pituitary hormones and the small bowel: effect of hypophysectomy on intestinal adaptation to small...

European Journal of Clinical Investigation (1 979) 9, 115-127

Pituitary hormones and the small bowel: effect of hypophysectomy on intestinal adaptation to small bowel resection in the rat*

BRYCE TAYLOR,?. GERARD M. MURPHY & R. HERMON DOWLING, Gastroenterology Unit, Guy’s Hospital and Medical School, London

Received 28 December 1978

Abstract. The influence of pituitary hormones on intestinal adaptation to small bowel resection was studied by examining jejunal and ileal structure and function in control and in sham-operated rats, and in animals with 50% proximal or distal resection which were divided into three main groups: normally-fed, hypophysectomized, and pair-fed. The pituitary was removed 2 weeks before intestinal surgery and gut structure and function were studied 4 weeks later. The effectiveness of hypophysectomy was confirmed by histological examination of the aspirated pituitary, and by showing a significant subse- quent reduction in weight of the testes and adrenals. Food intake and body weight fell significantly after removing the pituitary; intestinal surgery caused a transient further decrease in food intake.

Measurements of intestinal villus height and crypt depth, indices of mucosal mass (mucosal wet weight, protein and DNA contentlcm intestine), measurements of mucosal a-glucosidase activity, and in vivo galactose absorption/unit length of intestine all showed comparable results. In rats with an intact intestine, resection resulted in mucosal hyperplasia and increased segmental absorption. Following hypophysectomy, there was marked mucosal hypoplasia and hypofunction which seemed to be due largely to associated hypophagia since comparable changes were found in the pair-fed, sham- operated rats. However following pituitary removal, both distal jejunum and proximal ileum retained their capacity to regenerate though the magnitude of this adaptive change was much greater in the resected, pair- fed rats suggesting that hypophagia alone cannot explain the diminished adaptation to resection after hypo-

* Presented in part at the Medical Research Society Meeting, Hammersmith Hospital, London, December 1974 (Clin Sci Mol Med (1975) 48, 13P (abstract)) and at the British Society of Gastroenterology, University of Southampton, 1975 (Gut (1975) 16, 397 (abstract)).

Present address: Western Surgical Group, Toronto Western Medical Building, 25 Leonard Avenue, Toronto, Ontario MST 2R2, Canada.

Correspondence: Professor R. Hermon Dowling, Gastro- enterology Unit, Guy’s Hospital and Medical School, London SE19RT.

0 1979 Blackwell Scientific Publications 0014-2972/79/04004115$02.00

physectomy. By inference, pituitary hormones do influence the adaptive response to resection.

Key words. Pituitary hormones, hypophysectomy, small bowel resection, intestinal adaptation, hypophagia, enterotrophic hormones.

introduction

Following partial small bowel resection, the residual intestine develops both structural and functional adaptive changes. These include an increase in the calibre of the intestine and mucosal hyperplasia which combine to produce a greater absorptive surface associated with enhanced absorption and increased enzyme activity per unit length of intestine [ 11.

Initially, studies of intestinal adaptation were con- fined to investigating the phenomena of the hyperplastic response. More recently, research has concentrated on the mechanism for the structural and functional changes in the hope that an enterotrophic factor(s) could be isolated whch might have some therapeutic value for patients with malabsorption and/or malnutrition secon- dary to disease or resection of the small intestine.

There is much indirect evidence that food in the small bowel lumen is important for the development of these adaptive changes [2]. Whether the nutrients act directly on the mucosa or indirectly by releasing sys- temically or locally acting hormones (chalones or hormones of the paracrine system) which are trophic to the intestine, is unknown.

There is evidence that hormonal factors may not only affect the normal small bowel but may also play a role in intestinal adaptation. First, studies in parabiotic rats 13-51 and in cross circulated pigs [6 ] have shown that small bowel resection in the donor animal provokes an increased rate of cell proliferation and a greater intestinal weight in the recipient, suggesting that some trophic hormone must have stimulated intestinal growth. Secondly, during lactation rats develop hyperphagia, villus hyperplasia, and increased absorption per unit length of intestine. These adaptive changes d o not result from increased food intake as they occur equally in isolated Thiry-Vella intestinal loops, which are not in continuity with the bowel, and in the intact intestine [7].

115

1 16 BRYCE TAYLOR, GERARD M. MURPHY & R. HERMON DOWLING

This again suggests that some trophc hormonal sub- stance(s) was reaching the isolated intestine. Thirdly, since hyperplasia can be induced in a defunctioned, self- emptying blind loop of ileum by resecting a further segment of the intestine remaining in continuity [8], it has been concluded that a ‘systemic stimulator’ must have been responsible for the adaptive changes in the semi- isolated loop.

There are several hormonal candidates for the role of ‘enterotrophin’, including enteroglucagon [9, 101, gastrin [ l I ] , prolactin [I21 and growth hormone [13]. It is known, for example, that growth hormone not only affects the growth of peripheral tissues, but that it also causes visceromegaly in animals [I41 and in man [15]. Surprisingly, however, little is known about its effects on the gut. Prolactin is thought to increase both liver and kidney weight [ 161, and the decreased circulating prolactin concentrations seen during lactation [ 171 could explain the trophic changes seen in bypassed loops of intestine in lactating rats [7].

For all these reasons, it seemed important to study further the effect of pituitary hormones on the gut; although the effects of hypophysectomy on normal intestine have been studied before [ 18-24], the influence of hypophysectomy on the adaptive response to resection has not been tested previously. This paper describes studies of jejunal and ileal structure and function in normally-fed, hypophysectomized and pair-fed (to match the reduced food intake seen after removing the pituitary) rats and compares the changes in animals with an intact small bowel with those following jejunal or ileal resection.

