GASTROENTEROLOGY
1992;102:1949-1956
Truncated and Native Insulinlike Growth Factor I Enhance Mucosal Adaptation After Jejunoileal Resection JON A. VANDERHOOF, ROBERT H. McCUSKER, ROSS HAMID MOHAMMADPOUR, DARCY J. BLACKWOOD, RICHARD F. HARTY, and JUNG H. Y. PARK
CLARK,
Department of Pediatrics, Creighton University School of Medicine and University of Nebraska Medical Center, Omaha, Nebraska; Department of Medicine, University of North Carolina, Chapel Hill, North Carolina; and Department of Developmental Biology, Genentech, Inc., South San Francisco, California
It has been shown previously that insulinlike growth factors (IGFs) stimulate the proliferation of intestinal crypt cells in vitro. To examine the in vivo effects of IGF-I on mucosal adaptation, three groups of Sprague-Dawley rats underwent 80% jejunoileal resection. Miniosmotic pumps were then inserted under the skin immediately after resection to deliver vehicle (resected control), 1.5 mg/kg per day of IGF-I, or 1.5 mg/kg per day of des-(l-3)IGF-I (des-IGF-I). Des-IGF-I is a truncated form of IGF-I that binds as well to type I IGF receptors but less tightly to several forms of IGF-binding proteins (IGFBPs) than IGF-I. Ad libitum food intake did not differ among the three resected groups. Body weight gains were greater in animals receiving des-IGF-I than in those receiving IGF-I, which were greater than resected controls. All animals were killed 7 days postoperatively, and the remaining small intestine was removed and divided at the anastomotic site. Both IGF-I and des-IGF-I induced hyperplasia (increased DNA and protein content] in the duodenojejunum but not in the ileum. IGF-I and des-IGF-I were equally active. In contrast, sucrase, maltase, and leucine aminopeptidase activities were greater only in the ileum of animals receiving IGF-I and des-IGF-I than in resected controls. Although more potent in stimulating overall body weight gain, des-IGF-I was not more potent than IGFI when duodenal and ileal responses were determined. IGF infusion (IGF-I > des-IGF-I) increased the levels of circulating IGFBP-3 and IGFBP-2, which may act to modulate the biological effectiveness of the infused peptides. These results suggest that both IGFI and des-IGF-I may have potential as therapeutic agents for short bowel patients.
I
nsulinlike growth factors (IGFs) and insulin are important regulators of somatic growth.’ IGF-I, IGF-II, and insulin, when administered to IEC-6 cells in culture, cause stimulation of cellular proliferation. IEC-6 cells are an established line of intestinal
epithelial cells derived from rat jejunal crypts. Comparison of dose-response curves for these growth factors on proliferative indices in IEC-6 cells indicates that IGF-I is the most potent and efficacious peptide, suggesting that the effects are mediated by interaction with the type I IGF receptor.’ Rat intestinal epithelium contains both type I and type II IGF receptors. Studies that have examined the regional distribution of IGF receptors within the intestinal villus-crypt unit indicate a predominance of receptors in the proliferative crypt region.3 These findings suggest that IGF-I may be an important growth factor in the regulation of intestinal epithelial cell growth. Unlike insulin, IGFs are bound to specific IGFbinding proteins (IGFBPs) in plasma and other biological fluids. To date, six IGFBPs have been purified and at least partially sequenceda4” The precise role of IGFBPs in modulating the anabolic and mitogenic effects of the IGFs is unclear. There are conflicting reports on the biological role of the IGFBPs in modulating IGF action. IGFBP-1 enhances the biological response to IGF-I upon the DNA synthesis of cultured aortic smooth muscle cells and human fibroblasts.g This preparation of IGFBP was also shown to adhere to the smooth muscle cell surfaceslO possibly accounting for the enhanced IGF effect. In other studies, IGFBP-1 has been shown to prevent the action of IGFs, possibly by limiting access to cell surface reof fibroblasts with IGFBPceptors. lls*’ Preincubation 3 or coincubation with an equimolar ratio of IGFBP-3 and IGF-I potentiates IGF-I-stimulated thymidine incorporation, but coincubation of IGFBP-3 can also inhibit the IGF-I effect.‘3*‘4 In the same manner as IGFBP-1, IGFBP-3 adheres to cell surfaces.15*‘” These findings suggest that IGFBPs function in diverse ways to modulate the biological effects of the IGFs. An IGF-I derivative with glycine, proline and glu0
1992 by the American Gastroenterological 0016-5085/92/$3.00
Association
1950
VANDERHOOF
ET AL.
