- Email: [email protected]
Effect of Hypophysectomy and Growth Hormone on Serum and Antral Gastrin Levels in the Rat
1976 Copyright © 1976 by The Williams & Wilkins Co.
Vol. 70, No.5
GASTROENTEROLOGY 70:727~732,
Printed in U.S.A.
EFFECT OF HYPOPHYSECTOMY AND GROWTH HORMONE ON SERUM AND ANTRAL GASTRIN LEVELS IN THE RAT M. REBECCA ENOCHS, Ph.D., AND LEONARD R. JOHNSON, Ph.D. Department of Physiology, University of Texas Medical School, Houston, Texas
The effects of hypophysectomy and subsequent replacement therapy with growth hormone on serum and antral gastrin levels was investigated in both fasted and nonfasted pair-fed rats. Hypophysectomy caused a 57% decrease in serum and 47% decrease in antral gastrin content in 18-hr fasted animals. In nonfasted animals, hypophysectomy resulted in a 42% fall in serum gastrin and a 76% fall in antral gastrin. Animals were given injections of growth hormone over a lO-day period, then fasted 16 to 18 hr before being killed. Doses of 100 to 200 /-Lg per 100 g of body weight were ineffective, but a dose of 500 /-Lg per 100 g was sufficient to restore completely serum gastrin levels to intact control values. In nonfasted rats this same dose raised serum gastrin to intact levels and increased antral gastrin significantly over hypophysectomized levels, but did not increase it to control values. When given to pair-fed intact animals, growth hormone caused slight but not significant elevations in serum and antral gastrin. The effect of growth hormone on gastrin secretion and/or synthesis may be a significant physiological factor in the regulation of normal gastrointestinal function and growth. Hypophysectomy has been clearly documented as causing trophic changes in the gastrointestinal tract of several species, including man. These changes include a decrease in mucosal weight in the submaxillary glands, stomach, pancreas, and small intestine. 1, 2 This decrease in weight is accompanied by a decrease in RNA content, enzyme activity, and zymogen granules in the salivary glands. 1 In the small intestine, hypophysectomy leads to mucosal atrophy, decreased enzyme activity, and decreased mitotic rate. 1, 3, 4 The pancreas exhibits a decrease in RNA and protein content, decreased cytoplasmic granules, and depressed output of amylase and proteolytic enzymes. 1, 5, 6 In the stomach there is hypomotility, mucosal atrophy, involution of chief and parietal cells with an associated decrease in secretory volume, and acid and pepsin output. 1, 2, 7, 8 The growth of the gastric mucosa in hypophysectomized rats, as measured by mucosa volume, is partially restored by growth hormone (GH), but not by other pituitary hormones. 2 In fact adrenocorticotrophic horReceived August 4, 1975. Accepted November 10, 1975. Address correspondence to: Dr. M. Rebecca Enochs, Department of Physiology, University of Texas Medical School, P. O. Box 20708, 6400 W. Cullen Street, Houston, Texas 77025. This study was supported by National Institutes of Health Grant AM 16505. Dr. Johnson is the recipient of Research Career Development Award AM 28972 from the National Institutes of Health. The authors are grateful to Dr. John H. Walsh of the Department of Medicine, University of California at Los Angeles, for the generous gift of Ab 1296, raised in rabbits immunized with heptadecapeptide gastrin-I. In addition, the authors are indebted to Dr. John Walsh and Dr. G. Dockray of U.C.L.A., and Dr. Linda D. Sander of the University of Texas Medical School, Houston, Texas, for assistance in establishing the gastrin radioimmunoassay. 727
mone (ACTH) may have the reverse effect. 2, 9 Despite findings that ACTH depresses mucosa volume in hypophysectomized rats 2 and mitotic rate in intact dogs,9 cortisone has been found to increase gastric secretory function in hypophysectomized dogs. 8 This apparent discrepancy may be due to species difference, dose effects, or specific inhibitory action of ACTH on stomach growth. The effect of GH showed no such discrepancy. GH increased mucosa volume in the hypophysectomized rat 2 and increased histamine-stimulated gastric secretory function in hypophysectomized dogs. 8 Several lines of evidence suggest that GH effects on gastrointestinal growth and function may be mediated, at least in part by gastrin. The specific trophic action of gastrin on gastrointestinal tissues has been established well. 10, 11 Chronic administration of a pharmacological dose of pentagastrin to hypophysectomized rats has been shown to increase pancreatic weight, as well as RNA and DNA content. 5 Dorchester and Haist reported that the secretin content of rat small intestine, as determined by bioassay, is depressed after hypophysectomy.12 This depression was prevented by anterior pituitary extracts, growth hormone, or ACTH. 13 An effect of GH on gastrin release was suggested by observations that patients with acromegaly sometimes have abnormally high maximal acid output, 14 and may have high antral gastrin levels as well as hyperplasia of antral G cells, 15 although the latter report has since been retracted. 16 This investigation was undertaken to study the effect of GH on serum and antral gastrin levels in the rat. Materials and Methods All animals used in these studies, both intact and hypophysectomized, were male Sprague-Dawley rats weighing 140 to
728
Vol. 70, No . .5
ENOCHS AND JOHNSON
