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Glucagon and the paraventricular hypothalamus: modulation of energy balance
Brain Research, 630 (1993) 245-251 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
245
BRES 19500
Glucagon and the paraventricular hypothalamus: modulation of energy balance Dale M. Atrens *, Jos~ A. Men6ndez Department of Psychology, University of Sydney, NSW 2006, Australia (Accepted 20 July 1993)
Key words: Paraventricular nucleus of the hypothalamus; Glucagon; Energy balance; Energy expenditure; Thermogenesis; Respiratory quotient; Respiratory exchange ratio; Substrate utilization; Food intake; Feeding; Body weight; Blood glucose; Indirect calorimetry; Calorimetry; Obesity; Rat
The effects of glucagon injections (25, 100 and 200 ng) into the paraventricular nucleus of the hypothalamus (PVN) were investigated in an open-circuit calorimeter. Wistar rats were tested, with no food available during the tests. High doses of glucagon (100 and 200 ng) produced small and short-lasting increases in energy expenditure. The independence of these changes from changes in locomotor activity suggests that the thermogenesis represents a primary modulation and is not secondary to increased locomotion. All three doses of glucagon produced long-lasting and dose-related increases in respiratory quotient which were unrelated to any changes in locomotor activity. As with the changes in energy expenditure, this dissociation indicates that the effects are not secondary to changes in locomotor activity. These data constitute the first evidence that glucagon in the PVN modulates the metabolic parameters central to energy balance. In separate experiments, the three doses of glucagon increased blood glucose concentration over a one hour period, but they did not affect food and water intake and body weight over 24 h. These findings suggest that glucagon normally acts on the PVN in conditions of increased body fat to initiate autonomic mechanisms which increase glycemic levels, thermogenesis and carbohydrate utilization. These data constitute the first direct evidence for the involvement of the PVN in the regulation of energy balance by glucagon. The main effect of glucagon in the PVN is anabolic in that it increases dependence on carbohydrates as an energy substrate which results in a sparing of fat reserves.
INTRODUCTION T h e effects o f p e r i p h e r a l l y - o r i n t r a c e r e b r o v e n t r i c u l a r l y - a d m i n i s t e r e d g l u c a g o n on e n e r g y b a l a n c e a r e m e d i a t e d by n u m e r o u s a n d c o m p l e x effects o n c a r b o h y d r a t e , fat a n d p r o t e i n m e t a b o l i s m aL34'4° as well as by effects o n t h e r m o g e n e s i s z'3, b o d y w e i g h t 7'1~ a n d f e e d ing 7'9,11A2A9. W e have r e c e n t l y d e m o n s t r a t e d t h a t int r a p e r i t o n e a l injections o f g l u c a g o n i n c r e a s e e n e r g y expenditure and respiratory quotient (Men6ndez and A t r e n s , in p r e p a r a t i o n ) . W h e r e a s i n c r e a s e d e n e r g y exp e n d i t u r e is clearly catabolic, i n c r e a s e d r e s p i r a t o r y q u o t i e n t , which i n d i c a t e s a n i n c r e a s e d r e l i a n c e on carb o h y d r a t e s as e n e r g y s o u r c e a n d t h e s p a r i n g o f fat reserves, m a y b e c o n s i d e r e d to b e anabolic. G l u c a g o n ' s p r o n o u n c e d effects on t h e m e t a b o l i c p a r a m e t e r s o f e n e r g y b a l a n c e w h e n it is i n j e c t e d p e r i p h e r a l l y o r int r a c e r e b r o v e n t r i c u l a r l y c o n t r a s t with t h e c o m p l e t e lack o f d a t a for injections into d i s c r e t e b r a i n areas.
* Corresponding author. Fax: (61) (2) 692-2603.
It is likely t h a t t h e a d m i n i s t r a t i o n o f g l u c a g o n into t h e h y p o t h a l a m u s w o u l d affect t h e m e t a b o l i c p a r a m e t e r s of e n e r g y b a l a n c e . First, t h e r e is a high c o n c e n t r a tion o f g l u c a g o n i m m u n o r e a c t i v i t y in t h e h y p o t h a l a m u s 14'35. S e c o n d , i n t r a c e r e b r o v e n t r i c u l a r injections o f g l u c a g o n m o d u l a t e f o o d intake, m a c r o n u t r i e n t selection, b l o o d glucose levels a n d s y m p a t h e t i c activity 1'11'~3'18'3°. T h e l a t t e r findings i n d i c a t e t h e involvem e n t o f t h e brain, b u t t h e y d o n o t a d d r e s s t h e p r o b l e m o f which b r a i n a r e a ( s ) is(are) involved. T h i r d , s o m e o f t h e m e t a b o l i c effects o f g l u c a g o n a r e m e d i a t e d by the activation o f t h e s y m p a t h e t i c n e r v o u s system 18, a n d t h e h y p o t h a l a m u s is a m a j o r c e n t r e for s y m p a t h e t i c activity 6'21'26. T h e p a r a v e n t r i c u l a r n u c l e u s o f t h e h y p o t h a l a m u s ( P V N ) is p a r t i c u l a r l y likely to p a r t i c i p a t e in any c e n t r a l effect o f g l u c a g o n o n e n e r g y b a l a n c e since it has o n e o f t h e highest c o n c e n t r a t i o n s o f g l u c a g o n i m m u n o r e a c t i v i t y 14'35. T h e P V N also p a r t i c i p a t e s extensively in t h e r e g u l a t i o n o f feeding, b o d y w e i g h t a n d e n e r g y m e t a b o l i s m 6'2°,21'26-28,36. T h e r e are, however, no d a t a o n t h e m e t a b o l i c effects o f g l u c a g o n i n j e c t e d into the PVN.
246 The effects energy motor weight
present study was designed to determine the of injections of glucagon into the PVN on expenditure, energy substrate utilization, locoactivity, feeding and drinking behavior, body and blood glucose levels.
MATERIALS AND METHODS Subjects Ten male Wistar rats obtained from the University of Sydney breeding farm were used. They weighed between 300 and 400 g at the time of surgery. The rats were individually housed in clear acrylic cages, with food (Allied Rat & Mouse Kubes, Sydney) and tap water provided ad libitum. The colony room was maintained at 22_+ 2°C, with a 14/10 h light/dark cycle. Every rat was handled daily for one week before surgery and for two weeks after in order to minimize the stress of h u m a n contact.
Surgery and histology The rats were anaesthetized with 1 m l / k g Ketalar (100 m g / m l ketamine hydrochloride; Parke-Davis Pty. Ltd.) and 0.1 ml R o m p u n (20 m g / m l xylazine hydrochloride; Bayer Australia Ltd.), both injected intramuscularly. They were placed in a Kopf stereotaxic apparatus and implanted with a single, stainless-steel, 22-gauge guide cannula fitted with a d u m m y cannula (Plastics One, USA). T h e coordinates relative to bregma were: posterior 1.8, lateral 1.8 and ventral 7.2, with the cannulae being implanted at an angle of 10° off the midline 33. The tip of the cannula was aimed for a position 1 m m dorsal to the PVN, and the injector cannula extended 1 m m beyond the tip of the guide cannula. At the conclusion of testing, the rats were given a lethal dose of Nembutal, after which their brains were removed for histological analysis. The brains were frozen to - 12°C, sectioned at 4 0 / z m and stained with Toluidine blue O. Cannulae placements were determined microscopically with reference to the atlas of Paxinos and Watson 33.