tamic acid residues cleaved from the N terminus [des-(l-3)-IGF-I (des-IGF-I)] stimulates protein and DNA synthesis and inhibits protein breakdown at concentrations lower than those required of IGFL1’*” Des-IGF-I has normal affinity for receptors on human skin fibroblasts*g and L6 myoblasts.” An important characteristic of des-IGF-I is its reduced affinity for IGFBPs.” Because soluble IGFBPs block cell surface binding of IGF-I,‘l a reduction in affinity for IGFBPs could explain the elevated bioactivity of des-IGF-I. Short bowel syndrome is a malabsorptive disorder resulting from resection of a large percentage of the small intestine. After intestinal resection, a considerable increase in mucosal surface area occurs through a gradual lengthening of villi lining the remaining small intestine.22 Although recent advances in the use of home total parenteral nutrition have increased the long-term survival of many patients with short bowel syndrome, living without parenteral nutrition depends on the process of mucosal adaptation. Therefore, it is important to find trophic factors that can enhance the mucosal adaptation that normally occurs. The purpose of this study was to examine in vivo effects of exogenously administered IGF-I and des-IGF-I on mucosal adaptive responses following massive small bowel resection in rats. In addition, IGF-I and des-IGF-I were compared to test whether binding to IGFBPs could alter the ability of IGF-I to stimulate mucosal hyperplasia. Materials and Methods Materials Recombinant human IGF-I and des-IGF-I were provided by Genentech, Inc. (South San Francisco, CA). 1251IGF-I (specific activity, 2000 Ci/mmol), carrier-free Nalz51, and molecular weight standards were purchased from Amersham (Arlington Heights, IL). Rabbit anti-IGF-I serum (UB3-189) was a gift from Drs. L. E. Underwood and J, J, Van Wyk (University of North Carolina, Chapel Hill, NC) through the National Hormone and Pituitary Program of NIADDK (Baltimore, MD). This antibody has wide species cross-reactivity and has been used successfully in studies with rats.’ Recombinant rat IGF-I has been shown to be 90% cross-reactive with this antiserum compared with recombinant human IGF-I (personal communication, Dr. L. E. Underwood). PH 75 nitrocellulose sheets were obtained from Schleicher and Schuell, Inc. (Keene, NH). Human y-globulin was purchased as an 18% solution (Cutter Biological, Berkeley, CA). Unless otherwise stated, all other chemicals were obtained from Sigma Chemical (St. Louis, MO). Animals The animals used in this study were cared for according to the guidelines of the Animal Review Committee
GASTROENTEROLOGY
Vol. 102, No. 6
at the University of Nebraska Medical Center. Twentyseven male Sprague-Dawley rats (120-140 g) were purchased (Sasco, Omaha, NE) and housed individually in hanging stainless steel cages. They were provided Purina rat chow (Ralston-Purina, St. Louis, MO) and tap water ad libitum. After 3 days of acclimation to laboratory conditions, they were randomly divided into four groups. Three groups (8 rats/group) were subjected to jejunoileal resection.23 After a 36-hour fast, all bowel between the points 4 cm distal to the ligament of Treitz and 12 cm proximal to the ileocecal valve was removed. Miniosmotic pumps (Alzet model 2001, Palo Alto, CA) were inserted under the skin immediately after resection to deliver either vehicle (0.1 mol/L acetic acid), 1.5 mg/kg per day of IGF-I, or 1.5 mg/kg per day of des-IGF-I. D-Glucose (5%, wt/vol) and terramycin (O.OZZ%, wt/vol) were provided in the drinking water for 36 hours after surgery, after which animals were placed back on the chow food. Food intake was monitored daily. The fourth group (3 rats) was subjected to the same fasting and refeeding conditions but did not undergo surgery. All animals were killed 7 days later, and the small intestine from the pylorus to the ileocecal valve was removed and divided at the anastomosis. Portions of intestine 1 cm on either side of the anastomosis were discarded because of the surgically induced hyperplasia occurring in this region. The mucosa was scraped from underlying tissue with a glass slide for the determination of DNA, protein, and enzyme activity. Blood was drawn from the aorta at the time of death, and serum was prepared for the determination of IGF-I, insulin, glucose, and IGFBP levels. The proximal 2 cm of the duodenum and ileum was removed and fixed in buffered formalin for histological analysis. After fixation, the tissue was sectioned (4 pm thick), mounted on glass slides, and stained with H&E. The villi and crypts were measured under a microscope with a micrometer eyepiece to determine villus height and crypt depth.
Mucosal DNA, Protein, and Enzyme Assays Mucosal samples were homogenized in deionized water. Protein concentration was determined by the method of Lowry et a1.24 and expressed as milligrams of protein per centimeter of bowel. DNA was extracted by the method of Munro and Fleck.25 The resulting DNA fractions were assayed according to the method of Giles and Myers.” Disaccharidase activities were measured according to the methods of Dahlqvist.” We measured the activity of alkaline phosphatase according to the method of phosphate as a substrate. Sommer,28 using p-nitrophenyl Leucine aminopeptidase activity was estimated by the method of Goldbarg and Rutenburg.2g Determination of JGF-I, Insulin, Levels in Serum
and
Glucose
Serum insulin levels were measured using a radioimmunoassay kit (Diagnostic Product Corporation, Los Angeles, CA). Serum glucose was analyzed by the method of Bergmeyer et a1.30using hexokinase and glucose-6-phosphate dehydrogenase. Before assaying for IGF-I, the IGFBPs were eliminated by acid-ethanol precipitation as described by Daughaday et a1.31 IGF-I levels were esti-
IGF-I AND MUCOSAL ADAPTATION
June 1992
mated by radioimmunoassay as described by Furlanetto et a1.32using 1251-IGF-I.Recombinant human IGF-I was used as the unlabeled standard except that des-IGF-I was used as the unlabeled standard for the des-IGF-I group. Determination
200 -
Resected control IGF-I Des-IGF-I Unoperated control
A-A A-A e--e o-o
g
1951
175--
of IGFBPs in Serum
IGF-binding capacity. Binding capacity was quantified by determining the ability of serum aliquots to bind ‘Z51-IGF-Ias described by McCusker et a1.33This assay measures the quantity of unsaturated IGFBPs in sera. Briefly, 1 pL of serum was incubated for 1 hour with 20,000 cpm of 1251-IGF-I,and the bound ‘2SI-IGF-I was precipitated by centrifugation after adding 0.25% human y-globulin and 12.5% polyethylene glycol (PEG) 8000. Activity is expressed as nanogram equivalents per milliliter. Ligand blotting with ‘251-IGF-I. The proteins of serum were electrophoresed through 12.5% discontinuous sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions as described by McCusker et a1.34The proteins were transferred to 0.05 pm PH 75 nitrocellulose sheets by electroblotting using a Semiphor Semi-dry Transfer Unit (Hoefer Scientific Instrument, San Francisco, CA). Nonspecific binding of the tracer to the nitrocellulose paper was prevented by preincubating the sheets sequentially in 1% Nonidet P-40 (Sigma Chemical Co., St. Louis, MO), 0.1% bovine serum albumin, and 2% Tween20 (Sigma) for 10 minutes, 2 hours and 20 minutes, respectively. To detect IGFBPs, the nitrocellulose sheets were incubated overnight with 100,000 cpm/mL of ‘251-IGF-I. The sheets were then washed, and IGFBPs were visualized by autoradiography. Relative molecular weights were estimated by running prestained molecular weight standards in a parallel lane. The density of each band was measured using a Hoefer 350 program and densitometer. All data were calculated as mean + SEM. Statistically significant differences among group means were determined by Duncan’s New Multiple-Range Test.35
Results Growth curves of animals are sholvn in Figure 1.. All rats initially lost weight; this finding was attributed to the fasting because body weight was depressed even in unoperated controls at day 1. Resected rats infused with IGF-I and des-IGF-I grew faster than resected control rats (P < 0.05). Des-IGF-I was more effective in stimulating the body weight gain of resected rats than IGF-I (P < O.O5).The increased growth rate in the IGF-I- and des-IGF-I-infused rats was not caused by food consumption because food intake did not differ among the three resected groups (data not shown). Body weights were lower in resected rats than in unoperated controls that had been subjected to the same starvation and feeding protocol as resected rats. Food intake of unoperated rats (18 -+ 3 g/day) was higher (P < 0.05) than that of resected controls (15 of:1 g/day). Mucosal DNA, protein, crypt depth, and villus height are shown in Table 1. Both IGF-I and des-IGF-
1251 0
1
2
DAYS
3
AFTER
4
5
6
7
SURGERY
Figure 1. Changes in body weights of animals after 80% jejunoileal resection. Body weights were determined daily beginning the day of surgery. Values represent means + SEM.