170 g. Both intact and hypophysectomized rats were obtained from Charles Rivers, Wilmington, Massachusetts. Hypophysectomized rats were given 5% dextrose in their drinking water, and allowed to recover from surgery for at least 2 weeks before use. The rats were housed three per cage in a room with a fixed lighting schedule, 12 hr on, 12 hr off. During periods of fasting, animals were housed in cages with wide mesh bottoms. Bovine GH (Calbiochem, Los Angeles, Calif., cat. no. 369152, 1.0 USP U per mg) was administered as a saline solution, pH 8.5. The biological activity of the hormone preparations was ascertained by monitoring rat weight changes over the course of the experimental period. A significant increase in weight was considered an indication that the preparation was biologically active. The animals were grouped in two ways: 1. In experiments using hypophysectomized rats, the animals were divided into three groups: (1) hypophysectomized controls; (2) hypophysectomy + GH; (3) intact controls. In these experiments all groups received a daily ration of food equal to the daily caloric intake of the hypophysectomized control group. Hypophysectomized controls were given intraperitoneal injections of saline twice daily for 10 days. Hypophysectomized animals given GH received intraperitoneal injections of the specified doses twice daily for 10 days. Intact control rats received no injections. In those experiments which are termed fasted, all groups were removed from food 16 (0 18 hr before being killed. In those experiments termed nonfastE:;d, animals remained in the feeding cages up until the time of sacrifice. 2. In experiments using only intact rats, animals were divided into two groups: (1) intact controls; (2) intact + GH. In these experiments the GH-treated animals were given a daily ration of food equal to the caloric intake of the intact controls. Fasted animals were removed from food 16 to 18 hr before being killed. Nonfasted animals remained in feeding cages until sacrifice. In all experiments the animals were sacrified between 9:00 and 11:00 AM to minimize effects of diurnal variation. The animals were weighed and anesthetized with ether. A midline incision was made and blood was withdrawn directly from the heart. Antral tissue was prepared according to the method described by Lichtenberger et al. 17 The antrum was removed and carefully trimmed. It was then weighed and homogenized in 1 II!1 of deionized water. One-half milliliter of the homogenate was removed and immediately added to 2 ml of boiling deionized water. The antral extract was boiled for 20 min then centrifuged at 12,000 rpm for 30 min. Serum and antral extracts were frozen for gastrin radioimmunoassay. The gastrin radioimmunoassay was performed according to the method of Yalow and Berson 18 using the antibody characterized by Dockray and Walsh. 19 Synthetic human gastrin I (1-17), obtained from Imperial Chemical Industries, Ltd., England. was used as a standard. Gastrin levels are expressed as picograms per milliliter of serum and micrograms per gram wet weight of antrum. The difference between means was evaluated using Student's t-test for unpaired data. Differences were considered significant if P < 0.05.
Results Body weights of hypophysectomized animals receiving daily injections of saline dropped very slightly over the 10-day experimental period (table 1). Intact rats which were pair-fed with hypophysectomized rats for 10 days
TABLE
1. Body weights at beginning and end of IO-day injection period
po
Final
lniti al
Group (no.)
m ean ± SEM
Hypophysectomized control (19) Hypophysectomy (19) andGH (12) Intact control a
152.7
±
3.16
150.1
±
3.15
< 0.025
150.18
±
3.22
177.53
±
3.14
< 0.001
158.67
±
14.60
148.42
±
16.40
< 0.001
Values were obtained using the paired t -test.
lost about 10 g. This can be accounted for by the limitation of their normal food intake to the decreased level of hypophysectomized animals. GH-treated hypophysectomized rats consistently gained from 20 to 30 g over the lO-day period, indicating that the hormone preparation was biologically active. Hypophysectomy alone caused a marked decrease in both serum and antral gastrin levels. In fasted animals, serum gastrin dropped from 227 pg per ml in intact rats to 97 pg per ml in hypophysectomized rats, a decrease of 57 %. Antral gastrin dropped from 15.5 p.g per g wet weight, to 8.25 p.g per g, a decrease of 47 % (fig. 1). In fed animals, serum gastrin was decreased by 42 % from 293 to 170 pg per ml, and antral gastrin by 76% from 27.89 p.g per g to 6.56 p.g per g (fig. 2). Figure 3 shows the effects of three different doses of GH on fasted serum gastrin levels in hypophysectomized rats. Doses of 100 and 200 p.g of GH per 100 g of body weight caused no significant change in serum gastrin levels . Adose of 500 p.g per 100 g, however, was sufficient to raise serum gastrins in hypophysectomized animals up to that of intact pair-fed controls. Although there was considerable variability in the GH-treated rats, the mean value was significantly higher than that of hypophysectomized controls, but not significantly different from intact controls. Antral determinations were not made on these animals. Because the presence of food in the stomach has such dramatic effects on gastrin levels, we also looked at the effect of GH in pair-fed animals which were allowed to feed up until the moment they were killed. All animals, both hypophysectomized and intact, had varying amounts of food in their stomachs. Despite this, the three groups had serum gastrin levels which were qualitatively similar to those of fasted animals (fig. 4) . Quantitatively, as would be expected, their gastrin levels were higher. This increase was significant (P < 0.025) only in hypophysectomized controls. Again there was considerable variability in GH-treated animals, but they still demonstrated a significant increase in mean serum gastrin over hypophysectomized controls. Antral gastrin values were also elevated by GH ; however, they remained significantly (P < 0.05) lower than intact controls. Effects of exogenous GH on tissue growth are generally more easily demonstrated in hypophysectomized animals. To determine whether this also applied to the
729
GROWTH HORMONE AND GASTRIN
May 1976
stimulation of gastrin release, GH was injected into intact animals. Exogenous GH stimulated a slight but not significant increase in serum gastrin in both fasted and nonfasted animals (tables 2 and 3). Only fasted rats
showed an increase in antral gastrin. The experiment was not carried out for 10 days, so a direct comparison with the effect on hypophysectomized animals cannot be made.
