- Email: [email protected]
Pancreatic glucagon does not alter intrameal gastric emptying of milk in the rat
Physiology & Behawor, Vol 45, pp 1259-1261. ©MaxwellPergamon Macmillanplc, 1989 Pnnted m the U S A
0031-9384/89 $3,00 + 00
BRIEF COMMUNICATION
Pancreatic Glucagon Does Not Alter Intrameal Gastric Emptying of Milk in the Rat ZSOLT STOCKINGER AND NORI GEARY 1
Psychology Department, 402a Schermerhorn Hall Columbia University, New York, N Y 10027
R e c e i v e d 3 O c t o b e r 1988
STOCKINGER, Z. AND N GEARY. Pancreanc glucagon does not alter mtrameal gastric emptymg of milk m the rat PHYSIOL BEHAV 45(6) 1259-1261, 1989.--The hypothesis that pancreatic glucagon (laG) inhibits feeding by altering mtrameal gastric emptying was tested m nondepnved rats at midday, conditmns under which exogenous PG is thought to ehclt satiety Rats received intraperltoneal mjectmns of PG (400 or 800 p.g/kg), cholecystokmm (CCK, 0 5 or 1.0 I~g/kg) or saline and were given bolus mtragastnc mfusmns of 10 ml evaporated milk through chromc gasmc cannulae. PG did not affect volumes of stomach contents recovered 20 minutes postlnjection. In contrast, CCK mhthlted gasmc emptying m a dose-related manner. When rmlk was offered to the same rats to dnnk, 400 p.g/kg PG slgmficantly reduced meal size. These findings suggest that PG's satiety effect is not caused by as effect on stomach emptying. Glucagon
Cholecystoklnm
Food retake
Satiety
PANCREATIC GLUCAGON (PG) reduces meal size and duration in rats (6, 8, 13). The behavioral specificity of this effect suggests that PG may signal satiety (7, 8, 10, 13). PG also has several spasmolytic actions on enteric muscles, and under some conditmns alters gastric emptying (5,12). To test whether altered gastric emptying causes PG's satiety effect, we compared intrameal gastric emptying and meal size under conditions in which intraperitoneal injection of PG is known to inhibit feeding, specifically, in nondeprived rats given milk in the middle of the bright phase of the lighting cycle (7,8). An emptying effect must occur during the meal to cause a reduction in meal size or duration. Under these conditions, control meals last 10-20 minutes (7). Therefore, stomach emptying was measured at 20 minutes. The effect of cholecystokinin (CCK), which potently inhibits gastric emptying m rats (3, 11, 14), was tested to assess the sensitivity of the method used to measure gastric emptying.
maintained on pelleted rat chow (Purina 5001) and tap water under a 12:12 light-dark cycle (lights on at 0200). Each rat was equipped with a chronic gastric cannula, using the procedure of Antin et al. (1).
Procedure Stomach emptying. Following recovery from surgery, the rats were adapted to gastric lavage. Food was removed at 0900, the cannula opened, and the stomach flushed repeatedly with 10 ml of 0.9% NaC1 at about 30°C until the drainage was clear. A drainage tube, with a 1.0 ml dead space filled with evaporated milk and with the end clamped to prevent leakage, was then attached to the cannula. Each rat was then intraperitoneally injected with 1 ml/kg saline and placed into a hanging clear plastic cage, with the drainage tube passed through a slot in the floor. Twenty minutes later, rats were given evaporated milk for one hour. On consecutive experimental days, food was removed at 0900, the stomach flushed, and the drainage tube attached as described above. Each rat was then injected with 1 ml/kg of either 400 or 800 Ixg/ml PG (glucagon hydrochloride, Eli Lilly, Indianapolis, IN), or 0.5 or 1.0 p.g/ml CCK (synthetic CCK octapeptide, Squibb,
METHOD
Subjects Eleven male Sprague-Dawley rats weighing 458-816 g were
~Requests for repnnts should be addressed to N. Geary.
1259
1260
STOCKINGER AND GEARY
50
25 E
v
40
T
K
15 J¢
E
"o
20
u
]30
m
E
10 5
+
o e-
10
T
20
20 30 Time (mm)
40
o t_
FIG 2 Inhthltory effect of PG on milk retake Data are cumulative milk retakes after injection of saline (light bars) or 400 /a,g/kg PG (dark bars), mean-+s e *p<0 05, **p<0 005 vs saline
o.
10
0
......
