JOURNAL

OF

SURGICAL

RESEARCH

x,540-546

(1979)

lleal Adaptation following Proximal Intestinal Resection Characterized by Decreased Cellular Uptake of Amino Acid NEIL RUDO, M.D., Surgical

PH.D.,

CLIFFORD W. DEVENEY,

M.D.,

Is

AND LAWRENCE W. WAY, M.D.

Service, Veterans Administration Medical Center. San Frarzcisco. Californiu Department of Surgery, University of California, San Francisco. California Presented at the Annual Meeting of the Association for Academic Cleveland, Ohio, November 12- 15, 1978

94121( and the 94122

Surgery,

Ileal absorption was studied in male Wistar rats 6 weeks atter resection of ah small bowel distal to the ligament of Treitz except for 20 cm of ileum. Intestinal absorption of 3-0-methylglucose (3-O-MG) and 1-aminocyclopentanecarboxylic acid (ACPC) was measured by in viva perfusion and an in vitro ring technique. These substrates are actively transported but are not metabolized by the cell, so cellular degradation or utilization cannot intluence the rate of uptake. Following resection, heal mucosal cells took up less ACPC than unresected controls. There were no differences in cellular uptake of 3-O-MG, but active uptake of this compound was minimal. The decrease in active uptake of ACPC probably reflected immaturity of the proliferating mucosal cells in the adapting ileal remnant after proximal resection.

INTRODUCTION

After small bowel resection all layers of the small intestine become enlarged, but the most striking changes are seen in the mucosa. Increased villous height, crypt depth, and intestinal circumference occur [ 1.5,20,23] and contribute to enlargement of the entire mucosal mass. Morphologic adaptation is greatest after proximal intestina1 resection, and the degree of adaptive change is most pronounced near the anastomosis [ 1,4]. The magnitude of response is thought to be related to the amount of intestine resected 18, 231. Following massive resection, adaptation in the remaining intestine is accompanied by increased absorption of foodstuffs per unit length. In vivo perfusion studies of intestinal remnants have demonstrated increased uptake of fat, protein, and carbohydrate per unit length [2,4, 14, 1.5, 19, 201 and there is a reasonable correlation between the degree of mucosal hypertrophy and the amount of increased absorption. It is not clear, however, to what extent the increased absorption is due to greater 540

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Q 1979 by Academic Press, of reproduction in any form

Inc. reserved.

numbers of cells per unit length of gut or to functional changes within the cell. The purpose of this study was to determine if adaptation is accompanied by functional augmentation of the active transport system for monosaccharides and amino acids. Absorption and uptake studies were performed with two substrates, 3-O-methylglucose (3-O-MG) and l-aminocyclopentanecarboxylic acid (ACPC). These substrates are actively transported but not metabolized, so cell metabolism was excluded as a factor affecting the results. METHODS Animal

Preparation

Under intraperitoneal pentobarbital (40 mg/lOO g) anesthesia, all small bowel distal to the ligament of Treitz except 20 cm of distal ileum was resected in male Wistar rats weighing 150-200 g. Intestinal continuity was reestablished by anastomosing the distal ileum to the jejunum at the ligament of Treitz using a single layer of continuous 7-O silk. After operation, the animals were

RUDO,

DEVENEY,

AND WAY:

ADAPTIVE

CHANGES

given 10 ml of saline subcutaneously and were subsequently allowed nothing by mouth for 24 hr. The rats were then allowed only water for an additional 24 hr before regular feedings were begun. The resected rats were pair-fed with a group of unresected controls. Absorption studies were performed 6 to 8 weeks after resection. Weights of pair-fed control and resected rats did not differ significantly at the time of study.