I enhanced mucosal weight because of an amplification of duodenojejunal hyperplasia (increased DNA and protein content) above that which normally occurs after 80% small bowel resection. There were no significant differences in duodenojejunal crypt depth among the three resected groups. However, the villus height was significantly higher in rats receiving IGF-I and des-IGF-I than in resected control rats. In the ileum, neither IGF-I nor des-IGF-I affected mucosal wet weight, hyperplasia, or hypertrophy. Crypt depth was greater in the ileal mucosa of rats receiving IGF-I and des-IGF-I than in resected controls. There were no differences in ileal villus height among the three resected groups. In the duodenojejunal mucosa, the specific activities (micromoles per gram protein per minute) of digestive enzymes were not changed by either IGF-I or des-IGF-I except that alkaline phosphatase activity was lower in rats infused with des-IGF-I than in those infused with IGF-I (Table 2). By contrast, when the enzyme activity was expressed as micromoles per centimeter per minute, total sucrase, maltase, alkaline phosphatase, and leucine aminopeptidase activities were higher in IGF-I-infused group, but only maltase and leucine aminopeptidase were significantly elevated by des-IGF-I compared with resected controls. Lactase activity was not affected by IGF infusion. The specific activities (micromoles per gram protein per minute) of sucrase, maltase, and leucine aminopeptidase were higher in the ileal mucosa from rats receiving IGF than in resected control rats. Sucrase and maltase activities in des-IGF-I-injected rats were not statistically different from those of the resected controls. Infusion of these two peptides did not influence lactase or alkaline phosphatase activities in the ileal mucosa (Table 3). Like the effects in
1952VANDERHOOF ET AL.
Table 1, Effects of Jejunoileal
Resection
GASTROENTEROLOGY
and IGF-I Treatment
on
Vol. 102, No. 6
MucosafAdaptation Resected
Measurement
Control
Duodenojejunum Mucosal weight (me/cm)
115 + 5”
143 + 5b
149 * 5b
242 + 8’ 13 f 0.5” 120 + 6”
315 + 24b 20 + 1.6b 128 + 7’
313 f lob 18 + 0.4b 130 + go
170 f 5c 10 f o.4c 107 k 7R
838 + 22”
951 It 32b
984 + 22b
662 f 5”
118 481 14 147 578
132 497 16 186 648
130 500 16 193 637
46 209 6 134 260
DNA &g/cm)
Protein (mg/cm) Crypt depth (pm) Villus height @m) Ileum Mucosal weight (mg/cm) DNA @g/cm) Protein (mg/cm) Crypt depth @m) Villus height (pm)
NOTE. Data represent mean + SEM (n = 3-8). “-“Values within rows with different superscripts
IGF-I
+ 5’ T!Z20” + 0.6’ + 8” f 28’
+ 3a + 16’ + 0.3” +. 7b f 24”
+- 7a.b +- 29” + 0.9’ + gb + 15”
Unoperated 80 + 1’
+ t+ * +
4’ 2ob 0.2b 10” 10b
differ (P < 0.05) among different groups.
the duodenojejunum, IGF infusion increased suerase (IGF-I only), maltase, and leucine aminopeptidase total activity (nanomoles per centimeter per minute) in the ileum, but neither affected lactase activity. Serum levels of IGF-I, insulin, glucose, and IGFBPs are shown in Table 4. IGF-I levels were lower in resected control rats than in unoperated rats. As expected, there was an increase in IGF-I levels in rats receiving IGF-I and des-IGF-I compared with resected controls. Insulin levels were lower in resected rats than in unoperated rats and were suppressed further by des-IGF-I infusion but not by IGF-I. IGF-I, but not des-IGF-I, slightly depressed serum glucose levels. The 1Z51-IGF-Ibinding capacity of serum, an indicator of the amount of unsaturated IGFBPs present in
Table 2. Effects of Jejunoileal Resection
Des-IGF-I
serum, was highest in the des-IGF-I-infused group and lowest in the IGF-I-infused group (Table 4). To examine for changes in levels of specific forms of IGFBPs, IGFBPs were quantified by ligand blot analysis (Figure 2). By this analysis, three classes of IGFBPs were detectable in rat sera: (a) a doublet of bands migrating in the 43-39,000 molecular weight range (IGFBP-3), which appeared to increase in intensity following treatment with either IGF-I or des-IGF-I; (b) a 32,000 molecular weight form (IGFBP-2) whose level was elevated by IGF-I only; and (c) a 24,000 molecular weight band (IGFBP4). These and other ligand blots were subjected to scanning densitometry to determine the magnitude of the apparent response (Table 4). Both IGF-I and des-IGF-I treatments increased the serum levels of IGFBP-3 as determined by scanning densitometry of ligand blots. Infusion of
and IGF-I on Duodenojejunal
Mucosal Enzyme Activities Resected
Enzyme activity Sucrase @mol. g protein-’ - min-‘) (nmol - cm-l - min-I)
Maltase @mol. g protein-’ - min-‘) @mol. cm-l . min-‘) Lactase @moI =g protein-’ - min-‘) (nmol - cm-’ *min-‘) Alkaline phosphatase @mol. g protein-’ *min-‘) @moI - cm-’ . min-‘) Leucine aminopeptidase f,umoI - g protein-’ . min-‘) fnmol - cm-’ - min-‘1 NOTE. Data represent mean * SEM (n = 3-8). ‘+Values within rows with different superscripts
Control 54 740 345 4.7 7.0 91
+ + f * f +
3.2’ 62”,’ 13O.b 0.30 1.3” 15O
658 + 40avb 8.9 k 0.7a 39 + 1.6” 521 + 33”
IGF-I 50 1010 323 6.5 6.6 133
+ 2.4’ k 10Sb + 8’ + 0.5b z!z1.2O + 33O
Des-IGF-I
Unoperated
47 841 321 5.8 3.6 65
53 512 388 3.8 7.5 74
f f k + + k
2.8” 50asb 13’ 0.3b 0.9” 15’
681 + 29” 13.8 + 1.5b
563 k 35b 10.2 + 0.7’.b
38 + 1.4’ 758 + 72b
35 * 2.10 636 i: 35b
differ (P < 0.05) among different
groups.