Experimental Design, Animals Studied and Methods

Experimental design and animals studied

The experimental protocol and the sampling sites for histology, in vitro studies and in vivo perfusion are shown schematically in Fig. 1 .

There were three main groups of male Wistar rats with a mean starting body weight of 25 1 g (+ SEM 4).

(i) Twenty-nine control rats allowed unlimited access to food.

(ii) Twenty-eight hypophysectomized rats. As hypophysectomy leads to a reduced food intake [18, 241, which itself may modify the structure and function of the small bowel [25] as well as the adaptive mucosal response to resection [26] it was necessary t o study a further control group.

(iii) Twenty-nine pair-fed animals whose daily food intake was limited to match, 2 4 h later, the food taken by the hypophysectomized rats on the previous day.

The rats were allowed 2 weeks to recover from hypophysectomy before proceeding to small bowel resection. Each main group of rats were then divided into four sub-groups (Fig. 1) as follows:

(a) A proximal, sham-resected group (n = 22; eight normally-fed, seven hypophysectomized and seven pair-

fed) in which the intestine was simply transected 2-3 cm distal to the ligament of Treitz and re-anastomosed (controls for proximal resection).

(b) A distal, sham-resected group (transection 2 cm above the ileocaecal valve) of twenty-one animals (eight normally-fed, seven hypophysectomized and six pair- fed), controls for distal resection.

(c) A proximal resection group in which 38 f 6.0 cm of upper small intestine, starting 2-3 cm distal t o the ligament of Treitz, was resected with continuity re- established using an end-to-end anastomosis as previously described I271 (n = 26; seven normally-fed, eight hypophysectomized and eleven pair-fed).

(d) A distal resection group (n = 17; six normally-fed, six hypophysectomized and five pair-fed) in which the 42 f 1.6 cm resection began at the mid-point of the small bowel and ended 2-3 cm proximal to the ileocaecal valve.

Jejunal and ileal structure and function were then studied 4 weeks later by which time previous studies have shown that the adaptive mucosal changes to resection have developed fully [27].

Thus, there were twelve experimental groups, proximal and distal transection, and proximal and distal resection subgroups for each of the three main groups of normally-fed hypophysectomized and pair-fed rats.

Surgical techniques Hypophysectomy. The rats were anaesthetized with

ether and a small endotracheal tube inserted to ensure a patent airway. Hypophysectomy was carried out by the transcervical route as described previously [28]. The technique may be summarized as follows. The strap muscles of the neck, the trachea and the oesophagus were reflected to expose a cruciform interosseous sep- tum at the base of the skull. This acted as a landmark for access to the ventral floor of the pituitary fossa which was exposed using a size 8 rose burr and dental drill. The hypophysis was then aspirated by suction through a 1 cm length of polyethylene tubing attached to a syringe which was inserted into the pituitary fossa.

Small bowel resection. This was also carried out under ether anaesthesia using the technique [27] described in outline above. To minimize the stress of resection in hypophysectomized animals, and as prophylaxis against possible infection, single injections of hydro- cortisone and tetracycline were given intramuscularly at the time of the partial enterectomy. To ensue stan- dardized experimental conditions, comparable injections were given t o al l groups in the study,

Efficacy of hypophysectomy and its effect on organ weights

In all animals the aspirated hypophysis (both anterior and posterior) was examined histologically to ensure that the gland had been removed essentially intact. However, previous studies have shown that after this

EFFECT OF PITUITARY HORMONES ON INTESTINAL ADAPTATION 1 17

NORMALLY FED HYPOPHYSECTOMY PAIR FED

&-

50% RESECTION I I SHAM-OPERATED (Prox. o r distal

transeclion) CONTROLS

DISTAL 50% RESECTION

I I I I I I I I I t I I

I t I 1 I 1 I 1 I I I I

3 bowel

.,....: ...,.,. $$ . Quantitative histology + mucosal wet weight, prot , DNA and Ol-glucosidase

...?.. 1 in vivo ... : - El 7 galactose perfusion

Figure 1. Experimental protocol and intestinal sampling and perfusion sites. Schematic outline illustrating experimental protocol and intestinal sampling sites for in vitro studies of mucosal mass and quantitative histology, and for the in vivo perfusion studies to measure galactose absorption. In the sham-operated group, separate animals were used for the jejunal (controls for the distal resection group) and ileal (controls for the jejunectomized rats) studies.

operation any pituitary elements accidentally left behind in the pituitary fossa rapidly atrophy [28]. In practice, the effectiveness of hypophysectomy is simply, but re- liably, ensured by weighing the testes and adrenals at the final operation [29]. At the same time, and as a control for these studies, the fresh weights of the liver and both kidneys were also recorded.

Food intake and body weight

The rats, w h c h were housed in individual cages with wire mesh floors (in an attempt to minimize copro. phagia), were fed a commercial rat chow (diet 41B, E. Dixon & Son, Crane Mead Mills, Ware, Herfordshire) and were allowed unlimited access to water.

To facilitate healing of the intestinal anastomoses, solid food was withheld for the first 48 h after the resection/transection operations ; all rats (including the

normally-fed and pair-fed groups) were given only glucose/sahne by mouth during this 2 day period. Subse- quently, food intake was recorded daily, and the rats were weighed weekly.

Intestinal structure and finction

Intestinal structure and function were studied at a final operation when the rats were again anaesthetized briefly for insertion of jejunal and ileal perfusion cannu- lae in segments of intestine measuring approximately 30 cm (mean 28 k 0.5 cm) as described previously [27].

Immediately after completing the in vivo absorption/ perfusion studies (see below), the rats were killed by cervical dislocation. The abdomen was then quickly opened, the perfused segment isolated, its mesentery removed, and the length of the perfused segment measured against a vertical scale with a standard 5 g stretch.

