Gastrin releasing peptide-29 evokes feeding responses in the rat

Peptides 32 (2011) 241–245

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Gastrin releasing peptide-29 evokes feeding responses in the rat Martha C. Washington, Susan A. Wright, Ayman I. Sayegh ∗ Gastroenterology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, United States

a r t i c l e

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Article history: Received 15 September 2010 Received in revised form 18 October 2010 Accepted 19 October 2010 Available online 3 November 2010 Keywords: Satiety Satiation Meal size Intermeal interval Satiety ratio

a b s t r a c t In mammals, gastrin releasing peptide (GRP) 10 and 27 reduce food intake. In the current work, we test the hypothesis that GRP-29, the large molecular form of GRP in the rat, also evokes feeding responses consistent with a possible role in satiety. Here, we measured three feeding responses, size of first meal, intermeal interval (IMI, time between first and second meal) and satiety ratio (SR, satiation period for every unit of food consumed in the first meal), in overnight food deprived rats following GRP-10, 27 or 29 (0, 0.3, 1.0, 2.1, 4.1, 10.3, 17.2 nmol/kg) intraperitoneally and presentation of a 10% sucrose test diet. GRP-29 and GRP-27 reduced the size of the first meal, prolonged IMI and increased SR, but GRP-10 failed to exhibit similar feeding responses. The order of potency was GRP-29 = GRP-27 > GRP-10. The current data support a role for GRP-29 in the short-term regulation of food intake. Published by Elsevier Inc.

1. Introduction In mammals, there are two peptides that have strong amino acid sequence homologies with bombesin (Bn), the biologically potent tetradecapeptide isolated from the skin of the European frog Bombina bombina [4,27,32], gastrin releasing peptide (GRP) and neuromedin B (NMB). Gastrin releasing peptide was first isolated from the porcine stomach [26] as a 27-amino acid peptide. However, the C-terminal decapeptide of GRP (GRP-10 or GRP18–27), also referred to as neuromedin C [29], was first isolated from canine intestine [36]. On the other hand, in the rat, it has been shown that the large molecular form of GRP is GRP-29 and not GRP-27 [32,33]. In addition to gastrointestinal organs – stomach and intestine – GRP is also found in enteric neurons, brain, spinal cord, sympathetic ganglia, smooth muscles, glands and many other tissues. There are three receptors for GRP: BB1 (NMB-R), BB2 (GRP-R) and BB3 (BRS-3; or the ‘orphan’ receptor) [10]. BB1 receptor is 100 times more sensitive to binding NMB than GRP. BB2 is 14 times more sensitive to binding GRP than NMB. BB3 binds both GRP and NMB, although weakly [1]. GRP binding sites are located in the gut (on endocrine, muscular, or neural elements) [31,46] and the CNS [1,21]. As a result, GRP mediates various sensory functions [15,25,34], and causes hormonal release, smooth muscle contraction [35,43] and reduction of food intake [5,7,8,41]. Reduction of food intake in response to peripheral and central injection of GRP has been shown in different species [9]. In addition,

∗ Corresponding author. Tel.: +1 334 727 8149; fax: +1 334 727 8177. E-mail address: [email protected] (A.I. Sayegh). 0196-9781/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.peptides.2010.10.027

direct NTS injections of GRP decreased meal size [14] and infusion of highly selective GRP antagonists in 3rd [14,28] or 4th [23,24] ventricles attenuated this effect. The previous data demonstrate a clear role for GRP-10 and 27 in satiety. However, such a role by GRP-29 has not been tested. Therefore, the goal of this work is to test the hypothesis that GRP29 will evoke feeding responses consistent for a possible role of this peptide in the short-term regulation of food intake. Here, we determined three feeding measurements, size of the first meal, length of time between first and second meal, also known as intermeal interval or IMI, and satiety ratio (SR) or length of the IMI divided by the amount of food consumed in the first meal, following intraperitoneal (i.p.) injection of GRP-10, 27 or 29. The feeding measurements are determined after rating the min-tomin behaviors of each individual animal that lead to feeding, e.g., grooming, exploring or resting. A meal starts following the injection and presentation of the test diet and ends when the animal shows three resting periods during three consecutive minutes. This technique eliminates possible discrepancy that may occur in studies that measure cumulative food intake or pre-decided meal size or time because it depends on rating the behavior of each individual animal. In addition, accurate measurement of the size of two consecutive meals will also allow precise calculation of the IMI and SR (dividing IMI by size of first meal). These values, prolonged IMI and increased SR, are critical to test the effectiveness of a satiety peptide. Finally, the current study utilized a 10% sucrose test diet. We used the liquid diet because most of the data on food intake are done with liquid diets. In addition, a liquid diet will eliminate possible gastric emptying effects by the solid food.