* + + k + k
5.3a 58’ 7b 0.2” 1.5O 15a
950 f 25’ 9.2 f 0.4avb 25 + 0.7b 239 _+ 3c
IGF-I AND MUCOSAL ADAPTATION
Tune 1992
Table 3. Effects of Jejunoileal
Resection
1953
and IGF-I on Ileal Mucosal Enzyme Activities Resected
Enzyme activity
IGF-I
Control 14 206 147 2.1 3.7 51
Sucrase @moI . g protein-’ *min?) (nmol . cm-’ - min-‘) Maltase @mol. g protein-’ - min-‘) @mol. cm-’ . min-‘) Lactase @mol. g protein-’ - min-‘) (nmol . cm-’ - min-‘) Alkaline phosphatase @mol. g protein-‘. min-‘) (nmol - cm-‘. min-‘) Leucine aminopeptidase @mol. g protein-’ . min-‘) (nmol . cm-’ . min-‘) NOTE. Data represent mean f SEM (n = 3-8). “-“Values within rows with different superscripts
f f i + k f
2.3” 38’ 18’,’ 0.3O 0.5” 7O.b
26 403 213 3.3 4.4 68
+ f f + t f
2.8a.b 41“,b 20USb 0.3b 0.5” 6’
5 27 96 0.6 5.2 32
+_ 1.8’ + 10” + 22” + 0.1” +_ 0.5” k 4b
55 + 7.6’ 860 k 114O
43 * 4.5O 694 ?I 99”
45 f 7.0” 268 k 38’
13 f 2.0° 187 + 33”,’
22 iz 2.8b 343 + 41b
25 f 3.5b 334 f 49b
13 + 2.9” 77 f 16”
differ (P < 0.05) among different groups.
IGF-I may be potential therapeutic agents for short bowel patients. A probable role of IGF-I in controlling intestinal growth was shown when northern blot and in situ hybridization analyses revealed the presence of messenger RNA for both IGF-I and IGF-II in rat and human intestine.3”,37 Furthermore, mouse intestinal explants release IGF-I in vitro3’ and rat intestinal epithelium contains both IGF-I and IGF-II receptors.3 In addition, IGF-I is a potent stimulator of intestinal epithelial cell (IEC-6 cell) proliferation.’ We have also observed that fibroblasts isolated from the rat small intestine and IEC-6 cells produce IGF-like peptides and IGFBPs (unpublished data). This evidence suggests that IGFs and IGFBPs are physiologically important in the intestinal mucosa. In the present study, we have shown that IGF-I enhanced mucosal hyperplasia after small bowel resection in vivo. Unoperated rats subjected to the same fasting and refeeding protocol as the resected rats were included
Discussion An important finding of the present study is that IGF-I and des-IGF-I significantly enhance mucosal adaptation that normally occurs after massive small bowel resection. These changes were observed morphometrically by increased villus height in the duodenojejunal segment and increased crypt depth in the ileum. Furthermore, biochemical measures of intestinal hyperplasia (DNA and protein content) were increased significantly by IGF-I and des-IGF-I in the duodenojejunal segment of the remaining intestine. Cellular differentiation, as reflected by total disaccharidase activity within duodenojejunal and ileal segments, was also stimulated significantly by both IGF-I and des-IGF-I. The implications of these results are significant because both IGF-I and des-
Resection
19 281 205 3.1 4.1 63
Unoperated
50 f 2.5” 724 + 53’
IGF-I but not des-IGF-I increased the serum levels of IGFBP-2. Neither IGF treatments changed serum levels of the 24,000 molecular weight IGFBP-4.
Table 4. Effects of Jejunoileal
+ 3.3b + 5ob + 23b + 0.3b -Irl.oa + 14O.b
Des-IGF-I
and IGF-I Treatment
on Serum IGF-I and IGFBP Levels Resected
Measurement IGF-I (ng/mL) Insulin (pILJ/mL) Glucose (mg/lOO mL)
IGF-binding capacity (ngEq/mL) IGFBP-3 (43-39,000 mol wt)d IGFBP-2 (32,000 mol wt)d IGFBP-4 (24,000 mol wt)d
Control
IGF-I
328 f 23”
521 zk 23b 20 I? 2.5” 139 + 2b
20 k 1.3” 146 + 2’ 177 +_ 5” 100 -t 7O
100 f 7” 100 + 15”
NOTE. Data represent mean + SEM (n = 3-8). “-“Values within rows with different superscripts differ (P < 0.05). dValues for IGFBP-3, -2, and -4 are expressed in scanning densitometry
155 176 181 125
-+ 5b + 17b.C F 28b k 26a.b
Des-IGF-I 570 15 143 680 150 119 112
k 27b -t l.lb xk 2”.b + 120” f 13b t 21a.b rf: 12”
units relative to levels in resected
control rat sera.
Unoperated 515 f 8b 29 f 3.3c 153 f 4” 178 f 5”
209 t 6” 143 + 32n,b 141 + 23b
1954
VANDERHOOF ET AL.