SAL
CCK
PG
FIG 1 Effects of PG and CCK on gasmc emptying Data are volumes emptied from stomach 20 minutes after lntragasmc infusion of 10 ml condensed milk, expressed as a percentage of the milk load, mean_+ s e Unshaded bar, saline rejection, hght shading, CCK rejection (left, 0 5 p,g/kg, right, 1 0 Ixg/kg), dark shading, PG injection (left, 400 Ixg/kg, right, 800 ixg/kg) *p<0.05 vs sahne, **p<0 0005 vs saline, + p < 0 005 vs 0 5 p,g/kg CCK
Princeton, NJ) vs. 0.9% saline according to separate crossover designs, and placed into the test cage. Ten ml of evaporated milk was infused through the drainage tube into the stomach Twenty minutes after injection, the remaining milk was evacuated from the stomach through the drainage tube using a 50 cc syringe, and the volume measured. Cannulae were then closed, and evaporated milk presented for approxtmately one hour to simulate the feeding procedure described below Volume emptied from the stomach was estimated as the 10 ml infusion (plus the 1 ml milk in the drainage tube dead space) minus the volume recovered. Data from 9 rats were analysed using post hoc t-tests after an overall one-way A N O V A revealed a significant effect, F ( 4 , 4 0 ) = 7.67, p < 0 . 0 1 , data from 2 rats whose cannulae leaked or whose gastric contents contained chow were discarded Meal size. Feeding was tested 3 days after the completion of the stomach emptying experiment. At 0900, food was removed and stomachs flushed as above Cannulae were then closed, and rats injected with 1 ml/kg of either 400 txg/ml PG or saline according to a cross-over design. Rats were then placed into their test cages, and evaporated milk was presented in graduated drlnlong cylinders. Intakes were recorded every 10 minutes, for 40 minutes. Data from the 9 rats without missing data points were analysed using two-tailed t-tests. RESULTS
Gastrw Emptying CCK inhibited gastric emptying in a dose-related manner (Fig. 1). Rats injected with saline emptied 38.7 -+ 2.2% (mean -+ s.e. ) of
their milk loads in 20 minutes, versus 29 5 -+ 2 5% after 0.5 ~g/kg CCK, t ( 8 ) = 3 . 7 , p < 0 . 0 0 3 , and 2 0 . 7 -+ 1.6% after 1.0 p~g/kg CCK, t ( 8 ) = 6 7, p < 0 . 0 0 0 1 In contrast, neither 400 ~g/kg PG [37.8 -+ 3.1% emptied, t(8) = 0.36], nor 800 ttg/kg PG [34.6 -+ 3.4% emptwd, t ( 8 ) = 1.5], significantly altered gastric emptying. There was no correlation between body weight and any measure (all p's>0.3).
Meal Stze PG significantly reduced evaporated milk intake (Fig. 2). At 10 minutes, PG inhibited milk intake by 20.0-+ 7 7% (mean-+ s.c.); at 20 minutes, 23.6 -+ 7.8%, at 30 minutes, 20.5 -+ 7.2%, and at 40 minutes, 17.9_+ 9.4%; all p ' s < 0 . 0 5 DISCUSSION
Both 0.5 and 1.0 v,g/kg CCK inhibited 20 minute cumulative gastric emptying, the larger dose significantly more than the smaller These doses are in the lower end of the range reported to inhibit gastric emptying in rats by Wemgarten and his colleagues (3,14). Therefore, our technique appears to provide a valid measure of intrameal gastric emptying. Neither 400 nor 800 txg/kg PG altered gastric emptying of milk after lntragastnc test infusions The smaller PG dose inhibited milk intake under similar experimental conditions in the same rats. This indicates that PG did not inhibit feeding under these con&tlons by altenng intrameal gastnc emptying, a factor which has been hypothesized to signal satiety (4,11). Since PG's satiety effect is maximal in the first 20 minutes of a meal, a gastric emptying effect after 20 minutes would not contribute to PG's inhibition of food intake. Our measure of stomach contents is not corrected for gastnc secretion, and so may underestimate gastric emptying. However, as PG appears to inhibit gastric secretion (2,9), it seems unhkely that this problem contributed to our fatlure to detect a reduction of gastric emptying with PG. In summary, we found no effect of exogenous PG on gastric emptying in a situation m which PG ellotS satiety. This result ~mplies that PG does not elicit satiety by altering gastric emptying. ACKNOWLEDGEMENT
This work was supported by grant AM-32448 to N Geary
REFERENCES
Antra, J ; Gibbs, J , Srmth, G P Cholecystokanm interacts with pregastrlc food stimulation to eliot satiety m the rat. Physlol Behav 20 67-70. 1978
2 Chrlstlansen, J PancreaUc glucagon and gastric acid secretion Scand J Gastroenterol 15 257-258, 1980 3 Conover, K L , Collins, S M , Welngarten, H P Effect of
GLUCAGON AND GASTRIC EMPTYING
4. 5. 6.