118 BRYCE TAYLOR, GERARD M. MURPHY & R. HERMON DOWLING

An adjacent, non-perfused, 5 cm length of intestine, measured in the same way, was then excised for measurement of mucosal wet weight, protein, DNA and brush border enzyme activity, flushed with 0.15 M saline, blotted gently, and its mucosa expressed on to pre- weighed foil by scraping the bowel between two glass microscope slides. The efficacy of this procedure was checked in a few rats by histological examination of the scrapings whch showed only mucosal elements, while the residual bowel consisted of serosal and muscle coats alone. In the proximally-resected rats, the 5 cm segment was taken proximal to the perfused segment but at least 3-4 cm distal to the anastomosis (Fig. 1) t o avoid the local trophic changes induced by the anastomosis per se [30]. In the distal resection group, the mucosal homogenates were taken from below the perfused segment, but proximal to, and clear of, the anastomosis (Fig. 1). Thus, although the segments for these in vitro studies were called ‘jejunum’ and ‘ileum’ they were, in fact, taken from the distal half of the upper small intestine and the proximal portion of the lower small intestine respectively. They were, therefore, almost adjacent segments.

Mucosal mass. In addition to recording mucosal wet weight per cm length of intestine, the mucosal scrapings were homogenized and stored in a solution containing 0.3 mol sucrose/O.l mol EDTA/21 X mol ethanol/] at -20°C until portions could be analysed for protein content [31], DNA [32] and mucosal enzymes (see below). These results were also expressed as mg/cm intestine.

Histological measurements, At the final operation, additional 1-2 cm segments of intestine (taken from the same areas as those described for the mucosal enzyme studies-see above) were opened longitudinally, pinned flat on cork and fixed in 10% formol-saline. Later, 4-5 pm sections were cut parallel to the long axis of the intestine for hstological measurements of villus height and crypt depth using a calibrated eyepiece graticule as previously described [27]. The results for villus height (the mean of eight t o ten readings from the tallest villi in well- orientated sections) and crypt depth (again the mean of eight to ten readings) were recorded in microns.

Mucosal a-glucosidase activity. The activity of the brush border enzyme a-glucosidase or maltase (EC 3.2.1.20) was measured on other portions of the mucosal homogenates [33]. The enzyme results were expressed both as the total amount of the enzyme per unit length of intestine (which is affected by mucosal hyper- and hypoplasia) and as enzyme specific activity (units/ mg mucosal DNA which provides a rough index of the amount of enzyme per cell).

Galactose absorption in vivo. Galactose absorption, from 40 ml of a galactose solution (64 mmol/l) made isotonic with NaCl and containing 2.5 g/l polyethylene glycol (PEG-molecular weight 4000) and 10-20 pCi/g

of 14C: PEG (as a non-absorbable marker of net fluid movement), was measured over a 60 min period using a recirculation-perfusion technique as previously described [27]. Galactose was estimated using the enzyme method of Wallenfels [34] and Rommel [35] (galactose dehydrogenase-Boehringer, Mannheim, Germany).

Galactose was selected as the monosaccharide substrate since it is not metabolized by rat intestinal mucosa [36]. The single 64 mmol/l concentration was chosen because previous studies from our laboratory have shown that while resected small bowel absorbs more galactose/ cm intestine at all concentrations from 4, 8, 16, 32 and 64 to 128 mmol/l, the difference between control and test groups was most significant at the 64 mmol/l concentration [Jacobs & Dowling, unpublished]. ‘Absorp- tion’, or disappearance of substrate from the perfusion medium, was expressed as pmoles galactose absorbed cm intestine-’ h-’.

Statistical methods Although relatively small numbers of animals were

studied, ‘scattergrams’ of the results obtained showed that the data were not skewed. For this reason, para- metric statistics with Student’s non-paired r tests were used to estimate the significance of differences found between the results in the various groups.

Results

Efficacy of hypophysectomy and its effect on organ weigh ts

There were no major differences in mean liver and kidney weights between the three main groups. Simi- larly, there was no significant difference in testicular weight between normally-fed and pair-fed rats, the mean values ranging from 1.08 ? 0.04 to 1.28 k 0.05 g/lOO g BW. There was, however, a striking and consistent decrease in testicular weight after hypophysectomy in both the sham-resected rats (0.20 f 0.01; P < 0.001) and in those with proximal and distal small bowel resection (0.2 1 k 0.01 ; P < 0.001). There was also a highly significant reduction in adrenal wet weight in the hypophysectomized animals when compared with results in the normally-fed and pair-fed groups.

Two rats continued to eat normally after hypophysectomy. They were shown subsequently to have normal testicular and adrenal weights confirming in- effective hypophysectomy. These two animals were discarded from the study.

Food intake and body weight The results for food intake in the different experi-

mental groups are shown in the upper panel of Fig. 2 ; the associated changes in body weight, expressed as percentage increases or decreases in relation to the initial body weight, are shown in the lower panel of the figure.

EFFECT OF PITUITARY HORMONES ON INTESTINAL ADAPTATION 119

FOOD INTAKE 40 r

Normal feeding+ 30 -

I I , I I I

0 1 2 3 4 5 6

t Weeks t t

Hypophysectomy Intestinal s u r g e r y Studies

BODY WEIGHT Normal control 140 - * .z M 130

P

70L 1 : Hypophysectomy Intestinal s u r g e r y

4 Studies

I 1 I 1 I 1 I 0 1 2 3 4 5 6

Weeks

Figure 2. Food intake and body weight after hypophysectomy i intestinal resection. Mean values (+ SEM) for food intake (upper panel) and body weight (lower panel) in the various experimental groups (see Legend to Fig. 1 and text). The normal range for food intake in the normally-fed controls is shown as a stippled zone; the results for proximal and distal small bowel resection (Res.) have been pooled both for normally-fed and hypophysectomized (HYP) ra t s Since food intake in the pair-fed rats was matched to that of the hypophysectomized animals, these results are not shown. Body weights are expressed as percentages of original body weights. The lower four lines show the results for hypophysectomized (solid tines) and pair-fed (broken lines) resected and sham-operated rats- none of which differed significantly from each other.