Cntl
IGF-I
GASTROENTEROLOGY Vol. 102, No. 6
;;e,s_; Unop
Resected Figure 2. Ligand blot of serum with ‘Z51-IGF-I. Rat sera were electrophoresed through 12.5% sodium dodecyl sulfate-polyacrylamide gels, and Western blots were prepared. Ligand blot analysis was used to determine the size and relative amounts of the various IGFBPs present as described in Materials and Methods. A representative set (n = 3-6) is shown. Sizes (molecular weight X 10e3) of the IGFBPs were estimated relative to the mobility of molecular weight standards. The 43-39,660 molecular weight IGFBPs are glycoprotein variants of the same growth hormone-dependent form, IGFBP-3.’ IGFBP-2 was confirmed as such by immunoblotting the Western blots with antisera specific to this form of IGFBP (data not shown). Rat and human IGFBP-4 has recently been cloned and named,6 and the 24,000 molecular weight IGFBP in sera has an N-terminal sequence identical to this recently characterized protein.‘5
in this study. Although these animals are not the ideal control group for the resected animals, results from these unoperated animals provide some interesting information. Serum levels of IGF-I, IGFBP-3, and IGFBP-4 decreased in resected control rats compared with unoperated rats. This decrease may be caused partially by the slight decrease in either food intake or absorption in resected rats, which would explain the decrease in serum insulin and glucose levels. Other studies have indicated that circulating levels of IGF-I were reduced in fasted rats,3Q by ligand blot analysis the levels of 4%39,000 molecular weight IGFBP-3 and 24,000 molecular weight IGFBP4 are markedly reduced in fasted animals, and IGFBP-2 is not consistently depressed compared with contro1s.34 In addition, serum concentrations of IGFBP-3 in rats have been shown to be IGF-I dependent.40*41Thus, the reduced serum levels of IGFBPs in resected rats in this study may reflect serum levels of IGF-I. Levels of IGFBP-3 were returned toward the levels measured in the sera of unoperated controls by IGF infusion (IGF-I and des-IGF-I) (Table 4), confirming the hypothesis. Des-IGF-I did not significantly change either IGFBP-2 or IGFBP-4 levels although IGFBP-2 levels were elevated in IGF-I treated rats compared with resected controls (Table 4).
Despite similar immunoassayable serum IGF-I levels in IGF-I- and des-IGF-I-infused rats and a lower total induction of serum IGFBP levels by des-IGF-I, the ‘251-IGF-I-binding capacity of sera was greatest in des-IGF-I-infused rats (Table 4). This finding indicates that the infused des-IGF-I either is free or dissociates from the IGFBPs during the binding capacity assay because of its low affinity for IGFBPs. Thus, with unaltered IGFBP levels the degree of unsaturation of circulating IGFBPs appears to be elevated in des-IGF-I-infused animals. Dissociation of des-IGF-I appears to be a plausible explanation because the clearance of des-IGF-I from rat serum is approximately 2 hours42 due to des-IGF-I association with the l50- and SO-kilodalton IGFBP complexes in sera. Dissociation of des-IGF-I from the IGFBPs during measurement of IGF distribution in sera may explain the apparent lack of IGFBP binding of des-IGF-I that has been published previously.43 Differential distribution of des-IGF-I vs. IGF-I between the various forms of IGFBPs present in serum and differences in tissue availability would be expected to alter the bioactivity of des-IGF-I compared with IGF-I. Such differences were found. In the present study, des-IGF-I was a better stimulant of somatic growth (body weight) than IGF-I (Figure 1). These results are consistent with those of Lemmey et a1.,44 who compared these two analogues at the dosage of 0.96 mg/kg per day in the same animal model. In their studies, des-IGF-I was also more potent in stimulating increases in duodenal weight, kidney weight, and body length than IGF-I.44 In contrast, we found that des-IGF-I was either lower or equipotent to IGF-I in stimulating duodenojejunal mucosal adaptation and ileal mucosal enzyme activities (Tables 2 and 3). It is possible that the dose of both IGFs (1.5 mg/kgper day) used in the present studies is high enough to produce the maximal responses in the intestine, thus masking some of the possible differences in activity between the two forms. These results also suggest a degree of tissue specificity in the effects of des-IGF-I vs. IGF-I. The precise role of IGFBPs in controlling differential growth of various tissues is unclear at this time. Future studies will be needed to determine the role of IGF induction of serum or locally secreted IGFBPs in controlling these effects. In the duodenojejunum, infusion of IGF-I and desIGF-I resulted in increased DNA, protein, and villus height but no change in crypt depth. In the ileum, these growth factors increased crypt depth but did not affect DNA, protein, or villus height. The magnitude of increases in DNA, protein, and villus height in resected controls compared with unoperated controls was much greater in the ileum (greater than twofold in each case) than in the duodenojejunum (1.26-1.42-fold increase) (Table 1). One can speculate
June 1992
that the adaptive responses to resection were already maximal in the ileum so that exogenous IGF-I could not further enhance these responses. Alternatively, the increase in crypt depth in the ileum of IGF-infused animals suggests that the adaptation process in the ileum was still in progress 7 days after resection. If the rats had been killed later, the differences in crypt depth may have disappeared and an increase in villus height may have become apparent. In the duodenojejunum, infusion of IGF-I and desIGF-I increased DNA and protein contents without altering enzyme activities per unit protein. It is possible that the enzyme activities were already so high in the proximal intestine that they could not be increased further. By contrast, the activities of maltase, sucrase, and leucine aminopeptidase in the ileum were increased significantly by IGF infusion without changes in mucosal DNA and protein contents. One possible explanation for this result is that IGF infusion stimulated nutrient transport in the enterocytes of the ileum, which then induced these digestive enzymes. In fact, alkaline phosphatase and lactase, which are of minimal significance for nutrient digestion and absorption in adult rats, were not induced by infusion of either IGF-I or des-IGF-I. In conclusion, continuous infusion of IGF-I or desIGF-I enhanced mucosal adaptation after small bowel resection in rats. These results suggest that both IGF-I and des-IGF-I have potential as therapeutic agents for patients with short bowel syndrome. It remains to be determined through dose-response studies whether des-IGF-I or IGF-I is the more efficacious stimulant of mucosal adaptation. References 1.
2.
3.
4.
5.
6.
7.
Daughaday WH, Rotwein P. Insulin-like growth factor I and II. Peptide, messenger ribonucleic acid and gene structures, serum and tissue concentration. Endocr Rev 1989;10:68-91. Park JHY, Joekel CS, Hart MH, Harty RH, Vanderhoof JA. Establishment of serum-free medium for IEC-6 cell culture: comparative effects of growth factors (abstr). Gastroenterology 1989;96:A383. Laburthe M, Bouyer-Fessard C, Gammeltoft S. Receptors for insulin-like growth factors I and II in rat intestinal epithelium. Am J Physiol 1988;254:G457-G462. Wood WI, Cachianes G, Henzel WJ, Winslow CA, Spencer SA, Hellmiss R, Martin JL, Baxter RC. Cloning and expression of the GH dependent insulin like growth factor binding protein. Mol Endocrinol 1988;2:1176-1185. Brewer MT, Stetler GL, Squires CH, Thompson RC, Busby WH, Clemmons DR. Cloning, characterization and expression of a human insulin-like growth factor binding protein. Biothem Biophys Res Commun 1988;152:1289-1297. Shimasaki S, Uchiyama F, Shimonaka M, Ling N. Molecular cloning of the cDNAs encoding a novel insulin-like growth factor-binding protein from rat and human. Mol Endocrinol 1990;4:1451-1458. Shimasaki S, Gao L, Shimonaka M, Ling N. Isolation and molecular cloning of insulin-like growth factor-binding protein6. Mol Endocrinol 1991;5:938-948.
