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Satiety function of neurons containing a CCK-like substance in the dorsal parabrachial nucleus
Physiology&Behavior,Vol. 48, pp. 865-871. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0031-9384/90 $3.00 + .00
Satiety Function of Neurons Containing a CCK-like Substance in the Dorsal Parabrachial Nucleus AKIKO TAKAKI, KATSUYA NAGAI,I SHUNJI TAKAKI, NOBORU YANAIHARA* AND HACHIRO NAKAGAWA
Division of Protein Metabolism, Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan and *Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka 420, Japan
TAKAKI, A., K. NAGAI, S. TAKAKI, N. YANAH-IARA AND H. NAKAGAWA. Satietyfunction of neurons containing a CCK-like substance in the dorsalparabrachial nucleus. PHYSIOL BEHAV 48(6) 865-871, 1990.--Glutaryl-CCK-8 (Glt-CCK-8, 16-160 pmol) suppressed food intake dose dependently when injected into the ventromedial hypothalamus (VMH) bilaterally, but not when injected unilaterally. In contrast, CCK-8 (160 and 320 pmol) did not suppress food intake when injected into the VMH bilaterally. When injected intraperitoneally, Glt-CCK-8 significantly decreased food intake at a dose of 320 pmol, though not at a dose of 160 pmol, whereas CCK-8 significantly reduced food intake even at a dose of 160 pmol. Pretreatment with proghimide, an antagonist of CCK-8, counteracted the effect on food intake of CCK-8 injected intraperitoneally, but did not influence that of GItCCK-8 injected either into the VMH or intraperitoneally. However, CCK-8 (800 pmol) prevented the satiety action of Glt-CCK-8 when injected into the VMH before the latter. Since a large dose of CCK-8 injected into the VMH was reported to suppress food intake, these findings suggest that, among the receptors for the satiety action of CCK, intracranial receptor has lower affinity for CCK-8 than for Glt-CCK-8 and peripheral receptor has higher affinity for CCK-8 than for Glt-CCK-8. Furthermore, bilateral lesions of the lateral part of the dorsal parabrachial nucleus (LPBD), from which the neurons containing a CCK-8-1ike substance extend fibers to the VMH, enhanced the satiety action of Glt-CCK-8 injected into the VMH. These results support the idea that these neurons which project to the VMH are involved in the satiety action. Cholecystokinin
Satiety action
Central effect
Receptor
PREVIOUSLY, we observed that continuous intracranial infusions of glutaryl-cholecystokinin-8 (GIt-CCK-8) and pyroglutamylCCK-8, derivatives of a synthetic COOH-terminal octapeptide of cholecystokinin (CCK-8), reduced daily food intake under freefeeding conditions, but that similar infusion of CCK-8 did not (16). Neurons in the lateral part of the dorsal parabrachial nucleus (LPBD) in the pons of rat brain are reported to contain a CCK8-like immunoreactive substance and to project to the ventromedial hypothalamus (VMH) (10). Moreover, the VMH is known to be involved in satiety (4). From these findings we speculated that neurons containing the CCK-8-1ike substance in the LPBD might play a role in suppressing food intake through an action on the VMH. To examine this, we tested the effect of LPBD lesions and found that these caused hyperphagia and obesity (17). To clarify the central mechanism of the satiety induced by CCK, in this study we examined the effects of GIt-CCK-8 (1.61600 pmol) and CCK-8 (160-800 pmol) injected centrally (into
Rat
the VMH) or peripherally on the food intake of rats that had been starved for 24 hours and found that at the dose used in this experiment not CCK-8 but Glt-CCK-8 injected into the VMH bilaterally caused a satiety action. Since if the neurons in the LPBD elicit a satiety action with CCK as a mediator acting on the VMH, the satiety action of GIt-CCK-8 injected bilaterally into the VMH might be sensitized by the LPBD lesions. Thus, we examined the effect of the LPBD lesions on the satiety action of Glt-CCK-8 injected into the VMH. A part of this study has appeared in abstract form (18). METHOD
Animals Male Wistar rats, initially weighing 250-300 g, were used. Animals were kept in a room at 2 4 ± I°C and 6 0 ± 10% relative humidity and illuminated for 12 hours a day (0800-2000 h) with
~Requests for reprints should be addressed to Katsuya Nagai, M.D., Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamada-Oka, Suita, Osaka 565, Japan.
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artificial fluorescent light (80 lux). Animals had free access to laboratory chow pellets (type MF, Oriental Yeast Co., Tokyo) and water.
VMH Cannulation and Intra- VMH Injection A pair of 23 gauge stainless steel tubes (inner diameter, 0.33 mm; outer diameter, 0.63 mm; length, 14 mm) were held in parallel by a wire bridge 2 mm from the top of the tubes fixed with solder and used as outer cannulae. The distance between the axes of the two tubes was adjusted to 0.6 mm. The rats were anesthetized with pentobarbital (35 mg/kg). A pair of outer cannulae, which served as guide cannulae, were bilaterally inserted stereotaxically into the dorsal edges of the VMH with the following coordinates: AP, 6.0 mm rostral to the interaural line; L, +-0.3 mm lateral to the median line; V, 8.7 nun below the skull surface, according to the atlas of Paxinos and Watson (20). During the 10-day recovery period after the operation, the animals were handled at random during the light period every day to accustom them to experimental manipulations. On the experimental day, the inner cannulae filled with the experimental solution were inserted into the guide cannulae of unanesthetized rats until the tips of the inner cannulae reached 0.3 mm below the tips of the guide cannulae. The inner cannulae were made of 29 gauge stainless steel tubes (outer diameter 0.3 mm) with stoppers 14.3 mm above their lower tips and were connected to polyethylene tubes (PE-10, Clay Adams, NJ). For bilateral injections into the VMH, 1 Ixl of test solution of vehicle was injected through each of the inner cannulae. For unilateral injections of materials into the VMH, only the right cannula was used. For tests on antagonistic effects, 0.5 ixl of antagonistic agent was injected into the VMH and 15 min later the same volume of test solution was injected into the VMH through a new inner cannula inserted into the outer guide cannulae. Unilateral injection of a test solution took about 2 min and bilateral injection took about 5 min.
Lesions of the Lateral Part of the Dorsal Parabrachial Nucleus (LPBD) Lesions were made in the LPBD of male Wistar strain rats weighing initially 200-230 g. The rats were anesthetized and LPBD lesions were made stereotaxically, using the following coordinates: AP, 0.2 mm posterior to the interaural axis; L, ---2.3 mm lateral to the sagittal suture; V, 7.3 mm below the skull surface. An enamel-coated stainless steel, monopolar electrode of 0.5 mm diameter with no coating 0.5 nun from its tip was inserted into the LPBD bilaterally and an anodal current of 30 IxA was applied for 15 min with the cathode attached to the tail. Control animals were treated in the same way, but no current was applied. The LPBD lesions resulted in severe stress on the day of the operation, and only about 60% of the animals recovered. Moreover, most of the survivors gained weight rapidly during three weeks following recovery. Of the survivors, only animals whose weights exceeded the range of the mean plus twice the standar~t error of the weights of the control animals (N = 8) during 3 ~eeks following recovery, and which showed no observable ~eurological abnormalities except in eating behavior, were included in the experimental group (N = 18). The VMH was cannulated as described above within 4 weeks after making LPBD lesions.