Experiment 1. Determination of energy expenditure, substrate utilization and locomotor activity Apparatus Respiratory quotient (RQ) and energy expenditure (EE) were calculated after recording oxygen ( 0 2) consumption and carbon dioxide (CO 2) production in an open-circuit calorimeter. Two clear acrylic cylindrical chambers with stainless steel grid floors and a volume of 6.28 litres each were used. One was used for testing the rats and the other as a reference standard for calibration of the atmospheric air. Compressed atmospheric air at a flow rate of 1600 m l / m i n and a pressure of 8 kPa above atmospheric was continuously drawn through both chambers. A system of solenoids allowed the air leaving one of the chambers to be split and directed for analysis, while the air from the other chamber was exhausted to the atmosphere. A sample of 110 m l / m i n was directed through a Perma Pure (Toms River, N J, USA) permeation drier (model PD750-12PP), a CD-3A CO 2 analyzer and a S-3A O 2 analyzer (Applied Electrochemistry, USA). The rest of the air was exhausted to the room. T h e analyzers were calibrated daily with primary gravimetric standards (Commonwealth Industrial Gases, Sydney). Locomotor activity was recorded by placing the testing chamber on an electronic balance (Mettler PE-2000) and using the unintegrated signal from the strain gauge. The reliability and validity of this method has been demonstrated in other studies 24-28'36. A microcomputer system controlled and monitored the calorimeter. The computer provided minute-by-minute records of air flow, CO 2 production, 0 2 consumption and activity counts. The following calculations were made: E E (kJ) = mol O 2 (364+ 113 RQ); R Q = vol. CO 2 produced/vol. 0 2 consumed 6'8A°'16. Energy expenditure was expressed in J / g to account for different body weights.
Procedure Each rat was habituated to the metabolic apparatus in 60-min tests before and after surgery. In addition to providing baseline data on the metabolic and activity parameters this procedure allowed for the determination of any effect produced by the surgical procedure itself. The rats were also habituated to the injection procedure by introducing the injector cannula into the guide cannula on two separate occasions before any experimentation. The experiment itself began approximately two weeks after surgery. The test sessions were conducted in the light phase of the cycle (between 10.00 and 16.00 h). The rats were given a 30-min run in the testing chamber before each treatment. They were then removed and injected in a counterbalanced order with 0.25 /xl of either sterile saline (NaCI 0.9%) or one of three doses of glucagon: 25, 100 and 200 ng (Sigma, USA) dissolved in sterile saline. These doses were derived from those used in other studies ll'ls. The d u m m y cannula was removed, and the injections were performed over a one-minute period through a 28-gauge injector (Plastics One) which projected 1 m m beyond the guide cannula. The rats were unrestrained during the injection procedure. After the injection, the injector cannula was removed and the d u m m y cannula re-secured. The rats were then placed in the testing chamber, and respiratory exchange and activity were monitored for 60 min. Following this, the rats were returned to their home cages. Each recording session in the metabolic apparatus was preceded and followed by a 5-min analysis of air leaving the calibration chamber, in order to assess any drift in the analyzers. Food was not available during the test sessions. At least seven days elapsed between successive injections in the same rat.
Data analysis The data from each glucagon treatment were compared with those of the saline treatment by a two-way analysis of variance with repeated measures on the treatment and time factors. T h e level of significance was set at 0.05. In order to further investigate any relationships between changes in energy expenditure and locomotor activity these data were subject to an analysis of covariance.
Experiment 2. Determination of food intake, water intake and body weight Procedure Another group of 8 rats was tested for 24 h food intake, water intake and body weight, in a paradigm similar to previous studies 26. Control food and water intake and body weight were recorded for 5 consecutive days before the experiment and the daily averages calculated. At 12.00 h on the day of testing the rats received a 0.25 ~1 injection of either NaCI 0.9% or one of three doses of glucagon: 25, 100 and 200 ng. At 12.00 h on the following day the a m o u n t s of standard lab chow and tap water consumed were recorded. Body weight was measured immediately before the injection and at the end of the session. The injections were given in a counterbalanced order and at least 5 days separated each treatment.
Data analysis The data were compared by a one way analysis of variance with repeated measures, followed by post-hoc Newman-Keuis test. The level of significance was set at 0.05.
Experiment 3. Determination of blood glucose concentration Procedure A further group of 8 rats was tested for blood glucose concentration one hour after a 0.25 p,1 injection of either NaCI 0.9% or one of three doses of glucagon: 25, 100 and 200 ng. Testing was conducted between 10.00 and 16.00 h. The injections were given in a counterbalanced order and at least 5 days separated each treatment. Blood samples were taken by 'milking' the tail after removing 1 - 2 m m of its tip 25,26. The blood was collected in heparinized vials and immediately analyzed in an automated glucose analyzer (Model
247 23AM, Yellow Springs Instruments, USA). Two samples were taken from every rat, one immediately before and the other one hour after the injection. The rats were lightly anaesthetized with ethylic ether during sampling in order to minimize the stress of the procedure. A number of pilot experiments and the counterbalanced order of injections, which includes the vehicle solution, has allowed us to eliminate any confounding effect due to this use of ether.
+ • -
Blood glucose concentration was expressed in mmol/l. Two-tailed Student's t-tests were used to compare blood glucose concentrations before and after the injections. One-way analysis of variance with repeated measures, followed by post hoc Newman-Keuls test, was used for the comparison between pre-injection values and between post-injection values across treatments. The level of significance was set at 0.05.
PU I O R T 0'92
0.48
+
25 ng •
" 100
X 0.43
EP
/
NE EN R D 0.38 G I YT U R 0.33 E
0.28
I
I
I
I
I
I
I
t
I
I
I
2
3
4
5
6
7
8
9
10
11
12
TIME A F T E R INJECTION (block= o f 5 min)
Fig. 1. Mean energy expenditure (J/g) over a 60 min period immediately after the injection of either saline or one of three doses of glucagon into the PVN. For clarity of illustration the data are presented in blocks of 5 min and S.E.M. values (range 0.008-0.04) are not included.
saline
D
~O-- O
A t TE 0 NO.gO RT Y 0.88
1
N o dose of glucagon p r o d u c e d a significant overall t r e a t m e n t effect on E E [F1, 9 = 0.16, 1.12 and 0.58, P > 0.5, respectively for the 25, 100 and 200 ng doses] (Fig. 1). However, the 100 and 200 ng doses did produce significant t r e a t m e n t by time interactions [F59,531 = 1.64, P < 0.01, and /759,531 = 1.85, P < 0.001, respectively], which reflect the increase in E E seen during the first 30 min of the session (Fig. 1). O n average, E E was
200 ng
-
0.86 ~
Energy expenditure
100 ng -
0.94
SQ
N o statistical differences were f o u n d between the pre-surgery, post-surgery and saline injection data for energy expenditure, respiratory quotient or activity. These data rule out the possibility of any c o n f o u n d i n g due to the surgical trauma, the volume injected or the injection of saline. T h e possibility of c o n f o u n d i n g due to c a n n u l a - i n d u c e d tissue d a m a g e was also r e d u c e d by the fact that the histological analysis showed minimal incidental damage.
-o
0.96 R E
Data analysis
RESULTS
25 ng
~ 2
3
4
5
6
7
8
9
10
11
12
TIME A F T E R INJECTION lblocks o f 5 rain)
Fig. 2. Mean respiratory quotient over a 60 rain period immediately after the injection of either saline or one of three doses of glucagon into the PVN. For clarity of illustration the data are presented in blocks of five minutes and S.E.M. values (range 0.006-0.02) are not included.
increased by 4.5% by 100 ng of glucagon and 3.6% by 200 ng of glucagon c o m p a r e d to control. T h e 25 ng dose did not p r o d u c e a significant t r e a t m e n t by time interaction [F59,531 = 1.24, P > 0.05). T h e main effect of time on E E was significant for all three doses of glucagon [F59,531 = 20.43, 19.16 and 21.23, P < 0.001, respectively]. This reflects the fact that the initially high values for the glucagon and saline treatments declined steadily over the test session (see below for explanation).
