer cette diffirence de reponse, nous avons fait, li I'aide du [U-14C]glucose,une Ctude sur la dynarnique du mCtahlisme du glucose chez les deux groupes de rats. Nous avons observe d'une part que le taux de production du glucose est plus eleve chez les temoins que chez les rats traites a l'adrenaline apres 120 min d'infusion d'adrenaline. Nous avons trouve d'autre part que le taux d'utilisation du glucose chez les rats traites augmente des le debut de l'infusion d'adrenaline et se maintient a ce niveau eleve pendant les 3 h d'infusion, alors que chez les temoins l'utilisation augrnente de fai;on significative seulement pendant Bes deiix dernieres heures d'infusion. Quant au taux de la clearance metabolique, nous obsenrons qu'il n'est pas modifie chez les traites pendant l'infusion d'adrknaline, tandis que chez les temoins non seulement il diminue au cours de la premitre heure d'infusion d'adrenaline mais il est au depart infeneur B celui des traites avant l'infusion. I1 semble a partir de ces resultats que le rat s'adapte au traitement chronique a l'adrenaline en liberant moins de glucose aer cours d'une infusion d'adrenaline et en l'utilisant mieux, probablement a cause d'une diminution de l'effet inhibiteur de l'adrenaline sur la secretion d'insuline.
Oxygen consumption in response to noradrenaline (LeBlanc and Poulist 1964) and 'Supported by a grant from the Medical Research Council of Canada (MT-878). "Holder of a Fellowship of the Medical Research Council of Quebec. "ssociate of the Medical Research Csrancil of Canada.
to adrenaline (Villemaire 1970) is greatly increased in rats treated chronically with noradrenaline. Increase in oxygen consumption has been observed also in cold-ada~ted rats (Hsieh and Car'lson 1957; Himms-Hagen 1967), which secrete large quantities sf eatecholamines (LeBlanc and Nadcau 1961)- As this sensitization woarld be related to changes
125
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ROUSSEAU-MIGNERON E T AL.: EFFECT O F ADRENALINE ON METABOLISM
in metabolic processes (Himms-Hagen 1967), we (Lafrance et al. 1972) have previousIy studied some effects of catecholamines on lipid and carbohydrate metabolism. We have bbserved particularly that blood-glucose responses to a subcutaneous injection of adrenaline are lower in rats treated chronically with adrenaline than in control animals. The aim of the present experiments was to investigate further thd effects of adrenaline on the dynamics of glucose metabolism in rats treated chronically with this hormone. [U-14CCgGlucose was used in unanesthetized normal and treated rats to measure rates of glucose production and utilization before and during the infusion of additional adrenaline.
RIateriaIs and Methods Two groups of male Wistar rats with an average initial weight of 200 g were used. The rats of the first group served as controls and were injected twice a day with 1 ml of olive oil per kilogram; those of the second group, the adrenaline-treated group (A-treated), were injected twice a day with 300 pg of adrenaline base suspended in olive oil, per kilogram. All injections were given subcutaneously for a period of 28 days. The rats were maintained at 28 "C and supplied with blaster fox chow and tap water ad libitum. A few days before the end of the experiment, the rats were cannulated by the technique of Popovic and Popovic (1960), which consists of inserting a polyethylene cannula into the right jugular vein (PE 20) and one into the left common carotid artery (PE l o ) , and exteriorizing them through neck incisions; this method allows infusion of materials and withdrawal of blsod in unanethetized and unrestrained rats. Heparin ( 100 USP units ml) was used as anticoagulant. The injections were not given on the day of cannulation, but during the 4-5-day recovery period, the average body weight then being 95% of that at cannulation. In the preliminary study, three control rats and four A-treated rats were used on the last morning of the experiment, each non-fasted rat being put into a cage without food or water and the arterial cannula connected to an extenison (Depocas and Masironi 1960). After a I-h resting period, turo arterial-blood samples were taken for initial values After a subcutaneous injection of 300 pg of adrenaline base in saline (in the form of chloride), per kilogram, arterial-blood samples were withdrawn at intervals of 15 min during the first 90 min, and of 30 min for the subsequent hour. The blsod samples were transferred to chilled hepasinized tubes and centrifuged at 4 "C. The plasma was frozen, and glucose determinations (Nelson 1944) were m~lde later on Somogyi ( 1945) deproteinized-plasma filtrates. The main experiment was conducted similarly to the preliminary study; however, on the last morninq of the experiment, after the resting period, a priming
dose followed by a continuous infusion of trace amounts of [U-14C]glucose was injected via the jugular cannula, as described by Depocas ( 1959). The adrenaline infusion was started 1 h after administration of the labeled glucose, at a dose of 0.75 ~g kg-'min-', and continued for 3 h. Arterial-blood samples (0.30 ml) were withdrawn before the adrenaline infusion at 20-min intervals. then at 30-min intervals during the infusion. They were treated as in the preliminary study. Plasma glucose was isolated quantitatively by direct paper chromatography (Depocas 1959), its concentration was estimated in the eluate (Nelson 1944), and its radioactivity was determined by scintillation counting. Rates of glucose production (rate of appearance (Ra)) and rates of utilization (rate of disappearance (Rd)) , uncorrected for 14C recycling, were calculated according to the equations of Steele (1959) as simplified by I9e Bods c.t ul. ( 1963 ) ; the metabolic-clearance rate s f glucose (MCR) was calculated from the ratio of the Rd to glucose concentration (Riggs 1963). 7'he statistical analysis of each parameter studied was carried out by the method of pairing, an application of Student's t test.