Experimental Procedure Animals were transferred to new cages and food was withdrawn at 1300 h on the day before the experiment and the rats
TAKAKI ET AL.
were starved for 24 h. Then, vehicle was injected into either the VMH (2 p,l) or the peritoneal cavity (20 ~1). The rats were then given 3 pellets of laboratory chow, and their food consumptions during the first and second one-hour period were measured with an accuracy of 0.1 g. The spillage of food in the cage was collected and weighed. When food and its spillage became wet with water and urine, it was dried by a heater and dry weight of them was measured. Water was freely available throughout the experiment. After a recovery period of 5-6 days, a second experiment in which a test substance was injected into the VMH or the peritoneal cavity was carded out by the same protocol as for the first experiment. Antagonistic agents were injected into the VMH or peritoneal cavity in the same way. Each group consisted of 7 or 8 animals, but data on animals in which the positions of the tips of the inner cannulae were incorrect were eliminated.
Histological Examination After the experiment, animals were killed by an overdose of pentobarbital and their brains were perfused and fixed with 10% formalin, embedded in paraffin, and sectioned at 15 ixm, and the sections were stained with cresyl violet. The sections around the tips of the inner cannulae were then examined histologically. Figure 1 shows a photomicrograph of the brain of a rat in which vacant regions indicate the positions of the bilateral cannulae. The rostro-caudal and dorso-ventral positions of the tips of the cannulae were exactly the same in all animals, but lateral displacements were seen in some cases. On the basis of a calculation of the interaxial distance of double cannulae and the outer diameter of the guide cannulae, data for animals in which the cannulae were displaced more than 0.15 mm or had penetrated into the third cerebral ventricle were discarded. The sites of LPBD lesions, as well as the sections around the tips of the carmulae were examined and from the findings the data for 5 control animals and 10 with LPBD lesions were included in the experimental data.
Agents GIt-CCK-8 and CCK-8 were synthesized as described previously (23). They were dissolved in 7% sodium bicarbonate, and the solutions were adjusted to pH 7.4 with 0.1 N HC1 and made isotonic with NaC1 solution. The vehicle solution was prepared in the same way, but without test agents. On unilateral administration of compounds, double strength solutions were used to give the same total dose. Proglumide (sodium salt), supplied by Kaken Pharmaceutical Co., was dissolved in distilled water. For intraperitoneal injection, the solutions were prepared at one-tenth the concentration used for the intracranial injection and 10 times larger volumes were injected.
Data and Statistical Analyses Food was given to fasted animals immediately after administration of the vehicle, and food consumption for the ftrst and second hour was measured. Since total food consumption during the 2-hour period varied for different animals, the food consumption of the first hour and the second hour was expressed as a percentage of the total consumption in 2 hours. The second experiment was done with test agents 5 or 6 days after the f'LrSt experiment. The food consumption of animals when treated with test agents was expressed as a percentage of the total consumption in 2 hours (100%) of animals when treated with vehicle. The reduction in food consumption during the first hour after injection of a test agent was also calculated as a difference between values (%) in
SATIETY ACTION OF CCK NEURONS IN PBD
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FIG. 1. Photomicrograph ( x 25) of a brain section including the empty cavities formed by the cannulae. The brain section was stained with cresyl violet. Bilateral empty cavities correspond to the sites of the tips of cannulae. HI, third ventricle.
this period after injections of vehicle and the test agent. The data obtained are expressed as means -+ standard errors of means. Statistical analyses were performed by Student's t-test and analysis of variance (ANOVA). RESULTS
Effects of Intra-VMH Injections Food intake data after bilateral injection of the vehicle (1 pJ on each side) and 160 pmol of GIt-CCK-8 (80 pmol in 1 pd on each side) into the VMH are shown in Fig. 2A. Food intake in
A
Bilateral VMH
[] []
Vehicle (5) GIt-CCK-8(160pmol/2/d)
[] []
CCK.8(160pmol/2pl ) (5) GIt.CCK-8(160pmol/1/d ) (5) B Bilateral VMH
(5)
C Unilateral VMH
(%l 100,
| ~o
| lh
2h
T
iol lh 2h
T
lh
2h
T
FIG. 2. Effects of bilateral VMH injections of GIt-CCK-8 (A) and CCK8 (B), and unilateral VMH injection of GIt-CCK-8 (C) on food consumption during the first (1 h) and second (2 h) hours of feeding (T= total 2-h consumption). Food consumption is represented as a percentage of the total 2-h food consumption after vehicle injection (100%) in each rat. The mean _+standard error of the mean for food consumption during the 2-h period after vehicle injection was 7.0 +-0.6 g. Numbers in parentheses are numbers of animals used. Significance was analyzed by Student's t-test, and a significant difference from the value for the vehicle-treated group is shown by asterisks (**p<0.01).
control rats treated with vehicle was 5.3---0.7 g in the fhst hour but only 2.0-+0.5 g in the second hour. Bilateral injection of GIt-CCK-8 significantly (p<0.01) decreased food intake below that of control rats during the first hour but not during the second hour of feeding. Thus, GIt-CCK-8 suppressed the total food intake during 2 hours after 24 hours of fast (Fig. 2A). In contrast to GIt-CCK-8, CCK-8 did not suppress food intake when injected 160 pmol (Fig. 2B). We also examined the effect of unilateral injection of 160 pmol of GIt-CCK-8 into the VMH. As seen in Fig. 2C, it did not suppress food intake. Next, we examined the effects of bilateral injections of different doses of GIt-CCK-8 into the VMH. Glt-CCK-8 suppressed food intake during the first hour dose dependently at up to 1600 pmol (Fig. 3A). On the other hand, CCK-8 had no significant effect at either 160 or 320 pmol (Fig. 3A).
Effects of Intraperitoneal Injections Glt-CCK-8 (160 pmol) did not inhibit food intake during the first hour after intraperitoneal injection (Fig. 4A). In contrast to intracerebral injection of CCK-8, its intraperitoneal injection significantly (p<0.01) inhibited food intake in the first hour, thus during the total two-hour period (Fig. 4B). As seen in Fig. 2B, the decrease in food consumption during the first hour after the GIt-CCK-8 injection was low at doses of up to 160 pmol, but increased at a dose of 320 pmol. On the other hand, the decrease in food consumption after CCK-8 injection was high even at a dose of 160 pmol (Fig. 4B).
Effects of Proglumide and CCK-8 on Satiety Actions of CCK-8 and GIt-CCK-8 To elucidate the mechanism of the satiety actions of GIt-CCK8 and CCK-8 injected into the VMH bilaterally and into the peritoneal cavity, respectively, we examined the effects of pretreatment with proglumide on their actions. Bilateral injection of proglumide (300 pmol) into the VMH prior to injecting GIt-CCK-8 had no significant effect on the action of the Glt-CCK-8 (Fig. 5A). In
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_"
Intra-VMH
_- GIt-CCK-8 CCK-8
60.
(%) 100.