7 8
9
cholecystokmln, bombesm, and gastrin on gastric emptying and satiety in the rat Soc. Neuroscl. Abstr. 13'245.19; 1987. Deutsch, J. A. Dietary control and the stomach. Prog Neurobiol 20:313-332, 1983 Diament, B., Picazo, J. Spasmolytic action and chmcal use of glucagon In" LeFebvre, P J., ed. Handbook of expenmental pharmacology, vol 66/11. New York Spnnger-Verlag, 1983:611--643. Geary, N , Langhans, W , Scharrer, E Metabolic concomitants of glucagon-induced suppression of feeding m the rat Am J Physiol. 241 E330-E335, 1981 Geary, N.; Smith, G. P. Pancreatic glucagon and postprandial satiety in the rat. Physiol. Behav. 28(2)'313-322, 1982. Hinton, V , Esguerra, M , Farhoody, N., Granger, J , Geary, N. Epmephnne inhibits feeding nonspecifically in the rat. Physiol. Behav 40(1) 109-115, 1987 Loud, F B., Hoist, J J , Rehfeld, J F , Chnstiansen, J Inhibition of gasmc acid secretion in man by glucagon during euglycemIa, hyper-
1261
glycenua, and hypoglycenua Dig Dis. Sci. 33(5):530-534; 1988. 10 Martin, J. R., Novin, D. Decreased feeding m rats following hepatic-portal infusion of glucagon. Physlol Behav 19:461--466; 1977. 11 Moran, T H , McHugh, P R. Cholecystokmm suppresses food intake by inhibiting gastric emptying. Am. J. Physlol 242 R491-R497; 1982. 12 MuShy, S. N. S., Ganiban, G Effect of the secretln family of pepudes on gastric emptymg and small intestinal transit in rats Peptides 9(3).583-588, 1988 13 Rltter, S , Weatherford, S. C., Stone, S. L. Glucagon-lnduced inhthltion of feeding is impaired by hepatic portal alloxan injection. Am J Physiol. 250 R683-R690, 1986. 14. Wemgarten, H. P., Conover, K L , Collins, S M. Cholecystoklnln and gastric emptying m the rat. Proc. Int. Conf Physiol Food Fluid Intake 9 87, 1986.
0031-9384/89 $3,00 + 00
BRIEF COMMUNICATION
Pancreatic Glucagon Does Not Alter Intrameal Gastric Emptying of Milk in the Rat ZSOLT STOCKINGER AND NORI GEARY 1
Psychology Department, 402a Schermerhorn Hall Columbia University, New York, N Y 10027
R e c e i v e d 3 O c t o b e r 1988
STOCKINGER, Z. AND N GEARY. Pancreanc glucagon does not alter mtrameal gastric emptymg of milk m the rat PHYSIOL BEHAV 45(6) 1259-1261, 1989.--The hypothesis that pancreatic glucagon (laG) inhibits feeding by altering mtrameal gastric emptying was tested m nondepnved rats at midday, conditmns under which exogenous PG is thought to ehclt satiety Rats received intraperltoneal mjectmns of PG (400 or 800 p.g/kg), cholecystokmm (CCK, 0 5 or 1.0 I~g/kg) or saline and were given bolus mtragastnc mfusmns of 10 ml evaporated milk through chromc gasmc cannulae. PG did not affect volumes of stomach contents recovered 20 minutes postlnjection. In contrast, CCK mhthlted gasmc emptying m a dose-related manner. When rmlk was offered to the same rats to dnnk, 400 p.g/kg PG slgmficantly reduced meal size. These findings suggest that PG's satiety effect is not caused by as effect on stomach emptying. Glucagon
Cholecystoklnm
Food retake
Satiety
PANCREATIC GLUCAGON (PG) reduces meal size and duration in rats (6, 8, 13). The behavioral specificity of this effect suggests that PG may signal satiety (7, 8, 10, 13). PG also has several spasmolytic actions on enteric muscles, and under some conditmns alters gastric emptying (5,12). To test whether altered gastric emptying causes PG's satiety effect, we compared intrameal gastric emptying and meal size under conditions in which intraperitoneal injection of PG is known to inhibit feeding, specifically, in nondeprived rats given milk in the middle of the bright phase of the lighting cycle (7,8). An emptying effect must occur during the meal to cause a reduction in meal size or duration. Under these conditions, control meals last 10-20 minutes (7). Therefore, stomach emptying was measured at 20 minutes. The effect of cholecystokinin (CCK), which potently inhibits gastric emptying m rats (3, 11, 14), was tested to assess the sensitivity of the method used to measure gastric emptying.
maintained on pelleted rat chow (Purina 5001) and tap water under a 12:12 light-dark cycle (lights on at 0200). Each rat was equipped with a chronic gastric cannula, using the procedure of Antin et al. (1).