300
E
---
.e: C)
20
200
z
a: en I-
l> Z -I
15
«
~
l>
r
<.!l
G')
~
:::>
l>
en
10
100
a: w en
-I ~
Z
5
(n 14) HYPOX
(n-14) INTACT
(n 4) HYPOX
(n4) INT ACT
FASTED
SERUM
FASTED
ANTRUM
FIG. 1. Effect of hypophysectomy on serum and antral gastrin in fasted rats. Bars represent means and standard errors of the means. Serum gastrins represent the combined results of three experiments. Antral gastrins are from one experiment. A single asterisk equals P < 0.05, and a triple asterisk equals P < 0.005 compared to hypophysectomized controls.
•••
300
30
25
=-E ---
.e:
C)
20
200
l>
z
-I ~
Z
l>
r
a: en
15
I-
«
G')
l>
en
-I
<.!l
~
~
10
:::>
a: w en
z £
(Q
5
(n 9) HYPOX
FED
(n9) INTACT
SERUM
(n 5) HYPOX
FED
e---
(n=5) INTACT
ANTRUM
FIG. 2. The same as figure 1 except that gastrins were measured in fed rats. Double asterisk equals P 0.005 compared to hypophysectomized controls.
< 0.01 and triple asterisk equals P <
ENOCHS AND JOHNSON
730
Vol. 70, No.5
••
300
Z
a:
I-
CI)
«
C)
~
::l
a:
w
CI)
(n =10)
(n =3)
HYPOX
CONTROL
(n =3)
(n =6)
HYPOX
HYPOX
HYPOX
GH
GH
GH
200~g / 100gm
500~g / 100gm
+
IOD.ug/ IOOgm
+
+
(n =12) INTACT CONTROLS
FIG. 3. Growth hormone effect on serum gastrin in fasted hypophysectomized rats. Double asterisk equals P
P
< 0.005 compared to hypophysectomized controls .
< 0.01 and triple asterisk equals
•
...... 300 E ...... Cl a. .......
~
Z
-f JJ
Z
~
~
I-
en
«
r-
200
G"l ~
":!::::>
en
w
......
-f JJ
Z
~
c: (Q ...... (Q
en 100
(n=ll)
HYPOX
(n =9)
HYPOX
FED
+ GH
(n =12)
INTACT
SERUM
(n -= 5)
HYPOX
(n =5)
(n ' 5)
HYPOX INTACT
FED
+ GH
ANTRUM
FIG. 4. Growth hormone effect on serum and antral gastrin in fed hypophysectomized rats. Serum gastrins represent the combined results of two experiments. Antral gastrins are from one experiment. Single asterisk equals P < 0.05 and triple asterisk equals P < 0.005 compared to hypophysectomized controls.
TABLE
2. Serum and antral gastrin contents of intact fasted rats treated with saline or GH for 2 days
Group
Control GH
Antrum' No.
5 6
Mean
153.3 181.3
± ±
±
SEM
No.
18.52 25.47
Mean
18.08 29.2
6 6
±
± ±
SEM
3.08 3.52c
a Values expressed as pg/ml. • Values expressed as p.g/g. c p < 0.05 compared to controls.
TABLE
3. Serum and antral gastrin contents of intact fed rats treated with saline or GH for 4 days Group (no.)
Antrum' mean
Control (4) GH(4)
731
GROWTH HORMONE AND GASTRIN
May 1976
284.0 ± 37.24 357.40 ± 84.60
±
SEM
32.33 35.01
± ±
3.8 2.9
a Values expressed as pg/ml. 'Values expressed as !1-g/g.