40-
0
40
1:6 4:8
'o
B =
1"6 lib 160320 1600 Dose (pmol) 0
Intraperitoneal
I///t
60
!40
~ .~ 20
:,~o 1:6 4:8 01=
1"6 8'0 1603'20 1600 Dose (pmol)
FIG. 3. Dose-dependent effects of GIt-CCK-8 and CCK-8 on food consumption. Glt-CCK-8 and CCK-8 were injected into the VMH bilaterally (A) or into the intraperitoneal cavity (B). Decrease in food consumption during the fin'st hour after injection of agents is indicated as a difference between values (%) in this period after injections of vehicle and a test agent. Each dosage group (1.6, 4.8, 16, 80, 160, 320 and 1600 pmol) consisted of 5 rats. Data are expressed as means--standard errors of means. The EDso value is about 40 pmol. A statistically significant difference was observed between groups (1.6-160 pmol) of the decrease in food consumption after VMH injection of GIt-CCK-8 and those after intraperitoneal injection of Glt-CCK-8 by ANOVA, p<0.05, F=6.74, however, it was not between groups (160 and 320 pmol) of the decrease after intraperitoneal injection of Glt-CCK-8 and CCK-8, F= 1.32.
a preliminary experiment using 2 rats, bilateral injection of 800 pmol of CCK-8 into the VMH did not inhibit food intake (the changes in food consumption during the first hour after injection [ ] Vehicle [ ] GIt-CCK-8(160pmol/20/d )
A Intrap~ritoneel
B intraperitoneral
(%)
100"
o 50,
|
i lh
///
FIG. 5. Effects of proglumide and a high dose of CCK-8 on food consumption during the f'lrst hour after injections of GIt-CCK-8 and CCK-8 injections into the VMH bilaterally (A) and into the peritoneal cavity (B1 and B-2). Each group consisted of 5 rats. *p<0.05, **p<0.01 and ***p<0.001 indicate significant differences by the t-test. Veh, vehicle; Pgl., proglumide. The explanations under the x-axis are those of pretreatments, and those above the graph are those of treatments. Other explanations are the same as for Fig. 2.
of CCK-8 were - 4 % and +6%). Then, we had a question whether CCK-8 has an affinity, even a low affinity, for the receptor responsible for the satiety action of GIt-CCK-8 or not. Thus, we examined the effect of bilateral preinjection of 800 pmol of CCK-8 into the VMH on the satiety action of GIt-CCK-8. As shown in Fig. 5A, treatment with CCK-8 before Glt-CCK-8 completely eliminated the suppressive action of GIt-CCK-8 on food intake in the first hour after its administration. We also examined the effect of pretreatment with proglumide (1500 pmol) on the satiety actions of intraperitoneal injections of CCK-8 and GIt-CCK-8. As shown in Fig. 5B, injection of proglumide into the peritoneal cavity before CCK-8 (320 pmol) completely abolished the satiety action of the latter. In contrast, pretreatment with proglumide did not affect the satiety action of Glt-CCK-8 (320 pmol) injected intraperitoneally (Fig. 5B).
Effect of Bilateral Lesions of the LPBD on the Satiety Action of Glt-CCK-8
[ ] CCK-8(160pmol/20/~1 )
i
A InIrI-VMH
[ ] Vehicle [ ] G|t-CCK.8(80pmol) CCK.8 ( 320pmol) [ ] GIt-CCK-8(320pmol) B-1 intrapefitonelll O-2 llrl~llperitOrleal l~ee
Figures 6 and 7 show the change in body weight after LPBD lesions and the common area of lesions in the brain, respectively. As reported previously (17), the body weight of rats with bilateral lesions of the LPBD increased significantly. This finding is confn'med by the results in Fig. 6. Figure 8 shows the effect of LPBD lesions on the satiety action of Glt-CCK-8. As expected, bilateral lesions of the LPBD sensitized the satiety action; GltCCK-8 at a dose of 16 pmol did not affect the food intake in the first hour of sham-operated rats, but markedly suppressed the food intake of rats with LPBD lesions. DISCUSSION
2h
lh
2h
FIG. 4. Effects of intraperitoneat injection of Glt-CCK-8 (160 pmol) (A) and CCK-8 (160 pmol) (]3) on food consumption of rats fasted for 24 hours. Results are expressed as described in the legend to Fig. 2. **(p<0.01) indicates a significant difference between values for the groups Ireated with vehicle and CCK by the t-test. Other explanations are as for Fig. 2.
In the present study, the following findings were obtained in rats fasted for 24 hours (Table 1): 1) When injected into the VMH bilaterally, GIt-CCK-8 (160 pmol) suppressed food intake, but CCK-8 (160 pmol) did not. However, unilateral injection of GItCCK-8 into the VMH did not suppress food intake (Fig. 2). 2) The satiety action of GIt-CCK-8 on the VMH was dose dependent (Fig. 3A). 3) In contrast, CCK-8 (160 pmol) elicited satiation
SATIETY ACTION OF CCK NEURONS IN PBD
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400
7
1oo]
[]
sham-lesioned (5)
[]
LPeO-lesionea(a)
350
300
0 ned (10)
4.8
16
G I t - C C K - 8 (pmol)
FIG. 8. Effects of bilateral lesions of the LPBD on food consumption during the fLrSthour after injection of GIt-CCK-8 into the VMH bilaterally in rats fasted for 24 h. Other explanations are the same as for Fig. 2.
250
~ t's
200
0
Days after operation
FIG. 6. Body weight change after bilateral lesions of the LPBD. Numbers of animals are indicated in parentheses.
when injected intraperitoneally, whereas Glt-CCK-8 (160 pmol) did not (Figs. 3 and 4). 4) Intraperitoneal injection of Glt-CCK-8 had a satiety action only when injected at a high dose (320 pmol) (Fig. 3B). 5) Pretreatment with proglumide, a CCK-antagonist, inhibited the satiety action of CCK-8 injected intraperitoneally, but did not inhibit that of Glt-CCK-8 injected either into the VMH or the peritoneal cavity (Fig. 5). 6) Pretreatment of the VMH with a large dose of CCK-8 (800 pmol) did not have any effect on food intake, but blocked the satiety action of GIt-CCK-8 injected into the VMH (Fig. 5A). 7) Bilateral lesions of the LPBD sensitized the VMH to the satiety action of GIt-CCK-8 injected into the VMH (Fig. 8). Until recently, it was generally accepted that in rats CCK had a satiety action only when it was injected peripherally, since in-
Interaural 0.2 rnm
-0.3 m m
-0.8mm
traperitoneal injection of 1 p.g/kg of CCK-8 significantly inhibited food intake, but its intracerebroventricular injection inhibited food intake only when it was injected at a high dose (12 ttg/kg) (19). However, our previous study showed that derivatives of CCK-8 (GltoCCK-8 and pGlu-CCK-8, 0.8 pmol/h) reduced food intake in free-feeding rats when they were injected intracranially, whereas CCK-8 itself did not (16). Furthermore, the present study showed that GIt-CCK-8 (160 pmol) had a satiety action when injected into the VMH bilaterally, but not when injected into the peritoneal cavity, and that proglumide, a CCK antagonist, did not block the satiety effect of Glt-CCK-8 when it was injected either centrally (VMH) or peripherally (peritoneal cavity), although it blocked the peripheral satiety action of CCK-8, and that a high dose of CCK-8 (800 pmol) inhibited the satiety action of GItCCK-8. In Fig. 3B, there are no statistically significant differences between groups of decrease in food consumption after intraperitoneal injections of Glt-CCK-8 and CCK-8 by ANOVA, F = 1.32. However, in Fig. 4, when these effects were compared with those of vehicle CCK-8 but not GIt-CCK-8 showed a significant (p<0.01) decrease in food intake. Since drug effects usually are examined compared with those of vehicles, the results shown in Fig. 4 are meaningful and the decreases of food consumption seen in Fig. 3B show the tendency of the effect. Moreover, in this experiment, effects of vehicle and CCK were obtained in the same animals in Fig. 4, but the decrease in food consumption due to Glt-CCK-8 and CCK-8 were examined in different rats (Fig. 3). Thus, it is clear that CCK-8 had a satiety effect from 160 pmol, but GIt-CCK-8 had it from 320 pmol. It was reported that injection of a large dose (300 nmol) of proglumide into the lateral cerebral ventricle inhibited a satiety action of peripheral injection of CCK-8 (12). Thus, we examined the effect of VMH injection of higher doses of proglumide. However, intra-VMH injection of proglumide (over 600 pmol) itself elicited
TABLE I SUMMARYOF THE RESULTSIN THIS STUDY GIt-CCK-8
~ D FIG. 7. Areas of common lesions in 10 rats with bilateral lesions of the LPBD are shown in black. The lower figure shows the site of the LPBD.
