Vol. 131, No. 6 Printed in KS A
Effects of Coadministered Growth Hormone (GH)Releasing Hormone and GH-Releasing Hexapeptide Maladaptive Aspects of Obesity in Zucker Rats BARRY B. BERCU, SEI-WON RICHARD F. WALKER
YANG,
Departments of Pediatrics, Pharmacology, Tampa, Florida 33612; and All ChildrenS
RYUJI
MASUDA,
CHENG-SHIH
HU,
on
AND
and Therapeutics, University of South Florida, Hospital, St. Petersburg, Florida 33701
ABSTRACT The purpose of this study was to determine the effect of chronic pharmacological stimulation of the pituitary gland on GH hyposecretion and other maladaptive aspects of obesity. Obese Zucker rats were coadministered GH-releasing hormone (GHRH; 3 fig/kg) and GHreleasing hexapeptide (GHRP-6; 300 pg/kg), a potent combination of synergistic GH secretagogues, once daily for 60 consecutive days. Although pituitary weights and GH concentrations were higher in obese rats administered the peptides than in obese rats administered saline, stimulated GH secretion was lower in obese rats than in lean rats. However, compared to those in lean rats, plasma insulin-like growth factor-I and insulin concentrations were higher in the obese rats regardless of treatment. The GH secretagogues did not alter food intake or body weight gain in sexually mature obese rats, whereas body weight
gain was significantly increased when they were administered to prepubertal obese rats. Although glucose tolerance was impaired in both groups of obese rats, it improved in obese rats administered GHRH and GHRP-6 compared to that in obese rats administered saline. On the other hand, plasma cholesterol concentrations were elevated in obese rats administered the GH secretagogues but not saline. In conclusion, the results of this study suggest that hyposensitivity to GHRH and GHRP-6 in obese Zucker rats results from high concentrations of plasma insulin-like growth factor-1 that negatively feedback on stimulated GH secretion. Nonetheless, daily episodes of endogenous GH secretion resulting from chronic coadministration of GH secretagogues significantly influenced the pituitary gland as well as lipid and carbohydrate metabolism. (Endocrinology 131: 2800-2804, 1992)
S
mechanismto GHRH (11, 12). Thus, it was of interest to test the efficacy of the peptide combination in Zucker rats for stimulating endogenous GH secretion and its potential for reversing certain maladaptive aspectsof obesity.
TIMULATED and spontaneous GH secretion as well as pituitary GH content are significantly lower in obese animals compared to those that are lean (l-4). Since diminished GH secretion can be reversed by food restriction (3, 5), the endocrine defect is probably an effect, rather than a cause of obesity. However, since GH treatment reduced lipid deposition (6), GH hyposecretion may sustain obesity once it is established. GH deficiency may also contribute indirectly to the establishment of obesity by increasing appetite. This hypothesis is basedupon the fact that food consumption was decreased in rats administered GH-releasing hormone (GHRH) directly into the brain (7), and body weight gain/ hyperphagia was prevented in rats administered GH immediately after surgical destruction of the hypothalamic ventromedial nucleus (VMH; satiety center) (8). The purpose of the present study was to determine whether endogenous GH secretion could be increasedpharmacologically in genetically obeseZucker rats, and if chronic treatment with GH secretagogueswould reverse certain maladaptive changesassociatedwith obesity. Obese Zucker rats respond poorly to GHRH (1, 9). However, in a prior study, we (10) discovered that old rats hyporesponsive to individually administered GH secretagogueswere hyperresponsive to coadministered GHRH and GH-releasing hexapeptide (GHRP-6), a synthetic peptide unrelated in structure or Received July 20, 1992. Address all correspondence and requests for reprints to: Dr. Richard F. Walker, Division of Endocrinology, Box 690, All Children’s Hospital, 801 Sixth Street South, St. Petersburg, Florida 33731-8920.
Materials and Methods Animal
husbandry
Obese and lean female Zucker rats (30 and 80 days of age; Charles River Breeding Laboratories. Wilmington, NC) were acclimated to local conditions for 1 week before being used in the present study. The older rats weighed between 410-479 g, while the younger rats weighed between 183-216 g when the experiment began. Female rats were used because they respond more consistently and reproducibly to GHRP-6 than do conscious males (13). Housing conditions included alternating 12-h periods of light and darkness (0600-1800 h), 60 f 5% relative humidity, 72 f 2 F ambient temperature, and constantly available food (Purina rat chow, Ralston-Purina, Kalamazoo, MI) and tap water. The animal facility at All Children’s Hospital in which this study was performed is a fully accredited institutional member of the American Association of Laboratory Animal Sciences and complies with federal statutes and regulations for animal use in research.
Blood sampling Samples for sequential analysis of plasma GH concentrations were collected from anesthetized rats between 0900-1100 h, except when surgical procedures and sampling consumed a full day (-0800-1800 h). When collection intervals were extended, samples were drawn alternatively from the different groups so as to control for time of day variation. Sequential blood samples for analysis of temporal GH secretory profiles in anesthetized female rats were collected via cannulae that were surgically placed within the femoral artery after the administration of
2800
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
GH SECRETION
ketamine (60 mg/kg, im) and pentobarbital (40 mg/kg, ip). All rats in a given experiment were surgically prepared en masse, and treatments were alternated between individual animals so that anesthesia times for all groups were approximately the same. Peptide solutions (1 ml/kg) were administered as 30.set intraarterial infusions, and blood samples for GH analysis were collected at designated times into heparinized tubes. When administered SC, GHRP-6 is rapidly absorbed and stimulates GH release in a manner indistinguishable from that after iv administration (13, 14), precluding the need to restrain the rats for iv administration via the tail. These characteristics allow GHRP-6 administration with minimal stress and support the use of SC administration whenever possible.