Results Figure 1 shows that a subcutaneous injection of adrenaline (300 p.g/kg in saline) rapidly increased the blood-glucose concentration in both groups; however, 30 min following the injection, it continued to rise in the control g o u p whereas in the A-treated group it reached a plateau, the levels being then significantly smaller than those in control animals. Thus the previously observed (Lafrance et al. 1972) differences in glycemia are maintained, at least for 150 min after injection. An intravenous infusion of adrenaline (0.75 pg kg- min- l) into control rats resulted CpJ
Control A-Treated
Time
(28 days)
trn~n)
FIG. 1. Blood-glucose responses to a subcutaneous injection of adrenaline (300 pg/kg) in three control rats and in four rats treated chronically with adrenaline for 28 days. The arrow indicates the time of injection. Vertical lines, S.E.M.
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CAN. J. PHYSIOL. PHARMACOL. VOL. 53, 1975
in a prompt rise in blood-glucose concentration (Fig. 2 ) , which was evident within the first 30 min of infusion. The blood-glucose concentration continued to rise moderately during the first hour and thereafter tended to stabilize at the elevated level. In the rats treated chronically, however, the increase in blood glucose was much smaller than that in control rats and the concentration became significantly different ( B < 0.05) from initial values after only 90 min of infusion; it remained at this slightly elevated level during the next 30 min and was not significantly different from the value in the control period during the last hour. The same figure also shows that chronic treatment had no effect on the initial blood-glucose value. The values of gl;cose Ra, Rd, and MCR before and during adrenaline infusioil are summarized in Fig. 3. Statistical analysis was made on data averaged for each hour, as described by Altszuler et al. ( 1967). Initially, adrenaline infusion increased glucose production significantly in both groups, but the increase was greater in the control ( 102% ) than in the A-treated rats (23 % ) during the second hour of infusion. Glucose production dropped slightly in the control group during the third hour of infusion, but remained higher than during the pre-infusion period. As in the control animals, the failure of adrenaline to sustain elevated glucose production despite continued adrenaline infusion has also been observed in normal dogs (Altszuler el al.
C+C
Control A-Treated
(28d a y s )
FIG. 2. Effect of a prolonged infusion of adrenaline (0.75 , L L ~ kg-' min-l) on blood-glucose concentration in five control rats and in eight rats treated chronically with adrenaline for 28 days. Vertical lines, S.E.M.
u
I5
n
control
C d r e n a l ~ n e ( 2 8 days)
FIG.3. Effects s f prolonged adrenaline infusion on the rates of glucose production, utilization, and metabolic clearance in control rats and rats treated chronically with adrenaline for 28 days. Vertical lines, S.E.M.
1967). As for gIucosc utilization, Rd increased in the A-treated group during the first hour of infusion ( P < 0.05) and stayed at the elevated level during the following 2 h; however, the metabolic clearance rate did not differ significantly from controI vaIues at any time. In control animals, Rd did not siLgnificantly increase during the first hour of infusion, despite the elevated blood-glucose levels, hut it did so during the next 2 h of infusion. Moreover, the MCR in controls was significantly different from that in A-treated rats at all times: during the pre-infusion period the MCR was smaller than in A-treated rats ( P < 0.05), and during adrenaline infusion it dropped by 33% of the pre-infusion value during the first hour, but remained unchanged in the A-treated rats.