IGF-I AND MUCOSAL ADAPTATION
1955
8. Binkert C, Landwehr J, Mary J-L, Schwander J, Heinrich G. Cloning sequence analysis and expression of cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2). EMBO J 1989;8:2497-2502. 9. Elgin RG, Busby WH Jr, Clemmons DR. An insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I. Proc Nat1 Acad Sci USA 1987;84:3254-3258. 10. Busby WH, Klapper DG, Clemmons DR. Purification of a 31000 dalton insulin-like growth factor binding protein from human amniotic fluid. J Biol Chem 1988;263:14203-14210. 11. Rutanen E-M, Pekonen F, Makinen T. Soluble 34K binding protein inhibits the binding of insulin-like growth factor I to its cell receptor in human secretory phase endometrium: evidence for autocrine/paracrine regulation of growth factor action. J Clin Endocrinol Metab 1988;66:173-180. 12. Ritvos 0, Ranta T, Jalkanen J, Suikkari A-M, Voutilainen R, Bohn H, Rutanen E-M. Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells. Endocrinology 1988;2150-2157. 13. De Mellow JSM, Baxter RC. Growth hormone-dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I-stimulated DNA synthesis in human skin fibroblasts. Biochem Biophys Res Commun 1988; 156:199-204.
14. Blum WF, Jenne EW, Reppin F, Kietzmann K, Ranke MB, Bierich JR. Insulin-like growth factor I (IGF-I) binding protein complex is a better mitogen than free IGF-I. Endocrinology 1989;125:766-772.
C, Bayne ML, Cascieri MA, 15. McCusker RH, Camacho-Hubner Clemmons DR. Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs). J Cell Physiol 1990;144:244-253.
16. Clemmons DR, Elgin RG, Han VKM, Casella SJ, D’Ercole AJ, Van Wyk JJ. Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I. J Clin Invest 1986;77:1548-1556. 17. Ballard FJ, Francis GL, Ross M, Bagley CJ, May B, Wallace JC. Natural and synthetic forms of insulin-like growth factor-l (IGF-1) and the potent derivative, destripeptide IGF-1: biological activities and receptor binding. Biochem Biophys Res Commun 1987;149:398-404. 18. Francis GL, Read LC, Ballard FJ, Bagley CJ, Upton FM, Gravestock PM, Wallace JC. Purification and partial sequence analysis of insulin-like growth factor-l from bovine colostrum. Biothem J 1986;233:207-213. 19. Ballard FJ, Ross M, Upton FM, Francis GL. Specific binding of insulin-like growth factors 1 and 2 to the type 1 and 2 receptors respectively. Biochem J 1988;249:721-726. 20. Szabo L, Mottershead F, Ballard J, Wallace JC. The bovine insulin-like growth factor (IGF) binding protein purified from conditioned medium requires the N-terminal tripeptide in IGF-I for binding. Biochem Biophys Res Commun 1988; 151:207-214. 21. McCusker
RH, Busby WH, Dehoff MH, Camacho-Hubner C, Clemmons DR. Insulin-like growth factor binding to cell monolayers is directly modulated by the addition of insulinlike growth factor binding proteins. Endocrinology 1991;129:
939-949. 22. Dowling RH, Booth CC. Structural
and lowing small intestinal resection in 1967;32:139-149. 23. Vanderhoof JA. Park JHY, Grandjean prostaglandin synthesis after massive Am J Physiol 1988;254:G373-G377.
functional changes folthe rat. Clin Sci Lond CJ. Reduced mucosal small bowel resection.
1956
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ET AL.
GASTROENTEROLOGY
24. Lowry OH, Rosebrough
38. D’Ercole AJ, Applewhite
25.
39.
26. 27. 28.
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31.
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33.
34.
35. 36.
37.
NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275. Munro HN, Fleck A. Recent developments in the measurement of nucleic acids in biological materials. Analyst 1966;91:78-87. Giles KW, Myers A. Improved diphenylamine method for estimation of DNA. Nature 1965;206:93-94. Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964;7:18-25. Sommer AJ. The determination of acid and alkaline phosphatase using p-nitrophenyl phosphate as substrate. Am J Med Technol 1954;20:244-253. Goldbarg JA, Rutenburg AM. The calorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 1958;11:283-291, Bergmeyer HU, Bernt E, Schmit F, Stork H. o-Glucose. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis New York: Academic, 1974:1196-1201. Daughaday WH, Mariz IK, Blethen SL. Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites: A comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol-extracted serum. J Clin Endocrinol Metab 1980;51:781-788. Furlanetto RW, Underwood LE, Van Wyk JJ, D’Ercole AJ. Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay. J Clin Invest 1977;60:648-657. McCusker RH, Campion DR, Clemmons DR. The ontogeny and regulation of a 31,000 Mr insulin-like growth factor/somatomedin (IGF) binding protein in fetal porcine plasma and sera. Endocrinology 1988;122:2071-2079. McCusker RH, Campion DR, Jones WK, Clemmons DR. The insulin-like growth factor-binding proteins of porcine serum: endocrine and nutritional regulation. Endocrinology 1989; 125:501-509. Steel RGD, Torrie JH. Principles and procedures of statistics. 2nd ed. New York: McGraw-Hill, 1980. Lund PK, Moats-Staats BM, Hynes MA, Simmons JG, Jansen M, D’Ercole AJ, Van Wyk JJ. Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues. J Biol Chem 1986;261:14539-14544. Han VKM, Lund PK, Lee DC, D’Ercole AJ. Expression of somatomedin/insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution. J Clin Endocrinol 1988;66:422-429.
40.