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2. Materials and methods The Tuskegee University Animal Care and Use Committee approved all of the animal protocols for this study. Adult, male, Sprague Dawley rats weighing between 350 and 400 g and housed in clear cages to allow complete visualization necessary for behavioral rating were used in this study. The rats lived in a controlled environment (12 h dark/12 h light cycle – lights off at 18:00 h, 21.5 ◦ C, with at libitum water and pelleted rodent chow, (Teklad, WI)). To habituate the rats to the laboratory environment and experimental design, every day, and at the same time, each rat was weighed, handled for 10 min and received an intraperitoneal (i.p.) injection of saline. All injections were made in a volume of 0.5 ml and were given at 07:00 h, 1 h into the beginning of the light cycle. 2.1. Baseline food intake Before measuring food intake in response to GRP, a baseline of food intake was established for each rat by the following procedure. Animals were fasted from food but not water overnight at 17:00 h. The following morning, at 07:00 h (1 h into the light cycle) each rat received a saline injection i.p. followed immediately by presentation of a 10% sucrose test diet. Two, well-trained, examiners equipped with headphones, laptop computers and timers started rating the min-to-min behaviors of the rats and measuring food intake immediately following the presentation of sucrose. Animal behaviors included feeding (licking the sucrose bottle), licking (licking the water bottle), biting, grooming, locomotion (walking around the cage), rearing up (front paws on side of cage while hind paws on the floor of cage), standing (only hind paws touching floor of cage), sniffing and stretching and resting (no movement by animal) [2,11]. A meal started immediately following saline injection and presentation of test meal to the animals and was terminated following recording of three consecutive resting periods over three consecutive minutes [12,13]. The agreement rate between the examiners exceeded 98%. The examiners measured the size of the first meal, IMI (time between first and second meal) and calculated SR (length of IMI divided by size of first meal (min/ml)). The previous process was done daily for a total of 14 days until the intake of all rats was stabilized. Following this baseline the experiment with GRP started. 2.2. Food intake On the day prior to the experiment, animals were deprived of food but not water at 17:00 h. At 07:00 h the following day (an hour following lights on), they received an i.p. injection of GRP-10, 27, 29 (amino acid sequence of GRP-29 was provided by Dr. Joseph Reeve

Jr. CURE, LA, USA and synthesized by Bachem, USA), (0.3, 1, 2.1, 4.1, 10.3 and 17.2 nmol/kg) or saline control followed by presentation of a 10% sucrose test diet. Treatments were given in the odd days, saline was given in the even days and one day was reserved to maintain the cages. The results of the saline injection during the even days matched the baseline results. Immediately following the injections, two independent examiners rated the behaviors of each rat and measured food intake to determine the size of the first meal, IMI and SR as described above. All treatments were rotated between rats until each rat received all of the treatments, which insured that each animal served as a control for itself. This system has been used previously [18,19,37,44]. 2.3. Data analysis The data were analyzed by a two-way repeated measures analysis of variance (ANOVA), with treatment and dose as the two independent variables. Multiple comparisons were performed using the Holm–Sidak test. In addition, fitting curves were also generated for all peptides to compare their efficacy and potency (SigmaStat for Windows version 3.11, Systat system Software, Inc., 2004). All results were displayed as mean ± SEM. Data were considered significant if p < 0.05. 3. Results 3.1. Effect of GRP on meal size GRP-29 (2.1, 4.1, 10.3, and 17.2 nmol/kg), GRP-27 (1, 2.1, 4.1, 10.3, and 17.2 nmol/kg) and GRP-10 (2.1 nmol/kg) reduced the size of the first meal relative to saline (p < 0.001, F = 20.259, DF = 16) (Fig. 1). There was no difference between peptides or doses. 3.2. Effect of GRP on intermeal interval GRP-29 and 27 (0.3, 1, 2.1, 4.1, and 17.2 nmol/kg) and GRP10 (0.3 nmol/kg) prolonged the IMI relative to saline (p < 0.001, F = 18.824, DF = 16) with a difference between the peptides by GRP27 (0.3 nmol/kg) which prolonged IMI more than GRP-10 (p = 0.006) (Fig. 2). 3.3. Effect of GRP on satiety ratio GRP-29 and 27 (0.3, 1, 2.1, 4.1, 17.2 nmol/kg) and GRP-10 (2.1 nmol/kg) increased the satiety ratio relative to saline (p < 0.001, F = 15.933, DF = 16) and with no difference between the peptides (Fig. 3). GRP-27 and 29 reduced meal size, prolonged IMI and increased SR similarly, but more than GRP-10. There was no difference