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ROUSSEAU-MIGNERON ET AL.: EFFECT OF ADRENALINE ON METABOLISM
Discussion It is well known that adrenaline, by reducing liver glycogen synthetase and increasing phosphorylase activity (Su therland and Rall 1960), as well as by inhibiting insulin secretion (Porte et al. 1966; Altszuler et a?. 1947; Malaisse et al. 1967), produces an increase in bloodglucose concentration. Thus the decrease in the MCR observed during thc first hour of adrenaline infusion in the control animals is probably explained by impaired Rd, despite the mass-action effect of hyperglycemia. During the second and third hours of infusion, bloodglucose concentrations in the control rats remain unchanged at elevated levels and MCR of glucose returns towards normal levels, which is due to an increase in Rd rather than in Ra. Whether the enhanced utilization of glucose during the second and third hours of adrenaline infusion, compared with the first hour, is due to a higher insulin secretion remains to be demonstrated. The infusion of adrenaline into the A-treated rats gave different results. Blood-glucose concentrations increased significantly less than in the control animals, whereas the MCR remained unchanged throughout the cntire infusion period. These results are explained by an immediate and sustained increase in calculated Rd. Accordingly, the A-treated animals differ from the control animals in an enhanced capacity to utilize glucose, even during the first hour of adrenaline infusion. In ternls of insulin secretion, these results could suggest that the block of insulin secretion, reported by various authors in control animals, is removed in A-treated rats, thus causing an enhanced glucose uptake even during the first hour of adrenaline infusion. A difference was also noted in Ra between the experimental and control group. In both groups, adrenaline increased the Ra during the entire infusion period, but the increase was greater in the control than in the A-treated group during the second hour of infusion. These results suggest that the difference in R a between the two groups is possibly due to a decrease in the glycogenolytic effect of adrenaline in rats chronically treated with this hormone. Finally, the possibility that the lower bloodglucose levels observed in the A-treated ani-
127
mals could be due to an enhanced removal rate of infused adrenaline should be discussed. Past experiments have shown that repeated injections of catecholamines actually enhance rather than reduce their metabolic effects (LeBlanc et al. 1972n, 1972b; Villemaire 1970) and cause an increase rather than a decrease in their urinary cxcretion (LeBlanc and Pouliot 1 964; Villcmaire 1970). Consequently, it seems unlikely that the changes in glucose metabolism observed in rats treated chronically with adrenaline are due to an increased removal rate of adrenriline when it is infused into them. We may then conclude that animals adapt to a chronic exogenous supply of adren a1'me. At the beginning of chronic treatment, the administration of :drenaline probably rewlts in a prompt rise in blood glucose resulting from an increase in production and an inhibition of peripheral utilization, possibly mediated by the suppression of insulin secretion; then after a few days of chronic injections, the liver may adapt its glucogenic function to a supply of adrenaline by reducing the rate of glucose production while glucose utilization is improved. Adaptation to cold, like that to repeated injections of adrenaline, results in an increased exposure of the organism to adrenaline, as indicated by a marked elevation of adrenaline excretion (LeBlanc and Nadeau 1961) . Furthermore, in both cold-adapted animals exposed to cold (Depocas 1962) and A-treated animals infused with adrenaline (present study), there is enhanced gIucose utilization. Thus the results of the present experiment would tend to coilfirm the assumed importance of adrenaline in resistance to the stress of cold. Calorigenesis, however, as discussed by Himms-Hagen ( 1967), is the result of a great variety of adrenergic effects, including the mobilization of glucose, but also the mobilization of free fatty acids. ALTSZULER, N . , STEELE,R.,RATHGEB,I., and DE BODO, R. C. 1967. Glucose metabolism and plasma insulin level during epinephrine infusion in the dog. Am. J. Physiol. 212,677-682. DE BODO,R. C . , STEELE,R . , ALTSZULER, N., DUNN,A., and BISHOP, J. S. 1%3. On the hormonal regulation of carbohydrate metabolism; studies with GI4 glucose. Recent Frog. Horm. Res. 19,445488.