AFTER

INTESTINAL

[ACPClinf

RESECTION

541

7

where abs = absorbed, inf = infusate, and eff = effluent. The percentage of ACPC absorbed over six lo-min intervals was averaged and expressed as the percentage ACPC absorbed per centimeter of ileum and the percentage ACPC absorbed per milligram mucosal weight. Identical perfusion experiments were perMeasurement of Absorption formed in another group of rats using 10 A laparotomy was performed, and a mM 3-O-MG (Aldrich Chemicals) and trace lo-cm segment of the most distal ileum was 3-0-[3H]MG (New England Nuclear, NETisolated and was cannulated proximally and 379, 4-5 Ci/mmole with [14C]inulin (New distally with Silastic tubing (i.d. = 0.078 in.). England Nuclear NEC- 164B, l-3 mCi/g) Both cannulas were secured with a tie of as the nonabsorbable marker. Results were 4-O silk. The bowel was replaced into the expressed as percentage uptake per centiperitoneal cavity with the polyethylene meter and percentage uptake per milligram tubes exiting through the wound. The in- mucosal weight. After the perfusion experiments, the testinal segment was then perfused with Krebs-Ringer bicarbonate buffer contain- lo-cm segment of bowel was weighed. The ing 10 n&J ACPC (Aldrich Chemicals) with mucosa was stripped and weighed, and trace [14C]ACPC (New England Nuclear, mucosal DNA and protein concentrations NEC-297,20-40 mCi/mmole). The perfusate were determined. DNA concentration was determined by also contained [3H]PEG (New England Nuclear, NET-405, 0.5-2 mCi/g) as a non- the spectrophotofluorometric method of absorbable marker to enable correction for Kissane as modified by Hinegardner [lo]. absorption or secretion of water. The rate Protein concentration was measured by the of perfusion was 15 ml/hr (2.5 ml/l0 min). method of Lowry [12]. After a 30-min equilibration period, a relatively constant rate of absorption had been Measurement of Active Substrate Uptake achieved; samples were then collected at lo-min intervals. The relative concentraIn vitro incubation of everted intestinal tions of ACPC and PEG were determined segments was used to provide an index of by liquid scintillation counting. The rate active uptake of sugar and amino acid by of absorption (per 10 cm-10 min) was ex- mucosa. The method was similar to that pressed by the formula: used by Crane and Mandelstam [3], Olsen and Rosenberg [17], and Rudo et al. [18]. Segments of distal ileum were removed and ACPC,,,, = [ACPClin, - [ACPCleff i washed in chilled oxygenated Krebs-Ringer bicarbonate buffer. The intestine was everted PEGlinr e(2.5 ml). to expose the mucosa externally. The ’ F’W,,, intestinal segment was then divided into The percentage of ACPC absorbed equals 3-mm segments or rings, which were incuthe amount of ACPC absorbed divided by bated in 2.4 ml of the oxygenated Krebsthe amount of ACPC infused, or Ringer bicarbonate buffer containing 3-0-

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[3H]MG (3 pmole/ml) and [14C]ACPC (2.7 nmol/ml) in the incubation medium. After incubation for 40 min at 37°C in stoppered flasks containing an atmosphere of 95% 0, and 5% COZ, the rings were removed, rinsed, blotted, weighed, and then extracted in distilled water by boiling for 6 min. Aliquots of tissue fluid extract and incubation media were counted in a liquid scintillation spectrometer to determine the concentrations of 3-O-[3H]MG and [14C]ACPC in the media and tissue extract. The concentration of these substrates in the incubation media is called the extracellular fluid concentration (ECF). After making adjustments for extracellular space (inulin space) and for tissue water, the concentrations of 3-0[3H]MG and [14C]ACPC in the intracellular water could be calculated. The concentration in the intracellular water is called the intracellular fluid concentration (ICF). The ratio of the concentration of these substances in intracellular water compared with incubation medium (extracellular water) is called the intracellular to extracellular fluid concentration ratio (ICF/ECF). The ICF/ ECF ratio reflects the ability of the cell to actively transport these substances and was utilized as an index of active transport. A higher ratio reflects greater active transport. Inulin space was determined in a separate experiment by incubating intestinal rings in the same Krebs-Ringer bicarbonate buffer system in the presence of [14C]inulin. After 45 min the tissue was removed, blotted dry, and weighed. The tissue was then extracted for 10 min in boiling water and the total amount of inulin was determined by scintillation counting of the extract. The concentration of [14C]inulin in the incubation medium was also determined by scintillation counting. The inulin space was then calculated by dividing the total inulin extracted from the tissue by the concentration of inulin in the medium. The inulin space was expressed as milliliters per nanogram of tissue. Tissue water was determined by dehydrating preweighed pieces of tissue in a drying oven for 18 hr and then weighing to

VOL. 26, NO. 5, MAY

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determine the amount of weight loss. Tissue water was expressed as milliliters per milligram, or as percentage of tissue weight. Since tissue water equals inulin space plus intracellular water, then intracellular water equals tissue water minus inulin space water. Statistics Comparisons between resected and control groups were made using Student’s t test. Data are displayed as mean ? standard deviation. Unless otherwise specified, all differences observed were significant with a probability of less than 0.05. DATA