Respiratory quotient N o dose of glucagon p r o d u c e d a significant overall t r e a t m e n t effect on R Q [FI, 9 = 0 . 1 5 , 1.51 and 4.96, P > 0.05, respectively for the 25, 100 and 200 ng doses] (Fig. 2). However, all three doses of glucagon did p r o d u c e significant t r e a t m e n t b y time interactions [F59,531 = 1.93, 4.33 and 2.11, P < 0.001, respectively], which reflect the increased R Q seen from approximately 5 - 1 0 min after the injection and maintained t h r o u g h o u t the rest of the session (Fig. 2). O n average, R Q was increased 0.7% by 25 ng of glucagon, 2.2% by 100 ng of glucagon and 5.1% by 200 ng of glucagon c o m p a r e d to control. T h e main effect of time on R Q was significant for all three doses of glucagon [11759,531 ~--18.57, 16.89 and 16.74, P < 0.001, respectively] (see below for explanation).
Locomotor activity N o dose of glucagon p r o d u c e d a significant treatm e n t effect on activity [ E l , 9 = 0.12, 0.70 and 0.08, P >
248 TABLE I 100 ng
•
Effects on 24 h food intake, water intake and body weight gain of single injections of 25, 100 and 200 ng glucagon, and saline into the PVN
,
200 ng :
The values are presented as means_+S.E.M. The control condition involved no injection. None of the effects was significantly ( P < 0.05) different from saline.
saline
leo
25 ng 100ng 200ng Saline Control
50
0
1
%
I
!
i
~
~
2
3
4
5
6
7
-&
8
- -
Food intake (g)
Water intake (g)
Body weight gain (g)
32.16-+2.84 31.09+_4.87 30.25_+2.20 27.94 -+ 1.77 25.11_+0.97
23.68_+2.64 23.70-+2.33 23.89-+2.01 23.73 _+ 1.60 27.77_+1.11
-3.88_+2.21 -3.25_+1.63 -1.50-+2.20 - 3.75 _+2.43 0.06_+0.37
!
9
10
11
12
TIME AFTER INJECTION (blocks of 5 rain)
Fig. 3. Mean locomotor activity (activity counts) over a 60 min period immediately after the injection of either saline or one of three doses of glucagon into the PVN. For clarity of illustration the data are presented in blocks of five minutes and S.E.M. values (range 3.3116.3) are not included.
0.05, respectively for the 25, 100 and 200 ng doses], nor a significant treatment by time i n t e r a c t i o n [F59,531 = 0.80, 0.87 and 0.65, P > 0.05, respectively] (Fig. 3). The main effect of time on activity was significant for all three doses of glucagon [F59,531 = 5.15, 5.97 and 7.34, P < 0.001, respectively] indicating a general decrease in activity over the test sessions (see below for explanation). The tendency of EE (Fig. 1) and activity (Fig. 3) values, and to a lesser degree RQ (Fig. 2), to start at high levels and decrease over time has been repeatedly found in this paradigm, and has been ascribed to the stress of the handling and injection procedures 24-28'36. They are reflected in the significant time effect of glucagon on those three parameters as described above. The magnitude and time course of these stress effects are similar to those reported elsewhere 24-28'36. In order to investigate the possibility that the increases in energy expenditure may have been secondary to changes in locomotor activity, several analyses were undertaken. First, as described above and in contrast to the changes in energy expenditure, none of the changes in locomotor activity and none of the interactions over time were significant. Second, analyses of covariance were conducted using both raw and log transformed data. The covariance matrices, which are not presented here, indicated no significant covariance between the changes in energy expenditure and locomotor activity in any of the rats.
Food intake, water intake and body weight The data in Table I indicate that glucagon had no significant effects in any of these three parameters
[F4.28 = 1.25, 1.97 and 0.85, P > 0.05, for food intake, water intake and body weight, respectively]. The effects of saline on body weight were suggestive but not significant. Similar non-significant effects for saline on food intake and body weight have been reported by others 37.
Blood glucose concentration The data in Table II indicate that glucagon significantly increased blood glucose concentration. There were average increases of 13.9% [t 7 = 4.23, P < 0.01] for the 25 ng dose; 10.6% [t 7 = 4.70, P < 0.01] for the 100 ng dose; and 12.1% [t 7 = 3.05, P < 0.05] for the 200 ng dose. There was no significant change in blood glucose after the injection of saline [t 7 = 1.94, P > 0.05]. This demonstrates that the injection procedure itself and the vehicle solution had little impact on this parameter. Analysis of variance on the pre-injection values across treatments showed no significant differences [F3.21 = 0.64, P > 0.05]. Analysis of variance on the post-injection values across treatments showed significant differences [F3,2~ = 10.02, P < 0.01]. Post-hoc comparisons with the Newman-Keuls test on these post-injection values indicated that the three glucagon data points were significantly different from the saline data ( P < 0.05), but that they did not differ significantly among themselves ( P > 0.05).
T A B L E II
Effect on blood glucose concentration of single injections of 25, 100 and 200 ng glucagon, and saline into the PVN Values are expressed in m m o l / I , mean-+ S.E.M.
25 ng 100ng 200 ng Saline
Pre-injeetion
1 h post-injection
% Change
5.13+0.21 5.45+0.21 5.50_+0.26 5.40-+0,17
5.84+0.32 6.03+0.17 6.16_+0.20 5.74-+0.16
13.9% * 10.6% * 12.1% * 6.3%
• P < 0.05 compared to saline.
249 DISCUSSION The present study provides the first demonstration that injections of glucagon into the PVN affect the metabolic aspects of energy balance. Glucagon acutely increased energy expenditure, respiratory quotient and blood glucose concentration, without affecting locomotor activity, 24 h food and water intake or body weight. The shift towards the utilization of carbohydrates and subsequent sparing of fats, indicated by the increased respiratory quotient 5'7'15, reflects a state of anabolism and positive energy balance. On the other hand, the small and short-lasting increase in energy expenditure suggests catabolism and negative energy balance. Glucagon's variable effects on locomotor activity contrast with its orderly effects on energy expenditure and energy substrate utilization. This fundamental dissociation indicates that both metabolic effects of glucagon are primary and not secondary to changes in motor activity. Previous studies indicate that systemic glucagon enhances thermogenesis in brown adipose tissue 1'2. However, the thermogenic effect demonstrated in the present experiment provides the first evidence for the involvement of PVN glucagon in whole-body thermogenesis. It also shows a functional similarity of PVN glucagon and insulin, although insulin's thermogenic effect is larger both in terms of peak magnitude and duration 26. Glucagon increased respiratory quotient in a clear and dose-dependent manner. The maximum respiratory quotient for each of the three doses occured about 20-25 min after the injection. Under normal physiological conditions respiratory quotients of around 0.90 are produced by the mixed catabolism of carbohydrates, fats and proteins. Values approaching 0.70 reflect the exclusive catabolism of fats seen, for example, in fasting. Values approaching 1.00, such as those in the present experiment and commonly after a meal 5'7'15, reflect the exclusive catabolism of carbohydrates. The dose-dependent effect of glucagon on respiratory quotient suggests the desirability of studying even higher doses. However, interpreting the data from such high doses would be greatly complicated by the possibly damaging effects of high and un-physiologic concentrations of glucagon. The present findings may be relevant to the effects of PVN regulation on macronutrient selection and preference for carbohydrate consumption 2°. Given a fundamental metabolic shift toward carbohydrate utilization, it is reasonable that the animal would preferentially consume carbohydrates. The finding that intracerebroventricular glucagon decreases carbohydrate intake 3° suggests that such in-