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DEPOCAS.F. 1959. Turnover of plasma glucose in anesthetized warm- and cold-acclimated rats exposed to cold. Can. J. Biochern. Physiol. 37, 175-18 1. 1962. Body glucose as fuel in white rats exposed to cold: results with fasted rats. Am. J. Physiol. 202, 1015-1018. DEPOCAS, F., and MASIRONI, R. 1960. Body glucose as fuel for thermogenesis in the white rat exposed to cold. Am. J. Physiol. 199,1051-1055. HIMMS-HAGEN,J. 1967. Sympathetic regulation of metabolism. Pharmacol. Rev. 19,368-46 1. HSIEH,A. C. L., and CARLSON,L. D. 1957. Role of adrenaline and noradrenaline in chemical regulation of heat production. Am. J. Physiol. 190,243-246. LAFRANCE,L., ROUSSEAU,S., BEGIN-HEICH, N . , and LEBLANC, J . 1972. Blood glucose and free fatty acid responses to catecholamines in rats treated chronically with noradrenaline or adrenaline. Proc. Soc. Exp. Biol. Med. 139, 157-160. LEBLANC, J. A., and NADEAU, G. 1961. Urinary excretion of adrenaline and noradrenaline in normal and coldadapted animals. Can. J . Biochem. Physiol. 39, 215-217. LEBLANC, J., LAFRANCE, k., VILLEMAIRE, A., ROBERGE, J., and ROUSSEAU,S. 1 9 7 2 ~ . C., VALLI~RES, Catecholamines and cold adaptation. Proceedings of the International Symposium of Environmental Physiology (Bioenergetics). FASEB. pp. 7 1-76. LEBLANC,J., and P o u ~ r o M. ~ , 1964. Importance of noradrenaline in cold adaptation. Am. J. Physiol. 207, 853-856. LEBLANC,J., VALLI~RES, J., and VACHON,C. 1972b.
Beta-receptor sensitization by repeated injections of isoproterenol and by cold adaptation. Am. J. Physiol. 222,1043-1046. MALAISSE, W., MALAISSE-LAGAE, F.. WRIGHT,P. H . , and ASHMORE, J. 1%7. Effects of adrenergic and cholinergic agents upon insulin secretion in vitro. Endocrinology, 80,975-978. NELSON, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153,375-380. P o ~ o v r c V., , and POPOVIC,P. 1960. Permanent cannulation of aorta and vena cava in rats and ground squirrels. J. Appl. Physiol. 15,727-728. PORTE, D., JR., GRABER,A. L., KUZUYA,T., and WILLIAMS, R. PI. 1966. The efyect of epinephrine on immunoreactive insulin levels in man. J . Clin. Invest. 45,228-236. RIGGS. R. P. 1963. The mathematical approach to physiological problems. The Williams & Wilkins Co., Baltimore, Md. M. 1945. Determination of blood sugar. J. Biol. SOMOGYI, Chem. 160,69-73. STEELE,R. 1959. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann. N.Y. Acad. Sci. 82.420-430. SUTHERLAND, E. W., and RALL,T. W. 1960. The relation of adenosine-3',5'-phosphate and phosphoryiase to the actions of catecholamines and other hormones. Pharmacol. Rev. 12,265-299. VILLEMATRE, A. 1970. Doctoral thesis, Faculte de Medecine, Universite Laval, Quebec. Que.
Effects of Adrenaline on the Dynamics of Carbohydrate Metabolism in Rats Treated Chronically with Adrenaline1 SUZANNE WBUSSEAU-MIGNERON,~ JACQUESLEBEANC,~ A N D LOUISELAFRANCE Department of$hy.siology, School of Medicine, Lava1 b'nilversity, Qrreb~c,Que. GI V 4G2 Dm'va'sion($Ba'ological Sciences, National Researc-11Coztncil s f C a n ~ d uOtta~va, , Ccancada K I A OR6
Received June 26,1974 ~OUSSEAL~-MIGNERON, S . , EEBLANC,J., LAFRANCE, L., and DEPOCAS,F. 1975. Effects of adrenaline on the dynamics of carbohydrate metabolisna in rats treated chronically with adrenaline. Can. J . Physiol. Pharmacol. 53, 124-128. FoBlowing a subcutaneous injection of adrenaline (300 pglkg), blood-gliicose levels were lower in rats treated chronically with adrenaline (300 pglkg twice a day for 28 days) than in control rats during at least 2.5 h after the injection. To explain this difference of response, the turnover rate of glucose was measured in control and adrenaline-treated rats during adrenaline infusion (0.75 p g l k g l min-I), with [U-14C]lglucoseas tracer. It was found that the rate of appearance of glucose was greater in the control than in the adrenaline-treated group after a 120-nain infusion of adrenaline. The rate of disappearance of glucose in the treated rats increased during the first 60 min of infusion and stayed at this elevated level for a subsequent 2 h, whereas in