Food intake. The sham-resected control rats ate approximately 35 g of chow per day. Food intake after hypophysectomy dropped to approximately 50% of its pre-operative value. There was a further transient decrease in food intake after intestinal surgery (both transection and resection) but food intake gradually returned to reach the pre-laparotomy levels about 10 days after surgery in the sham-resected group. Although it did not quite return to normal after hypophysectomy and resection, the difference between these two groups was never statistically significant (Fig. 2, upper panel).

Body weight. The mean body weight in the control rats increased by 40.0 * 5.2% over the 6 weeks of the study (Fig. 2, lower panel). The resected rats fed ad libitum also gained weight and in the 4 weeks after resection, there was a 14% increase in mean body weight

compared to a 25% increase in the normally-fed controls over the corresponding period.

The lower food intake after hypophysectomy was associated with a 10-15% decrease in mean body weight during the first 2 weeks after removing the pituitary. This seemed to be due mainly to the reduced food intake since there was a comparable percentage reduction in mean body weight in the pair-fed controls. Although these groups continued to lose weight during the subse- quent 4 weeks of the study, the rate of weight loss in the resected and transected, hypophysectomized and pair-fed animals diminished so that by 6 weeks (4 weeks after intestinal surgery) the overall weight loss amounted to approximately 20% of the pre-study baseline value. There was no significant difference in the mean percentage change in body weight between any of the pair-fed and hypophysectomized groups during the 6 weeks of the study.


intestinal structure

Mucosal mass In general, the pattern of results for mucosal wet

weight, protein, and DNA content/unit length of intestine were comparable. The overall results are shown in Table 1.

Distal jejunum. In normally fed rats, the distal part of the jejunum remaining after distal resection showed an obvious and significant increase in mucosal wet weight (P < 0.05), protein (P < 0.001) and DNA content/cm intestine (P < 0.01). Although all three variables showed a roughly comparable pattern, the mean percentage increase in mucosal wet weight (31%) was less marked than the corresponding increases in mucosal protein (83%) and DNA (63%).

After hypophysectomy, the sham-resected control ‘jejunum’ showed a clear-cut and statistically significant fall in the results for all three variables compared to the results for the distal jejunum in the normally-fed controls. The reductions in mucosal protein and DNA/cm intestine were comparable in both the hypophysectomized (68% for protein and 63% for DNA) and pair-fed

(67% and 59% respectively) sham-operated controls, suggesting that the distal jejunal mucosal hypoplasia occurring after removing the pituitary in animals with an intact intestine was mainly due to the associated reduction in food intake. The pattern and magnitude of change was usually comparable for all three indices studied, but unlike the results for distal jejunal protein and DNA where there was no difference between the mean values in the hypophysectomized and pair-fed rats, the mucosal wet weight was greater in the pair-fed rats than in the corresponding hypophysectomized group (Table 1). The difference was not statistically significant.

In spite of the lower baseline values (as a result of the mucosal hypoplasia) in the sham-operated, hypophysectomized controls, the distal part of the jejunal remnant retained its capacity to adapt t o resection, there being a significant increase in mucosal protein (P < 0.05).

In general, however, the magnitude of the regenerative response after resection was less after hypophysectomy than in the corresponding pair-fed rats. For example, the distal jejunal mucosal protein after resection and hypophysectomy was 93% greater than that in the hypophysectomy and transection controls, which compares with a 261% increase in the corresponding pair-fed groups. The corresponding figures for DNA were 17%

Table 1. Indices of mucosal m a s (wet weight, protein and DNA content in mg/cm intestine: mean f SEM for both jejunum and ileum in the different experimental groups (see text)

Jejunum Ileum

Normal feeding Hypophysect. Pair-feeding Normal feeding Hypophysect. Pair-feeding

Dist. Dist. Dist. Prox. Prox. Prox. Variables studied Cont. res Sham res. Sham res. Cont. res. Sham res. Sham res.

Mucosal wet (mg/cm)

wt 32.3 42.4 f 2.6 f 1.6 ***

18.6 29.0 27.7 29.1 2 2.3 i 5 . 1 f 3.7 f 7.6

ns ns

ns

59.4 17.1 31.5 29.2 40.8 f 4.2 f 2.8 f 3.7 f 5.6 f 5.5

29.7 5 1.0

ns *** ** *

Protein (mg/cm) 3.01 5.49 t 0.28 f 0.22 ***

DNA (mg/cm) 0.32 0.52 f 0.03 f 0.04 **

ns ns

0.95 1.83 1.01 3.65 -t 0.19 f 0.22 f 0.19 f 0.07 * ***

ns

3.1 7 6.04 1.14 2.51 1.38 4.51 i 0.15 i 0.32 k 0.18 i 0.43 f 0.10 f 0 . 8 3

ns * * ***

ns

***

0.12 0.14 0.13 0.24 t 0.02 2 0.01 f 0.02 f 0.03

ns *

*

0.54 0.11 0.19 0.13 0.38 0.30 i 0.02 i 0.03 f 0.02 f 0.04 k 0.02 f 0.06 ** ns ***

ns ns

* *

ns = not significant; * P < 0.05 ; ** P < 0.01 ; *** P < 0.001.