When initially operated, all rats weighed between 180 and 190 g (mean 183 f 2.5 g). Six to eight weeks later, the unresected rats weighed 344 + 40 g, and the resected rats weighed 304 ? 24 g. The difference was not significant. The rats were pair-fed, so food consumption was the samefor the two groups (24 + 5 g/day for controls, and 24 + 4 g/day for resected rats). The morphologic changes in the remaining ileum of resected rats, which are presented in Table 1, confirmed the findings of others. Bowel weight per unit length was greatly increased, with the majority of the increase secondary to mucosal enlargement. Disproportionate enlargement of the mucosa was reflected by an increase in the ratio of mucosal weight to total bowel weight in the resected animals. The mucosal DNA per centimeter, and the DNA per 100 mg of mucosa were larger in the resected animals but the DNA per milligram of protein was not different. Tissue water and inulin space in controls and resected animals were not significantly different (see Table 2). The perfusion studies demonstrated an increased uptake of both 3-O-MG and ACPC in resected animals when the uptake was measured per segment; however, when uptake was expressed per gram of mucosa, the ratios were reversed, with the control

RUDO,

DEVENEY,

AND WAY: ADAPTIVE

CHANGES TABLE

MORPHOLOGIC

AFTER

INTESTINAL

1

OBSERVATIONP

Control 36.2 62.0 0.5 13.8 437.0 8.18 57.0

Mucosa (mgicm ileum) Bowel weight (mg/cm) Ratio of mucosal wt/bowel wt Circumference (mm) mg mucosa) DNA (&lo0 Protein (m&O0 mg mucosa) DNA (p.g/mg protein) a Observations otherwise stated.

in six control

and six resected

INULIN

Inulin space Tissue water n Measurements animals.

12.7 ? 1.8 75.0 2 3.3 in four control

and four resected

are significant

2 2 t t t 2 k

20.0 18.0 0.07 1.6 29.0 0.89 3.0 (NS) (P < 0.05) unless

DISCUSSION

Following small bowel resection, the remaining intestine hypertrophies with the most marked enlargement in the mucosa. This is accompanied by changes in function, which include increased water, electrolyte, and nutrient transport per unit length [19]. The mucosal enlargement is secondary to TABLE UPTAKE

OF AMINO

ACID

3

AND MONOSACCHARIDE”

Control

Resected

Percentage uptake per 10 cm ileum ACPC 3-O-MG

35* 18-c

7 1

44& 222

3 6

Percentage uptake pergram mucosa

Resected 10.8 f 2.7 (NS) 76.0 f 3.8 (NS)

92.7 145.0 0.65 18.2 647.0 10.34 61.0

incubation of rings in 3-O-MG and ACPC. For ACPC, the ICF/ECF ratios were greater than 1 for both control and resected animals, but the ratio was higher in the control animals (9.3 vs 5.9). This signifies a greater ability of the cell to actively take up ACPC in the unresected animals. Because the ICF/ ECF ratios for 3-O-MG were so close to 1, it is likely that little, if any, active transport of this substrate occurred.

WATERY

Percentage of tissue wt Control

12.0 13.0 0.06 0.8 80.0 1.58 6.0

Substrate

2

SPACE AND TISSUE

+ k k + + + 2

Resected

animals. All differences

animals showing greater uptake. Table 3 compares uptake per unit length with uptake per gram of mucosa. The uptake of ACPC per mucosal weight was greater in unresected animals; the uptake of 3-0-MG was not different. The in vitro incubation of intestinal rings also measured total uptake of substrates but was primarily used as an index of active uptake. If the intracellular to extracellular fluid ratios are 1 or less, this suggests that the substrate in the medium is equilibrating with intracellular fluid and active transport is not occurring. An intracellular to extracellular fluid ratio (ICF/ECF) of greater than 1 implies active transport. In a separate experiment, we determined that the uptake of ACPC and 3-O-MG was linear over 1 hr (see Fig. 1). This implies that the tissue was viable and that transport was occurring at a constant rate during this time. Table 4 presents the results of in vitro TABLE

543

RESECTION

ACPC 3-O-MG

118 -+ 12 46 2 18

43 k 10 29 2 6.2 (NS)

“ Differences are significant P < 0.05 unless otherwise stated. Measurements in three control and three resected animals.