Discussion Most hormones secreted by the anterior pituitary are trophic, in that they stimulate growth in their target organs. ACTH, thyrotrophic hormone, and luteinizing hormone also act to increase release of peripheral hormones. GH has been shown to affect biosynthetic processes related to tissue growth in a number of body tissues, but has not been implicated previously in the stimulation of release of hormones other than its functional mediator, somatomedin. 20 , 21 In this study evidence has been presented which indicates that GH is necessary for the maintenance of serum and antral gastrin levels. It is possible that the decrease in gastric secretion 1,2,7,8 and the hypotrophy of the stomach,1. 2 small intestine,3, 4 and pancreas,5, 6 which accompanies hypophysectomy, result from the observed decrease in serum and antral gastrin. This supposition is supported by evidence that GH6 and pentagastrin 11,5 cause similar increases in nucleic acid contents of the pancreas and weights of the duodenum of hypophysectomized rats. The results obtained in this study suggest that some of the trophic actions of GH in gastrointestinal tissues are mediated by gastrin. The factors regulating the synthesis of gastrin are largely unknown. Numerous observations, however, suggest that the release of gastrin is necessary to maintain antral levels of the hormone. Food deprivation in the rat results not only in decreased gastrin release, as indicated by depressed serum levels, but also in decreased antral gastrin and decreased numbers of antral G-cells. 22 The same findings occur in rats fed solely by intravenous alimentation. 23 The extreme sensitivity of both serum and antral gastrin levels to feeding was controlled in the present series of studies by pair-feeding. Since feeding is so important in determining gastrin levels, the experiments were also done in both fasted and fed animals. The current data indicate that GH as well as feeding (or a stimulus for gastrin release) is necessary to maintain
gastrin levels. It is likely that normal circulating levels of GH exert a necessary permissive effect on gastrin synthesis. In conclusion it appears that the absence of pituitary hormones is accompanied by hyposecretion of the antral G-cells, and possibly a decreased rate of synthesis of gastrin as shown by lowered serum and antral gastrin levels, respectively. Supplemental hormonal replacement with chronic injections of GH reverses these effects, returning antral function toward normal. It is hypothesized that GH is permissive for gastrin synthesis and, therefore, necessary for normal gastrin release. It is possible in view of these results that the maintenance of gastrointestinal function and structure is dependent on a pituitary-antral axis in much the same way as the adrenal gland depends on the pituitary adrenal axis. Whether the effects of GH on endogenous gastrin are related to the release of the somatomedins· and other growth factors 21 remains an interesting speculation. REFERENCES 1. Schapiro H, Wruble LD, Britt LG: The effect of hypophysectomy on the gastrointestinal tract. A review of the literature. Am J Dig Dis 15:1019-1030, 1970 2. Crean GP: The endocrine system and the stomach. Vitam Horm 21:215-280, 1963 3. Levin RJ: The effects of hormones on the absorptive, metabolic and digestive functions of the small intestine. J Endocrinol 45:339-341, 1969 4. Leblond CPo Carriere R: The effect of growth hormone and thyroxine on the mitotic rate of the intestinal mucosa of the rat. Endocrinology 56:261-266, 1955 5. Mayston PD, Barrowman JA: Influence of chronic adminstration of pentagastrin on the pancreas in hypophysectomized rats. Gastroenterology 64:391-399, 1973 6. Sesso A, Valeri V: Nucleic acid patterns in the pancreas of hypophysectomized rats after administration of growth hormone and of thyroxine. Exp Cell Res 14:201-203, 1958 7. Snapper I, Groen J, Hunter D, et al: Achlorhydria, anemia and subacute combined degeneration in pituitary and gonadal insufficiency. Q J Med 6: 195, 1937 8. Jacobson ED, Magnani TJ: Some effects of hypophysectomy on gastrointestinal function and structure. Gut 5:473-479, 1964 9. Martin M, Menguy R: Influence of adrenocorticotropin, cortisone, aspirin, and phenylbutazone on the rate of exfoliation and the rate of renewal of gastric mucosal cells. Gastroenterology 58:329-336, 1970 10. Johnson LR: Gut hormones on growth of gastrointestinal mucosa. In Endocrinology of the Gut. Edited by WY Chey and FP Brooks. Thorofare, N. J., Charles B. Slack, 1974, p 163-177 11. Mayston PD, Barrowman JA: The influence of chronic adminstration of pentagastrin upon the rat pancreas. Q J Exp Physiol 56: 113-122, 1971 12. Dorchester JE, Haist RE: The secretin content of the intestine in normal and hypophysectomized rats. J Physiol (Lond) 118: 188-195, 1952 13. Dorchester JR, Haist RE: The effect of anterior pituitary extracts, dessicated thyroid, growth hormone preparations and ACTH on the extractable secretin of the intestines of hypophysectomized and intact rats. J Physiol (Lond) 119:266-273, 1953 14. Hall WH: The parietal cell mass: growth hormone relationship in man. AM J Dig Dis 16:139-143, 1975 15. Creutzfeldt W, Arnold R, Creutzfeldt C, et al: Gastrin and G-cells in the antral mucosa of patients with pernicious anemia, acromeg-
732
16.
17.
18. 19.
ENOCHS AND JOHNSON
aly and hyperparathyroidism and in a Zollinger-Ellison tumor of the pancreas. Eur J Clin Invest 1:361- 479, 1971 Creutzfeldt W, Crutzfeldt C, Arnold R: Gastrin-producing cells. In Endocrinology of the Gut. Edited by WY Chey and P Brooks. Thorofare, N. J., Charles B Slack, 1974, p 35-62 Lichtenberger L, Welch JD, Johnson LR: Relationship between the changes in gastrin levels and intestinal properties in the starved rat. Am J Dig Dis (in press) Yalow RS, Berson SA: Radioimmunoassay of gastrin. Gastroenterology 58:1-41 , 70 Dockray GJ, Walsh JH: Amino terminal gastrin fragment in serum of Zollinger-Ellison syndrome patients. Gastroenterology 68:222- 230. 1975
Vol. 70. No . .5
20. Drezner MK, Eisenbarth GS, Neelon FA, et al: Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum. Biochem Biophys Acta 381:384- 396, 1975 21. Hall K, Luft R: Growth hormone and somatomedin. In Advances in Metabolic Disorders. Edited by R Levine and R Luft. New York, Academic Press, 1974, p 1-31 22. Lichtenberger LM , Lechago J, Johnson LR: Depression of antral and serum gastrin concentration by food deprivation in the rat. Gastroenterology 68: 1473- 1479, 1975 23. Johnson LR, Copeland EM, Dudrick SJ. et al: Structural and hormonal alterations in the gastrointestinal tract of parenterally fed rats. Gastroenterology 68: 1177 -1183, 1975
Vol. 70, No.5
GASTROENTEROLOGY 70:727~732,
Printed in U.S.A.