EFFECT OF PITUITARY HORMONES ON INTESTINAL ADAPTATION 12 1

after removing the pituitary and 85% in the pair-fed animals. In fact, the distal jejunal mucosal mass ulti- mately achieved in pair-fed, resected rats was inter- mediate in magnitude between that seen for normally- fed, resected and hypophysectomized, resected rats (Fig. 3); the exception lay in the results for mucosal wet weight where the pair-fed, resected group showed a large scatter of results about the mean (Table 1).

Proximal ileum. The pattern of response for mucosal mass was, broadly speaking, comparable to that described in detail for the ‘jejunum’. In rats with an intact intestine (sham-resected controls), hypophysectomy again led to marked proximal ileal mucosal hypoplasia. This seemed to be due largely to the associated reduction in food intake since, with the exception of mucosal wet weight (which was significantly greater in the pair-fed rats than in the hypophysectomized animals (P < 0.05)), there was no significant difference between hypophysectomized and pair-fed controls in the indices of ileal mucosal massicm intestine.

The proximal ileal adaptive response to jejunectomy is normally much greater than that seen in the ‘jejunum’ following distal small bowel resection [27, 371. This pattern of response was confirmed in the present studies by the indices of mucosal mass in the normally-fed rats. But once again, the capacity of the proximal ileum to adapt to jejunal resection was retained after hypophysectomy even though the degree of adaptation was significantly less in the rats without a pituitary (a 120% increase in mucosal protein (P = NS) and a 73% increase in mucosal DNA, P = NS) than in the resected rats

N-F

whose food intake was restricted to a comparable extent, which showed a 227% increase in protein (P< 0.01) and a 192% increase in DNA (P < 0.01).

Histological m earn rem ents

A representative example of proximd ileal mucosal histology in the three main groups of jejunectomized rats (controls, hypophysectomized and pair-fed) is shown in Fig. 3. The overall results for measurement of villus height and crypt depth are shown for the ‘jejunum’ and the ‘ileum’ in Fig. 4.

The pattern of results noted for the variables o f mucosal mass is again clearly seen in the results of quantitative hstology. In normally-fed rats distal jejunal villus height increased from a mean of 350k 17 pm in the controls t o 382 k 2 2 after ileectomy (a 9% increase which was not statistically significant) while proximal ileal villus height increased from 428 f 10 to 528 k 20 pm (a 23% increase, P < 0.01) as a result of jejunal resection. There were corresponding increases in the thick- ness of both ‘jejunal’ and ‘ileal’ proliferative zones (crypt depth) after resection although these differences did not reach statistical significance.

In the rats with an intact intestine, the degree of villus hypoplasia was comparable after hypophysect omy and pair-feeding (272 k 23 pm after hypophysectomy when compared to normally-fed controls; P < 0.05 and 310 k 16 pm in the pair-fed rats (P = NS) for distal jejunum; 305 * 22; P < 0.001 and 304 f 22 pm; P < 0.001 respectively for the proximal ileum). The corresponding measurements of crypt depth showed a similar

P- F

Figure 3. Ileal mucosal histology in jejunectomized rats. Representative histological sections from the residual ileum following 50% proximal small bowel resection in normally-fed (N-F), hypophysectomized (Hypox) and pair-fed (P-F) rats showing marked mucosal hypoplasia in the hypophysectomized animals which had, nonetheless, shown a regenerative response to resection when compared with the hypophysectomized sham-operated controls. The ileum in the pair-fed jejunectomized rats shows a modest reduction in villus size and crypt depth when compared with the normally-fed jejunectomized controls.


I 6oo r Normal feeding Hypophysectorny I Pair feeding

i I I I I I I

4- I

I I I

300 I

6oo Normal feeding Hypophysectomy P a i r feeding I

500

400

300

200

100

E 3 0

100

200

300 I I

Figure 4. Histological measurements of villus height and crypt depth. Mean values (r SEM) for villus height (plotted in pm above the zero line) and crypt depth (shown below) for jejunum (upper panel) and ileum (lower panel). C = control; Res. = 50% small bowelresection (proximal or distal); sham = sham-operated controls with simple intestinal transection. The numbers in vertical columns indicate the numbers of animals studied. For statistical analysis of the results, see text.

pattern. In the resected, hypophysectomized rats, the 15% increase in mean ‘jejunal’ villus height from 272 f 23 to 312 * 31 pm and the 19% increase in the average height of the ‘ileal’ villi from 305 f 22 to 361 f 13 pm (when compared to the corresponding hypophysectom ized controls) were significantly less (P < 0.001 and P < 0.01 respectively) than the post-resection increases of 51% and 64% in the mean ‘jejunal’ (from 310 * 16 to 472 f 27) and ‘ileal’ (from 305 f 22 to 500 f 15) villus heights of the pair-fed rats. In fact, the villus height in the pair-fed resected ‘jejunum’ (472 f 27) was

actually greater than that in the normally-fed illeectom- ized group (382 f 22). Once again, the pattern of results for crypt depth showed comparable, but less marked, changes than those of villus height.

These data suggest that although the reduction in food intake seen after hypophysectomy may explain partly the lower levels of mucosal mass attained in response to either proximal or distal small bowel resection, hypophagia alone cannot be the sole cause since it does not explain the discrepancy between the post resection results in the hypophysectomized and pair-fed rats. By inference, therefore, the removal of the pituitary does impair the adaptive response to resection, presumably by abolishing pituitary hormone secretion.