the control rats. it remained unchanged at the beginning of adrenaline infusion and significantly increased only duidng the second and third hours of infusion. In addition, the metabolic-clearance rate of glucose was not modified by adrenaline in the treated group, belt in the controlgroup, the initial clearance rate was significantly less tHan in the treated group, and decreased during the first hour of adrenaline infusion even though blood glucose reached values of 244 mg1108 ml. From these data, it is suggested that rats adapt to a chronic exogenous supply of adrenaline by a reduced increase in glucose production in response to adrenaline infusion and a better glucose utilization, which possibly indicates a decrease in the inhibitory effect of adrenaline on insulin secretion. ROUSSEAU-MIGNERON, S., LEBLANC,J., LAFRANCE, L. et DEPOCAS,F. 1975. EfTects of adrenaline on the dynamics of carbohydrate metabolism in rats treated chronically with adrenaline. Can. J. Physiol. Pharrnacol. 53, 124-128. Nous avons mesure la concentration du glucose sanguin apres une injection sous-cutade d'adrenaline (300 pglkg) chez des rats tCmoins et des rats traites chroniquernent B l'adrenaline (300 pglkg deux fois par jour. pendant 28 jours); nous avons remarque au cours des 150 min d'observation que la concentration du glucose sanguin est beaucoup plus faible chez les rats trait& que chez les ternoins. Afin d'expliquer cette diffirence de reponse, nous avons fait, li I'aide du [U-14C]glucose,une Ctude sur la dynarnique du mCtahlisme du glucose chez les deux groupes de rats. Nous avons observe d'une part que le taux de production du glucose est plus eleve chez les temoins que chez les rats traites a l'adrenaline apres 120 min d'infusion d'adrenaline. Nous avons trouve d'autre part que le taux d'utilisation du glucose chez les rats traites augmente des le debut de l'infusion d'adrenaline et se maintient a ce niveau eleve pendant les 3 h d'infusion, alors que chez les temoins l'utilisation augrnente de fai;on significative seulement pendant Bes deiix dernieres heures d'infusion. Quant au taux de la clearance metabolique, nous obsenrons qu'il n'est pas modifie chez les traites pendant l'infusion d'adrknaline, tandis que chez les temoins non seulement il diminue au cours de la premitre heure d'infusion d'adrenaline mais il est au depart infeneur B celui des traites avant l'infusion. I1 semble a partir de ces resultats que le rat s'adapte au traitement chronique a l'adrenaline en liberant moins de glucose aer cours d'une infusion d'adrenaline et en l'utilisant mieux, probablement a cause d'une diminution de l'effet inhibiteur de l'adrenaline sur la secretion d'insuline.
Oxygen consumption in response to noradrenaline (LeBlanc and Poulist 1964) and 'Supported by a grant from the Medical Research Council of Canada (MT-878). "Holder of a Fellowship of the Medical Research Council of Quebec. "ssociate of the Medical Research Csrancil of Canada.
to adrenaline (Villemaire 1970) is greatly increased in rats treated chronically with noradrenaline. Increase in oxygen consumption has been observed also in cold-ada~ted rats (Hsieh and Car'lson 1957; Himms-Hagen 1967), which secrete large quantities sf eatecholamines (LeBlanc and Nadcau 1961)- As this sensitization woarld be related to changes
125
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ROUSSEAU-MIGNERON E T AL.: EFFECT O F ADRENALINE ON METABOLISM
in metabolic processes (Himms-Hagen 1967), we (Lafrance et al. 1972) have previousIy studied some effects of catecholamines on lipid and carbohydrate metabolism. We have bbserved particularly that blood-glucose responses to a subcutaneous injection of adrenaline are lower in rats treated chronically with adrenaline than in control animals. The aim of the present experiments was to investigate further thd effects of adrenaline on the dynamics of glucose metabolism in rats treated chronically with this hormone. [U-14CCgGlucose was used in unanesthetized normal and treated rats to measure rates of glucose production and utilization before and during the infusion of additional adrenaline.