Procedure Stomach emptying. Following recovery from surgery, the rats were adapted to gastric lavage. Food was removed at 0900, the cannula opened, and the stomach flushed repeatedly with 10 ml of 0.9% NaC1 at about 30°C until the drainage was clear. A drainage tube, with a 1.0 ml dead space filled with evaporated milk and with the end clamped to prevent leakage, was then attached to the cannula. Each rat was then intraperitoneally injected with 1 ml/kg saline and placed into a hanging clear plastic cage, with the drainage tube passed through a slot in the floor. Twenty minutes later, rats were given evaporated milk for one hour. On consecutive experimental days, food was removed at 0900, the stomach flushed, and the drainage tube attached as described above. Each rat was then injected with 1 ml/kg of either 400 or 800 Ixg/ml PG (glucagon hydrochloride, Eli Lilly, Indianapolis, IN), or 0.5 or 1.0 p.g/ml CCK (synthetic CCK octapeptide, Squibb,
METHOD
Subjects Eleven male Sprague-Dawley rats weighing 458-816 g were
~Requests for repnnts should be addressed to N. Geary.
1259
1260
STOCKINGER AND GEARY
50
25 E
v
40
T
K
15 J¢
E
"o
20
u
]30
m
E
10 5
+
o e-
10
T
20
20 30 Time (mm)
40
o t_
FIG 2 Inhthltory effect of PG on milk retake Data are cumulative milk retakes after injection of saline (light bars) or 400 /a,g/kg PG (dark bars), mean-+s e *p<0 05, **p<0 005 vs saline
o.
10
0
......
SAL
CCK
PG
FIG 1 Effects of PG and CCK on gasmc emptying Data are volumes emptied from stomach 20 minutes after lntragasmc infusion of 10 ml condensed milk, expressed as a percentage of the milk load, mean_+ s e Unshaded bar, saline rejection, hght shading, CCK rejection (left, 0 5 p,g/kg, right, 1 0 Ixg/kg), dark shading, PG injection (left, 400 Ixg/kg, right, 800 ixg/kg) *p<0.05 vs sahne, **p<0 0005 vs saline, + p < 0 005 vs 0 5 p,g/kg CCK
Princeton, NJ) vs. 0.9% saline according to separate crossover designs, and placed into the test cage. Ten ml of evaporated milk was infused through the drainage tube into the stomach Twenty minutes after injection, the remaining milk was evacuated from the stomach through the drainage tube using a 50 cc syringe, and the volume measured. Cannulae were then closed, and evaporated milk presented for approxtmately one hour to simulate the feeding procedure described below Volume emptied from the stomach was estimated as the 10 ml infusion (plus the 1 ml milk in the drainage tube dead space) minus the volume recovered. Data from 9 rats were analysed using post hoc t-tests after an overall one-way A N O V A revealed a significant effect, F ( 4 , 4 0 ) = 7.67, p < 0 . 0 1 , data from 2 rats whose cannulae leaked or whose gastric contents contained chow were discarded Meal size. Feeding was tested 3 days after the completion of the stomach emptying experiment. At 0900, food was removed and stomachs flushed as above Cannulae were then closed, and rats injected with 1 ml/kg of either 400 txg/ml PG or saline according to a cross-over design. Rats were then placed into their test cages, and evaporated milk was presented in graduated drlnlong cylinders. Intakes were recorded every 10 minutes, for 40 minutes. Data from the 9 rats without missing data points were analysed using two-tailed t-tests. RESULTS
Gastrw Emptying CCK inhibited gastric emptying in a dose-related manner (Fig. 1). Rats injected with saline emptied 38.7 -+ 2.2% (mean -+ s.e. ) of
their milk loads in 20 minutes, versus 29 5 -+ 2 5% after 0.5 ~g/kg CCK, t ( 8 ) = 3 . 7 , p < 0 . 0 0 3 , and 2 0 . 7 -+ 1.6% after 1.0 p~g/kg CCK, t ( 8 ) = 6 7, p < 0 . 0 0 0 1 In contrast, neither 400 ~g/kg PG [37.8 -+ 3.1% emptied, t(8) = 0.36], nor 800 ttg/kg PG [34.6 -+ 3.4% emptwd, t ( 8 ) = 1.5], significantly altered gastric emptying. There was no correlation between body weight and any measure (all p's>0.3).