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GT, Underwood LE. Evidence that somatomedin is synthesized by multiple tissues in the fetus. Dev Biol 1980;75:315-328. Maes M, Underwood LE, Ketelslegers J-M. Plasma somatomedin-C in fasted and refed rats: close relationship with changes in liver somatogenic but not lactogenic binding site. J Endocrinol 1983;97:243-252. Clemmons DR, Thissen JP, Maes M, Ketelslegers JM, Underwood LE. Insulin-like growth factor-I (IGF-I) infusion into hypophysectomized or protein-deprived rats induces specific IGF binding proteins in serum. Endocrinology 1989;125:29672972. Zapf J, Hauri C, Waldvogel M, Futo E, Hasler K, Binz K, Guler HP, Schmid C, Froesch ER. Recombinant human insulin-like growth factor I induces its own specific carrier protein in hypophysectomized and diabetic rats. Proc Nat1 Acad Sci USA 1989;86:3813-3817. Drakenberg K, Ostenson C-G, Sara V. Circulating forms and biological activity of intact and truncated insulin-like growth factor I in adult and neonatal rats. Acta Endocrinol 1990;123:43-50. Ballard FJ, Knowles SE, Walton PE, Edson K, Owens PC, Mohler MA, Ferraiolo BL. Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(l-3)IGF-I in rats. J Endocrinol 1991;128:197-204. Lemmey AB, Martin AA, Read LC, Tomas FM, Owens PC, Ballard FJ. IGF-I and the truncated analogue des-(l-3)-IGF-I enhance growth in rats after gut resection. Am J Physiol 1991;260:E213-E219.
45. Zapf J, Born W, Chang JY, James P, Froesch
ER, Fisher JA. Isolation and NH,-terminal amino acid sequences of rat serum carrier proteins for insulin-like growth factors. Biothem Biophys Res Commun 1988;1187-1194.
Received July 17,199l. Accepted November 121991. Address requests for reprints to: Jung H. Y. Park, Ph.D., Swanson Hall Room 3049, 502 South 44th Street, Omaha, Nebraska 68105-1065. Supported by a grant from Caremark, Inc., Deerfield, Illinois. The authors are grateful to Drs. L. E. Underwood and J. J. Van Wyk (University of North Carolina, Chapel Hill, North Carolina) and the National Hormone and Pituitary Program (Baltimore, Maryland) for the provision of rabbit insulinlike growth factor I antiserum. They thank Dr. L. E. Underwood for the “‘I-insulinlike growth factor I used for ligand blot analysis.
1992;102:1949-1956
Truncated and Native Insulinlike Growth Factor I Enhance Mucosal Adaptation After Jejunoileal Resection JON A. VANDERHOOF, ROBERT H. McCUSKER, ROSS HAMID MOHAMMADPOUR, DARCY J. BLACKWOOD, RICHARD F. HARTY, and JUNG H. Y. PARK
CLARK,
Department of Pediatrics, Creighton University School of Medicine and University of Nebraska Medical Center, Omaha, Nebraska; Department of Medicine, University of North Carolina, Chapel Hill, North Carolina; and Department of Developmental Biology, Genentech, Inc., South San Francisco, California
It has been shown previously that insulinlike growth factors (IGFs) stimulate the proliferation of intestinal crypt cells in vitro. To examine the in vivo effects of IGF-I on mucosal adaptation, three groups of Sprague-Dawley rats underwent 80% jejunoileal resection. Miniosmotic pumps were then inserted under the skin immediately after resection to deliver vehicle (resected control), 1.5 mg/kg per day of IGF-I, or 1.5 mg/kg per day of des-(l-3)IGF-I (des-IGF-I). Des-IGF-I is a truncated form of IGF-I that binds as well to type I IGF receptors but less tightly to several forms of IGF-binding proteins (IGFBPs) than IGF-I. Ad libitum food intake did not differ among the three resected groups. Body weight gains were greater in animals receiving des-IGF-I than in those receiving IGF-I, which were greater than resected controls. All animals were killed 7 days postoperatively, and the remaining small intestine was removed and divided at the anastomotic site. Both IGF-I and des-IGF-I induced hyperplasia (increased DNA and protein content] in the duodenojejunum but not in the ileum. IGF-I and des-IGF-I were equally active. In contrast, sucrase, maltase, and leucine aminopeptidase activities were greater only in the ileum of animals receiving IGF-I and des-IGF-I than in resected controls. Although more potent in stimulating overall body weight gain, des-IGF-I was not more potent than IGFI when duodenal and ileal responses were determined. IGF infusion (IGF-I > des-IGF-I) increased the levels of circulating IGFBP-3 and IGFBP-2, which may act to modulate the biological effectiveness of the infused peptides. These results suggest that both IGFI and des-IGF-I may have potential as therapeutic agents for short bowel patients.
I
nsulinlike growth factors (IGFs) and insulin are important regulators of somatic growth.’ IGF-I, IGF-II, and insulin, when administered to IEC-6 cells in culture, cause stimulation of cellular proliferation. IEC-6 cells are an established line of intestinal
epithelial cells derived from rat jejunal crypts. Comparison of dose-response curves for these growth factors on proliferative indices in IEC-6 cells indicates that IGF-I is the most potent and efficacious peptide, suggesting that the effects are mediated by interaction with the type I IGF receptor.’ Rat intestinal epithelium contains both type I and type II IGF receptors. Studies that have examined the regional distribution of IGF receptors within the intestinal villus-crypt unit indicate a predominance of receptors in the proliferative crypt region.3 These findings suggest that IGF-I may be an important growth factor in the regulation of intestinal epithelial cell growth. Unlike insulin, IGFs are bound to specific IGFbinding proteins (IGFBPs) in plasma and other biological fluids. To date, six IGFBPs have been purified and at least partially sequenceda4” The precise role of IGFBPs in modulating the anabolic and mitogenic effects of the IGFs is unclear. There are conflicting reports on the biological role of the IGFBPs in modulating IGF action. IGFBP-1 enhances the biological response to IGF-I upon the DNA synthesis of cultured aortic smooth muscle cells and human fibroblasts.g This preparation of IGFBP was also shown to adhere to the smooth muscle cell surfaceslO possibly accounting for the enhanced IGF effect. In other studies, IGFBP-1 has been shown to prevent the action of IGFs, possibly by limiting access to cell surface reof fibroblasts with IGFBPceptors. lls*’ Preincubation 3 or coincubation with an equimolar ratio of IGFBP-3 and IGF-I potentiates IGF-I-stimulated thymidine incorporation, but coincubation of IGFBP-3 can also inhibit the IGF-I effect.‘3*‘4 In the same manner as IGFBP-1, IGFBP-3 adheres to cell surfaces.15*‘” These findings suggest that IGFBPs function in diverse ways to modulate the biological effects of the IGFs. An IGF-I derivative with glycine, proline and glu0
1992 by the American Gastroenterological 0016-5085/92/$3.00
Association
1950
VANDERHOOF
ET AL.