Suppressive effect of food intake Blocking effect of proglumide Blocking effect of CCK-8 Sensitizing effect of LPBD-lesions
VMH ip VMH ip VMH VMH
+ +, marked; +, moderate; - , none.
++ + --+ ++
CCK-8
++
870
TAKAKI ET AL.
a satiety action (data is not shown here). Thus, we could not examine the central effect of higher doses of proglumide than 300 pmol. These results suggest that in the central and peripheral site there are at least 2 types of CCK receptor in rats and that GltCCK-8 has high affinity for the central one and low affinity for the peripheral one, and CCK-8 has high affinity for the peripheral one and low affinity for the central one. Consistent with the latter suggestion, it was reported that in rats 1) CCK receptors in the brain and peripheral tissue such as the pancreas are different (11), 2) a higher dose (2.6 nmol/rat) of CCK-8 injection into the later cerebral ventricle causes a satiety action (14). In this connection, it was reported that bilateral injection of CCK-8 (40 pmol) into the VMH suppressed the ingestion of sweet noncaloric drink (9). Moreover, recently two kinds of nonpeptidal CCK antagonists were synthesized (3, 5, 7, 13), and one (MK-329) was found to have a high affinity for a peripheral receptor, whereas the other (L365,260) had high affinity for the central CCK receptor. There are also reports that in the brain the two types of CCK receptors could be identified (15), and that in studies using MK-329, a peripheral type of CCK receptor was located in areas such as the area postrema, the nucleus tractus solitarius and the interpeduncular nucleus (8). Peripheral administrations of these antagonists were shown to increase food intake of partially satiated rats and postpone the onset of satiety, and L365,260 was found to be 100 times more potent than MK-329, which has high affinity for the peripheral CCK receptor (3, 5-7, 13). These findings suggest that the brain type CCK receptor is involved in the satiety action of endogenous CCK. These results also support the above suggestion that GIt-CCK-8 and CCK-8 acted on different receptors, the central and peripheral types of CCK receptor, respectively. Previously we found that intracranial infusions of Glt-CCK-8 and pGlu-CCK-8 had satiety actions in rats fed ad lib (16), and that bilateral lesions of the LPBD induced hyperphagia and obesity (17), probably due to elimination of CCK-8-1ike immunoreactive terminals in the VMH. These findings and a report that neurons containing a CCK-8-1ike immunoreactive substance in the LPBD project fibers to the ipsilateral VMH (10), together
with the present findings suggest that release of the CCK-8-1ike substance in the VMH from the terminals of the neurons from the LPBD is involved in satiety. In this connection, it is noteworthy that bilateral injection of caerulein (0.1 ixg/kg), a decapeptide with cholecytokinin activity, into the VMH caused short-term suppression of feeding in the dark period, and that bilateral lesions of the VMH eliminated its action (22). Moreover, high densities of CCK receptors, which might be the brain type (6, 8, 15) were found in the VMH (1), and CCK was shown to increase the activities of neurons in the VMH (21). These findings also support the satiety function of neurons containing CCK-8-1ike substance in the LPBD that project fibers to the VMH. In accordance bilateral lesions of the LPBD enhanced the satiety effect of GItCCK-8 (Fig. 8) and a large dose of CCK-8 blocked the satiety action of Glt-CCK-8 (Fig. 5). In this connection, if CCK-8 has low affinity for central receptor for CCK, bilateral lesions of the LPBD might sensitize the action of CCK-8. Thus, it must be examined whether CCK-8 (1.6-800 pmol) has a satiety action in rats with bilateral lesions of the LPBD in future. The brain type of CCK receptor was also shown to be present in the paraventricular nucleus (PVN) of the hypothalamus (6). Thus, the problems of whether neurons containing CCK project fibers to the PVN, and whether these elicit a satiety action also require examination. In this study done in the middle of the light period, about 7090% of the food intake during the first 2 hours after fasting for 24 hours was consumed during the first hour. After intra-VMH injection of Glt-CCK-8 marked reduction of food intake was observed during this first hour, and there was no compensatory increase in food intake during the second hour. Similar phenomena were observed previously (2). These findings show that GltCCK-8 injection still had some effect during the second hour, or that in the second hour the phase of the ultradian rhythm of appetite of these rats did not permit further eating. These possibilities must be examined. In conclusion these findings support the idea that neurons containing a CCK-8-1ike substance in the LPBD suppress food intake through the effect of this substance on the VMH.
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13. 14.
15.
radiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective nonpeptide CCK antagonists. J. Neurosci. 7:2967-2976; 1987. Hughes, A. M.; Everitt, B. J.; Herbert, J. Selective effects of 13-endorphin infused into the hypothaiamus, preoptic area and bed nucleus of the stria terminalis on the sexual and ingestive behaviour of male rats. Neuroscience 23:1063-1073; 1987. Inagaki, S.; Shiotani, Y.; Yamano, M.; Shiosaka, S.; Takagai, H.; Tateishi, K.; Hashimura, E.; Hamaoka, T.; Tohyama, M. Distribution, origin and fine structure of cholecystokinin-8-1ike immunoreactive terminal in the nucleus ventromedialis hypothalami of the rat. J. Neurosci. 4:1289-1299; 1984. Innis, R. B.; Snyder, S. H. Distinct cholecystokinin receptors in brain and pancreas. Proc. Natl. Acad. Sci. USA 77:6917-6921; 1980. Linden, A.; Uvn~is, K.; Forsberg, G.; Bednar, I.; S6dersten, P. Plasma concentrations of cholecystokinin octapeptide and food intake in male rats treated with cholecystokinin octapeptide. J. Endocrinol. 121:59-65; 1989. Lotti, V. J.; Chang, R. S. L. A new potent and selective nonpeptide gastrin antagonist and brain cholecystokinin receptor (CCK-8) ligand: L-365,260. Eur. J. Pharmacol. 162:273-280; 1989. Makovec, F.; Bani, M.; Chiste, R.; Revel, L.; Rovati, L. C.; Setnikar, I. Different peripheral and central antagonistic activity of new glutaramic acid derivatives on satiety induced by cholecystokinin in rats. Regul. Peptides 16:281-290; 1986. Moran, T. H.; Robinson, P. H.; Goldrich, M. S.; McHugh, P. R. Two brain cholecyctokinin receptors: Implications for behavior ac-
SATIETY ACTION OF CCK NEURONS IN PBD
tions. Brain Res. 362:175-179; 1986. 16. Mori, T.; Nagai, K.; Nakagawa, H.; Yanaihara, N. Intracranial infusion of CCK-8 derivatives suppresses food intake in rats. Am. J. Physiol. 251:R718-R723; 1986. 17. Nagai, K.; Ino, H.; Nakagawa, H.; Yamano, M.; Tohyama, M.; Shiosaka, S.; Shiotani, Y.; Inagaki, S.; Kitoh, S. Lesions of the lateral part of dorsal parabrachial nucleus caused hyperphagia and obesity. J. Clin. Biochem. Nutr. 3:103-112; 1987. 18. Nagal, K.; Ino, H.; Takagi, A.; Inagaki, S.; Tnhyama, M.; Yanaihara, N.; Nakagawa, H. Satiety function of neurons containing CCK-like substance in the dorsal parabrachial nucleus. Appetite 12:226; 1989. 19. Nemeroff, C. B.; Osbehr, A. J., HI; Bissette, G.; Jahnke, G.; Lipton, A.; Prange, A. J. Cholecystokinin inhibits tail pinch-induced
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eating in rats. Science 200:793-794; 1978. 20. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Sydney: Academic Press; 1982. 21. Shiraishi, T.; Simpson, A. D. Various neuropeptide effects on ventromedial hypothalamic neurons. J. Physiol. Soc. Jpn. 44:381; 1982. 22. Stem, J. J.; Cudillo, C. A.; Kruper, J. Ventromedial hypothalamus and short-term feeding suppression by caerulein in male rats. J. Comp. Physiol. Psychol. 90:484--490; 1976. 23. Yanaihara, N.; Sugiura, N.; Kishimoto, K.; Naruse, T.; Yanaihara, C.; Solomon, T. Synthetic and structure-function study on C-terminal segments of CCK and gastfin. In: Ragnarsson, U., ed. Peptides. Stockholm: Almqvist and Wiksell; 1984:373-378.
0031-9384/90 $3.00 + .00
Satiety Function of Neurons Containing a CCK-like Substance in the Dorsal Parabrachial Nucleus AKIKO TAKAKI, KATSUYA NAGAI,I SHUNJI TAKAKI, NOBORU YANAIHARA* AND HACHIRO NAKAGAWA
Division of Protein Metabolism, Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan and *Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka 420, Japan
TAKAKI, A., K. NAGAI, S. TAKAKI, N. YANAH-IARA AND H. NAKAGAWA. Satietyfunction of neurons containing a CCK-like substance in the dorsalparabrachial nucleus. PHYSIOL BEHAV 48(6) 865-871, 1990.--Glutaryl-CCK-8 (Glt-CCK-8, 16-160 pmol) suppressed food intake dose dependently when injected into the ventromedial hypothalamus (VMH) bilaterally, but not when injected unilaterally. In contrast, CCK-8 (160 and 320 pmol) did not suppress food intake when injected into the VMH bilaterally. When injected intraperitoneally, Glt-CCK-8 significantly decreased food intake at a dose of 320 pmol, though not at a dose of 160 pmol, whereas CCK-8 significantly reduced food intake even at a dose of 160 pmol. Pretreatment with proghimide, an antagonist of CCK-8, counteracted the effect on food intake of CCK-8 injected intraperitoneally, but did not influence that of GItCCK-8 injected either into the VMH or intraperitoneally. However, CCK-8 (800 pmol) prevented the satiety action of Glt-CCK-8 when injected into the VMH before the latter. Since a large dose of CCK-8 injected into the VMH was reported to suppress food intake, these findings suggest that, among the receptors for the satiety action of CCK, intracranial receptor has lower affinity for CCK-8 than for Glt-CCK-8 and peripheral receptor has higher affinity for CCK-8 than for Glt-CCK-8. Furthermore, bilateral lesions of the lateral part of the dorsal parabrachial nucleus (LPBD), from which the neurons containing a CCK-8-1ike substance extend fibers to the VMH, enhanced the satiety action of Glt-CCK-8 injected into the VMH. These results support the idea that these neurons which project to the VMH are involved in the satiety action. Cholecystokinin
Satiety action
Central effect
Receptor
PREVIOUSLY, we observed that continuous intracranial infusions of glutaryl-cholecystokinin-8 (GIt-CCK-8) and pyroglutamylCCK-8, derivatives of a synthetic COOH-terminal octapeptide of cholecystokinin (CCK-8), reduced daily food intake under freefeeding conditions, but that similar infusion of CCK-8 did not (16). Neurons in the lateral part of the dorsal parabrachial nucleus (LPBD) in the pons of rat brain are reported to contain a CCK8-like immunoreactive substance and to project to the ventromedial hypothalamus (VMH) (10). Moreover, the VMH is known to be involved in satiety (4). From these findings we speculated that neurons containing the CCK-8-1ike substance in the LPBD might play a role in suppressing food intake through an action on the VMH. To examine this, we tested the effect of LPBD lesions and found that these caused hyperphagia and obesity (17). To clarify the central mechanism of the satiety induced by CCK, in this study we examined the effects of GIt-CCK-8 (1.61600 pmol) and CCK-8 (160-800 pmol) injected centrally (into
Rat
the VMH) or peripherally on the food intake of rats that had been starved for 24 hours and found that at the dose used in this experiment not CCK-8 but Glt-CCK-8 injected into the VMH bilaterally caused a satiety action. Since if the neurons in the LPBD elicit a satiety action with CCK as a mediator acting on the VMH, the satiety action of GIt-CCK-8 injected bilaterally into the VMH might be sensitized by the LPBD lesions. Thus, we examined the effect of the LPBD lesions on the satiety action of Glt-CCK-8 injected into the VMH. A part of this study has appeared in abstract form (18). METHOD
Animals Male Wistar rats, initially weighing 250-300 g, were used. Animals were kept in a room at 2 4 ± I°C and 6 0 ± 10% relative humidity and illuminated for 12 hours a day (0800-2000 h) with
~Requests for reprints should be addressed to Katsuya Nagai, M.D., Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamada-Oka, Suita, Osaka 565, Japan.
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artificial fluorescent light (80 lux). Animals had free access to laboratory chow pellets (type MF, Oriental Yeast Co., Tokyo) and water.
VMH Cannulation and Intra- VMH Injection A pair of 23 gauge stainless steel tubes (inner diameter, 0.33 mm; outer diameter, 0.63 mm; length, 14 mm) were held in parallel by a wire bridge 2 mm from the top of the tubes fixed with solder and used as outer cannulae. The distance between the axes of the two tubes was adjusted to 0.6 mm. The rats were anesthetized with pentobarbital (35 mg/kg). A pair of outer cannulae, which served as guide cannulae, were bilaterally inserted stereotaxically into the dorsal edges of the VMH with the following coordinates: AP, 6.0 mm rostral to the interaural line; L, +-0.3 mm lateral to the median line; V, 8.7 nun below the skull surface, according to the atlas of Paxinos and Watson (20). During the 10-day recovery period after the operation, the animals were handled at random during the light period every day to accustom them to experimental manipulations. On the experimental day, the inner cannulae filled with the experimental solution were inserted into the guide cannulae of unanesthetized rats until the tips of the inner cannulae reached 0.3 mm below the tips of the guide cannulae. The inner cannulae were made of 29 gauge stainless steel tubes (outer diameter 0.3 mm) with stoppers 14.3 mm above their lower tips and were connected to polyethylene tubes (PE-10, Clay Adams, NJ). For bilateral injections into the VMH, 1 Ixl of test solution of vehicle was injected through each of the inner cannulae. For unilateral injections of materials into the VMH, only the right cannula was used. For tests on antagonistic effects, 0.5 ixl of antagonistic agent was injected into the VMH and 15 min later the same volume of test solution was injected into the VMH through a new inner cannula inserted into the outer guide cannulae. Unilateral injection of a test solution took about 2 min and bilateral injection took about 5 min.