Meal Stze PG significantly reduced evaporated milk intake (Fig. 2). At 10 minutes, PG inhibited milk intake by 20.0-+ 7 7% (mean-+ s.c.); at 20 minutes, 23.6 -+ 7.8%, at 30 minutes, 20.5 -+ 7.2%, and at 40 minutes, 17.9_+ 9.4%; all p ' s < 0 . 0 5 DISCUSSION
Both 0.5 and 1.0 v,g/kg CCK inhibited 20 minute cumulative gastric emptying, the larger dose significantly more than the smaller These doses are in the lower end of the range reported to inhibit gastric emptying in rats by Wemgarten and his colleagues (3,14). Therefore, our technique appears to provide a valid measure of intrameal gastric emptying. Neither 400 nor 800 txg/kg PG altered gastric emptying of milk after lntragastnc test infusions The smaller PG dose inhibited milk intake under similar experimental conditions in the same rats. This indicates that PG did not inhibit feeding under these con&tlons by altenng intrameal gastnc emptying, a factor which has been hypothesized to signal satiety (4,11). Since PG's satiety effect is maximal in the first 20 minutes of a meal, a gastric emptying effect after 20 minutes would not contribute to PG's inhibition of food intake. Our measure of stomach contents is not corrected for gastnc secretion, and so may underestimate gastric emptying. However, as PG appears to inhibit gastric secretion (2,9), it seems unhkely that this problem contributed to our fatlure to detect a reduction of gastric emptying with PG. In summary, we found no effect of exogenous PG on gastric emptying in a situation m which PG ellotS satiety. This result ~mplies that PG does not elicit satiety by altering gastric emptying. ACKNOWLEDGEMENT
This work was supported by grant AM-32448 to N Geary
REFERENCES
Antra, J ; Gibbs, J , Srmth, G P Cholecystokanm interacts with pregastrlc food stimulation to eliot satiety m the rat. Physlol Behav 20 67-70. 1978
2 Chrlstlansen, J PancreaUc glucagon and gastric acid secretion Scand J Gastroenterol 15 257-258, 1980 3 Conover, K L , Collins, S M , Welngarten, H P Effect of
GLUCAGON AND GASTRIC EMPTYING
4. 5. 6.
7 8
9
cholecystokmln, bombesm, and gastrin on gastric emptying and satiety in the rat Soc. Neuroscl. Abstr. 13'245.19; 1987. Deutsch, J. A. Dietary control and the stomach. Prog Neurobiol 20:313-332, 1983 Diament, B., Picazo, J. Spasmolytic action and chmcal use of glucagon In" LeFebvre, P J., ed. Handbook of expenmental pharmacology, vol 66/11. New York Spnnger-Verlag, 1983:611--643. Geary, N , Langhans, W , Scharrer, E Metabolic concomitants of glucagon-induced suppression of feeding m the rat Am J Physiol. 241 E330-E335, 1981 Geary, N.; Smith, G. P. Pancreatic glucagon and postprandial satiety in the rat. Physiol. Behav. 28(2)'313-322, 1982. Hinton, V , Esguerra, M , Farhoody, N., Granger, J , Geary, N. Epmephnne inhibits feeding nonspecifically in the rat. Physiol. Behav 40(1) 109-115, 1987 Loud, F B., Hoist, J J , Rehfeld, J F , Chnstiansen, J Inhibition of gasmc acid secretion in man by glucagon during euglycemIa, hyper-
1261
glycenua, and hypoglycenua Dig Dis. Sci. 33(5):530-534; 1988. 10 Martin, J. R., Novin, D. Decreased feeding m rats following hepatic-portal infusion of glucagon. Physlol Behav 19:461--466; 1977. 11 Moran, T H , McHugh, P R. Cholecystokmm suppresses food intake by inhibiting gastric emptying. Am. J. Physlol 242 R491-R497; 1982. 12 MuShy, S. N. S., Ganiban, G Effect of the secretln family of pepudes on gastric emptymg and small intestinal transit in rats Peptides 9(3).583-588, 1988 13 Rltter, S , Weatherford, S. C., Stone, S. L. Glucagon-lnduced inhthltion of feeding is impaired by hepatic portal alloxan injection. Am J Physiol. 250 R683-R690, 1986. 14. Wemgarten, H. P., Conover, K L , Collins, S M. Cholecystoklnln and gastric emptying m the rat. Proc. Int. Conf Physiol Food Fluid Intake 9 87, 1986.