EFFECT OF HYPOPHYSECTOMY AND GROWTH HORMONE ON SERUM AND ANTRAL GASTRIN LEVELS IN THE RAT M. REBECCA ENOCHS, Ph.D., AND LEONARD R. JOHNSON, Ph.D. Department of Physiology, University of Texas Medical School, Houston, Texas
The effects of hypophysectomy and subsequent replacement therapy with growth hormone on serum and antral gastrin levels was investigated in both fasted and nonfasted pair-fed rats. Hypophysectomy caused a 57% decrease in serum and 47% decrease in antral gastrin content in 18-hr fasted animals. In nonfasted animals, hypophysectomy resulted in a 42% fall in serum gastrin and a 76% fall in antral gastrin. Animals were given injections of growth hormone over a lO-day period, then fasted 16 to 18 hr before being killed. Doses of 100 to 200 /-Lg per 100 g of body weight were ineffective, but a dose of 500 /-Lg per 100 g was sufficient to restore completely serum gastrin levels to intact control values. In nonfasted rats this same dose raised serum gastrin to intact levels and increased antral gastrin significantly over hypophysectomized levels, but did not increase it to control values. When given to pair-fed intact animals, growth hormone caused slight but not significant elevations in serum and antral gastrin. The effect of growth hormone on gastrin secretion and/or synthesis may be a significant physiological factor in the regulation of normal gastrointestinal function and growth. Hypophysectomy has been clearly documented as causing trophic changes in the gastrointestinal tract of several species, including man. These changes include a decrease in mucosal weight in the submaxillary glands, stomach, pancreas, and small intestine. 1, 2 This decrease in weight is accompanied by a decrease in RNA content, enzyme activity, and zymogen granules in the salivary glands. 1 In the small intestine, hypophysectomy leads to mucosal atrophy, decreased enzyme activity, and decreased mitotic rate. 1, 3, 4 The pancreas exhibits a decrease in RNA and protein content, decreased cytoplasmic granules, and depressed output of amylase and proteolytic enzymes. 1, 5, 6 In the stomach there is hypomotility, mucosal atrophy, involution of chief and parietal cells with an associated decrease in secretory volume, and acid and pepsin output. 1, 2, 7, 8 The growth of the gastric mucosa in hypophysectomized rats, as measured by mucosa volume, is partially restored by growth hormone (GH), but not by other pituitary hormones. 2 In fact adrenocorticotrophic horReceived August 4, 1975. Accepted November 10, 1975. Address correspondence to: Dr. M. Rebecca Enochs, Department of Physiology, University of Texas Medical School, P. O. Box 20708, 6400 W. Cullen Street, Houston, Texas 77025. This study was supported by National Institutes of Health Grant AM 16505. Dr. Johnson is the recipient of Research Career Development Award AM 28972 from the National Institutes of Health. The authors are grateful to Dr. John H. Walsh of the Department of Medicine, University of California at Los Angeles, for the generous gift of Ab 1296, raised in rabbits immunized with heptadecapeptide gastrin-I. In addition, the authors are indebted to Dr. John Walsh and Dr. G. Dockray of U.C.L.A., and Dr. Linda D. Sander of the University of Texas Medical School, Houston, Texas, for assistance in establishing the gastrin radioimmunoassay. 727
mone (ACTH) may have the reverse effect. 2, 9 Despite findings that ACTH depresses mucosa volume in hypophysectomized rats 2 and mitotic rate in intact dogs,9 cortisone has been found to increase gastric secretory function in hypophysectomized dogs. 8 This apparent discrepancy may be due to species difference, dose effects, or specific inhibitory action of ACTH on stomach growth. The effect of GH showed no such discrepancy. GH increased mucosa volume in the hypophysectomized rat 2 and increased histamine-stimulated gastric secretory function in hypophysectomized dogs. 8 Several lines of evidence suggest that GH effects on gastrointestinal growth and function may be mediated, at least in part by gastrin. The specific trophic action of gastrin on gastrointestinal tissues has been established well. 10, 11 Chronic administration of a pharmacological dose of pentagastrin to hypophysectomized rats has been shown to increase pancreatic weight, as well as RNA and DNA content. 5 Dorchester and Haist reported that the secretin content of rat small intestine, as determined by bioassay, is depressed after hypophysectomy.12 This depression was prevented by anterior pituitary extracts, growth hormone, or ACTH. 13 An effect of GH on gastrin release was suggested by observations that patients with acromegaly sometimes have abnormally high maximal acid output, 14 and may have high antral gastrin levels as well as hyperplasia of antral G cells, 15 although the latter report has since been retracted. 16 This investigation was undertaken to study the effect of GH on serum and antral gastrin levels in the rat. Materials and Methods All animals used in these studies, both intact and hypophysectomized, were male Sprague-Dawley rats weighing 140 to
728
Vol. 70, No . .5
ENOCHS AND JOHNSON