Experimental
design
Obese and lean Zucker rats were randomly assigned to 3 groups (12 rats/group) designated to receive daily SC injections of GHRH and GHRP-6 (1 group of obese rats) or saline (1 group of obese and 1 group of lean rats). Solutions (1 ml/kg) of GHRP-6 and rat GHRH (300 and 3 rg/kg, respectively) or saline were administered at approximately 0900 h for 60 consecutive days. In a preliminary dose-range study, the dose of 300 pg/kg GHRP-6 was the ED ,oD in conscious 3-month-old female rats (14). The dose of GHRH (3 pg/kg) was selected from published data showing that the peptide was efficacious when administered SC at a dose of 10 fig/kg (15). The lower dose was used in the present study because of the known synergistic activity of GHRH and GHRP-6 and its efficacy in GHRH-resistant rats (10). A preliminary analysis of individually administered GHRH and coadministered GHRH/GHRP-6 efficacy in obese rats was performed on the first day of the study. Lean and obese rats (six rats per group) were anesthetized, and their right femoral arteries were cannulated. F’eptides were administered, and blood samples (-250 ~1) were collected in heparinized tubes before and at selected intervals after administration of the GH secretagogues. The rats were exsanguinated at the last blood sample, and plasma was subsequently analyzed for GH concentrations. Since individually administered GHRH was ineffective in obese rats (Fig. l), the remaining animals received daily SC injections of saline or GHRP-6/GHRH for a total period of 60 consecutive days. Body weight and food consumption were determined at regular intervals, and lean body mass was determined after 30 days of treatment. Lean body mass was estimated using the ratio of water displacement volume to dry body weight. The rats were immersed in a water-filled tank containing a series of concentric rings that acted as baffles to reduce surface turbulence
T
IN OBESE RATS
2801
resulting from swimming. Thus, at the tank perimeter, the water rose without turbulence, and spilled through a channel into a collecting vessel. This apparatus allowed accurate and reproducible measurement of water displaced by each rat, which was then weighed (1 gm Hz0 = 1 cc) and divided by the animal’s dry body weight (volume of Hz0 displaced/body weight; V/W) to provide a body composition index (BCI) or estimate of lean body mass. Since fat is less dense than muscle, decreasing the V/W indicated increasing lean body mass, A glucose tolerance test was performed after 40 days of treatment to determine the effects of daily episodic release of endogenous GH on carbohydrate metabolism in obese rats. A 30% aqueous solution of glucose (1 g/kg; 3 ml/kg) was administered as an ip injection to unanesthetized rats from which food was withheld for approximately 8 h. Blood samples were taken from a tail vein before the glucose injection and at 15-min intervals thereafter. Blood glucose concentrations were determined using an ExacTech Blood Glucose Sensor (MediSense, Inc., Cambridge, MA). On the final day of treatment, all rats were anesthetized with ketaminepentobarbital and administered GHRH (1 rg/kg) and GHRP-6 (30 fig/ kg), iv, to determine the effects of long term treatment on stimulated GH secretion, serum nonesterified FFA (blood was collected into nonheparinized tubes), plasma insulin-like growth factor-l (IGF-I), insulin, and cholesterol concentrations. After exsanguination, the pituitary glands were rapidly removed, weighed, and frozen on dry ice for subsequent estimation of GH concentrations, All procedures in this experiment were described in a protocol that was approved by the University of South Florida Institutional Animal Care and Use Committee.
Quantitation
of
hormone
concentrations
Plasma GH, IGF-I, and insulin concentrations were estimated by RIA. Blood samples were centrifuged, and plasma was collected for RIA analysis of GH concentrations, Pituitaries were homogenized in 0.1 N NaOH-saline and diluted, and aliquots were analyzed by RIA for GH concentrations. GH concentrations were estimated in duplicate by RIA and expressed in terms of the reference preparation rat GH RP-2 from the NIDDK. Radiolabeled rat GH was purchased from Chemicon lnternational, Inc. (Temecula, CA), and precipitating antibody (goat antimonkey y,y[I’-41) was purchased from Antibodies, Inc. (Davis, CA) and used at a concentration of 1:lO. lntra- and interassay coefficients of variation were less than 5% and less than 8%, respectively. IGF-I concentrations were estimated after acid-ethanol extraction from plasma, using methods provided in a commercially available kit (Nichols Institute, San Juan Capistrano, CA). Plasma insulin concentrations were determined according to the method of Yalow and Berson (16), using Lilly pork insulin I’] 5682 as a reference standard. Serum FFA and cholesterol were measured using commercially available reagents from Wako Chemicals USA (Dallas, TX; kit 40-2100) and Sigma Chemical Co. (St. Louis, MO; kit 352), respectively.
Chemicals GHRP-6 (His-o-Trp-Ala-Trp-o-Phe-Lys-NH,) and rat GHRH were purchased from Peninsula Laboratories (Belmont, CA). Controls received saline administered in an equal volume and by the same route as the peptides.
Data analysis
0
B
15
TIME
20
25
30
(minutes)
Frc. 1. Comparison of GH secretion after a provocative dose of GHRP6 (A and A) or GHRH and GHRP-6 (0 and 0) in obese rats (A and 0) and lean rats (A and 0). Rats were never exposed to the GH secretagogues, either alone or in combination, before the provocative dose was administered.
Pituitary and plasma hormone concentrations as well as tissue weights and body composition indices were compared among the groups of young and old rats using analysis of variance with Duncan’s multiple range test. Changes in peptide-stimulated plasma GH concentrations, plasma glucose, and body weight change over time were compared using repeated measures analysis. Results were expressed as the mean f SEM. P < 0.05 was used to define statistically significant differences.
Results
Coadministered GHRH and GHRP-6 were a lesseffective stimulus for GH secretion in obeserats than in lean rats on
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
2802
GH SECRETION IN OBESE RATS
the first and last days of treatment. However, as shown in Fig. 1, the combination of peptides was synergistic in the obese rats, stimulating significantly more endogenous GH secretion than GHRH alone. Since GHRH was essentially ineffective in the obese rats, only the combination of GH secretagogueswas administered for the remainder of the study. Although GHRH and GHRP-6 remained effective after 45 consecutive days of administration to the obeserats, the GH secretory responseswere not altered by duration of treatment, and peak plasma GH concentrations remained approximately one third of those in lean rats throughout the study (Fig. 2). Basal plasma GH concentrations were comparable in lean and obeserats. As shown in Table 1, plasma IGF-I concentrations were significantly higher in both groups of obeserats than in lean rats, despite the lack of pituitary stimulation in response to saline or lower stimulated GH secretion in responseto GHRH and GHRP-6. Furthermore, the elevated IGF-I concentrations were not strongly correlated with the administration of GH secretagogues.Although mean IGF-I levels in obeserats coadministered GHRH and GHRP-6 were higher than those in obeserats administered saline, the differences were not statistically significant. Similar trends were observed for serum nonesterified FFA and insulin concentrations, which were significantly greater in 1,200
-
l,fJ@J s‘
E 3 5
6W-
5
600-
s $ 400 200
Endo. Vol131.
1992 No 6
both groups of obese rats than in lean rats. In contrast, plasma cholesterol concentrations were significantly greater in obese rats coadministered GHRH and GHRP-6 than in obeserats or lean rats administered saline, in which cholesterol concentrations were comparable (Table 1). The changes in cholesterol concentrations were attributed either directly to the GH secretagoguesor indirectly to the increase in endogenousGH that they produced. The data presented in Fig. 3 show that body weight gain was not reduced in 80-day-old obese rats coadministered GHRH and GHRP-6 for 45 consecutive days despite their relatively high plasma concentrations of stimulated GH and basal IGF-I. However, when peptide administration was begun at 30 days of age, body weight was significantly increasedcompared to that of saline-treated obesecontrols. Although statistical significance was not reached, the mean BCI, a measureof lean body mass,was reduced in obeserats administered GH secretagogues,suggestingthat the peptides contributed to protein, rather than fat, deposition. BCI in the heterozygous Zucker rats was significantly lower, indicating leaner body massfor these rats than either group of homozygous Zucker rats (Fig. 4). BCI varied more after coadministration of GHRH and GHRP-6 than before treatment began, suggestingthat quantitative differences occurred in the responsesof individual rats to the GH secretagogues. The data presented in Table 2 show that increased body weight was accompanied by greater food intake in obese rats. Mean food intake per day was remarkably constant in lean and obese animals, suggesting that the hypothalamic satiety center had a higher set-point in the latter group, which corresponded to their greater body weights. Apparently, that set-point was not altered by peripherally administered GHRH, GHRP-6, stimulated endogenous GH, or elevated plasma IGF-I. As alsoshown in Table 2, pituitary weight and GH content, which were significantly lower in saline-treated obese rats than in lean rats, were significantly higher in GH secreta-
0
0
6
15
25
35
600
TIME (MINUTES) FIG. 2. GH secretion in response to a provocative dose of GHRH and GHRP-6 in lean rats (A) or obese rats (0) previously administered saline compared to that in obese rats (0) previously coadministered GHRH and GHRP-6 for 60 consecutive days. The response in lean rats is significantly greater (P < 0.01) than that in obese rats. Differences in stimulated GH secretion between the two groups of obese rats are not statistically significant. n = 6 rats/group.