Lesions of the Lateral Part of the Dorsal Parabrachial Nucleus (LPBD) Lesions were made in the LPBD of male Wistar strain rats weighing initially 200-230 g. The rats were anesthetized and LPBD lesions were made stereotaxically, using the following coordinates: AP, 0.2 mm posterior to the interaural axis; L, ---2.3 mm lateral to the sagittal suture; V, 7.3 mm below the skull surface. An enamel-coated stainless steel, monopolar electrode of 0.5 mm diameter with no coating 0.5 nun from its tip was inserted into the LPBD bilaterally and an anodal current of 30 IxA was applied for 15 min with the cathode attached to the tail. Control animals were treated in the same way, but no current was applied. The LPBD lesions resulted in severe stress on the day of the operation, and only about 60% of the animals recovered. Moreover, most of the survivors gained weight rapidly during three weeks following recovery. Of the survivors, only animals whose weights exceeded the range of the mean plus twice the standar~t error of the weights of the control animals (N = 8) during 3 ~eeks following recovery, and which showed no observable ~eurological abnormalities except in eating behavior, were included in the experimental group (N = 18). The VMH was cannulated as described above within 4 weeks after making LPBD lesions.
Experimental Procedure Animals were transferred to new cages and food was withdrawn at 1300 h on the day before the experiment and the rats
TAKAKI ET AL.
were starved for 24 h. Then, vehicle was injected into either the VMH (2 p,l) or the peritoneal cavity (20 ~1). The rats were then given 3 pellets of laboratory chow, and their food consumptions during the first and second one-hour period were measured with an accuracy of 0.1 g. The spillage of food in the cage was collected and weighed. When food and its spillage became wet with water and urine, it was dried by a heater and dry weight of them was measured. Water was freely available throughout the experiment. After a recovery period of 5-6 days, a second experiment in which a test substance was injected into the VMH or the peritoneal cavity was carded out by the same protocol as for the first experiment. Antagonistic agents were injected into the VMH or peritoneal cavity in the same way. Each group consisted of 7 or 8 animals, but data on animals in which the positions of the tips of the inner cannulae were incorrect were eliminated.
Histological Examination After the experiment, animals were killed by an overdose of pentobarbital and their brains were perfused and fixed with 10% formalin, embedded in paraffin, and sectioned at 15 ixm, and the sections were stained with cresyl violet. The sections around the tips of the inner cannulae were then examined histologically. Figure 1 shows a photomicrograph of the brain of a rat in which vacant regions indicate the positions of the bilateral cannulae. The rostro-caudal and dorso-ventral positions of the tips of the cannulae were exactly the same in all animals, but lateral displacements were seen in some cases. On the basis of a calculation of the interaxial distance of double cannulae and the outer diameter of the guide cannulae, data for animals in which the cannulae were displaced more than 0.15 mm or had penetrated into the third cerebral ventricle were discarded. The sites of LPBD lesions, as well as the sections around the tips of the carmulae were examined and from the findings the data for 5 control animals and 10 with LPBD lesions were included in the experimental data.
Agents GIt-CCK-8 and CCK-8 were synthesized as described previously (23). They were dissolved in 7% sodium bicarbonate, and the solutions were adjusted to pH 7.4 with 0.1 N HC1 and made isotonic with NaC1 solution. The vehicle solution was prepared in the same way, but without test agents. On unilateral administration of compounds, double strength solutions were used to give the same total dose. Proglumide (sodium salt), supplied by Kaken Pharmaceutical Co., was dissolved in distilled water. For intraperitoneal injection, the solutions were prepared at one-tenth the concentration used for the intracranial injection and 10 times larger volumes were injected.
Data and Statistical Analyses Food was given to fasted animals immediately after administration of the vehicle, and food consumption for the ftrst and second hour was measured. Since total food consumption during the 2-hour period varied for different animals, the food consumption of the first hour and the second hour was expressed as a percentage of the total consumption in 2 hours. The second experiment was done with test agents 5 or 6 days after the f'LrSt experiment. The food consumption of animals when treated with test agents was expressed as a percentage of the total consumption in 2 hours (100%) of animals when treated with vehicle. The reduction in food consumption during the first hour after injection of a test agent was also calculated as a difference between values (%) in
SATIETY ACTION OF CCK NEURONS IN PBD
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FIG. 1. Photomicrograph ( x 25) of a brain section including the empty cavities formed by the cannulae. The brain section was stained with cresyl violet. Bilateral empty cavities correspond to the sites of the tips of cannulae. HI, third ventricle.
this period after injections of vehicle and the test agent. The data obtained are expressed as means -+ standard errors of means. Statistical analyses were performed by Student's t-test and analysis of variance (ANOVA). RESULTS
Effects of Intra-VMH Injections Food intake data after bilateral injection of the vehicle (1 pJ on each side) and 160 pmol of GIt-CCK-8 (80 pmol in 1 pd on each side) into the VMH are shown in Fig. 2A. Food intake in
A
Bilateral VMH
[] []
Vehicle (5) GIt-CCK-8(160pmol/2/d)
[] []
CCK.8(160pmol/2pl ) (5) GIt.CCK-8(160pmol/1/d ) (5) B Bilateral VMH
(5)
C Unilateral VMH
(%l 100,
| ~o
| lh
2h
T
iol lh 2h
T
lh
2h
T
FIG. 2. Effects of bilateral VMH injections of GIt-CCK-8 (A) and CCK8 (B), and unilateral VMH injection of GIt-CCK-8 (C) on food consumption during the first (1 h) and second (2 h) hours of feeding (T= total 2-h consumption). Food consumption is represented as a percentage of the total 2-h food consumption after vehicle injection (100%) in each rat. The mean _+standard error of the mean for food consumption during the 2-h period after vehicle injection was 7.0 +-0.6 g. Numbers in parentheses are numbers of animals used. Significance was analyzed by Student's t-test, and a significant difference from the value for the vehicle-treated group is shown by asterisks (**p<0.01).
control rats treated with vehicle was 5.3---0.7 g in the fhst hour but only 2.0-+0.5 g in the second hour. Bilateral injection of GIt-CCK-8 significantly (p<0.01) decreased food intake below that of control rats during the first hour but not during the second hour of feeding. Thus, GIt-CCK-8 suppressed the total food intake during 2 hours after 24 hours of fast (Fig. 2A). In contrast to GIt-CCK-8, CCK-8 did not suppress food intake when injected 160 pmol (Fig. 2B). We also examined the effect of unilateral injection of 160 pmol of GIt-CCK-8 into the VMH. As seen in Fig. 2C, it did not suppress food intake. Next, we examined the effects of bilateral injections of different doses of GIt-CCK-8 into the VMH. Glt-CCK-8 suppressed food intake during the first hour dose dependently at up to 1600 pmol (Fig. 3A). On the other hand, CCK-8 had no significant effect at either 160 or 320 pmol (Fig. 3A).