170 g. Both intact and hypophysectomized rats were obtained from Charles Rivers, Wilmington, Massachusetts. Hypophysectomized rats were given 5% dextrose in their drinking water, and allowed to recover from surgery for at least 2 weeks before use. The rats were housed three per cage in a room with a fixed lighting schedule, 12 hr on, 12 hr off. During periods of fasting, animals were housed in cages with wide mesh bottoms. Bovine GH (Calbiochem, Los Angeles, Calif., cat. no. 369152, 1.0 USP U per mg) was administered as a saline solution, pH 8.5. The biological activity of the hormone preparations was ascertained by monitoring rat weight changes over the course of the experimental period. A significant increase in weight was considered an indication that the preparation was biologically active. The animals were grouped in two ways: 1. In experiments using hypophysectomized rats, the animals were divided into three groups: (1) hypophysectomized controls; (2) hypophysectomy + GH; (3) intact controls. In these experiments all groups received a daily ration of food equal to the daily caloric intake of the hypophysectomized control group. Hypophysectomized controls were given intraperitoneal injections of saline twice daily for 10 days. Hypophysectomized animals given GH received intraperitoneal injections of the specified doses twice daily for 10 days. Intact control rats received no injections. In those experiments which are termed fasted, all groups were removed from food 16 (0 18 hr before being killed. In those experiments termed nonfastE:;d, animals remained in the feeding cages up until the time of sacrifice. 2. In experiments using only intact rats, animals were divided into two groups: (1) intact controls; (2) intact + GH. In these experiments the GH-treated animals were given a daily ration of food equal to the caloric intake of the intact controls. Fasted animals were removed from food 16 to 18 hr before being killed. Nonfasted animals remained in feeding cages until sacrifice. In all experiments the animals were sacrified between 9:00 and 11:00 AM to minimize effects of diurnal variation. The animals were weighed and anesthetized with ether. A midline incision was made and blood was withdrawn directly from the heart. Antral tissue was prepared according to the method described by Lichtenberger et al. 17 The antrum was removed and carefully trimmed. It was then weighed and homogenized in 1 II!1 of deionized water. One-half milliliter of the homogenate was removed and immediately added to 2 ml of boiling deionized water. The antral extract was boiled for 20 min then centrifuged at 12,000 rpm for 30 min. Serum and antral extracts were frozen for gastrin radioimmunoassay. The gastrin radioimmunoassay was performed according to the method of Yalow and Berson 18 using the antibody characterized by Dockray and Walsh. 19 Synthetic human gastrin I (1-17), obtained from Imperial Chemical Industries, Ltd., England. was used as a standard. Gastrin levels are expressed as picograms per milliliter of serum and micrograms per gram wet weight of antrum. The difference between means was evaluated using Student's t-test for unpaired data. Differences were considered significant if P < 0.05.
Results Body weights of hypophysectomized animals receiving daily injections of saline dropped very slightly over the 10-day experimental period (table 1). Intact rats which were pair-fed with hypophysectomized rats for 10 days
TABLE
1. Body weights at beginning and end of IO-day injection period
po
Final
lniti al
Group (no.)
m ean ± SEM
Hypophysectomized control (19) Hypophysectomy (19) andGH (12) Intact control a
152.7
±
3.16
150.1
±
3.15
< 0.025
150.18
±
3.22
177.53
±
3.14
< 0.001
158.67
±
14.60
148.42
±
16.40
< 0.001
Values were obtained using the paired t -test.
lost about 10 g. This can be accounted for by the limitation of their normal food intake to the decreased level of hypophysectomized animals. GH-treated hypophysectomized rats consistently gained from 20 to 30 g over the lO-day period, indicating that the hormone preparation was biologically active. Hypophysectomy alone caused a marked decrease in both serum and antral gastrin levels. In fasted animals, serum gastrin dropped from 227 pg per ml in intact rats to 97 pg per ml in hypophysectomized rats, a decrease of 57 %. Antral gastrin dropped from 15.5 p.g per g wet weight, to 8.25 p.g per g, a decrease of 47 % (fig. 1). In fed animals, serum gastrin was decreased by 42 % from 293 to 170 pg per ml, and antral gastrin by 76% from 27.89 p.g per g to 6.56 p.g per g (fig. 2). Figure 3 shows the effects of three different doses of GH on fasted serum gastrin levels in hypophysectomized rats. Doses of 100 and 200 p.g of GH per 100 g of body weight caused no significant change in serum gastrin levels . Adose of 500 p.g per 100 g, however, was sufficient to raise serum gastrins in hypophysectomized animals up to that of intact pair-fed controls. Although there was considerable variability in the GH-treated rats, the mean value was significantly higher than that of hypophysectomized controls, but not significantly different from intact controls. Antral determinations were not made on these animals. Because the presence of food in the stomach has such dramatic effects on gastrin levels, we also looked at the effect of GH in pair-fed animals which were allowed to feed up until the moment they were killed. All animals, both hypophysectomized and intact, had varying amounts of food in their stomachs. Despite this, the three groups had serum gastrin levels which were qualitatively similar to those of fasted animals (fig. 4) . Quantitatively, as would be expected, their gastrin levels were higher. This increase was significant (P < 0.025) only in hypophysectomized controls. Again there was considerable variability in GH-treated animals, but they still demonstrated a significant increase in mean serum gastrin over hypophysectomized controls. Antral gastrin values were also elevated by GH ; however, they remained significantly (P < 0.05) lower than intact controls. Effects of exogenous GH on tissue growth are generally more easily demonstrated in hypophysectomized animals. To determine whether this also applied to the
729
GROWTH HORMONE AND GASTRIN
May 1976
stimulation of gastrin release, GH was injected into intact animals. Exogenous GH stimulated a slight but not significant increase in serum gastrin in both fasted and nonfasted animals (tables 2 and 3). Only fasted rats
showed an increase in antral gastrin. The experiment was not carried out for 10 days, so a direct comparison with the effect on hypophysectomized animals cannot be made.