TABLE consecutive substances
1. Effects
of GHRH and GHRP-6 coadministration for 4.5 days on serum and plasma concentrations of various in obese and lean Zucker rats
Group and treatment Lean (saline) Obese (GHRH + GHRP-6) Obese (saline)
Plasma IGF-I Wml)
Plasma insulin NJ/ml)
Plasma cholesterol (mg/dl)
Serum FFA (meqjliter)
753 + 48” 1033 + 43
12.6 f 3” 48.4 f 7
54.1 + 7.7 118.1 f 10.6”
0.38 + 0.06” 1.9 + 0.8
957 + 59
39.4 I? 6
74.6 + 4.9
0.87 + 0.2
Values represent the mean + SEM for determinations from six rats per group. ” Significantly different (P < 0.05) from values for obese rats.
-5oc-
E 8 ; ii 3
400-
300
-
$I $ 200 -
loo
‘, 0
I 5
I 10
I 15
I 20
I 25
I 30
I 40
I 60
I 60
DAYS WC. 3. Body weight changes in rats administered saline (0, A, and n ) or GHRH and GHRP-6 (0, A, and 0). Treatment was begun in lean and obese rats at 80 days of age (W, 0 and 0, 0, respectively) or in obese rats at 30 days of age (A and A). Values represent the mean + SEM for six rats per group. *, Significantly different (P < 0.05) from saline treatment in same age group.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
GH SECRETION
IN OBESE RATS
2803
1.00
0.95
-
0.9il
-
0.65 -
-+-
0.80 -
0.75 -
0.70 0
0.65
-
0.60
0
GHRH + GHRP-6
Saline
Obese
GHRH + GHRP-S
Saline Lean
FIG. 4. Body composition in obese and lean rats administered (m and Cl, respectively) or coadministered GHRH and GHRP-6 0, respectively) for 30 consecutive days. Values represent the (horizontal lines), SEs (rectangles), and ranges (vertical lines) rats per group. *, Significantly different (P < 0.05) from obese treated rats.
saline ( means for six saline-
TABLE 2. Effect of GHRH and GHRP-6 coadministration for 45 consecutive days on food consumption and the pituitary gland in obese and lean Zucker rats Group and treatment
‘,
Food wt consumed Pituitary bd k/day) 14 f 0.9” 11.3 * 0.4 23 + 1.3 10.1 f 0.7
G~~~n~~nt (d&d) 91 + 1.9 79 + 2.0
Pituitary GH cow. b&w) 8.2 f 1.8 9.4 + 3.5
Lean (saline) Obese (GHRH + GHRP-6) 6.1 f 0.4” 66 + 1.1’ 10.9 + 1.3 Obese (saline) 22 + 1.0 Values represent the mean + SEM for six rats per group. ’ Significantly different from other groups (P < 0.05).
gogue-treated obeserats. Despite group differences in pituitary weight and GH content, pituitary GH concentrations were comparable in all lean and obeserats. Glucose intolerance, as indicated by prolonged periods of elevated blood glucose concentrations after ip administration of the sugar, was observed in obesesaline-treated rats, whereas glucose tolerance tests improved in obese GH secretagogue-treated rats (Fig. 5). These data suggestthat obesity-associatedglucoseintolerance was partially resolved by daily episodesof endogenousGH secretion. Discussion The main finding of this study was that coadministered GHRH and GHRP-6 were an effective stimulus, albeit less efficacious than in lean rats, for endogenousGH secretion in obese rats. Nonetheless, the GH secretagoguesincreased pituitary weight and pituitary GH content, suggestingthat GH hyposecretion in the obese rats resulted from reduced pituitary stimulation and/or a functional defect in the mechanism for GH release,as proposed by Renier et al. (1). It is possiblethat high plasma IGF-I concentrations in the obese
I
I
I
I
15
30
45
90
TIME (MINUTES) FIG. 5. Glucose tolerance tests in lean rats (A) and obese rats (Cl) previously administered saline or previously coadministered GHRH and GHRP-6 (0) for 60 consecutive days. Changes in blood glucose as a function of time in obese saline-pretreated rats are significantly different (P < 0.05) from those in lean saline-pretreated rats. Values represent the mean f SEM for six rats per group.