Effects of Intraperitoneal Injections Glt-CCK-8 (160 pmol) did not inhibit food intake during the first hour after intraperitoneal injection (Fig. 4A). In contrast to intracerebral injection of CCK-8, its intraperitoneal injection significantly (p<0.01) inhibited food intake in the first hour, thus during the total two-hour period (Fig. 4B). As seen in Fig. 2B, the decrease in food consumption during the first hour after the GIt-CCK-8 injection was low at doses of up to 160 pmol, but increased at a dose of 320 pmol. On the other hand, the decrease in food consumption after CCK-8 injection was high even at a dose of 160 pmol (Fig. 4B).
Effects of Proglumide and CCK-8 on Satiety Actions of CCK-8 and GIt-CCK-8 To elucidate the mechanism of the satiety actions of GIt-CCK8 and CCK-8 injected into the VMH bilaterally and into the peritoneal cavity, respectively, we examined the effects of pretreatment with proglumide on their actions. Bilateral injection of proglumide (300 pmol) into the VMH prior to injecting GIt-CCK-8 had no significant effect on the action of the Glt-CCK-8 (Fig. 5A). In
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_"
Intra-VMH
_- GIt-CCK-8 CCK-8
60.
(%) 100.
40-
0
40
1:6 4:8
'o
B =
1"6 lib 160320 1600 Dose (pmol) 0
Intraperitoneal
I///t
60
!40
~ .~ 20
:,~o 1:6 4:8 01=
1"6 8'0 1603'20 1600 Dose (pmol)
FIG. 3. Dose-dependent effects of GIt-CCK-8 and CCK-8 on food consumption. Glt-CCK-8 and CCK-8 were injected into the VMH bilaterally (A) or into the intraperitoneal cavity (B). Decrease in food consumption during the fin'st hour after injection of agents is indicated as a difference between values (%) in this period after injections of vehicle and a test agent. Each dosage group (1.6, 4.8, 16, 80, 160, 320 and 1600 pmol) consisted of 5 rats. Data are expressed as means--standard errors of means. The EDso value is about 40 pmol. A statistically significant difference was observed between groups (1.6-160 pmol) of the decrease in food consumption after VMH injection of GIt-CCK-8 and those after intraperitoneal injection of Glt-CCK-8 by ANOVA, p<0.05, F=6.74, however, it was not between groups (160 and 320 pmol) of the decrease after intraperitoneal injection of Glt-CCK-8 and CCK-8, F= 1.32.
a preliminary experiment using 2 rats, bilateral injection of 800 pmol of CCK-8 into the VMH did not inhibit food intake (the changes in food consumption during the first hour after injection [ ] Vehicle [ ] GIt-CCK-8(160pmol/20/d )
A Intrap~ritoneel
B intraperitoneral
(%)
100"
o 50,
|
i lh
///
FIG. 5. Effects of proglumide and a high dose of CCK-8 on food consumption during the f'lrst hour after injections of GIt-CCK-8 and CCK-8 injections into the VMH bilaterally (A) and into the peritoneal cavity (B1 and B-2). Each group consisted of 5 rats. *p<0.05, **p<0.01 and ***p<0.001 indicate significant differences by the t-test. Veh, vehicle; Pgl., proglumide. The explanations under the x-axis are those of pretreatments, and those above the graph are those of treatments. Other explanations are the same as for Fig. 2.
of CCK-8 were - 4 % and +6%). Then, we had a question whether CCK-8 has an affinity, even a low affinity, for the receptor responsible for the satiety action of GIt-CCK-8 or not. Thus, we examined the effect of bilateral preinjection of 800 pmol of CCK-8 into the VMH on the satiety action of GIt-CCK-8. As shown in Fig. 5A, treatment with CCK-8 before Glt-CCK-8 completely eliminated the suppressive action of GIt-CCK-8 on food intake in the first hour after its administration. We also examined the effect of pretreatment with proglumide (1500 pmol) on the satiety actions of intraperitoneal injections of CCK-8 and GIt-CCK-8. As shown in Fig. 5B, injection of proglumide into the peritoneal cavity before CCK-8 (320 pmol) completely abolished the satiety action of the latter. In contrast, pretreatment with proglumide did not affect the satiety action of Glt-CCK-8 (320 pmol) injected intraperitoneally (Fig. 5B).
Effect of Bilateral Lesions of the LPBD on the Satiety Action of Glt-CCK-8
[ ] CCK-8(160pmol/20/~1 )
i
A InIrI-VMH
[ ] Vehicle [ ] G|t-CCK.8(80pmol) CCK.8 ( 320pmol) [ ] GIt-CCK-8(320pmol) B-1 intrapefitonelll O-2 llrl~llperitOrleal l~ee
Figures 6 and 7 show the change in body weight after LPBD lesions and the common area of lesions in the brain, respectively. As reported previously (17), the body weight of rats with bilateral lesions of the LPBD increased significantly. This finding is confn'med by the results in Fig. 6. Figure 8 shows the effect of LPBD lesions on the satiety action of Glt-CCK-8. As expected, bilateral lesions of the LPBD sensitized the satiety action; GltCCK-8 at a dose of 16 pmol did not affect the food intake in the first hour of sham-operated rats, but markedly suppressed the food intake of rats with LPBD lesions. DISCUSSION
2h
lh
2h
FIG. 4. Effects of intraperitoneat injection of Glt-CCK-8 (160 pmol) (A) and CCK-8 (160 pmol) (]3) on food consumption of rats fasted for 24 hours. Results are expressed as described in the legend to Fig. 2. **(p<0.01) indicates a significant difference between values for the groups Ireated with vehicle and CCK by the t-test. Other explanations are as for Fig. 2.
In the present study, the following findings were obtained in rats fasted for 24 hours (Table 1): 1) When injected into the VMH bilaterally, GIt-CCK-8 (160 pmol) suppressed food intake, but CCK-8 (160 pmol) did not. However, unilateral injection of GItCCK-8 into the VMH did not suppress food intake (Fig. 2). 2) The satiety action of GIt-CCK-8 on the VMH was dose dependent (Fig. 3A). 3) In contrast, CCK-8 (160 pmol) elicited satiation
SATIETY ACTION OF CCK NEURONS IN PBD
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400
7
1oo]
[]
sham-lesioned (5)
[]
LPeO-lesionea(a)
350
300
0 ned (10)
4.8
16
G I t - C C K - 8 (pmol)
FIG. 8. Effects of bilateral lesions of the LPBD on food consumption during the fLrSthour after injection of GIt-CCK-8 into the VMH bilaterally in rats fasted for 24 h. Other explanations are the same as for Fig. 2.