RIateriaIs and Methods Two groups of male Wistar rats with an average initial weight of 200 g were used. The rats of the first group served as controls and were injected twice a day with 1 ml of olive oil per kilogram; those of the second group, the adrenaline-treated group (A-treated), were injected twice a day with 300 pg of adrenaline base suspended in olive oil, per kilogram. All injections were given subcutaneously for a period of 28 days. The rats were maintained at 28 "C and supplied with blaster fox chow and tap water ad libitum. A few days before the end of the experiment, the rats were cannulated by the technique of Popovic and Popovic (1960), which consists of inserting a polyethylene cannula into the right jugular vein (PE 20) and one into the left common carotid artery (PE l o ) , and exteriorizing them through neck incisions; this method allows infusion of materials and withdrawal of blsod in unanethetized and unrestrained rats. Heparin ( 100 USP units ml) was used as anticoagulant. The injections were not given on the day of cannulation, but during the 4-5-day recovery period, the average body weight then being 95% of that at cannulation. In the preliminary study, three control rats and four A-treated rats were used on the last morning of the experiment, each non-fasted rat being put into a cage without food or water and the arterial cannula connected to an extenison (Depocas and Masironi 1960). After a I-h resting period, turo arterial-blood samples were taken for initial values After a subcutaneous injection of 300 pg of adrenaline base in saline (in the form of chloride), per kilogram, arterial-blood samples were withdrawn at intervals of 15 min during the first 90 min, and of 30 min for the subsequent hour. The blsod samples were transferred to chilled hepasinized tubes and centrifuged at 4 "C. The plasma was frozen, and glucose determinations (Nelson 1944) were m~lde later on Somogyi ( 1945) deproteinized-plasma filtrates. The main experiment was conducted similarly to the preliminary study; however, on the last morninq of the experiment, after the resting period, a priming
dose followed by a continuous infusion of trace amounts of [U-14C]glucose was injected via the jugular cannula, as described by Depocas ( 1959). The adrenaline infusion was started 1 h after administration of the labeled glucose, at a dose of 0.75 ~g kg-'min-', and continued for 3 h. Arterial-blood samples (0.30 ml) were withdrawn before the adrenaline infusion at 20-min intervals. then at 30-min intervals during the infusion. They were treated as in the preliminary study. Plasma glucose was isolated quantitatively by direct paper chromatography (Depocas 1959), its concentration was estimated in the eluate (Nelson 1944), and its radioactivity was determined by scintillation counting. Rates of glucose production (rate of appearance (Ra)) and rates of utilization (rate of disappearance (Rd)) , uncorrected for 14C recycling, were calculated according to the equations of Steele (1959) as simplified by I9e Bods c.t ul. ( 1963 ) ; the metabolic-clearance rate s f glucose (MCR) was calculated from the ratio of the Rd to glucose concentration (Riggs 1963). 7'he statistical analysis of each parameter studied was carried out by the method of pairing, an application of Student's t test.
Results Figure 1 shows that a subcutaneous injection of adrenaline (300 p.g/kg in saline) rapidly increased the blood-glucose concentration in both groups; however, 30 min following the injection, it continued to rise in the control g o u p whereas in the A-treated group it reached a plateau, the levels being then significantly smaller than those in control animals. Thus the previously observed (Lafrance et al. 1972) differences in glycemia are maintained, at least for 150 min after injection. An intravenous infusion of adrenaline (0.75 pg kg- min- l) into control rats resulted CpJ
Control A-Treated
Time
(28 days)
trn~n)
FIG. 1. Blood-glucose responses to a subcutaneous injection of adrenaline (300 pg/kg) in three control rats and in four rats treated chronically with adrenaline for 28 days. The arrow indicates the time of injection. Vertical lines, S.E.M.
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CAN. J. PHYSIOL. PHARMACOL. VOL. 53, 1975
in a prompt rise in blood-glucose concentration (Fig. 2 ) , which was evident within the first 30 min of infusion. The blood-glucose concentration continued to rise moderately during the first hour and thereafter tended to stabilize at the elevated level. In the rats treated chronically, however, the increase in blood glucose was much smaller than that in control rats and the concentration became significantly different ( B < 0.05) from initial values after only 90 min of infusion; it remained at this slightly elevated level during the next 30 min and was not significantly different from the value in the control period during the last hour. The same figure also shows that chronic treatment had no effect on the initial blood-glucose value. The values of gl;cose Ra, Rd, and MCR before and during adrenaline infusioil are summarized in Fig. 3. Statistical analysis was made on data averaged for each hour, as described by Altszuler et al. ( 1967). Initially, adrenaline infusion increased glucose production significantly in both groups, but the increase was greater in the control ( 102% ) than in the A-treated rats (23 % ) during the second hour of infusion. Glucose production dropped slightly in the control group during the third hour of infusion, but remained higher than during the pre-infusion period. As in the control animals, the failure of adrenaline to sustain elevated glucose production despite continued adrenaline infusion has also been observed in normal dogs (Altszuler el al.