rats inhibited GH releaseeither by direct negative feedback at the pituitary or indirectly and in concert with GHRP-6 by releasingsomatostatin (13, 17, 18). This is the first documentation of elevated in vivo plasma IGF-I concentrations in the obesefemale Zucker rat, and its causeis unknown. However, elevated IGF-I is seen clinically in obesechildren with suppressed GH secretion, and it may simply be related to increasedinsulin concentrations associatedwith hyperphagia. Hyperinsulinimia (2) and elevated plasma triglycerides (19) in obese Zucker rats have also been associatedwith GH hyposecretion (20,21) and, asshown in this study, correlated with increasedfood intake. The logic for coadministering GHRH and GHRP-6 to reduce food intake and maintain normal body weight was based upon the fact that hyperphagia and obesity resulting from destruction of the VMH (satiety center) in rats can be prevented with exogenousGH (8). Although obeserats were hyposensitive to GHRH, the peptide was coadministered with GHRP-6 in this study becausetheir actions are synergistic (22), causingepisodesof endogenousGH secretionthat achieved peak plasma concentrations of approximately 350 rig/ml. However, the peptide combination was ineffective in reducing food intake and body weight gain in obeseZucker rats. In fact, body weight gain in the homozygous rats was significantly increased when treatment was begun in weanling rats compared to adults. Additional weight gain in the younger rats was attributed to acceleration of the increase in plasma IGF-I that occurs spontaneously during maturation in obeseZucker rats (23). The data were insufficient to determine whether the additional weight gain in young rats chronically administered GHRH and GHRP-6 resulted from an increasein lean body mass.Although the mean BCI in obeserats coadministered GHRH and GHRP-6 was slightly lower than that in obese rat administered saline, the differences were not statistically significant. The lack of a significant change in BCI in the obeserats coadministered GHRH and GHRP-6 was attrib-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
GH SECRETION
uted to limits of the resolving power of the water displacement method. In heterozygotes, which were much leaner than the homozygotes, the differences in BCI were statistically significant. Increased weight in obese peptide-treated rats could alsobe attributed to altered lipid metabolism, since their plasma cholesterol concentrations were significantly higher than those in the other obeseor lean groups. Alternatively, increased plasma cholesterol may have occurred secondarily to GH-mediated renal dysfunction (24). However, if GHRH- and GHRP-6-stimulated endogenous GH has the potential to cause renal dysfunction, obese Zucker rats may be uniquely vulnerable, since similar effects were not observed in female Fischer 344 rats administered the peptides for 60 consecutive days (our unpublished data). One objective of the present study was to determine whether established obesity could be reversed by elevating endogenous GH, not to prevent weight gain or decrease hyperphagia before they occurred. The data from this study suggestthat stimulated GH is not effective for that purpose. Perhaps, pharmacologically stimulated GH would have prevented hyperphagia and reduced body weight gain in the obeseZucker rat if treatment was begun in preobeseneonates (~5 days of age), since VMH lesion-induced obesity was prevented when GH was administered immediately after surgery (8). Furthermore, the route of administration may have prevented GHRH from reducing food intake, as it did when administered directed into the brain (7). An unexpected finding of this study was that obesityassociatedglucose intolerance in saline-treated homozygous Zucker rats was partially corrected in GH secretagoguetreated rats. This effect, which is not consistent with a diabetogenic effect of GH, may reflect normalization of the combined influences of insulin and GH on carbohydrate metabolismthat were restored by coadministration of GHRH and GHRP-6. In conclusion, the results of this study confirm attenuated GH secretion in obeserats administered synergistic GH secretagogues for up to 45 consecutive days. Nonetheless, coadministration of GHRH and GHRP-6 significantly increased plasma GH concentrations. Although the resulting increase in endogenous GH did not reduce hyperphagia or body weight gain in the genetically obeserats, changes in carbohydrate and lipid metabolism suggestthat the animals were responsive to episodesof stimulated endogenous GH secretion and support further studies into the therapeutic potential of GH secretagoguesin obesity. References
3. Williams T, Berelowitz M, Joffe SN, Thorner MO, Rivier J, Vale W, Frohman L 1984 Impaired growth hormone responses to growth
RATS
hormone-releasing factor in obesity: with weight reduction. N Engl J Med 4.
5.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
19 20 21
in genetically
obese
24
rats.
Cheng K, Chan WW-S, Barreta A, Convey DM, Smith RG 1991 Evidence for a role of protein kinase-C in His-o-Trp-Ala-Trp-o-I’heLys-NH,-induced growth hormone release from rat primary pituitary cells. Endocrinology 129:3337-3342 Bercu BB, Weideman CA, Walker RF 1991 Sex differences in growth hormone (GH) secretion by rats administered GH releasing hexapeptide. Endocrinology 129:2592-2598 Bercu BB, Yang S-W, Masuda R, Walker RF 1992 Role of selected endogenous peptides in growth hormone releasing hexapeptide (GHRP-6) activity: analysis of GHRH, TRH and GnRH. Endocrinology 130:2579-2586 Shulman DI, Sweetland M, Duckett G, Root AW 1987 Age-related differences in the growth hormone secretory response to hGHRH l-44 in male rats from infancy through puberty. Zri vivo and in vifro studies. Acta Endocrinol (Copenh) 116:138-144 Yalow RS, Berson SA 1964 Immunoassay of plasma insulin. Methods Biochem Anal 12:69-76
Berelowitz
M, Szabo M, Frohman LH, Fireston S, Chu L 1981 hormone negative feedback by and pituitary. Science 212:1279-
Sheppard
MC, Shapira B, Hudson A 1980 Tissue and serum somatostatin-like immunoreactivity in lean and obese Zucker rats. Horm Metab Res 12:236-239 Barry WS, Bray GA 1969 Plasma triglycerides in genetically obese rats. Metabolism 18:833-839 Melmed S 1984 Insulin suppresses growth hormone secretion by rat pituitary cells. J Clin Invest 73:1425-1433 Imaki T, Shibasaki T, Masuda A, Hotta M, Yamauchi N, Demura H, Shizume K, Wakabayashi I, Ling N 1984 The effect of glucose (GH)-releasing 118:2390-2394
factor-
Bowers CY, Reynolds GH, Durham D, Barrera CM, Pezzoli SS, Thorner MO 1990 Growth hormone (GH)-releasing peptide stimulates GH release releasing hormone.
23
Zucker
AZ, Canary JJ 1972 Growth hormone reobese. J Clin Endocrinol Metab 34:498-511 Martin RJ, Jeanrenaud B 1985 Growth hormone in obesity and diabetes: inappropriate hypothalamic control of secretion, Int J Obesity 9:99-104 Vaccarino FJ, Bloom FE, Rivier J, Vale1 W, Koob GF 1985 Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor. Nature (Lond) 314:167-168 York DA, Bray GA 1972 Dependence of hypothalamic obesity on insulin, the pituitary and the adrenal gland. Endocrinology 90:885894 Gaudreau I’, Renier G, Prazeau P 1987 rGHR(l-29)NH,-induced growth hormone secretion in genetically and dietary obese rats. Sot Neurosci Abstr 13:667 Walker RF, Yang S-W, Bercu BB 1991 Robust growth hormone (GH) secretion in aged female rats co-administered GH-releasing hexapeptide (GHRP-6) and GH-releasing hormone (GHRH). Life Sci 49:1499-1504 Codd EE, Shu AYL, Walker RF 1989 Binding of the growth hormone releasing peptide GHRP-6 to specific hypothalamic and pituitary sites. Neuropharmacology 28:1139-l 144
and free fatty acids on growth hormone mediated GH secretion in rats. Endocrinology 22
reversible
in the thinned
Somatomedin-C mediates growth effects on both the hypothalamus 1281 18.
defect
Ball MF, el-Khodary sponse
6.
a pituitary 311:1403-1407
Finkelstein JA, Bervois P, Mendue M, Willougby JO 1986 Growth hormone and prolactin secretion Endocrinology 118:1233-1236
1. Renier G, Gaudreau
2.