Intestinal function

Mucosal a-glucosidase activity

The results for a-glucosidase activity are shown in Table 2. When expressed per unit length of intestine, the pattern of results was comparable to that seen for the indices of mucosal mass. This was particularly true in the ileum, where jejunal resection resulted in increased amounts of maltase in the normally-fed (P<O.O2), hypophysectomized (P = NS), and pair-fed rats (P < 0.001), compared to their corresponding controls. Once again, the extent of adaptive response was most marked in the pair-fed animals. In contrast, when the results were expressed as enzyme activity per mg mucosal DNA (i.e. specific activity) there was a paradoxical response. In the normally-fed rats, although the total amount of enzyme present in the intestine increased, the specific activity fell significantly (P < 0.05) in the hyperplastic mucosa seen after jejunectomy. Conversely, in the non- resected animals with a hypoplastic mucosa resulting from hypophysectomy or pair-feeding, there was a marked increase in maltase specific activity which was significantly greater than that seen in the normally-fed controls (P < 0.001) This reverted towards normal when the hypoplasia of either pituitary removal or partial starvation was offset, at least in part, by the hyperplasia induced by resection.

In the jejunum the changes in a-glucosidase activity (activity/unit length intestine) were less striking than

Table 2. a-Glucosidase (EC 3.2.1.20) u/cm intestine and u/mg DNA; mean i SEM for both distal jejunum and proximal ileum

Jejunum Ileum

Main groups: Normal feeding Hypophysectomy Pair-feeding Normal feeding Hypophysectomy Pair-feeding

Dist. Dist. Dist. Prox. Prox. Prox. Sham res. Cont. res. Sham res. Sham res. Sub groups: Cont. res. Sham res.

31.2 29.8 21.6 39.1 36.2 44.7 29.8 39.6 23.7 28.1 30.2 41.7 u/cm k 1.0 f 2.1 i 3.7 k 2.0 * 4.0 k 1.9 * 2.8 k 2.9 i 3.0 c 3.2 i 1.9 k 3.6

u/mgDNA 104.6 62.1 197.9 317.0 293.0 204.7 102.0 72.8 239.1 182.0 259.3 126.9 c 9.8 * 3.2 k 50.2 t40.8 i 19.9 * 40.6 * 8.6 f 5.8 i 28.0 I: 35.6 5 42.9 t 23.4


those in the ileum. In rats with an intact intestine, hypophysectomy again led to a reduction in enzyme activity (P<O.O5) although removal of the pituitary did not prevent an increase of enzyme activity in the resected animals (P<O.O2). In terms of specific activity, increased values were again associated with the hypoplastic mucosa of the hypophysectomized (P= NS) and the pair-fed rats (P < 0.00 l), while the hyperplasia induced by resection resulted in a decrease in specific activity (P = NS) except in the hypophysectomized group where there was a significant increase (P < 0.02).

Galactose absorption in vivo

The results of segmental galactose absorption, expressed per unit length of intestine, are shown in Fig. 5 , with jejunal data on the upper panel and ileal data below.

In general, as with the markers of intestinal structure, the in vivo galactose absorption data showed a similar pattern of results. In the sham-resected controls the mean jejunal galactose absorption (pmol cm intestine-' h-') fell from 10.2 f 1.5 in normally-fed rats, to 7.6 f 1.9 in the hypophysectomized animals (P = NS) but increased significantly to 19.2 f 2.9 in the pair-fed animals (P < 0.05). For the ileum, the corresponding values were 13.1 f 0.9 in normally-fed rats, 11.5 rt 1.5 after hypophysectomy, and 7.0 f 1.8 in the pair-fed group. Thus, there were minor variations in the pattern of these

Hypophysectomy feeding P a i r feeding

40 1 I I I I I

!-I

' k 30 c

!-I I

,E 20 10 6 g 10

C. Res . Sham Res . S h a m R e s .

Normal Pair feeding Hypophysectomy feeding

d 30 1 I I .. 'L. I I I c'

E 4 20

3 10 - 0

3 0

C. Res . Sham Res . Sham R e s .

Figure 5. In vivo galactose for jejunum and ileum. In vrvo galactose absorption results (mean k SEM; pmol cm intestine-' h-') as measured by luminal disappearance of galactose, corrected for net fluid transfer, from a 64 mM galactose solution. See Legend to Fig. 4 and text.

results when compared with the overall pattern for the indices of gut structure and function; jejunal absorption in the pair-fed controls was somewhat higher, and ileal absorption somewhat lower, than in the rats with an intact intestine and hypophysectomy.

After resection in the hypophysectomized rats, both the jejunum (9.9 f 2.0 pmol; P = NS) and the ileum (17.7 i 2.4; P = NS) retained their capacity to develop functional adaptation with 29% and 64% increases in mean galactose absorption when compared to the corresponding values in sham-resected hypophysectomized controls, but once again the degree of functional adaptation to resection was greater in the pair-fed group. The mean jejunal galactose absorption of 29.3 f 3.0 pmol (P = NS) and ileal absorption of 19.5 f 1.8 pmol (P < 0.01) in the pair-fed resected rats represented 53% and 1 17% increases above their appropriate sham-operated controls. In fact, the results in the pair-fed ileectomized rats were actually greater than the post-resection values in the normally-fed, distal resected animals (P < 0.02).

Discussion

This paper shows once again that in normally-fed rats both the jejunum and the ileum show adaptive mucosal changes after proximal and distal small bowel resection [27]. It also confirms that, in rats with an intact small intestine, hypophysectomy causes mucosal hypoplasia with diminished enzyme activity and reduced absorption per unit length of intestine [ 18,231. In the sham-resected rats the degree of mucosal hypoplasia and hypofunction in the hypophysectomized group was comparable to that seen in the pair-fed group, suggesting that the small bowel changes in hypophysectomized rats with an intact intestine are almost entirely due to the associated reduction in food intake. In this respect, hypophysectomy causes intestinal mucosal changes similar to those seen during partial starvation [25].