C+C
Control A-Treated
(28d a y s )
FIG. 2. Effect of a prolonged infusion of adrenaline (0.75 , L L ~ kg-' min-l) on blood-glucose concentration in five control rats and in eight rats treated chronically with adrenaline for 28 days. Vertical lines, S.E.M.
u
I5
n
control
C d r e n a l ~ n e ( 2 8 days)
FIG.3. Effects s f prolonged adrenaline infusion on the rates of glucose production, utilization, and metabolic clearance in control rats and rats treated chronically with adrenaline for 28 days. Vertical lines, S.E.M.
1967). As for gIucosc utilization, Rd increased in the A-treated group during the first hour of infusion ( P < 0.05) and stayed at the elevated level during the following 2 h; however, the metabolic clearance rate did not differ significantly from controI vaIues at any time. In control animals, Rd did not siLgnificantly increase during the first hour of infusion, despite the elevated blood-glucose levels, hut it did so during the next 2 h of infusion. Moreover, the MCR in controls was significantly different from that in A-treated rats at all times: during the pre-infusion period the MCR was smaller than in A-treated rats ( P < 0.05), and during adrenaline infusion it dropped by 33% of the pre-infusion value during the first hour, but remained unchanged in the A-treated rats.
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ROUSSEAU-MIGNERON ET AL.: EFFECT OF ADRENALINE ON METABOLISM
Discussion It is well known that adrenaline, by reducing liver glycogen synthetase and increasing phosphorylase activity (Su therland and Rall 1960), as well as by inhibiting insulin secretion (Porte et al. 1966; Altszuler et a?. 1947; Malaisse et al. 1967), produces an increase in bloodglucose concentration. Thus the decrease in the MCR observed during thc first hour of adrenaline infusion in the control animals is probably explained by impaired Rd, despite the mass-action effect of hyperglycemia. During the second and third hours of infusion, bloodglucose concentrations in the control rats remain unchanged at elevated levels and MCR of glucose returns towards normal levels, which is due to an increase in Rd rather than in Ra. Whether the enhanced utilization of glucose during the second and third hours of adrenaline infusion, compared with the first hour, is due to a higher insulin secretion remains to be demonstrated. The infusion of adrenaline into the A-treated rats gave different results. Blood-glucose concentrations increased significantly less than in the control animals, whereas the MCR remained unchanged throughout the cntire infusion period. These results are explained by an immediate and sustained increase in calculated Rd. Accordingly, the A-treated animals differ from the control animals in an enhanced capacity to utilize glucose, even during the first hour of adrenaline infusion. In ternls of insulin secretion, these results could suggest that the block of insulin secretion, reported by various authors in control animals, is removed in A-treated rats, thus causing an enhanced glucose uptake even during the first hour of adrenaline infusion. A difference was also noted in Ra between the experimental and control group. In both groups, adrenaline increased the Ra during the entire infusion period, but the increase was greater in the control than in the A-treated group during the second hour of infusion. These results suggest that the difference in R a between the two groups is possibly due to a decrease in the glycogenolytic effect of adrenaline in rats chronically treated with this hormone. Finally, the possibility that the lower bloodglucose levels observed in the A-treated ani-
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mals could be due to an enhanced removal rate of infused adrenaline should be discussed. Past experiments have shown that repeated injections of catecholamines actually enhance rather than reduce their metabolic effects (LeBlanc et al. 1972n, 1972b; Villemaire 1970) and cause an increase rather than a decrease in their urinary cxcretion (LeBlanc and Pouliot 1 964; Villcmaire 1970). Consequently, it seems unlikely that the changes in glucose metabolism observed in rats treated chronically with adrenaline are due to an increased removal rate of adrenriline when it is infused into them. We may then conclude that animals adapt to a chronic exogenous supply of adren a1'me. At the beginning of chronic treatment, the administration of :drenaline probably rewlts in a prompt rise in blood glucose resulting from an increase in production and an inhibition of peripheral utilization, possibly mediated by the suppression of insulin secretion; then after a few days of chronic injections, the liver may adapt its glucogenic function to a supply of adrenaline by reducing the rate of glucose production while glucose utilization is improved. Adaptation to cold, like that to repeated injections of adrenaline, results in an increased exposure of the organism to adrenaline, as indicated by a marked elevation of adrenaline excretion (LeBlanc and Nadeau 1961) . Furthermore, in both cold-adapted animals exposed to cold (Depocas 1962) and A-treated animals infused with adrenaline (present study), there is enhanced gIucose utilization. Thus the results of the present experiment would tend to coilfirm the assumed importance of adrenaline in resistance to the stress of cold. Calorigenesis, however, as discussed by Himms-Hagen ( 1967), is the result of a great variety of adrenergic effects, including the mobilization of glucose, but also the mobilization of free fatty acids. ALTSZULER, N . , STEELE,R.,RATHGEB,I., and DE BODO, R. C. 1967. Glucose metabolism and plasma insulin level during epinephrine infusion in the dog. Am. J. Physiol. 212,677-682. DE BODO,R. C . , STEELE,R . , ALTSZULER, N., DUNN,A., and BISHOP, J. S. 1%3. On the hormonal regulation of carbohydrate metabolism; studies with GI4 glucose. Recent Frog. Horm. Res. 19,445488.