Fig. 1. Effect of gastrin releasing peptides on the size of the first meal. Food deprived male rats (n = 17) received an injection of gastrin releasing peptide (GRP) 10, 27, or 29 (0.3, 1, 2.1, 4.1, 10.3 and 17.2 nmol/kg) or saline i.p. (1 h into the light cycle) followed by presentation with a 10% sucrose test diet and the size of the first meal was determined. GRP-29 (2.1, 4.1, 10.3, 17.2 nmol/kg), GRP-27 (1, 2.1, 4.1, 10.3, 17.2 nmol/kg) and GRP-10 (2.1 nmol/kg) reduced the size of the first meal relative to saline (*). p < 0.05.

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Fig. 2. Effect of gastrin releasing peptides on the length of the intermeal interval. Food deprived rats (n = 17) received an injection of gastrin releasing peptide (GRP) 10, 27, or 29 (0.3, 1, 2.1, 4.1, 10.3 and 17.2 nmol/kg) or saline i.p. 1 h into the light cycle. Immediately following the injection rats were presented with a 10% sucrose test diet and their min-to-min feeding behaviors were rated to determine the time between first and second meals or intermeal interval (IMI). All doses of GRP-29 and GRP-27 except (10.3 nmol/kg), and GRP-10 (0.3 nmol/kg) prolonged IMI relative to saline (*) and GRP-27 (0.3 nmol) prolonged it more than the same dose of GRP-10 (†). p < 0.05.

Fig. 3. Effect of gastrin releasing peptides on the satiety ratio. Food deprived rats (n = 17) received an injection of gastrin releasing peptide (GRP) 10, 27, or 29 (0.3, 1, 2.1, 4.1, 10.3 and 17.2 nmol/kg) or saline i.p. 1 h into the light cycle. Following the injection they were presented with a 10% sucrose test diet and their min-to-min behaviors were rated to determine the size of the first meal and time between first and second meals. These values were used to calculate the satiety ratio by dividing the time between the two meals by the size of the first meal. All doses of GRP-10 and GRP-27 and GRP-29 (2.1 nmol/kg) increased satiety ratio relative to saline (*). p < 0.05.

between GRP-27 and GRP-29 in these responses (Fig. 4 and significant values are shown in Table 1). GRP-27 and 29 had a tendency to reduce meal size more when doses where increased. In addition, the low doses of GRP-27 and 29 had a tendency to prolong IMI and increase satiety ratio compared to higher doses. 4. Discussion The current work provides feeding measurements, including size of first meal, IMI and SR, for GRP-29, the large molecular form of GRP in the rat [32]. Our results show that GRP-29 and GRP-27, the major molecular forms of GRP in mammals other than rats, are similar in reducing the size of the first meal, prolonging IMI and increasing the satiety ratio. Therefore, GRP-29 evokes feeding responses consistent with a possible role in the short-term regulation of food intake. Short-term control of food intake is mainly regulated by gastrointestinal peptides, e.g., GRP (secreted from the stomach) and cholecystokinin (CCK, secreted from the small intestine). This control mechanism consists of two components, regulation of meal size and IMI. A satiety peptide reduces meal size (causes satiety) and prolongs the time between two consecutive meals or IMI (evokes satiation). The results of the current work showed that GRP-29 reduces meal size and prolongs the IMI. Therefore, this peptide has a role in regulation of short-term food intake.