hibitory effects are mediated by circumventricular areas other than the PVN. Glucagon injected into the PVN increased blood glucose concentration. The effect was not dose-dependent. Blood glucose levels and respiratory quotient are regulated, at least in part, by sympathetic activation26. They are interrelated aspects of the overall energy balance and changes in one are normally concordant with changes in the other as it is the case for insulin injections into the PVN 26. The present study shows constant high levels of glycemia coexisting with increasing levels of respiratory quotient. This suggests, as discussed below, that other neurochemical/neurohumoral systems are likely to participate, together with the sympathetic system, in the effects of glucagon. Finally, acute injections of glucagon into the PVN had no effects on feeding, drinking or body weight. Glucagon has been variously shown to inhibit 9'11' 13,19,22,41, enhance11,12,30 and have no effects3'11 on feeding, and that it reduces body weight 3'7'11. However, many of these studies were based on chronic and systemic administration of glucagon3'7'19'22'41, in contrast to the single PVN injection used in the present study. On the other hand, intracerebroventricular infusions of glucagon in doses similar to those used here have been reported to suppress 24 h food intake 13. The negative findings of the present study suggest that, as is the case for the previously reported inhibition of carbohydrate intake 3°, the centrally-mediated inhibitory effects of glucagon on feeding are likely mediated by circumventricular areas other than the PVN. Some considerations regarding similarities and differences between glucagon and insulin are pertinent. First, it is known that the relative concentrations of insulin and glucagon are determinants of the body weight set point 7. Second, glucagon and insulin are elevated in obesity 15,26,38. In this context insulin signals increased body fat to the PVN 26. It is likely, then, that glucagon also stimulates the PVN in similar circumstances. Third, the present study shows that glucagon and insulin have similar thermogenic and hyperglycemic effects, even though their magnitudes are different 26. Fourth, glucagon increases respiratory quotient in a manner similar to low doses of insulin. However, at high doses insulin decreases respiratory quotient 26. Several lines of evidence point to the sympathetic nervous system (SNS) as a mediator of the common effects of glucagon and insulin in the PVN. The PVN has anatomical and functional links with the SNS 2°'21. We have postulated that the PVN is a major integrative centre for metabolic signals from the periphery with SNS-mediated neurohumoral control of energy
250 intake, energy substrate utilization and thermogenesis 26. The activation of the PVN by signals of excessive body fat, such as insulin, shifts whole-body metabolism toward a state of catabolism26. The SNS mediates this catabolism by increasing thermogenesis, fat combustion and blood glucose, while inhibiting food intake 26. It is known that the intracerebroventricular administration of g!uc~gon stimulates sympathetic activity TM and that this accounts for the glucagon-induced hyperglycemia L~8. It is likely, then, that SNS stimulation mediates the thermogeniC and hyperglycaemic effects of PVN glucagon. The increase in respiratory quotient produced by glucagon cannot be explained through the activation of the SNS, since SNS activation with insulin decreases respiratory quotient 26. This suggests that the effects observed here represent parasympathetic activation. The likelihood of this mode of action is increased by the fact that the PVN has anatomical and functional links with the vagus n e r v e 4'21'29'31'32'34. Therefore, the parasympathetic system may mediate the effects of glucagon on respiratory quotient. This means that glucagon in the PVN may simultaneously activate the sympathetic and parasympathetic systems, acting either as a neurotransmitter or as a neuromodulator 13. The fact that at least two different populations of PVN neurons participate in the opposite actions of many other feeding-regulating factors as well as in a variety of neuroendocrine functions ~7 also supports the above-postulated mode of action. In summary, the present data constitute the first direct evidence that glucagon in the PVN modulates the metabolic parameters relevant to maintaining energy balance. Glucagon injected into the PVN produces a brief and small increase in thermogenesis, whereas it produces a long-lasting and large magnitude increase in respiratory quotient. Locomotor activity remains unaffected. Blood glucose levels are similarly increased by the different doses of glucagon, whereas 24 h ingestive behavior and body weight remain unaffected. These data suggest that glucagon in the PVN has a primary role as a modulator of substrate utilization by inducing the preferential utilization of carbohydrates and the sparing of fat reserves. Acknowledgements. This research was supported by grants from the Australian Research Council to D.M.A. REFERENCES 1 Amir, S., Central glucagon-induced hyperglycemia is mediated by combined activation of the adrenal medulla and sympathetic nerve endings, Physiol. Behav., 37 (1986) 563-566.
2 Billington, C.J., Bartness, T.J., Briggs, J., Levine, A.S. and Morley, J.E., Glucagon stimulation of brown adipose tissue growth and thermogenesis, Am. J. Physiol., 252 (1987) R160-R165. 3 Billington, C.J., Briggs, J.E., Link, J.G. and Levine, A.S., Glucagon in physiological concentrations stimulates brown fat thermogenesis in vivo, Am. J. Physiol., 261 (1991) R501-R507. 4 Blundell, J., Pharmacological approaches to appetite suppression, Trends Pharmacol. Sci., 12 (1991) 147-157. 5 Bravo, E., L., Tarazi, R.C. and Gifford, R.W., Circulating and urinary catecholamines in pheochromocytoma. Diagnostic and pathophysiologic implications, N. Engl. J. Med., 301 (1979) 682686. 6 Bray, G.A., Nutrient balance and obesity: an approach to control of food intake in humans, Med. Clin. N. Am., 73 (1989) 29-45. 7 de Castro, J.M., Paullin, S.K. and DeLugas, G.M., Insulin and glucagon as determinants of body weight set point and microregulation in rats, J. Comp. Physiol. PsychoL, 92 (1978) 571-579. 8 Ferranini, E., The theoretical bases of indirect calorimetry: a review, Metabolism, 37 (1988) 287-301. 9 Geary, N., Pancreatic glucagon signals postprandial satiety, Neurosci. Biobehav. Rev., 14 (1990) 323-338. 10 Gleeson, M., Brown, J.F., Waring, J.J. and Stock, M.J., The effects of physical exercise on metabolic rate and dietary-induced thermogenesis, Br. J. Nutr., 47 (1982) 173-181. 11 Grossman, S.P., The role of glucose, insulin and glucagon in the regulation of food intake and body weight, Neurosci. Biobehav. Rev., 10 (1986) 295-315. 12 Hell, N.S. and Timo-Iaria, C., Increase of food intake induced by glucagon in the rat, Physiol. Behav., 34 (1985) 39-44. 13 Inokuchi, A., Oomura, Y. and Nishimura, H., Effect of intracerebroventricularly infused glucagon on feeding behavior, Physiol. Behav., 33 (1984) 397-400. 14 Jin, S.-L.C., Han, V.K., Simmons, J.G., Towle, A.C., Lauder, J.M. and Lund, P.K., Distribution of glucagon-like peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study, J. Comp. Neurol., 271 (1988) 519-532. 15 Kalkhoff, R.K., Gossain, V.V. and Matute, M.L., Plasma glucagon in obesity: response to arginine, glucose, and protein administration, N. Engl. J. Med., 289 (1973) 465-467. 