P, Deslauriers C, Brazeau P 1989 In vitro and irl vivo growth hormone responsiveness to growth hormonereleasing factor in male and female Zucker rats. Neuroendocrinology 50:454-459 Davies RR, Turner SJ, Cook D 1985 The response of obese subjects to continuous infusion of human pancreatic growth hormone-releasing factor l-44. Clin Endocrinol (Oxf) 23:521-525
IN OBESE
in normal men and acts synertistically with GHJ Clin Endocrinol Metab 70:975-982 Leidy Jr JW, Roman0 TM, Millard WJ, Developmental changes of the growth hormone axis in lean and obese Zucker male and female rats. 74th Annual Meeting of The Endocrine Society, San Antonio TX, 1992 (Abstract 463) Selye H 1951 Role of somatotropic hormone in the production of malignant nephrosclerosis, periateritis nodosa and hypertensive disease. Br Med J 1:263-273
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
Effects of Coadministered Growth Hormone (GH)Releasing Hormone and GH-Releasing Hexapeptide Maladaptive Aspects of Obesity in Zucker Rats BARRY B. BERCU, SEI-WON RICHARD F. WALKER
YANG,
Departments of Pediatrics, Pharmacology, Tampa, Florida 33612; and All ChildrenS
RYUJI
MASUDA,
CHENG-SHIH
HU,
on
AND
and Therapeutics, University of South Florida, Hospital, St. Petersburg, Florida 33701
ABSTRACT The purpose of this study was to determine the effect of chronic pharmacological stimulation of the pituitary gland on GH hyposecretion and other maladaptive aspects of obesity. Obese Zucker rats were coadministered GH-releasing hormone (GHRH; 3 fig/kg) and GHreleasing hexapeptide (GHRP-6; 300 pg/kg), a potent combination of synergistic GH secretagogues, once daily for 60 consecutive days. Although pituitary weights and GH concentrations were higher in obese rats administered the peptides than in obese rats administered saline, stimulated GH secretion was lower in obese rats than in lean rats. However, compared to those in lean rats, plasma insulin-like growth factor-I and insulin concentrations were higher in the obese rats regardless of treatment. The GH secretagogues did not alter food intake or body weight gain in sexually mature obese rats, whereas body weight
gain was significantly increased when they were administered to prepubertal obese rats. Although glucose tolerance was impaired in both groups of obese rats, it improved in obese rats administered GHRH and GHRP-6 compared to that in obese rats administered saline. On the other hand, plasma cholesterol concentrations were elevated in obese rats administered the GH secretagogues but not saline. In conclusion, the results of this study suggest that hyposensitivity to GHRH and GHRP-6 in obese Zucker rats results from high concentrations of plasma insulin-like growth factor-1 that negatively feedback on stimulated GH secretion. Nonetheless, daily episodes of endogenous GH secretion resulting from chronic coadministration of GH secretagogues significantly influenced the pituitary gland as well as lipid and carbohydrate metabolism. (Endocrinology 131: 2800-2804, 1992)
S
mechanismto GHRH (11, 12). Thus, it was of interest to test the efficacy of the peptide combination in Zucker rats for stimulating endogenous GH secretion and its potential for reversing certain maladaptive aspectsof obesity.
TIMULATED and spontaneous GH secretion as well as pituitary GH content are significantly lower in obese animals compared to those that are lean (l-4). Since diminished GH secretion can be reversed by food restriction (3, 5), the endocrine defect is probably an effect, rather than a cause of obesity. However, since GH treatment reduced lipid deposition (6), GH hyposecretion may sustain obesity once it is established. GH deficiency may also contribute indirectly to the establishment of obesity by increasing appetite. This hypothesis is basedupon the fact that food consumption was decreased in rats administered GH-releasing hormone (GHRH) directly into the brain (7), and body weight gain/ hyperphagia was prevented in rats administered GH immediately after surgical destruction of the hypothalamic ventromedial nucleus (VMH; satiety center) (8). The purpose of the present study was to determine whether endogenous GH secretion could be increasedpharmacologically in genetically obeseZucker rats, and if chronic treatment with GH secretagogueswould reverse certain maladaptive changesassociatedwith obesity. Obese Zucker rats respond poorly to GHRH (1, 9). However, in a prior study, we (10) discovered that old rats hyporesponsive to individually administered GH secretagogueswere hyperresponsive to coadministered GHRH and GH-releasing hexapeptide (GHRP-6), a synthetic peptide unrelated in structure or Received July 20, 1992. Address all correspondence and requests for reprints to: Dr. Richard F. Walker, Division of Endocrinology, Box 690, All Children’s Hospital, 801 Sixth Street South, St. Petersburg, Florida 33731-8920.
Materials and Methods Animal
husbandry
Obese and lean female Zucker rats (30 and 80 days of age; Charles River Breeding Laboratories. Wilmington, NC) were acclimated to local conditions for 1 week before being used in the present study. The older rats weighed between 410-479 g, while the younger rats weighed between 183-216 g when the experiment began. Female rats were used because they respond more consistently and reproducibly to GHRP-6 than do conscious males (13). Housing conditions included alternating 12-h periods of light and darkness (0600-1800 h), 60 f 5% relative humidity, 72 f 2 F ambient temperature, and constantly available food (Purina rat chow, Ralston-Purina, Kalamazoo, MI) and tap water. The animal facility at All Children’s Hospital in which this study was performed is a fully accredited institutional member of the American Association of Laboratory Animal Sciences and complies with federal statutes and regulations for animal use in research.
Blood sampling Samples for sequential analysis of plasma GH concentrations were collected from anesthetized rats between 0900-1100 h, except when surgical procedures and sampling consumed a full day (-0800-1800 h). When collection intervals were extended, samples were drawn alternatively from the different groups so as to control for time of day variation. Sequential blood samples for analysis of temporal GH secretory profiles in anesthetized female rats were collected via cannulae that were surgically placed within the femoral artery after the administration of
2800
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
GH SECRETION
ketamine (60 mg/kg, im) and pentobarbital (40 mg/kg, ip). All rats in a given experiment were surgically prepared en masse, and treatments were alternated between individual animals so that anesthesia times for all groups were approximately the same. Peptide solutions (1 ml/kg) were administered as 30.set intraarterial infusions, and blood samples for GH analysis were collected at designated times into heparinized tubes. When administered SC, GHRP-6 is rapidly absorbed and stimulates GH release in a manner indistinguishable from that after iv administration (13, 14), precluding the need to restrain the rats for iv administration via the tail. These characteristics allow GHRP-6 administration with minimal stress and support the use of SC administration whenever possible.