300
E
---
.e: C)
20
200
z
a: en I-
l> Z -I
15
«
~
l>
r
<.!l
G')
~
:::>
l>
en
10
100
a: w en
-I ~
Z
5
(n 14) HYPOX
(n-14) INTACT
(n 4) HYPOX
(n4) INT ACT
FASTED
SERUM
FASTED
ANTRUM
FIG. 1. Effect of hypophysectomy on serum and antral gastrin in fasted rats. Bars represent means and standard errors of the means. Serum gastrins represent the combined results of three experiments. Antral gastrins are from one experiment. A single asterisk equals P < 0.05, and a triple asterisk equals P < 0.005 compared to hypophysectomized controls.
•••
300
30
25
=-E ---
.e:
C)
20
200
l>
z
-I ~
Z
l>
r
a: en
15
I-
«
G')
l>
en
-I
<.!l
~
~
10
:::>
a: w en
z £
(Q
5
(n 9) HYPOX
FED
(n9) INTACT
SERUM
(n 5) HYPOX
FED
e---
(n=5) INTACT
ANTRUM
FIG. 2. The same as figure 1 except that gastrins were measured in fed rats. Double asterisk equals P 0.005 compared to hypophysectomized controls.
< 0.01 and triple asterisk equals P <
ENOCHS AND JOHNSON
730
Vol. 70, No.5
••
300
Z
a:
I-
CI)
«
C)
~
::l
a:
w
CI)
(n =10)
(n =3)
HYPOX
CONTROL
(n =3)
(n =6)
HYPOX
HYPOX
HYPOX
GH
GH
GH
200~g / 100gm
500~g / 100gm
+
IOD.ug/ IOOgm
+
+
(n =12) INTACT CONTROLS
FIG. 3. Growth hormone effect on serum gastrin in fasted hypophysectomized rats. Double asterisk equals P
P
< 0.005 compared to hypophysectomized controls .
< 0.01 and triple asterisk equals
•
...... 300 E ...... Cl a. .......
~
Z
-f JJ
Z
~
~
I-
en
«
r-
200
G"l ~
":!::::>
en
w
......
-f JJ
Z
~
c: (Q ...... (Q
en 100
(n=ll)
HYPOX
(n =9)
HYPOX
FED
+ GH
(n =12)
INTACT
SERUM
(n -= 5)
HYPOX
(n =5)
(n ' 5)
HYPOX INTACT
FED
+ GH
ANTRUM
FIG. 4. Growth hormone effect on serum and antral gastrin in fed hypophysectomized rats. Serum gastrins represent the combined results of two experiments. Antral gastrins are from one experiment. Single asterisk equals P < 0.05 and triple asterisk equals P < 0.005 compared to hypophysectomized controls.
TABLE
2. Serum and antral gastrin contents of intact fasted rats treated with saline or GH for 2 days
Group
Control GH
Antrum' No.
5 6
Mean
153.3 181.3
± ±
±
SEM
No.
18.52 25.47
Mean
18.08 29.2
6 6
±
± ±
SEM
3.08 3.52c
a Values expressed as pg/ml. • Values expressed as p.g/g. c p < 0.05 compared to controls.
TABLE
3. Serum and antral gastrin contents of intact fed rats treated with saline or GH for 4 days Group (no.)
Antrum' mean
Control (4) GH(4)
731
GROWTH HORMONE AND GASTRIN
May 1976
284.0 ± 37.24 357.40 ± 84.60
±
SEM
32.33 35.01
± ±
3.8 2.9
a Values expressed as pg/ml. 'Values expressed as !1-g/g.