250
~ t's
200
0
Days after operation
FIG. 6. Body weight change after bilateral lesions of the LPBD. Numbers of animals are indicated in parentheses.
when injected intraperitoneally, whereas Glt-CCK-8 (160 pmol) did not (Figs. 3 and 4). 4) Intraperitoneal injection of Glt-CCK-8 had a satiety action only when injected at a high dose (320 pmol) (Fig. 3B). 5) Pretreatment with proglumide, a CCK-antagonist, inhibited the satiety action of CCK-8 injected intraperitoneally, but did not inhibit that of Glt-CCK-8 injected either into the VMH or the peritoneal cavity (Fig. 5). 6) Pretreatment of the VMH with a large dose of CCK-8 (800 pmol) did not have any effect on food intake, but blocked the satiety action of GIt-CCK-8 injected into the VMH (Fig. 5A). 7) Bilateral lesions of the LPBD sensitized the VMH to the satiety action of GIt-CCK-8 injected into the VMH (Fig. 8). Until recently, it was generally accepted that in rats CCK had a satiety action only when it was injected peripherally, since in-
Interaural 0.2 rnm
-0.3 m m
-0.8mm
traperitoneal injection of 1 p.g/kg of CCK-8 significantly inhibited food intake, but its intracerebroventricular injection inhibited food intake only when it was injected at a high dose (12 ttg/kg) (19). However, our previous study showed that derivatives of CCK-8 (GltoCCK-8 and pGlu-CCK-8, 0.8 pmol/h) reduced food intake in free-feeding rats when they were injected intracranially, whereas CCK-8 itself did not (16). Furthermore, the present study showed that GIt-CCK-8 (160 pmol) had a satiety action when injected into the VMH bilaterally, but not when injected into the peritoneal cavity, and that proglumide, a CCK antagonist, did not block the satiety effect of Glt-CCK-8 when it was injected either centrally (VMH) or peripherally (peritoneal cavity), although it blocked the peripheral satiety action of CCK-8, and that a high dose of CCK-8 (800 pmol) inhibited the satiety action of GItCCK-8. In Fig. 3B, there are no statistically significant differences between groups of decrease in food consumption after intraperitoneal injections of Glt-CCK-8 and CCK-8 by ANOVA, F = 1.32. However, in Fig. 4, when these effects were compared with those of vehicle CCK-8 but not GIt-CCK-8 showed a significant (p<0.01) decrease in food intake. Since drug effects usually are examined compared with those of vehicles, the results shown in Fig. 4 are meaningful and the decreases of food consumption seen in Fig. 3B show the tendency of the effect. Moreover, in this experiment, effects of vehicle and CCK were obtained in the same animals in Fig. 4, but the decrease in food consumption due to Glt-CCK-8 and CCK-8 were examined in different rats (Fig. 3). Thus, it is clear that CCK-8 had a satiety effect from 160 pmol, but GIt-CCK-8 had it from 320 pmol. It was reported that injection of a large dose (300 nmol) of proglumide into the lateral cerebral ventricle inhibited a satiety action of peripheral injection of CCK-8 (12). Thus, we examined the effect of VMH injection of higher doses of proglumide. However, intra-VMH injection of proglumide (over 600 pmol) itself elicited
TABLE I SUMMARYOF THE RESULTSIN THIS STUDY GIt-CCK-8
~ D FIG. 7. Areas of common lesions in 10 rats with bilateral lesions of the LPBD are shown in black. The lower figure shows the site of the LPBD.
Suppressive effect of food intake Blocking effect of proglumide Blocking effect of CCK-8 Sensitizing effect of LPBD-lesions
VMH ip VMH ip VMH VMH
+ +, marked; +, moderate; - , none.
++ + --+ ++
CCK-8
++
870
TAKAKI ET AL.
a satiety action (data is not shown here). Thus, we could not examine the central effect of higher doses of proglumide than 300 pmol. These results suggest that in the central and peripheral site there are at least 2 types of CCK receptor in rats and that GltCCK-8 has high affinity for the central one and low affinity for the peripheral one, and CCK-8 has high affinity for the peripheral one and low affinity for the central one. Consistent with the latter suggestion, it was reported that in rats 1) CCK receptors in the brain and peripheral tissue such as the pancreas are different (11), 2) a higher dose (2.6 nmol/rat) of CCK-8 injection into the later cerebral ventricle causes a satiety action (14). In this connection, it was reported that bilateral injection of CCK-8 (40 pmol) into the VMH suppressed the ingestion of sweet noncaloric drink (9). Moreover, recently two kinds of nonpeptidal CCK antagonists were synthesized (3, 5, 7, 13), and one (MK-329) was found to have a high affinity for a peripheral receptor, whereas the other (L365,260) had high affinity for the central CCK receptor. There are also reports that in the brain the two types of CCK receptors could be identified (15), and that in studies using MK-329, a peripheral type of CCK receptor was located in areas such as the area postrema, the nucleus tractus solitarius and the interpeduncular nucleus (8). Peripheral administrations of these antagonists were shown to increase food intake of partially satiated rats and postpone the onset of satiety, and L365,260 was found to be 100 times more potent than MK-329, which has high affinity for the peripheral CCK receptor (3, 5-7, 13). These findings suggest that the brain type CCK receptor is involved in the satiety action of endogenous CCK. These results also support the above suggestion that GIt-CCK-8 and CCK-8 acted on different receptors, the central and peripheral types of CCK receptor, respectively. Previously we found that intracranial infusions of Glt-CCK-8 and pGlu-CCK-8 had satiety actions in rats fed ad lib (16), and that bilateral lesions of the LPBD induced hyperphagia and obesity (17), probably due to elimination of CCK-8-1ike immunoreactive terminals in the VMH. These findings and a report that neurons containing a CCK-8-1ike immunoreactive substance in the LPBD project fibers to the ipsilateral VMH (10), together
with the present findings suggest that release of the CCK-8-1ike substance in the VMH from the terminals of the neurons from the LPBD is involved in satiety. In this connection, it is noteworthy that bilateral injection of caerulein (0.1 ixg/kg), a decapeptide with cholecytokinin activity, into the VMH caused short-term suppression of feeding in the dark period, and that bilateral lesions of the VMH eliminated its action (22). Moreover, high densities of CCK receptors, which might be the brain type (6, 8, 15) were found in the VMH (1), and CCK was shown to increase the activities of neurons in the VMH (21). These findings also support the satiety function of neurons containing CCK-8-1ike substance in the LPBD that project fibers to the VMH. In accordance bilateral lesions of the LPBD enhanced the satiety effect of GItCCK-8 (Fig. 8) and a large dose of CCK-8 blocked the satiety action of Glt-CCK-8 (Fig. 5). In this connection, if CCK-8 has low affinity for central receptor for CCK, bilateral lesions of the LPBD might sensitize the action of CCK-8. Thus, it must be examined whether CCK-8 (1.6-800 pmol) has a satiety action in rats with bilateral lesions of the LPBD in future. The brain type of CCK receptor was also shown to be present in the paraventricular nucleus (PVN) of the hypothalamus (6). Thus, the problems of whether neurons containing CCK project fibers to the PVN, and whether these elicit a satiety action also require examination. In this study done in the middle of the light period, about 7090% of the food intake during the first 2 hours after fasting for 24 hours was consumed during the first hour. After intra-VMH injection of Glt-CCK-8 marked reduction of food intake was observed during this first hour, and there was no compensatory increase in food intake during the second hour. Similar phenomena were observed previously (2). These findings show that GltCCK-8 injection still had some effect during the second hour, or that in the second hour the phase of the ultradian rhythm of appetite of these rats did not permit further eating. These possibilities must be examined. In conclusion these findings support the idea that neurons containing a CCK-8-1ike substance in the LPBD suppress food intake through the effect of this substance on the VMH.
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