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CAN. J. PHYSIOL. PHARMACOL. VOL. 53, 1975
DEPOCAS.F. 1959. Turnover of plasma glucose in anesthetized warm- and cold-acclimated rats exposed to cold. Can. J. Biochern. Physiol. 37, 175-18 1. 1962. Body glucose as fuel in white rats exposed to cold: results with fasted rats. Am. J. Physiol. 202, 1015-1018. DEPOCAS, F., and MASIRONI, R. 1960. Body glucose as fuel for thermogenesis in the white rat exposed to cold. Am. J. Physiol. 199,1051-1055. HIMMS-HAGEN,J. 1967. Sympathetic regulation of metabolism. Pharmacol. Rev. 19,368-46 1. HSIEH,A. C. L., and CARLSON,L. D. 1957. Role of adrenaline and noradrenaline in chemical regulation of heat production. Am. J. Physiol. 190,243-246. LAFRANCE,L., ROUSSEAU,S., BEGIN-HEICH, N . , and LEBLANC, J . 1972. Blood glucose and free fatty acid responses to catecholamines in rats treated chronically with noradrenaline or adrenaline. Proc. Soc. Exp. Biol. Med. 139, 157-160. LEBLANC, J. A., and NADEAU, G. 1961. Urinary excretion of adrenaline and noradrenaline in normal and coldadapted animals. Can. J . Biochem. Physiol. 39, 215-217. LEBLANC, J., LAFRANCE, k., VILLEMAIRE, A., ROBERGE, J., and ROUSSEAU,S. 1 9 7 2 ~ . C., VALLI~RES, Catecholamines and cold adaptation. Proceedings of the International Symposium of Environmental Physiology (Bioenergetics). FASEB. pp. 7 1-76. LEBLANC,J., and P o u ~ r o M. ~ , 1964. Importance of noradrenaline in cold adaptation. Am. J. Physiol. 207, 853-856. LEBLANC,J., VALLI~RES, J., and VACHON,C. 1972b.
Beta-receptor sensitization by repeated injections of isoproterenol and by cold adaptation. Am. J. Physiol. 222,1043-1046. MALAISSE, W., MALAISSE-LAGAE, F.. WRIGHT,P. H . , and ASHMORE, J. 1%7. Effects of adrenergic and cholinergic agents upon insulin secretion in vitro. Endocrinology, 80,975-978. NELSON, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153,375-380. P o ~ o v r c V., , and POPOVIC,P. 1960. Permanent cannulation of aorta and vena cava in rats and ground squirrels. J. Appl. Physiol. 15,727-728. PORTE, D., JR., GRABER,A. L., KUZUYA,T., and WILLIAMS, R. PI. 1966. The efyect of epinephrine on immunoreactive insulin levels in man. J . Clin. Invest. 45,228-236. RIGGS. R. P. 1963. The mathematical approach to physiological problems. The Williams & Wilkins Co., Baltimore, Md. M. 1945. Determination of blood sugar. J. Biol. SOMOGYI, Chem. 160,69-73. STEELE,R. 1959. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann. N.Y. Acad. Sci. 82.420-430. SUTHERLAND, E. W., and RALL,T. W. 1960. The relation of adenosine-3',5'-phosphate and phosphoryiase to the actions of catecholamines and other hormones. Pharmacol. Rev. 12,265-299. VILLEMATRE, A. 1970. Doctoral thesis, Faculte de Medecine, Universite Laval, Quebec. Que.