Discussion Most hormones secreted by the anterior pituitary are trophic, in that they stimulate growth in their target organs. ACTH, thyrotrophic hormone, and luteinizing hormone also act to increase release of peripheral hormones. GH has been shown to affect biosynthetic processes related to tissue growth in a number of body tissues, but has not been implicated previously in the stimulation of release of hormones other than its functional mediator, somatomedin. 20 , 21 In this study evidence has been presented which indicates that GH is necessary for the maintenance of serum and antral gastrin levels. It is possible that the decrease in gastric secretion 1,2,7,8 and the hypotrophy of the stomach,1. 2 small intestine,3, 4 and pancreas,5, 6 which accompanies hypophysectomy, result from the observed decrease in serum and antral gastrin. This supposition is supported by evidence that GH6 and pentagastrin 11,5 cause similar increases in nucleic acid contents of the pancreas and weights of the duodenum of hypophysectomized rats. The results obtained in this study suggest that some of the trophic actions of GH in gastrointestinal tissues are mediated by gastrin. The factors regulating the synthesis of gastrin are largely unknown. Numerous observations, however, suggest that the release of gastrin is necessary to maintain antral levels of the hormone. Food deprivation in the rat results not only in decreased gastrin release, as indicated by depressed serum levels, but also in decreased antral gastrin and decreased numbers of antral G-cells. 22 The same findings occur in rats fed solely by intravenous alimentation. 23 The extreme sensitivity of both serum and antral gastrin levels to feeding was controlled in the present series of studies by pair-feeding. Since feeding is so important in determining gastrin levels, the experiments were also done in both fasted and fed animals. The current data indicate that GH as well as feeding (or a stimulus for gastrin release) is necessary to maintain
gastrin levels. It is likely that normal circulating levels of GH exert a necessary permissive effect on gastrin synthesis. In conclusion it appears that the absence of pituitary hormones is accompanied by hyposecretion of the antral G-cells, and possibly a decreased rate of synthesis of gastrin as shown by lowered serum and antral gastrin levels, respectively. Supplemental hormonal replacement with chronic injections of GH reverses these effects, returning antral function toward normal. It is hypothesized that GH is permissive for gastrin synthesis and, therefore, necessary for normal gastrin release. It is possible in view of these results that the maintenance of gastrointestinal function and structure is dependent on a pituitary-antral axis in much the same way as the adrenal gland depends on the pituitary adrenal axis. Whether the effects of GH on endogenous gastrin are related to the release of the somatomedins· and other growth factors 21 remains an interesting speculation. REFERENCES 1. Schapiro H, Wruble LD, Britt LG: The effect of hypophysectomy on the gastrointestinal tract. A review of the literature. Am J Dig Dis 15:1019-1030, 1970 2. Crean GP: The endocrine system and the stomach. Vitam Horm 21:215-280, 1963 3. Levin RJ: The effects of hormones on the absorptive, metabolic and digestive functions of the small intestine. J Endocrinol 45:339-341, 1969 4. Leblond CPo Carriere R: The effect of growth hormone and thyroxine on the mitotic rate of the intestinal mucosa of the rat. Endocrinology 56:261-266, 1955 5. Mayston PD, Barrowman JA: Influence of chronic adminstration of pentagastrin on the pancreas in hypophysectomized rats. Gastroenterology 64:391-399, 1973 6. Sesso A, Valeri V: Nucleic acid patterns in the pancreas of hypophysectomized rats after administration of growth hormone and of thyroxine. Exp Cell Res 14:201-203, 1958 7. Snapper I, Groen J, Hunter D, et al: Achlorhydria, anemia and subacute combined degeneration in pituitary and gonadal insufficiency. Q J Med 6: 195, 1937 8. Jacobson ED, Magnani TJ: Some effects of hypophysectomy on gastrointestinal function and structure. Gut 5:473-479, 1964 9. Martin M, Menguy R: Influence of adrenocorticotropin, cortisone, aspirin, and phenylbutazone on the rate of exfoliation and the rate of renewal of gastric mucosal cells. Gastroenterology 58:329-336, 1970 10. Johnson LR: Gut hormones on growth of gastrointestinal mucosa. In Endocrinology of the Gut. Edited by WY Chey and FP Brooks. Thorofare, N. J., Charles B. Slack, 1974, p 163-177 11. Mayston PD, Barrowman JA: The influence of chronic adminstration of pentagastrin upon the rat pancreas. Q J Exp Physiol 56: 113-122, 1971 12. Dorchester JE, Haist RE: The secretin content of the intestine in normal and hypophysectomized rats. J Physiol (Lond) 118: 188-195, 1952 13. Dorchester JR, Haist RE: The effect of anterior pituitary extracts, dessicated thyroid, growth hormone preparations and ACTH on the extractable secretin of the intestines of hypophysectomized and intact rats. J Physiol (Lond) 119:266-273, 1953 14. Hall WH: The parietal cell mass: growth hormone relationship in man. AM J Dig Dis 16:139-143, 1975 15. Creutzfeldt W, Arnold R, Creutzfeldt C, et al: Gastrin and G-cells in the antral mucosa of patients with pernicious anemia, acromeg-
732
16.
17.
18. 19.
ENOCHS AND JOHNSON
aly and hyperparathyroidism and in a Zollinger-Ellison tumor of the pancreas. Eur J Clin Invest 1:361- 479, 1971 Creutzfeldt W, Crutzfeldt C, Arnold R: Gastrin-producing cells. In Endocrinology of the Gut. Edited by WY Chey and P Brooks. Thorofare, N. J., Charles B Slack, 1974, p 35-62 Lichtenberger L, Welch JD, Johnson LR: Relationship between the changes in gastrin levels and intestinal properties in the starved rat. Am J Dig Dis (in press) Yalow RS, Berson SA: Radioimmunoassay of gastrin. Gastroenterology 58:1-41 , 70 Dockray GJ, Walsh JH: Amino terminal gastrin fragment in serum of Zollinger-Ellison syndrome patients. Gastroenterology 68:222- 230. 1975
Vol. 70. No . .5
20. Drezner MK, Eisenbarth GS, Neelon FA, et al: Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum. Biochem Biophys Acta 381:384- 396, 1975 21. Hall K, Luft R: Growth hormone and somatomedin. In Advances in Metabolic Disorders. Edited by R Levine and R Luft. New York, Academic Press, 1974, p 1-31 22. Lichtenberger LM , Lechago J, Johnson LR: Depression of antral and serum gastrin concentration by food deprivation in the rat. Gastroenterology 68: 1473- 1479, 1975 23. Johnson LR, Copeland EM, Dudrick SJ. et al: Structural and hormonal alterations in the gastrointestinal tract of parenterally fed rats. Gastroenterology 68: 1177 -1183, 1975