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cellular hyperplasia [4, 9, 161 and includes (1) increased cell numbers, (2) increased cell proliferation and migration rate, and (3) increased DNA synthesis and total DNA content [ 141.Evidence of hyperplasia occurs as early as 2 days and reaches a plateau 12 days postresection [9, 161. Hyperplasia follows removal of as little as 20% of the intestine and the degree of hyperplasia is influenced by the amount of intestine removed [8]. A slight shortening of the cell cycle occurs [7, 11, 141, and is accompanied by increased cell turnover as demonstrated by greater extrusion of DNA into the intestinal lumen [21]. In this experiment we studied the rats 6 weeks postoperatively, by which time the adaptive changes should have been complete. Our morphologic data are consistent with the findings of others. Small bowel enlargement involved all layers of the bowel, but the most striking change occurred in the mucosa as manifested by an increased ratio of mucosal weight to bowel weight (see Table 1). The parallel increase in total DNA and in DNA per mucosal weight is another indication that the enlargement was due to cellular hyperplasia. The fact that DNA and protein both increased per weight of adapted bowel suggests that the cells may be smaller after resection. The increase in intestinal circumference from 13 to 18 mm also shows that the mucosal surface area is greater. It has been suggested that cellular hyperplasia leads to the production of more immature cells and thus to decreased funcTABLE

4

INVITRO ACTIVETRANSPORTOF AMINO ACID AND SUGAR FOLLOWING MASSIVE RESECTION" ICFIECF Substrate

Control

Resected

ACPC 3-O-MC

9.3 -t 0.8 1.4 k 0.6

5.9 + 0.9 (P < 0.05) 1.0 ” 0.4 (NS)

a Measurements animals.

in five control

and five resected

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I 60

Incubotmn

FIG. 1. The rate of uptake of [‘“CIACPC. Between 10 and 60 min uptake was linear. A similar rate was seen with 3-O-MG. Incubation time with 3-O-MG and ACPC was 40 min.

tional capacity of the cell. In vitro studies using cell suspensions have demonstrated decreased uptake of [14C]glucose and [‘“Clleucine in the cells from the remaining ileum after resection [20]. In addition, the cells from the remnant were smaller than those from the controls [20]. After resection, levels of brush border enzymes per milligram of DNA were found to be decreased, suggesting that the mucosal cells were metabolically immature [6, 13, 201. Our perfusion and in vitro ring studies demonstrated that uptake per mucosal weight or per DNA was decreased in the remaining ileum after proximal bowel resection. This signified decreased active cellular uptake and is consistent with cellular immaturity in the remnant after resection. The studies with 3-O-MG revealed no differences in uptake when measured per tissue weight. Because the ICF/ECF was so close to 1, however, this substrate was not actively transported. Other in vitro studies in rats have found a decrease in uptake of glucose and amino acid per cell after resection [20]. In vitro studies in dogs measuring ileal uptake of amino acid per weight failed to demonstrate any differences between resected and control animals. However, the dogs had a 50% bowel resection, which is less than the 80% resection performed in our rats [5]. In

RUDO, DEVENEY,

AND WAY: ADAPTIVE

addition, both of these studies used substrates that could be metabolized by the cell. Nonmetabolizable substrates were used in this study so intracellular degradation or utilization could not influence uptake. The results indicated that the increased absorption of both 3-O-MG and ACPC in the ileal remnant after resection was entirely a result of mucosal enlargement and was not owing to increased cellular transport. SUMMARY

This study demonstrated that cellular hyperplasia occurred in the mucosa of the ileal remnant after proximal intestinal resection, which resulted in a large increase in mucosal mass and an overall increase in absorption of sugar (3-O-MG) and amino acid (ACPC) per segment. Active uptake of amino acid per weight of mucosa and per cell decreased after resection, probably reflecting cellular immaturity. There was no difference in the absorption of 3-O-MG per mucosal weight, nor was there any difference in active uptake between controls and resected animals. Active uptake of 3-O-MG was minimal. Therefore, increased ileal absorption of amino acid (ACPC) and sugar (3-O-MG) after proximal resection resulted from increased mucosal mass and not from increased capacity of the cell to actively transport these substrates. ACKNOWLEDGMENTS We wish to acknowledge the technical assistance of Anny Wong and the expert secretarial assistance of Dorothy Archibald and Hulda Shollar.