In spite of the intestinal mucosal hypoplasia induced by pituitary removal, the small bowel of the hypophysectomized rats retained its capacity to regenerate after resection. However, the pair-fed animals showed an even greater adaptive response t o resection in spite of the fact that the degree of pre-resection mucosal hypoplasia and hypofunction was comparable in pair-fed and hypophysectomized rats. This difference in the magnitude of the adaptive response suggests that the hypophysis, presumably via its hormones, played some role in modifying the extent of regeneration after jejunectomy and ileectomy.

Experimental design

In this and previous studies of intestinal adaptation, we have stressed the importance of an integrated, multi- faceted approach with measurements of mucosal mass, histology, enzyme activity, and absorptive function at the same time and in the same animal. This approach has obvious advantages, but it does limit the depth of study of any single parameter. For example, since only half

124 BRYCE TAYLOR, GERARD M. MURPHY & R. HEFWON DOWLING

the bowel remained after resection, and since approximately 30 cm of this were used for the in viuo perfusion studies, it was not possible to sample for histology or for the markers of mucosal mass at regular intervals along the intestine. In studies of jejunum, for example, we had to choose arbitrarily whether to sample from proximal or distal to the perfusion segment. The fact that we selected the most distal site undoubtedly explains the quite marked adaptive changes seen after ileectomy, since it is known that there is a build up in the magnitude of adaptive change on either side of the anastomosis which reaches a peak where the intestinal segments have been joined [30]. Therefore the more distal the jejunal and the more proximal the ileal sampling sites, the greater will be the adaptive response. Indeed, in agreement with this concept, the percentage increases in the indices of mucosal mass after resection were generally greater than those for in vivo absorption.

The other theoretical disadvantage of the multi- faceted approach is that when measuring, as in the present study, eight different parameters of intestinal structure and function, inevitably there will be minor variations in the degree of change shown by the variables. For example, the percentage increase in distal jejunal mucosal protein after distal resection in the normally- fed rats was greater than that for mucosal wet weight. But the strength of the multi-faceted approach lies in the concordance between the variables, and in fact the overall pattern of results was remarkably consistent in the present studies.

We found that rats tolerated simultaneous hypophysectomy and small bowel resection poorly. There- fore, either we had to resect the small bowel first followed, some time later, by hypophysectomy or to start by removing the pituitary and follow this with a partial enterectomy. Since the aim was to study the effect of the pituitary on the adaptive response to resection, rather than the effect of hypophysectomy on an already adapted intestine, we chose to carry out the jejunal and ileal resections 2 weeks after hypophysectomy. By removing the pituitary first, one might speculate that a hypoplastic mucosa would already have developed 2 weeks later. In the present study we did not examine the speed of onset of the adaptive changes in gut structure and function but it is known from the results of other studies that when the intestine has been partially or completely deprived of food, it rapidly becomes hypoplastic 138-411. If we assume, therefore, that both jejunum and ileum were already hypoplastic at the time of resection, it could be argued that the adaptation to resection in these rats was simply a scaled-down replica of the adaptive response seen in normally-fed rats. Indeed the magnitude of adaptive change after resection was essentially the same for hypophysectomized ‘jejunum’ and ‘ileum’ as for the intestine of the normally-fed rats. However, the fact remains that the adaptive response to resection was much greater in the pair-fed rats than in the hypophysectomized group and since food intake in these latter two groups was the same, the difference in the magnitude of adaptation

must have been due to removal of the pituitary. The observation that the magnitude of the re-

generative response to resection was greater in the pair- fed rats than in the normally-fed animals is new, but the mechanism for this phenomenon remains unknown. One might speculate that a mucosa made hypoplastic by the partial starvation of pair-feeding would be ‘hypersensi- tive’ to any undiluted hormonal response resulting from resection in non-hypophysectomized rats. However, the effects of partial starvation are complex, and conceiv- ably pair-feeding could alter other factors known to have a trophic effect on the intestine such as pancreatico- biliary secretions [42, 591 or mucosal blood flow [43] which have not been studied here. Furthermore, the effects of a 50% reduction in food intake may well be different after hypophysectomy (which decreases ap- petite) from those in pair-fed animals which, being partially starved by deliberately restricting food intake, seem ravenously hungry.

Adaptation to resection Although the present results confirm that adaptive

changes develop after both distal and proximal resection, there were, nonetheless, differences in the adaptive response from those previously reported, since in this study the degree of adaptation was almost as great in ‘jejunum’ as in ‘ileum’. It is generally accepted that the ileum shows a much greater adaptive response to resection than does the jejunum [27, 31, 381, the sort of response shown for mucosal wet weight in the present study. The apparent discrepancy between the present and previous results for the other markers of intestinal structure may be due to the sampling sites for the studies of intestinal morphology whch, as explained in the Methods section, were almost adjacent, coming from just above and just below the mid-point of the small intestine.

Normally, there is a clear-cut proximal to distal gradient for villus height, crypt depth and indices of mucosal mass [44]. However, as shown in Table 1, mucosal wet weight, protein and DNA levels were almost the same in control ‘jejunum’ as in control ‘ileum’ and, paradoxically, mean villus height was actually higher in ‘ileum’ than in ‘jejunum’.

Adaptive response of the normal small bowel to hypophysectomy

Structure. The distal jejunal and proximal ileal mucosal hypoplasia described in the present paper after removing the pituitary agrees well with the results of many other studies of intestine after hypophysectomy [for review of this subject see references 45 and 461. A decreased small bowel diameter with mucosal ‘atrophy’ and shortened villi have all been described before [20, 23, 24, 281 while Leblond & Carrikre [47] found a decreased mitotic index in the duodenal crypts of hypophysectomized rats. However, in most of these investi- gations the importance of the associated reduction in food intake was not fully appreciated and pair-fed con-


trols were not studied. Riecken and colleagues [24] rightly emphasized the importance of including a pair- fed control group when studying hypophysectomized rats, but, unlike the results of the present study, they showed that in rats with an intact intestine, the reduction in villus height, crypt depth and segmental glucose absorption was less marked in the pair-fed rats than in the hypophysectomized animals. Nishikawara [48] also studied both pair-fed and hypophysectomized rats and showed that intestinal mucosal hexokinase and phosphatase activities were reduced to a greater extent after removing the pituitary than in pair-fed controls.