Experimental
design
Obese and lean Zucker rats were randomly assigned to 3 groups (12 rats/group) designated to receive daily SC injections of GHRH and GHRP-6 (1 group of obese rats) or saline (1 group of obese and 1 group of lean rats). Solutions (1 ml/kg) of GHRP-6 and rat GHRH (300 and 3 rg/kg, respectively) or saline were administered at approximately 0900 h for 60 consecutive days. In a preliminary dose-range study, the dose of 300 pg/kg GHRP-6 was the ED ,oD in conscious 3-month-old female rats (14). The dose of GHRH (3 pg/kg) was selected from published data showing that the peptide was efficacious when administered SC at a dose of 10 fig/kg (15). The lower dose was used in the present study because of the known synergistic activity of GHRH and GHRP-6 and its efficacy in GHRH-resistant rats (10). A preliminary analysis of individually administered GHRH and coadministered GHRH/GHRP-6 efficacy in obese rats was performed on the first day of the study. Lean and obese rats (six rats per group) were anesthetized, and their right femoral arteries were cannulated. F’eptides were administered, and blood samples (-250 ~1) were collected in heparinized tubes before and at selected intervals after administration of the GH secretagogues. The rats were exsanguinated at the last blood sample, and plasma was subsequently analyzed for GH concentrations. Since individually administered GHRH was ineffective in obese rats (Fig. l), the remaining animals received daily SC injections of saline or GHRP-6/GHRH for a total period of 60 consecutive days. Body weight and food consumption were determined at regular intervals, and lean body mass was determined after 30 days of treatment. Lean body mass was estimated using the ratio of water displacement volume to dry body weight. The rats were immersed in a water-filled tank containing a series of concentric rings that acted as baffles to reduce surface turbulence
T
IN OBESE RATS
2801
resulting from swimming. Thus, at the tank perimeter, the water rose without turbulence, and spilled through a channel into a collecting vessel. This apparatus allowed accurate and reproducible measurement of water displaced by each rat, which was then weighed (1 gm Hz0 = 1 cc) and divided by the animal’s dry body weight (volume of Hz0 displaced/body weight; V/W) to provide a body composition index (BCI) or estimate of lean body mass. Since fat is less dense than muscle, decreasing the V/W indicated increasing lean body mass, A glucose tolerance test was performed after 40 days of treatment to determine the effects of daily episodic release of endogenous GH on carbohydrate metabolism in obese rats. A 30% aqueous solution of glucose (1 g/kg; 3 ml/kg) was administered as an ip injection to unanesthetized rats from which food was withheld for approximately 8 h. Blood samples were taken from a tail vein before the glucose injection and at 15-min intervals thereafter. Blood glucose concentrations were determined using an ExacTech Blood Glucose Sensor (MediSense, Inc., Cambridge, MA). On the final day of treatment, all rats were anesthetized with ketaminepentobarbital and administered GHRH (1 rg/kg) and GHRP-6 (30 fig/ kg), iv, to determine the effects of long term treatment on stimulated GH secretion, serum nonesterified FFA (blood was collected into nonheparinized tubes), plasma insulin-like growth factor-l (IGF-I), insulin, and cholesterol concentrations. After exsanguination, the pituitary glands were rapidly removed, weighed, and frozen on dry ice for subsequent estimation of GH concentrations, All procedures in this experiment were described in a protocol that was approved by the University of South Florida Institutional Animal Care and Use Committee.
Quantitation
of
hormone
concentrations
Plasma GH, IGF-I, and insulin concentrations were estimated by RIA. Blood samples were centrifuged, and plasma was collected for RIA analysis of GH concentrations, Pituitaries were homogenized in 0.1 N NaOH-saline and diluted, and aliquots were analyzed by RIA for GH concentrations. GH concentrations were estimated in duplicate by RIA and expressed in terms of the reference preparation rat GH RP-2 from the NIDDK. Radiolabeled rat GH was purchased from Chemicon lnternational, Inc. (Temecula, CA), and precipitating antibody (goat antimonkey y,y[I’-41) was purchased from Antibodies, Inc. (Davis, CA) and used at a concentration of 1:lO. lntra- and interassay coefficients of variation were less than 5% and less than 8%, respectively. IGF-I concentrations were estimated after acid-ethanol extraction from plasma, using methods provided in a commercially available kit (Nichols Institute, San Juan Capistrano, CA). Plasma insulin concentrations were determined according to the method of Yalow and Berson (16), using Lilly pork insulin I’] 5682 as a reference standard. Serum FFA and cholesterol were measured using commercially available reagents from Wako Chemicals USA (Dallas, TX; kit 40-2100) and Sigma Chemical Co. (St. Louis, MO; kit 352), respectively.
Chemicals GHRP-6 (His-o-Trp-Ala-Trp-o-Phe-Lys-NH,) and rat GHRH were purchased from Peninsula Laboratories (Belmont, CA). Controls received saline administered in an equal volume and by the same route as the peptides.
Data analysis
0
B
15
TIME
20
25
30
(minutes)
Frc. 1. Comparison of GH secretion after a provocative dose of GHRP6 (A and A) or GHRH and GHRP-6 (0 and 0) in obese rats (A and 0) and lean rats (A and 0). Rats were never exposed to the GH secretagogues, either alone or in combination, before the provocative dose was administered.
Pituitary and plasma hormone concentrations as well as tissue weights and body composition indices were compared among the groups of young and old rats using analysis of variance with Duncan’s multiple range test. Changes in peptide-stimulated plasma GH concentrations, plasma glucose, and body weight change over time were compared using repeated measures analysis. Results were expressed as the mean f SEM. P < 0.05 was used to define statistically significant differences.
Results
Coadministered GHRH and GHRP-6 were a lesseffective stimulus for GH secretion in obeserats than in lean rats on
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
2802
GH SECRETION IN OBESE RATS
the first and last days of treatment. However, as shown in Fig. 1, the combination of peptides was synergistic in the obese rats, stimulating significantly more endogenous GH secretion than GHRH alone. Since GHRH was essentially ineffective in the obese rats, only the combination of GH secretagogueswas administered for the remainder of the study. Although GHRH and GHRP-6 remained effective after 45 consecutive days of administration to the obeserats, the GH secretory responseswere not altered by duration of treatment, and peak plasma GH concentrations remained approximately one third of those in lean rats throughout the study (Fig. 2). Basal plasma GH concentrations were comparable in lean and obeserats. As shown in Table 1, plasma IGF-I concentrations were significantly higher in both groups of obeserats than in lean rats, despite the lack of pituitary stimulation in response to saline or lower stimulated GH secretion in responseto GHRH and GHRP-6. Furthermore, the elevated IGF-I concentrations were not strongly correlated with the administration of GH secretagogues.Although mean IGF-I levels in obeserats coadministered GHRH and GHRP-6 were higher than those in obeserats administered saline, the differences were not statistically significant. Similar trends were observed for serum nonesterified FFA and insulin concentrations, which were significantly greater in 1,200
-
l,fJ@J s‘
E 3 5
6W-
5
600-
s $ 400 200
Endo. Vol131.
1992 No 6
both groups of obese rats than in lean rats. In contrast, plasma cholesterol concentrations were significantly greater in obese rats coadministered GHRH and GHRP-6 than in obeserats or lean rats administered saline, in which cholesterol concentrations were comparable (Table 1). The changes in cholesterol concentrations were attributed either directly to the GH secretagoguesor indirectly to the increase in endogenousGH that they produced. The data presented in Fig. 3 show that body weight gain was not reduced in 80-day-old obese rats coadministered GHRH and GHRP-6 for 45 consecutive days despite their relatively high plasma concentrations of stimulated GH and basal IGF-I. However, when peptide administration was begun at 30 days of age, body weight was significantly increasedcompared to that of saline-treated obesecontrols. Although statistical significance was not reached, the mean BCI, a measureof lean body mass,was reduced in obeserats administered GH secretagogues,suggestingthat the peptides contributed to protein, rather than fat, deposition. BCI in the heterozygous Zucker rats was significantly lower, indicating leaner body massfor these rats than either group of homozygous Zucker rats (Fig. 4). BCI varied more after coadministration of GHRH and GHRP-6 than before treatment began, suggestingthat quantitative differences occurred in the responsesof individual rats to the GH secretagogues. The data presented in Table 2 show that increased body weight was accompanied by greater food intake in obese rats. Mean food intake per day was remarkably constant in lean and obese animals, suggesting that the hypothalamic satiety center had a higher set-point in the latter group, which corresponded to their greater body weights. Apparently, that set-point was not altered by peripherally administered GHRH, GHRP-6, stimulated endogenous GH, or elevated plasma IGF-I. As alsoshown in Table 2, pituitary weight and GH content, which were significantly lower in saline-treated obese rats than in lean rats, were significantly higher in GH secreta-
0
0
6
15
25
35
600
TIME (MINUTES) FIG. 2. GH secretion in response to a provocative dose of GHRH and GHRP-6 in lean rats (A) or obese rats (0) previously administered saline compared to that in obese rats (0) previously coadministered GHRH and GHRP-6 for 60 consecutive days. The response in lean rats is significantly greater (P < 0.01) than that in obese rats. Differences in stimulated GH secretion between the two groups of obese rats are not statistically significant. n = 6 rats/group.