16 Kleiber, M., The Fire of Life; an Introduction to Animal Energetics, Robert R. Krieger, New York, 1975. 17 Kow, L.-M. and Pfaff, D.W., Responses of hypothalamic paraventricular neurons in vitro to norepinephrine and other feeding-relevant agents, Physiol. Behav., 46 (1989) 265-271. 18 Krzeski, R., Czyzyk-Krzeska, M.F., Trzebski, A. and Millhorn, D.E., Centrally administered glucagon stimulates sympathetic nerve activity in rat, Brain Res., 504 (1989) 297-300. 19 Le Sauter, J. and Geary, N., Redundant vagal mediation of the synergistic satiety effect of pancreatic glucagon and cholecystokinin in sham feeding rats, J. Auton. Nerv. System, 30 (1990) 13-22. 20 Leibowitz, S.F., Hypothalamic paraventricular nucleus: interaction between a2-noradrenergic system and circulating hormones and nutrients in relation to energy balance, Neurosci. Biobehav. Rev., 12 (1988) 101-109. 21 Luiten, P.G., ter Horst, G.J. and Steffens, A.B., The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism, Prog. Neurobiol., 28 (1987) 1-54. 22 Martin, J.R. and Novin, D., Decreased feeding in rats following hepatic-portal infusion of glucagon, Physiol. Behav., 19 (1977) 461-466. 23 Mayer, J., Glucostatic mechanism of regulation of food intake, N. Engl. J. Med., 249 (1953) 13-16. 24 McGregor, I.S., Men6ndez, J.A. and Atrens, D.M., Metabolic effects obtained from excitatory amino acid stimulation of the sulcal prefrontal cortex, Brain Res., 529 (1990) 1-6. 25 Men6ndez, J.A. and Atrens, D.M., Insulin increases energy expenditure and respiratory quotient in the rat, Pharmacol. Biochem. Behav., 34 (1989) 765-768. 26 Men6ndez, J.A. and Atrens, D.M., Insulin and the paraventricu-
251
27 28
29 30
31 32 33 34
lar hypothalamus: modulation of energy balance, Brain Res., 555 (1991) 193-201. Mem~ndez, J.A., Atrens, D.M. and Leibowitz, S.F., Metabolic effects of galanin injections into the paraventricular nucleus of the hypothalamus, Peptides, 13 (1992) 323-327. Men6ndez, J.A., McGregor, I.S., Healey, P.A., Atrens, D.M. and Leibowitz, S.F., Metabolic effects of neuropeptide Y injections into the paraventricular nucleus of the hypothalamus, Brain Res., 516 (1990) 8-14. Morley, J.E. and Levine, A.S., The pharmacology of eating behavior, Annu. Rev. Pharmacol. Toxicol., 25 (1985) 127-146. Nagai, K., Thibault, L., Nishikawa, K., Hashida, A., Ootani, K. and Nakagawa, H., Effect of glucagon in macronutrient selfselection: glucagon-enhanced protein intake, Brain Res. Bull., 27 (1991) 409-415. Novin, D., Gustatory and visceral modulation of feeding. In J.E. Morley, M.B. Sterman and J.H. Walsh (Eds.), Nutritional Modulation of Neural Function, Academic Press, CA, 1988, pp. 67-86. Oomura, Y., Chemical and neuronal control of feeding motivation, Physiol. Behav., 44 (1988) 555-560. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. Porte Jr., D. and Woods, S.C., Neural regulation of islet hormones and its role in energy balance and stress hyperglycemia. In M. Ellenberg and H. Rifkin (Eds.), Diabetes Mellitus, Theory and
35 36 37
38 39
40
41
Practice, Vol. 1, 3rd edn., Medical Examination Publishing Co., New York, 1983, pp. 267-294. Salazar, I. and Vaillant, C., Glucagon-like immunoreactivity in rat hypothalamus, Regul. PeptMes, 22 (1988) 430. Siviy, S.M., Kritikos, A., Atrens, D.M. and Shepherd, A., Effects of norepinephrine infused in the paraventricular hypothalamus on energy expenditure in the rat, Brain Res., 487 (1989) 79-88. Stanley, B.G., Kyrkouli, S.E., Lampert, S. and Leibowitz, S.F., Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity, Peptides, 7 (1986) 1189-1192. Starke, A.A., Erhardt, G., Berger, M. and Zimmermann, H., Elevated pancreatic glucagon in obesity, Diabetes, 33 (1984) 277280. Steffens, A.B., Scheurink, A.J., Luiten, P.G. and Bohus, B., Hypothalamic food intake regulating areas are involved in the homeostasis of blood glucose and plasma FFA levels, Physiol. Behav., 44 (1988) 581-589. Unger, R.H. and Dobbs, R.E., Insulin, glucagon, and somatostatin secretion in the regulation of metabolism, Annu. Rev. Physiol., 40 (1978) 307-343, Vanderweele, D.A., Geiselman, P.J. and Novin, D., Pancreatic glucagon, food deprivation and feeding in intact and vagotomized rabbits, Physiol. Behav., 23 (1979) 155-158.
245
BRES 19500
Glucagon and the paraventricular hypothalamus: modulation of energy balance Dale M. Atrens *, Jos~ A. Men6ndez Department of Psychology, University of Sydney, NSW 2006, Australia (Accepted 20 July 1993)
Key words: Paraventricular nucleus of the hypothalamus; Glucagon; Energy balance; Energy expenditure; Thermogenesis; Respiratory quotient; Respiratory exchange ratio; Substrate utilization; Food intake; Feeding; Body weight; Blood glucose; Indirect calorimetry; Calorimetry; Obesity; Rat
The effects of glucagon injections (25, 100 and 200 ng) into the paraventricular nucleus of the hypothalamus (PVN) were investigated in an open-circuit calorimeter. Wistar rats were tested, with no food available during the tests. High doses of glucagon (100 and 200 ng) produced small and short-lasting increases in energy expenditure. The independence of these changes from changes in locomotor activity suggests that the thermogenesis represents a primary modulation and is not secondary to increased locomotion. All three doses of glucagon produced long-lasting and dose-related increases in respiratory quotient which were unrelated to any changes in locomotor activity. As with the changes in energy expenditure, this dissociation indicates that the effects are not secondary to changes in locomotor activity. These data constitute the first evidence that glucagon in the PVN modulates the metabolic parameters central to energy balance. In separate experiments, the three doses of glucagon increased blood glucose concentration over a one hour period, but they did not affect food and water intake and body weight over 24 h. These findings suggest that glucagon normally acts on the PVN in conditions of increased body fat to initiate autonomic mechanisms which increase glycemic levels, thermogenesis and carbohydrate utilization. These data constitute the first direct evidence for the involvement of the PVN in the regulation of energy balance by glucagon. The main effect of glucagon in the PVN is anabolic in that it increases dependence on carbohydrates as an energy substrate which results in a sparing of fat reserves.
INTRODUCTION T h e effects o f p e r i p h e r a l l y - o r i n t r a c e r e b r o v e n t r i c u l a r l y - a d m i n i s t e r e d g l u c a g o n on e n e r g y b a l a n c e a r e m e d i a t e d by n u m e r o u s a n d c o m p l e x effects o n c a r b o h y d r a t e , fat a n d p r o t e i n m e t a b o l i s m aL34'4° as well as by effects o n t h e r m o g e n e s i s z'3, b o d y w e i g h t 7'1~ a n d f e e d ing 7'9,11A2A9. W e have r e c e n t l y d e m o n s t r a t e d t h a t int r a p e r i t o n e a l injections o f g l u c a g o n i n c r e a s e e n e r g y expenditure and respiratory quotient (Men6ndez and A t r e n s , in p r e p a r a t i o n ) . W h e r e a s i n c r e a s e d e n e r g y exp e n d i t u r e is clearly catabolic, i n c r e a s e d r e s p i r a t o r y q u o t i e n t , which i n d i c a t e s a n i n c r e a s e d r e l i a n c e on carb o h y d r a t e s as e n e r g y s o u r c e a n d t h e s p a r i n g o f fat reserves, m a y b e c o n s i d e r e d to b e anabolic. G l u c a g o n ' s p r o n o u n c e d effects on t h e m e t a b o l i c p a r a m e t e r s o f e n e r g y b a l a n c e w h e n it is i n j e c t e d p e r i p h e r a l l y o r int r a c e r e b r o v e n t r i c u l a r l y c o n t r a s t with t h e c o m p l e t e lack o f d a t a for injections into d i s c r e t e b r a i n areas.
* Corresponding author. Fax: (61) (2) 692-2603.