Although there was no statistically significant difference between sham-resected, pair-fed and sham- resected, hypophysectomized rats for most of the variables in the present study, examination of the results shows that the mean values for almost all the parameters studied were, in fact, slightly higher in the pair-fed than in the hypophysectomized rats. Thus, although in general the present findings agree with the results of previous studies, they do, nonetheless, suggest that in animals with an intact small intestine the effects of hypophysectomy are mainly due t o the associated reduction in food intake.

Function. Many previous investigators have studied intestinal function following hypophysectomy but with apparently conflicting results. As long ago as the mid 1930s, Bennett [49] and Phillips & Robb [50] showed that intestinal glucose absorption was 30-35% lower in hypophysectomized rats than in controls. Similarly, Samuels & Reinecke [5 I ] found increased faecal fat and nitrogen excretion in hypophysectomized rats suggesting, indirectly, decreased intestinal absorption. Riecken and colleagues [24] using an in vivo recirculation perfusion system similar t o that used in the present study, found a 44% decrease in jejunal glucose absorption. Decreased water, glucose, and net electrolyte absorption have also been found when in vivo absorption techniques have been used [45]. However, Houssay & Foglia [52] could find no change in sugar absorption by the intestine of hypophysectomized toads. Furthermore, Finkelstein & Schachter [53] showed that net sodium flux was u n c h ~ g e d in hypophysectomized rats, as measured by the everted sac technique, and Somkin & Levitan [54] showed no change in intestinal fat absorption after pituitary removal in rats. Conversely, Havivi & Levitan [22] found a transient increase in rat small bowel glucose and galactose absorption 5 days after hypophysectomy which had returned to normal 6 days later. These authors also showed an increase in intestinal mucosal hexokinase activity following pituitary removal [2 11. Other workers have also found enhanced glucose absorption [55], a greater transport of iron, proline, and calcium [53], more effective intestinal fatty acid incorporation into esters [56], and increased bidirectional fluxes of glucose, histidine, and proline in jejunal sacs of hypophysectomized rats [57].

The reason for these apparently conflicting results may be due, at least in part, to the techniques used to

measure absorption. Many of the studies showing normal or increased absorption after hypophysectomy depended on in vitro techniques while results in the majority of the studies using in vivo methods show diminished absorption. This suggests that when studying hypoplastic intestine, in vitro methods such as the everted sac technique [58] may yield artefactual results, analogous to the situation seen during partial o r complete starvation where in vitro methods actually suggested enhanced absorption while in vivo techniques showed that absorption was, in fact, diminished [25].

Adaptive response to resection in hypophysectomized rats

If one accepts that the difference in the adaptive response to resection between hypophysectomized and pair-fed rats is indeed due to the pituitary, then this difference could be due directly to decreased circulating levels of a pituitary hormone(s) or it could be mediated indirectly by the effects of hypophysectomy on exocrine pancreatic secretions or gut hormones both of which may be trophic to the intestine [59-63 1.

Considering the hypothetical indirect mechanisms first, Tiscomia & Dreiling [64] showed that hypophysectomy led to marked reductions in pancreatic weight and in exocrine pancreatic secretions in the dog. If Altmann’s theories are correct [59], a corresponding decrease in pancreatic secretions in the present study might have affected the adaptive response t o resection after hypophysectomy. This mechanism may also be important in the adaptive changes seen in the intestine during exclusive parenteral nutrition. We have shown recently that daily stimulation of the pancreas with cholecystokinin and secretin [66] completely prevents the mucosal hypoplasia and hypofunction which would otherwise have occurred as a result of excluding luminal nutrition by total parental feeding in the dog [40,65].

With regard to the effects of hypophysectomy on the release of enterotrophic hormones, little is known about the effects of pituitary removal on hormones other than those secreted by the target organs. However, Dorchester & Haist [ 181 did show that the amount of total extractable secretin was reduced in the intestine of hypophysectomized rats. Recent evidence suggests that, if anything, secretin is antitrophic to the intestine [67] but hypophysectomy might well have comparable effects on other hypothetical enterotrophins such as gastrin [ 111 and enteroglucagon [9, lo]. Indeed, Enochs & Johnson have shown recently that both serum and antral mucosal gastrin levels fall after hypophysectomy in the rat and that injection of growth hormone restores the serum gastrin levels to near normal [68].

Returning to the effects of the pituitary hormones themselves, many studies have examined the effect of single or multiple hormonal replacement in hypophysectomized animals. Growth hormone, prolactin, thyroxin and ACTH (or prednisolone and/or cortisone) have all been shown to prevent completely or partially the intestinal hypoplasia induced by removing the hypo-


physis. Similar hormonal replacement studies have not yet been carried out after combined resection and hypophysectomy, but such experiments are planned to ex- tend the work described in this paper. These studies will form the basis for future publications.

Acknowledgments

Thanks are due to the staff of the animal house, Guy’s Hospital and Medical School, for their care of the animals, Mr Bob Francis for preparing the histological sections, Professor K. W. Weinbren and colleagues at the Royal Postgraduate Medical School for their advice on hypophysectomy and to Miss Margaret Henderson who typed the script.

B.T. was supported by an Ontaria Health grant and partially by a Roscoe-Graham travelling fellowship.

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pituitary hormones and the small bowel: effect of hypophysectomy on intestinal adaptation to small...

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