TABLE consecutive substances
1. Effects
of GHRH and GHRP-6 coadministration for 4.5 days on serum and plasma concentrations of various in obese and lean Zucker rats
Group and treatment Lean (saline) Obese (GHRH + GHRP-6) Obese (saline)
Plasma IGF-I Wml)
Plasma insulin NJ/ml)
Plasma cholesterol (mg/dl)
Serum FFA (meqjliter)
753 + 48” 1033 + 43
12.6 f 3” 48.4 f 7
54.1 + 7.7 118.1 f 10.6”
0.38 + 0.06” 1.9 + 0.8
957 + 59
39.4 I? 6
74.6 + 4.9
0.87 + 0.2
Values represent the mean + SEM for determinations from six rats per group. ” Significantly different (P < 0.05) from values for obese rats.
-5oc-
E 8 ; ii 3
400-
300
-
$I $ 200 -
loo
‘, 0
I 5
I 10
I 15
I 20
I 25
I 30
I 40
I 60
I 60
DAYS WC. 3. Body weight changes in rats administered saline (0, A, and n ) or GHRH and GHRP-6 (0, A, and 0). Treatment was begun in lean and obese rats at 80 days of age (W, 0 and 0, 0, respectively) or in obese rats at 30 days of age (A and A). Values represent the mean + SEM for six rats per group. *, Significantly different (P < 0.05) from saline treatment in same age group.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
GH SECRETION
IN OBESE RATS
2803
1.00
0.95
-
0.9il
-
0.65 -
-+-
0.80 -
0.75 -
0.70 0
0.65
-
0.60
0
GHRH + GHRP-6
Saline
Obese
GHRH + GHRP-S
Saline Lean
FIG. 4. Body composition in obese and lean rats administered (m and Cl, respectively) or coadministered GHRH and GHRP-6 0, respectively) for 30 consecutive days. Values represent the (horizontal lines), SEs (rectangles), and ranges (vertical lines) rats per group. *, Significantly different (P < 0.05) from obese treated rats.
saline ( means for six saline-
TABLE 2. Effect of GHRH and GHRP-6 coadministration for 45 consecutive days on food consumption and the pituitary gland in obese and lean Zucker rats Group and treatment
‘,
Food wt consumed Pituitary bd k/day) 14 f 0.9” 11.3 * 0.4 23 + 1.3 10.1 f 0.7
G~~~n~~nt (d&d) 91 + 1.9 79 + 2.0
Pituitary GH cow. b&w) 8.2 f 1.8 9.4 + 3.5
Lean (saline) Obese (GHRH + GHRP-6) 6.1 f 0.4” 66 + 1.1’ 10.9 + 1.3 Obese (saline) 22 + 1.0 Values represent the mean + SEM for six rats per group. ’ Significantly different from other groups (P < 0.05).
gogue-treated obeserats. Despite group differences in pituitary weight and GH content, pituitary GH concentrations were comparable in all lean and obeserats. Glucose intolerance, as indicated by prolonged periods of elevated blood glucose concentrations after ip administration of the sugar, was observed in obesesaline-treated rats, whereas glucose tolerance tests improved in obese GH secretagogue-treated rats (Fig. 5). These data suggestthat obesity-associatedglucoseintolerance was partially resolved by daily episodesof endogenousGH secretion. Discussion The main finding of this study was that coadministered GHRH and GHRP-6 were an effective stimulus, albeit less efficacious than in lean rats, for endogenousGH secretion in obese rats. Nonetheless, the GH secretagoguesincreased pituitary weight and pituitary GH content, suggestingthat GH hyposecretion in the obese rats resulted from reduced pituitary stimulation and/or a functional defect in the mechanism for GH release,as proposed by Renier et al. (1). It is possiblethat high plasma IGF-I concentrations in the obese
I
I
I
I
15
30
45
90
TIME (MINUTES) FIG. 5. Glucose tolerance tests in lean rats (A) and obese rats (Cl) previously administered saline or previously coadministered GHRH and GHRP-6 (0) for 60 consecutive days. Changes in blood glucose as a function of time in obese saline-pretreated rats are significantly different (P < 0.05) from those in lean saline-pretreated rats. Values represent the mean f SEM for six rats per group.