REFERENCES 1. Booth, C. C., Evans, K. T., Menzies, T., et al. Intestinal hypertrophy following partial resection of the small bowel in the rat. Brir. J. Surg. 46: 403, 1959. 2. Bury, K. D. Carbohydrate digestion and absorption after massive resection of the small intestine. Surg. Gynecol.

Obstet. 135: 177, 1972.

3. Crane, R. R., and Mandelstam, P. An effect of alloxan diabetes in the active transport of sugar by rat small intestine in vitro. Biochim. Biophys. Acta 45: 460, l%O.

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4. Dowling, R. H., and Booth, C. C. Structural and functional changes following small intestinal resection in the rat. C&z. Sci. 32: 139, 1967. 5. Feldman, E. J., Dowling, R. H., McNaughton, J., and Peters, T. J. Effects of oral versus intravenous nutrition on intestinal adaptation after small bowel 70: 712, resection in the dog. Gasfroenferology 1976. Gleeson, M. H., Dowling, R. H., and Peters, T. J. Biochemical changes in intestinal mucosa after experimental small bowel bypass in the rat. Clin. Sci. 43: 743, 1972. Hanson, W. R., and Osborne, J. W. Epithelial cell kinetics in the small intestine of the rat 60 days after resection of 70 percent of the ileum and 60: 1087, 1971. jejunum. Gastroenterology Hanson, W. R., Osborne, J. W., and Sharp, J. G. Compensation by the residual intestine after intestinal resection in the rat. I. Influence of the amount of tissue removed. Gastroenterology 72: 692, 1977. 9. Hanson, W. R., Osborne, J. W., and Sharp, J. G. Compensation by the residual intestine after intestinal resection in the rat. Gasfroenferology 72: 701, 1977. 10. Hinegardner, R. T. An improved fluorometric assay for DNA. Anal. Biochem. 39: 197, 1971. 11. Loran, M. R., and Cracker, T. T. Population dynamics of intestinal epithelia in the rat 2 months after partial resection of the ileum. J. Cell Biol. 19: 285, 1963. 12 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Eio/. Chem. 193: 265, 1951. 13. McCarthy, D. M., and Kim, Y. S. Changes in sucrose enterokinase, and peptide hydrolase after intestinal resection, the association of cellular hyperplasia and adaptation. J. C&z. Invest. 52: 942, 1973. 14. McDermott, F. F., and Roudnew, B. IleaI crypt cell population kinetics after 40 percent small bowel resection. Autoradiographic studies in the rat. Gastroenterology 70: 707, 1976. 1.5. Nygaard, K. Resection of the small intestine in rats. Acta Chir. Stand. 133: 233, 1%7. 16. Obertop, H., Nundy, S., Malamud, D., and Malt, R. Onset of cell proliferation in the shortened gut rapid hyperplasia after jejunal resection. Gastroenterology

72: 267, 1977.

17. Olsen, W. A., Rosenberg,. I. H. Intestinal transport of amino acids and sugars in diabetic rats. J. Clin. Invest. 49: 2256, 1970.’ 18. Rudo, N. D., Lawrence, A. M., and Rosenberg, I. H. Treatment with glucagon-binding antibodies alters the intestinal response to starvation in the rat. Gastroenterology 69: 265, 1975. 19. Weinstein, D. L., Shoemaker, C. P., Hersh, T., et

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al. Enhanced intestinal absorption after small bowel resection in man. Arch. Surg. 99: 560, 1969. 20. Weser, E., and Hemandez, M. H. Studies of small bowel adaptation after intestinal resection in the rat. Gastroenterology 60: 69, 1971. 21. Weser, E., and Tawil, T. Epithelial cell loss in remaining intestine after small bowel resection in the rat. Gastroenferology 71: 412, 1976.

22. Williamson, R. C. N., Bauer, F. L. R., Ross, J. S., et al. Proximal enterectomy stimulates distal hyperplasia more than bypass of pancreaticobiliary diversion. Gastroenterology 72: 692, 1977. 23. Wilmore, D. W., Dudrick, S. J., Daly, J. M., et al. The role of nutrition in the adaptation of the small intestine after massive resection. Surg. Gynecol. Obstet. 132: 673, 1971.