It is likely t h a t t h e a d m i n i s t r a t i o n o f g l u c a g o n into t h e h y p o t h a l a m u s w o u l d affect t h e m e t a b o l i c p a r a m e t e r s of e n e r g y b a l a n c e . First, t h e r e is a high c o n c e n t r a tion o f g l u c a g o n i m m u n o r e a c t i v i t y in t h e h y p o t h a l a m u s 14'35. S e c o n d , i n t r a c e r e b r o v e n t r i c u l a r injections o f g l u c a g o n m o d u l a t e f o o d intake, m a c r o n u t r i e n t selection, b l o o d glucose levels a n d s y m p a t h e t i c activity 1'11'~3'18'3°. T h e l a t t e r findings i n d i c a t e t h e involvem e n t o f t h e brain, b u t t h e y d o n o t a d d r e s s t h e p r o b l e m o f which b r a i n a r e a ( s ) is(are) involved. T h i r d , s o m e o f t h e m e t a b o l i c effects o f g l u c a g o n a r e m e d i a t e d by the activation o f t h e s y m p a t h e t i c n e r v o u s system 18, a n d t h e h y p o t h a l a m u s is a m a j o r c e n t r e for s y m p a t h e t i c activity 6'21'26. T h e p a r a v e n t r i c u l a r n u c l e u s o f t h e h y p o t h a l a m u s ( P V N ) is p a r t i c u l a r l y likely to p a r t i c i p a t e in any c e n t r a l effect o f g l u c a g o n o n e n e r g y b a l a n c e since it has o n e o f t h e highest c o n c e n t r a t i o n s o f g l u c a g o n i m m u n o r e a c t i v i t y 14'35. T h e P V N also p a r t i c i p a t e s extensively in t h e r e g u l a t i o n o f feeding, b o d y w e i g h t a n d e n e r g y m e t a b o l i s m 6'2°,21'26-28,36. T h e r e are, however, no d a t a o n t h e m e t a b o l i c effects o f g l u c a g o n i n j e c t e d into the PVN.
246 The effects energy motor weight
present study was designed to determine the of injections of glucagon into the PVN on expenditure, energy substrate utilization, locoactivity, feeding and drinking behavior, body and blood glucose levels.
MATERIALS AND METHODS Subjects Ten male Wistar rats obtained from the University of Sydney breeding farm were used. They weighed between 300 and 400 g at the time of surgery. The rats were individually housed in clear acrylic cages, with food (Allied Rat & Mouse Kubes, Sydney) and tap water provided ad libitum. The colony room was maintained at 22_+ 2°C, with a 14/10 h light/dark cycle. Every rat was handled daily for one week before surgery and for two weeks after in order to minimize the stress of h u m a n contact.
Surgery and histology The rats were anaesthetized with 1 m l / k g Ketalar (100 m g / m l ketamine hydrochloride; Parke-Davis Pty. Ltd.) and 0.1 ml R o m p u n (20 m g / m l xylazine hydrochloride; Bayer Australia Ltd.), both injected intramuscularly. They were placed in a Kopf stereotaxic apparatus and implanted with a single, stainless-steel, 22-gauge guide cannula fitted with a d u m m y cannula (Plastics One, USA). T h e coordinates relative to bregma were: posterior 1.8, lateral 1.8 and ventral 7.2, with the cannulae being implanted at an angle of 10° off the midline 33. The tip of the cannula was aimed for a position 1 m m dorsal to the PVN, and the injector cannula extended 1 m m beyond the tip of the guide cannula. At the conclusion of testing, the rats were given a lethal dose of Nembutal, after which their brains were removed for histological analysis. The brains were frozen to - 12°C, sectioned at 4 0 / z m and stained with Toluidine blue O. Cannulae placements were determined microscopically with reference to the atlas of Paxinos and Watson 33.
Experiment 1. Determination of energy expenditure, substrate utilization and locomotor activity Apparatus Respiratory quotient (RQ) and energy expenditure (EE) were calculated after recording oxygen ( 0 2) consumption and carbon dioxide (CO 2) production in an open-circuit calorimeter. Two clear acrylic cylindrical chambers with stainless steel grid floors and a volume of 6.28 litres each were used. One was used for testing the rats and the other as a reference standard for calibration of the atmospheric air. Compressed atmospheric air at a flow rate of 1600 m l / m i n and a pressure of 8 kPa above atmospheric was continuously drawn through both chambers. A system of solenoids allowed the air leaving one of the chambers to be split and directed for analysis, while the air from the other chamber was exhausted to the atmosphere. A sample of 110 m l / m i n was directed through a Perma Pure (Toms River, N J, USA) permeation drier (model PD750-12PP), a CD-3A CO 2 analyzer and a S-3A O 2 analyzer (Applied Electrochemistry, USA). The rest of the air was exhausted to the room. T h e analyzers were calibrated daily with primary gravimetric standards (Commonwealth Industrial Gases, Sydney). Locomotor activity was recorded by placing the testing chamber on an electronic balance (Mettler PE-2000) and using the unintegrated signal from the strain gauge. The reliability and validity of this method has been demonstrated in other studies 24-28'36. A microcomputer system controlled and monitored the calorimeter. The computer provided minute-by-minute records of air flow, CO 2 production, 0 2 consumption and activity counts. The following calculations were made: E E (kJ) = mol O 2 (364+ 113 RQ); R Q = vol. CO 2 produced/vol. 0 2 consumed 6'8A°'16. Energy expenditure was expressed in J / g to account for different body weights.
Procedure Each rat was habituated to the metabolic apparatus in 60-min tests before and after surgery. In addition to providing baseline data on the metabolic and activity parameters this procedure allowed for the determination of any effect produced by the surgical procedure itself. The rats were also habituated to the injection procedure by introducing the injector cannula into the guide cannula on two separate occasions before any experimentation. The experiment itself began approximately two weeks after surgery. The test sessions were conducted in the light phase of the cycle (between 10.00 and 16.00 h). The rats were given a 30-min run in the testing chamber before each treatment. They were then removed and injected in a counterbalanced order with 0.25 /xl of either sterile saline (NaCI 0.9%) or one of three doses of glucagon: 25, 100 and 200 ng (Sigma, USA) dissolved in sterile saline. These doses were derived from those used in other studies ll'ls. The d u m m y cannula was removed, and the injections were performed over a one-minute period through a 28-gauge injector (Plastics One) which projected 1 m m beyond the guide cannula. The rats were unrestrained during the injection procedure. After the injection, the injector cannula was removed and the d u m m y cannula re-secured. The rats were then placed in the testing chamber, and respiratory exchange and activity were monitored for 60 min. Following this, the rats were returned to their home cages. Each recording session in the metabolic apparatus was preceded and followed by a 5-min analysis of air leaving the calibration chamber, in order to assess any drift in the analyzers. Food was not available during the test sessions. At least seven days elapsed between successive injections in the same rat.
Data analysis The data from each glucagon treatment were compared with those of the saline treatment by a two-way analysis of variance with repeated measures on the treatment and time factors. T h e level of significance was set at 0.05. In order to further investigate any relationships between changes in energy expenditure and locomotor activity these data were subject to an analysis of covariance.
Experiment 2. Determination of food intake, water intake and body weight Procedure Another group of 8 rats was tested for 24 h food intake, water intake and body weight, in a paradigm similar to previous studies 26. Control food and water intake and body weight were recorded for 5 consecutive days before the experiment and the daily averages calculated. At 12.00 h on the day of testing the rats received a 0.25 ~1 injection of either NaCI 0.9% or one of three doses of glucagon: 25, 100 and 200 ng. At 12.00 h on the following day the a m o u n t s of standard lab chow and tap water consumed were recorded. Body weight was measured immediately before the injection and at the end of the session. The injections were given in a counterbalanced order and at least 5 days separated each treatment.
Data analysis The data were compared by a one way analysis of variance with repeated measures, followed by post-hoc Newman-Keuis test. The level of significance was set at 0.05.