rats inhibited GH releaseeither by direct negative feedback at the pituitary or indirectly and in concert with GHRP-6 by releasingsomatostatin (13, 17, 18). This is the first documentation of elevated in vivo plasma IGF-I concentrations in the obesefemale Zucker rat, and its causeis unknown. However, elevated IGF-I is seen clinically in obesechildren with suppressed GH secretion, and it may simply be related to increasedinsulin concentrations associatedwith hyperphagia. Hyperinsulinimia (2) and elevated plasma triglycerides (19) in obese Zucker rats have also been associatedwith GH hyposecretion (20,21) and, asshown in this study, correlated with increasedfood intake. The logic for coadministering GHRH and GHRP-6 to reduce food intake and maintain normal body weight was based upon the fact that hyperphagia and obesity resulting from destruction of the VMH (satiety center) in rats can be prevented with exogenousGH (8). Although obeserats were hyposensitive to GHRH, the peptide was coadministered with GHRP-6 in this study becausetheir actions are synergistic (22), causingepisodesof endogenousGH secretionthat achieved peak plasma concentrations of approximately 350 rig/ml. However, the peptide combination was ineffective in reducing food intake and body weight gain in obeseZucker rats. In fact, body weight gain in the homozygous rats was significantly increased when treatment was begun in weanling rats compared to adults. Additional weight gain in the younger rats was attributed to acceleration of the increase in plasma IGF-I that occurs spontaneously during maturation in obeseZucker rats (23). The data were insufficient to determine whether the additional weight gain in young rats chronically administered GHRH and GHRP-6 resulted from an increasein lean body mass.Although the mean BCI in obeserats coadministered GHRH and GHRP-6 was slightly lower than that in obese rat administered saline, the differences were not statistically significant. The lack of a significant change in BCI in the obeserats coadministered GHRH and GHRP-6 was attrib-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
STIMULATED
GH SECRETION
uted to limits of the resolving power of the water displacement method. In heterozygotes, which were much leaner than the homozygotes, the differences in BCI were statistically significant. Increased weight in obese peptide-treated rats could alsobe attributed to altered lipid metabolism, since their plasma cholesterol concentrations were significantly higher than those in the other obeseor lean groups. Alternatively, increased plasma cholesterol may have occurred secondarily to GH-mediated renal dysfunction (24). However, if GHRH- and GHRP-6-stimulated endogenous GH has the potential to cause renal dysfunction, obese Zucker rats may be uniquely vulnerable, since similar effects were not observed in female Fischer 344 rats administered the peptides for 60 consecutive days (our unpublished data). One objective of the present study was to determine whether established obesity could be reversed by elevating endogenous GH, not to prevent weight gain or decrease hyperphagia before they occurred. The data from this study suggestthat stimulated GH is not effective for that purpose. Perhaps, pharmacologically stimulated GH would have prevented hyperphagia and reduced body weight gain in the obeseZucker rat if treatment was begun in preobeseneonates (~5 days of age), since VMH lesion-induced obesity was prevented when GH was administered immediately after surgery (8). Furthermore, the route of administration may have prevented GHRH from reducing food intake, as it did when administered directed into the brain (7). An unexpected finding of this study was that obesityassociatedglucose intolerance in saline-treated homozygous Zucker rats was partially corrected in GH secretagoguetreated rats. This effect, which is not consistent with a diabetogenic effect of GH, may reflect normalization of the combined influences of insulin and GH on carbohydrate metabolismthat were restored by coadministration of GHRH and GHRP-6. In conclusion, the results of this study confirm attenuated GH secretion in obeserats administered synergistic GH secretagogues for up to 45 consecutive days. Nonetheless, coadministration of GHRH and GHRP-6 significantly increased plasma GH concentrations. Although the resulting increase in endogenous GH did not reduce hyperphagia or body weight gain in the genetically obeserats, changes in carbohydrate and lipid metabolism suggestthat the animals were responsive to episodesof stimulated endogenous GH secretion and support further studies into the therapeutic potential of GH secretagoguesin obesity. References
3. Williams T, Berelowitz M, Joffe SN, Thorner MO, Rivier J, Vale W, Frohman L 1984 Impaired growth hormone responses to growth
RATS
hormone-releasing factor in obesity: with weight reduction. N Engl J Med 4.
5.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
19 20 21
in genetically
obese
24
rats.
Cheng K, Chan WW-S, Barreta A, Convey DM, Smith RG 1991 Evidence for a role of protein kinase-C in His-o-Trp-Ala-Trp-o-I’heLys-NH,-induced growth hormone release from rat primary pituitary cells. Endocrinology 129:3337-3342 Bercu BB, Weideman CA, Walker RF 1991 Sex differences in growth hormone (GH) secretion by rats administered GH releasing hexapeptide. Endocrinology 129:2592-2598 Bercu BB, Yang S-W, Masuda R, Walker RF 1992 Role of selected endogenous peptides in growth hormone releasing hexapeptide (GHRP-6) activity: analysis of GHRH, TRH and GnRH. Endocrinology 130:2579-2586 Shulman DI, Sweetland M, Duckett G, Root AW 1987 Age-related differences in the growth hormone secretory response to hGHRH l-44 in male rats from infancy through puberty. Zri vivo and in vifro studies. Acta Endocrinol (Copenh) 116:138-144 Yalow RS, Berson SA 1964 Immunoassay of plasma insulin. Methods Biochem Anal 12:69-76
Berelowitz
M, Szabo M, Frohman LH, Fireston S, Chu L 1981 hormone negative feedback by and pituitary. Science 212:1279-
Sheppard
MC, Shapira B, Hudson A 1980 Tissue and serum somatostatin-like immunoreactivity in lean and obese Zucker rats. Horm Metab Res 12:236-239 Barry WS, Bray GA 1969 Plasma triglycerides in genetically obese rats. Metabolism 18:833-839 Melmed S 1984 Insulin suppresses growth hormone secretion by rat pituitary cells. J Clin Invest 73:1425-1433 Imaki T, Shibasaki T, Masuda A, Hotta M, Yamauchi N, Demura H, Shizume K, Wakabayashi I, Ling N 1984 The effect of glucose (GH)-releasing 118:2390-2394
factor-
Bowers CY, Reynolds GH, Durham D, Barrera CM, Pezzoli SS, Thorner MO 1990 Growth hormone (GH)-releasing peptide stimulates GH release releasing hormone.
23
Zucker
AZ, Canary JJ 1972 Growth hormone reobese. J Clin Endocrinol Metab 34:498-511 Martin RJ, Jeanrenaud B 1985 Growth hormone in obesity and diabetes: inappropriate hypothalamic control of secretion, Int J Obesity 9:99-104 Vaccarino FJ, Bloom FE, Rivier J, Vale1 W, Koob GF 1985 Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor. Nature (Lond) 314:167-168 York DA, Bray GA 1972 Dependence of hypothalamic obesity on insulin, the pituitary and the adrenal gland. Endocrinology 90:885894 Gaudreau I’, Renier G, Prazeau P 1987 rGHR(l-29)NH,-induced growth hormone secretion in genetically and dietary obese rats. Sot Neurosci Abstr 13:667 Walker RF, Yang S-W, Bercu BB 1991 Robust growth hormone (GH) secretion in aged female rats co-administered GH-releasing hexapeptide (GHRP-6) and GH-releasing hormone (GHRH). Life Sci 49:1499-1504 Codd EE, Shu AYL, Walker RF 1989 Binding of the growth hormone releasing peptide GHRP-6 to specific hypothalamic and pituitary sites. Neuropharmacology 28:1139-l 144
and free fatty acids on growth hormone mediated GH secretion in rats. Endocrinology 22
reversible
in the thinned
Somatomedin-C mediates growth effects on both the hypothalamus 1281 18.
defect
Ball MF, el-Khodary sponse
6.
a pituitary 311:1403-1407
Finkelstein JA, Bervois P, Mendue M, Willougby JO 1986 Growth hormone and prolactin secretion Endocrinology 118:1233-1236
1. Renier G, Gaudreau
2.
P, Deslauriers C, Brazeau P 1989 In vitro and irl vivo growth hormone responsiveness to growth hormonereleasing factor in male and female Zucker rats. Neuroendocrinology 50:454-459 Davies RR, Turner SJ, Cook D 1985 The response of obese subjects to continuous infusion of human pancreatic growth hormone-releasing factor l-44. Clin Endocrinol (Oxf) 23:521-525
IN OBESE
in normal men and acts synertistically with GHJ Clin Endocrinol Metab 70:975-982 Leidy Jr JW, Roman0 TM, Millard WJ, Developmental changes of the growth hormone axis in lean and obese Zucker male and female rats. 74th Annual Meeting of The Endocrine Society, San Antonio TX, 1992 (Abstract 463) Selye H 1951 Role of somatotropic hormone in the production of malignant nephrosclerosis, periateritis nodosa and hypertensive disease. Br Med J 1:263-273
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 16:38 For personal use only. No other uses without permission. . All rights reserved.