This role has been demonstrated previously for GRP-27. Central and peripheral GRP reduce food intake in numerous species e.g., turkey, mouse, rat, wolf, pig, baboon and humans [3,6,7,40,41,47]. Intraperitoneal injections of GRP decreased meal size in rats [8]. Acute and chronic intravenous injections of GRP-27 (1, 2, 4, and 8 ␮g/kg) reduced food intake in baboons [5]. Finally, systemic GRP failed to suppress food intake in GRP-KO mice [20]. The current work demonstrated that intraperitoneal injections of GRP-29 reduced meal size and prolonged intermeal interval similarly to GRP-27, indicating that both peptides probably activate the same receptor subtypes or BB2 receptors. The mechanism by which GRP reduces meal size and/or prolongs IMI is not completely understood. However, the anorectic effect of peripheral administration of Bn is blocked by lesioning both vagal and spinal visceral nerves, but not by either one alone [30,42]. These results were confirmed by systemic capsaicin treatment [22,23] which ablates sensory afferents. In addition, electrical or chemical stimulation of enteric neurons in isolated, vascularly perfused, rat stomachs released endogenous GRP [38], and electrical stimulation of the vagus also released GRP-like immunoreactivity in gastric venous efferents in the cat [45], pig [16,17] and isolated rat stomach [39]. As such, it is possible that, since GRP is released from enteric neurons in the stomach, the enteric nervous system of the gut may have a role in the feeding responses which GRP-29 evokes.

Table 1 Correlations for the effect of GRP-10, 27 and 29 on meal size, intermeal interval and satiety ratio. Peptide

Meal size correlation

p Value

IMI correlation

p Value

SR correlation

p Value

GRP-10 GRP-27 GRP-29

0.46 −0.46 −0.445

1.0 1.0 1.0

−0.469 −0.939 −0.617

1.0 0.066 1.0

0.821 −0.528 −0.314

0.544 1.0 1.0

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M.C. Washington et al. / Peptides 32 (2011) 241–245 GRP-10 GRP-10 Regression line GRP-27 GRP-27 Regression line GRP-29 GRP-29 Regression Line

14

Grants Support: The Birmingham Racing Commission, HRSA/COE D34HP00001-22-00 and NIH, NCMHD SC21MD000102-09.

12

Acknowledgments

Intake (ml)

10

The authors express their gratitude to Dr. Joseph Reeve Jr. CURE, LA for providing the sequence for rat GRP-29, Mrs. Carol Williams for her valuable editorial corrections and Drs. John Heath (Tuskegee University) and James Cox (University of Alabama at Birmingham) for their assistance in the statistical analysis.

8 6 4

References

2 0 0.3

1

2.06

4.12

10.31

17.2

Dose(nmol/kg) GRP-10 GRP-10 Regression line GRP-27 GRP-27 Regression line GRP-29 GRP-29 Regression Line

50

Time (min)

40

30

20

10

0 0.3

1

2.06

4.12

10.31

17.2

Dose(nmol/kg)

Intake per unit time (min/ml)

10

Sat GRP-10 GRP-10 Regression line Sat GRP-27 GRP-27 Regression line Sat GRP-29 GRP-29 Regression Line

8

6

4

2

0 0.3

1

2.06

4.12

10.31

17.2

Dose(nmol/kg) Fig. 4. Fitting curves for the effect of GRP-10, 27 and 29 on meal size (upper panel), intermeal interval (IMI, middle panel) and satiety ratio (SR, lower panel) showing the regression lines for these effects. GRP-27 and 29 reduced meal size, prolonged IMI and increased SR similarly but GRP-10 failed to affect these parameters. In addition, GRP-27 and 29 had a tendency to reduce meal size when doses are increased. Significance is shown in Table 1. In addition, the low doses of GRP-27 and 29 had a tendency to prolong IMI and increase satiety ratio compared to higher doses.

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