Experiment 3. Determination of blood glucose concentration Procedure A further group of 8 rats was tested for blood glucose concentration one hour after a 0.25 p,1 injection of either NaCI 0.9% or one of three doses of glucagon: 25, 100 and 200 ng. Testing was conducted between 10.00 and 16.00 h. The injections were given in a counterbalanced order and at least 5 days separated each treatment. Blood samples were taken by 'milking' the tail after removing 1 - 2 m m of its tip 25,26. The blood was collected in heparinized vials and immediately analyzed in an automated glucose analyzer (Model
247 23AM, Yellow Springs Instruments, USA). Two samples were taken from every rat, one immediately before and the other one hour after the injection. The rats were lightly anaesthetized with ethylic ether during sampling in order to minimize the stress of the procedure. A number of pilot experiments and the counterbalanced order of injections, which includes the vehicle solution, has allowed us to eliminate any confounding effect due to this use of ether.
+ • -
Blood glucose concentration was expressed in mmol/l. Two-tailed Student's t-tests were used to compare blood glucose concentrations before and after the injections. One-way analysis of variance with repeated measures, followed by post hoc Newman-Keuls test, was used for the comparison between pre-injection values and between post-injection values across treatments. The level of significance was set at 0.05.
PU I O R T 0'92
0.48
+
25 ng •
" 100
X 0.43
EP
/
NE EN R D 0.38 G I YT U R 0.33 E
0.28
I
I
I
I
I
I
I
t
I
I
I
2
3
4
5
6
7
8
9
10
11
12
TIME A F T E R INJECTION (block= o f 5 min)
Fig. 1. Mean energy expenditure (J/g) over a 60 min period immediately after the injection of either saline or one of three doses of glucagon into the PVN. For clarity of illustration the data are presented in blocks of 5 min and S.E.M. values (range 0.008-0.04) are not included.
saline
D
~O-- O
A t TE 0 NO.gO RT Y 0.88
1
N o dose of glucagon p r o d u c e d a significant overall t r e a t m e n t effect on E E [F1, 9 = 0.16, 1.12 and 0.58, P > 0.5, respectively for the 25, 100 and 200 ng doses] (Fig. 1). However, the 100 and 200 ng doses did produce significant t r e a t m e n t by time interactions [F59,531 = 1.64, P < 0.01, and /759,531 = 1.85, P < 0.001, respectively], which reflect the increase in E E seen during the first 30 min of the session (Fig. 1). O n average, E E was
200 ng
-
0.86 ~
Energy expenditure
100 ng -
0.94
SQ
N o statistical differences were f o u n d between the pre-surgery, post-surgery and saline injection data for energy expenditure, respiratory quotient or activity. These data rule out the possibility of any c o n f o u n d i n g due to the surgical trauma, the volume injected or the injection of saline. T h e possibility of c o n f o u n d i n g due to c a n n u l a - i n d u c e d tissue d a m a g e was also r e d u c e d by the fact that the histological analysis showed minimal incidental damage.
-o
0.96 R E
Data analysis
RESULTS
25 ng
~ 2
3
4
5
6
7
8
9
10
11
12
TIME A F T E R INJECTION lblocks o f 5 rain)
Fig. 2. Mean respiratory quotient over a 60 rain period immediately after the injection of either saline or one of three doses of glucagon into the PVN. For clarity of illustration the data are presented in blocks of five minutes and S.E.M. values (range 0.006-0.02) are not included.
increased by 4.5% by 100 ng of glucagon and 3.6% by 200 ng of glucagon c o m p a r e d to control. T h e 25 ng dose did not p r o d u c e a significant t r e a t m e n t by time interaction [F59,531 = 1.24, P > 0.05). T h e main effect of time on E E was significant for all three doses of glucagon [F59,531 = 20.43, 19.16 and 21.23, P < 0.001, respectively]. This reflects the fact that the initially high values for the glucagon and saline treatments declined steadily over the test session (see below for explanation).
Respiratory quotient N o dose of glucagon p r o d u c e d a significant overall t r e a t m e n t effect on R Q [FI, 9 = 0 . 1 5 , 1.51 and 4.96, P > 0.05, respectively for the 25, 100 and 200 ng doses] (Fig. 2). However, all three doses of glucagon did p r o d u c e significant t r e a t m e n t b y time interactions [F59,531 = 1.93, 4.33 and 2.11, P < 0.001, respectively], which reflect the increased R Q seen from approximately 5 - 1 0 min after the injection and maintained t h r o u g h o u t the rest of the session (Fig. 2). O n average, R Q was increased 0.7% by 25 ng of glucagon, 2.2% by 100 ng of glucagon and 5.1% by 200 ng of glucagon c o m p a r e d to control. T h e main effect of time on R Q was significant for all three doses of glucagon [11759,531 ~--18.57, 16.89 and 16.74, P < 0.001, respectively] (see below for explanation).
Locomotor activity N o dose of glucagon p r o d u c e d a significant treatm e n t effect on activity [ E l , 9 = 0.12, 0.70 and 0.08, P >
248 TABLE I 100 ng
•
Effects on 24 h food intake, water intake and body weight gain of single injections of 25, 100 and 200 ng glucagon, and saline into the PVN
,
200 ng :
The values are presented as means_+S.E.M. The control condition involved no injection. None of the effects was significantly ( P < 0.05) different from saline.
saline
leo
25 ng 100ng 200ng Saline Control
50
0
1
%
I
!
i
~
~
2
3
4
5
6
7
-&
8
- -
Food intake (g)
Water intake (g)
Body weight gain (g)
32.16-+2.84 31.09+_4.87 30.25_+2.20 27.94 -+ 1.77 25.11_+0.97
23.68_+2.64 23.70-+2.33 23.89-+2.01 23.73 _+ 1.60 27.77_+1.11
-3.88_+2.21 -3.25_+1.63 -1.50-+2.20 - 3.75 _+2.43 0.06_+0.37
!
9
10
11
12
TIME AFTER INJECTION (blocks of 5 rain)
Fig. 3. Mean locomotor activity (activity counts) over a 60 min period immediately after the injection of either saline or one of three doses of glucagon into the PVN. For clarity of illustration the data are presented in blocks of five minutes and S.E.M. values (range 3.3116.3) are not included.
0.05, respectively for the 25, 100 and 200 ng doses], nor a significant treatment by time i n t e r a c t i o n [F59,531 = 0.80, 0.87 and 0.65, P > 0.05, respectively] (Fig. 3). The main effect of time on activity was significant for all three doses of glucagon [F59,531 = 5.15, 5.97 and 7.34, P < 0.001, respectively] indicating a general decrease in activity over the test sessions (see below for explanation). The tendency of EE (Fig. 1) and activity (Fig. 3) values, and to a lesser degree RQ (Fig. 2), to start at high levels and decrease over time has been repeatedly found in this paradigm, and has been ascribed to the stress of the handling and injection procedures 24-28'36. They are reflected in the significant time effect of glucagon on those three parameters as described above. The magnitude and time course of these stress effects are similar to those reported elsewhere 24-28'36. In order to investigate the possibility that the increases in energy expenditure may have been secondary to changes in locomotor activity, several analyses were undertaken. First, as described above and in contrast to the changes in energy expenditure, none of the changes in locomotor activity and none of the interactions over time were significant. Second, analyses of covariance were conducted using both raw and log transformed data. The covariance matrices, which are not presented here, indicated no significant covariance between the changes in energy expenditure and locomotor activity in any of the rats.
Food intake, water intake and body weight The data in Table I indicate that glucagon had no significant effects in any of these three parameters
[F4.28 = 1.25, 1.97 and 0.85, P > 0.05, for food intake, water intake and body weight, respectively]. The effects of saline on body weight were suggestive but not significant. Similar non-significant effects for saline on food intake and body weight have been reported by others 37.
Blood glucose concentration The data in Table II indicate that glucagon significantly increased blood glucose concentration. There were average increases of 13.9% [t 7 = 4.23, P < 0.01] for the 25 ng dose; 10.6% [t 7 = 4.70, P < 0.01] for the 100 ng dose; and 12.1% [t 7 = 3.05, P < 0.05] for the 200 ng dose. There was no significant change in blood glucose after the injection of saline [t 7 = 1.94, P > 0.05]. This demonstrates that the injection procedure itself and the vehicle solution had little impact on this parameter. Analysis of variance on the pre-injection values across treatments showed no significant differences [F3.21 = 0.64, P > 0.05]. Analysis of variance on the post-injection values across treatments showed significant differences [F3,2~ = 10.02, P < 0.01]. Post-hoc comparisons with the Newman-Keuls test on these post-injection values indicated that the three glucagon data points were significantly different from the saline data ( P < 0.05), but that they did not differ significantly among themselves ( P > 0.05).
T A B L E II
Effect on blood glucose concentration of single injections of 25, 100 and 200 ng glucagon, and saline into the PVN Values are expressed in m m o l / I , mean-+ S.E.M.
25 ng 100ng 200 ng Saline
Pre-injeetion
1 h post-injection
% Change
5.13+0.21 5.45+0.21 5.50_+0.26 5.40-+0,17
5.84+0.32 6.03+0.17 6.16_+0.20 5.74-+0.16
13.9% * 10.6% * 12.1% * 6.3%
• P < 0.05 compared to saline.
249 DISCUSSION The present study provides the first demonstration that injections of glucagon into the PVN affect the metabolic aspects of energy balance. Glucagon acutely increased energy expenditure, respiratory quotient and blood glucose concentration, without affecting locomotor activity, 24 h food and water intake or body weight. The shift towards the utilization of carbohydrates and subsequent sparing of fats, indicated by the increased respiratory quotient 5'7'15, reflects a state of anabolism and positive energy balance. On the other hand, the small and short-lasting increase in energy expenditure suggests catabolism and negative energy balance. Glucagon's variable effects on locomotor activity contrast with its orderly effects on energy expenditure and energy substrate utilization. This fundamental dissociation indicates that both metabolic effects of glucagon are primary and not secondary to changes in motor activity. Previous studies indicate that systemic glucagon enhances thermogenesis in brown adipose tissue 1'2. However, the thermogenic effect demonstrated in the present experiment provides the first evidence for the involvement of PVN glucagon in whole-body thermogenesis. It also shows a functional similarity of PVN glucagon and insulin, although insulin's thermogenic effect is larger both in terms of peak magnitude and duration 26. Glucagon increased respiratory quotient in a clear and dose-dependent manner. The maximum respiratory quotient for each of the three doses occured about 20-25 min after the injection. Under normal physiological conditions respiratory quotients of around 0.90 are produced by the mixed catabolism of carbohydrates, fats and proteins. Values approaching 0.70 reflect the exclusive catabolism of fats seen, for example, in fasting. Values approaching 1.00, such as those in the present experiment and commonly after a meal 5'7'15, reflect the exclusive catabolism of carbohydrates. The dose-dependent effect of glucagon on respiratory quotient suggests the desirability of studying even higher doses. However, interpreting the data from such high doses would be greatly complicated by the possibly damaging effects of high and un-physiologic concentrations of glucagon. The present findings may be relevant to the effects of PVN regulation on macronutrient selection and preference for carbohydrate consumption 2°. Given a fundamental metabolic shift toward carbohydrate utilization, it is reasonable that the animal would preferentially consume carbohydrates. The finding that intracerebroventricular glucagon decreases carbohydrate intake 3° suggests that such in-
hibitory effects are mediated by circumventricular areas other than the PVN. Glucagon injected into the PVN increased blood glucose concentration. The effect was not dose-dependent. Blood glucose levels and respiratory quotient are regulated, at least in part, by sympathetic activation26. They are interrelated aspects of the overall energy balance and changes in one are normally concordant with changes in the other as it is the case for insulin injections into the PVN 26. The present study shows constant high levels of glycemia coexisting with increasing levels of respiratory quotient. This suggests, as discussed below, that other neurochemical/neurohumoral systems are likely to participate, together with the sympathetic system, in the effects of glucagon. Finally, acute injections of glucagon into the PVN had no effects on feeding, drinking or body weight. Glucagon has been variously shown to inhibit 9'11' 13,19,22,41, enhance11,12,30 and have no effects3'11 on feeding, and that it reduces body weight 3'7'11. However, many of these studies were based on chronic and systemic administration of glucagon3'7'19'22'41, in contrast to the single PVN injection used in the present study. On the other hand, intracerebroventricular infusions of glucagon in doses similar to those used here have been reported to suppress 24 h food intake 13. The negative findings of the present study suggest that, as is the case for the previously reported inhibition of carbohydrate intake 3°, the centrally-mediated inhibitory effects of glucagon on feeding are likely mediated by circumventricular areas other than the PVN. Some considerations regarding similarities and differences between glucagon and insulin are pertinent. First, it is known that the relative concentrations of insulin and glucagon are determinants of the body weight set point 7. Second, glucagon and insulin are elevated in obesity 15,26,38. In this context insulin signals increased body fat to the PVN 26. It is likely, then, that glucagon also stimulates the PVN in similar circumstances. Third, the present study shows that glucagon and insulin have similar thermogenic and hyperglycemic effects, even though their magnitudes are different 26. Fourth, glucagon increases respiratory quotient in a manner similar to low doses of insulin. However, at high doses insulin decreases respiratory quotient 26. Several lines of evidence point to the sympathetic nervous system (SNS) as a mediator of the common effects of glucagon and insulin in the PVN. The PVN has anatomical and functional links with the SNS 2°'21. We have postulated that the PVN is a major integrative centre for metabolic signals from the periphery with SNS-mediated neurohumoral control of energy
250 intake, energy substrate utilization and thermogenesis 26. The activation of the PVN by signals of excessive body fat, such as insulin, shifts whole-body metabolism toward a state of catabolism26. The SNS mediates this catabolism by increasing thermogenesis, fat combustion and blood glucose, while inhibiting food intake 26. It is known that the intracerebroventricular administration of g!uc~gon stimulates sympathetic activity TM and that this accounts for the glucagon-induced hyperglycemia L~8. It is likely, then, that SNS stimulation mediates the thermogeniC and hyperglycaemic effects of PVN glucagon. The increase in respiratory quotient produced by glucagon cannot be explained through the activation of the SNS, since SNS activation with insulin decreases respiratory quotient 26. This suggests that the effects observed here represent parasympathetic activation. The likelihood of this mode of action is increased by the fact that the PVN has anatomical and functional links with the vagus n e r v e 4'21'29'31'32'34. Therefore, the parasympathetic system may mediate the effects of glucagon on respiratory quotient. This means that glucagon in the PVN may simultaneously activate the sympathetic and parasympathetic systems, acting either as a neurotransmitter or as a neuromodulator 13. The fact that at least two different populations of PVN neurons participate in the opposite actions of many other feeding-regulating factors as well as in a variety of neuroendocrine functions ~7 also supports the above-postulated mode of action. In summary, the present data constitute the first direct evidence that glucagon in the PVN modulates the metabolic parameters relevant to maintaining energy balance. Glucagon injected into the PVN produces a brief and small increase in thermogenesis, whereas it produces a long-lasting and large magnitude increase in respiratory quotient. Locomotor activity remains unaffected. Blood glucose levels are similarly increased by the different doses of glucagon, whereas 24 h ingestive behavior and body weight remain unaffected. These data suggest that glucagon in the PVN has a primary role as a modulator of substrate utilization by inducing the preferential utilization of carbohydrates and the sparing of fat reserves. Acknowledgements. This research was supported by grants from the Australian Research Council to D.M.A. REFERENCES 1 Amir, S., Central glucagon-induced hyperglycemia is mediated by combined activation of the adrenal medulla and sympathetic nerve endings, Physiol. Behav., 37 (1986) 563-566.
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