tamic acid residues cleaved from the N terminus [des-(l-3)-IGF-I (des-IGF-I)] stimulates protein and DNA synthesis and inhibits protein breakdown at concentrations lower than those required of IGFL1’*” Des-IGF-I has normal affinity for receptors on human skin fibroblasts*g and L6 myoblasts.” An important characteristic of des-IGF-I is its reduced affinity for IGFBPs.” Because soluble IGFBPs block cell surface binding of IGF-I,‘l a reduction in affinity for IGFBPs could explain the elevated bioactivity of des-IGF-I. Short bowel syndrome is a malabsorptive disorder resulting from resection of a large percentage of the small intestine. After intestinal resection, a considerable increase in mucosal surface area occurs through a gradual lengthening of villi lining the remaining small intestine.22 Although recent advances in the use of home total parenteral nutrition have increased the long-term survival of many patients with short bowel syndrome, living without parenteral nutrition depends on the process of mucosal adaptation. Therefore, it is important to find trophic factors that can enhance the mucosal adaptation that normally occurs. The purpose of this study was to examine in vivo effects of exogenously administered IGF-I and des-IGF-I on mucosal adaptive responses following massive small bowel resection in rats. In addition, IGF-I and des-IGF-I were compared to test whether binding to IGFBPs could alter the ability of IGF-I to stimulate mucosal hyperplasia. Materials and Methods Materials Recombinant human IGF-I and des-IGF-I were provided by Genentech, Inc. (South San Francisco, CA). 1251IGF-I (specific activity, 2000 Ci/mmol), carrier-free Nalz51, and molecular weight standards were purchased from Amersham (Arlington Heights, IL). Rabbit anti-IGF-I serum (UB3-189) was a gift from Drs. L. E. Underwood and J, J, Van Wyk (University of North Carolina, Chapel Hill, NC) through the National Hormone and Pituitary Program of NIADDK (Baltimore, MD). This antibody has wide species cross-reactivity and has been used successfully in studies with rats.’ Recombinant rat IGF-I has been shown to be 90% cross-reactive with this antiserum compared with recombinant human IGF-I (personal communication, Dr. L. E. Underwood). PH 75 nitrocellulose sheets were obtained from Schleicher and Schuell, Inc. (Keene, NH). Human y-globulin was purchased as an 18% solution (Cutter Biological, Berkeley, CA). Unless otherwise stated, all other chemicals were obtained from Sigma Chemical (St. Louis, MO). Animals The animals used in this study were cared for according to the guidelines of the Animal Review Committee
GASTROENTEROLOGY
Vol. 102, No. 6
at the University of Nebraska Medical Center. Twentyseven male Sprague-Dawley rats (120-140 g) were purchased (Sasco, Omaha, NE) and housed individually in hanging stainless steel cages. They were provided Purina rat chow (Ralston-Purina, St. Louis, MO) and tap water ad libitum. After 3 days of acclimation to laboratory conditions, they were randomly divided into four groups. Three groups (8 rats/group) were subjected to jejunoileal resection.23 After a 36-hour fast, all bowel between the points 4 cm distal to the ligament of Treitz and 12 cm proximal to the ileocecal valve was removed. Miniosmotic pumps (Alzet model 2001, Palo Alto, CA) were inserted under the skin immediately after resection to deliver either vehicle (0.1 mol/L acetic acid), 1.5 mg/kg per day of IGF-I, or 1.5 mg/kg per day of des-IGF-I. D-Glucose (5%, wt/vol) and terramycin (O.OZZ%, wt/vol) were provided in the drinking water for 36 hours after surgery, after which animals were placed back on the chow food. Food intake was monitored daily. The fourth group (3 rats) was subjected to the same fasting and refeeding conditions but did not undergo surgery. All animals were killed 7 days later, and the small intestine from the pylorus to the ileocecal valve was removed and divided at the anastomosis. Portions of intestine 1 cm on either side of the anastomosis were discarded because of the surgically induced hyperplasia occurring in this region. The mucosa was scraped from underlying tissue with a glass slide for the determination of DNA, protein, and enzyme activity. Blood was drawn from the aorta at the time of death, and serum was prepared for the determination of IGF-I, insulin, glucose, and IGFBP levels. The proximal 2 cm of the duodenum and ileum was removed and fixed in buffered formalin for histological analysis. After fixation, the tissue was sectioned (4 pm thick), mounted on glass slides, and stained with H&E. The villi and crypts were measured under a microscope with a micrometer eyepiece to determine villus height and crypt depth.
Mucosal DNA, Protein, and Enzyme Assays Mucosal samples were homogenized in deionized water. Protein concentration was determined by the method of Lowry et a1.24 and expressed as milligrams of protein per centimeter of bowel. DNA was extracted by the method of Munro and Fleck.25 The resulting DNA fractions were assayed according to the method of Giles and Myers.” Disaccharidase activities were measured according to the methods of Dahlqvist.” We measured the activity of alkaline phosphatase according to the method of phosphate as a substrate. Sommer,28 using p-nitrophenyl Leucine aminopeptidase activity was estimated by the method of Goldbarg and Rutenburg.2g Determination of JGF-I, Insulin, Levels in Serum
and
Glucose
Serum insulin levels were measured using a radioimmunoassay kit (Diagnostic Product Corporation, Los Angeles, CA). Serum glucose was analyzed by the method of Bergmeyer et a1.30using hexokinase and glucose-6-phosphate dehydrogenase. Before assaying for IGF-I, the IGFBPs were eliminated by acid-ethanol precipitation as described by Daughaday et a1.31 IGF-I levels were esti-
IGF-I AND MUCOSAL ADAPTATION
June 1992
mated by radioimmunoassay as described by Furlanetto et a1.32using 1251-IGF-I.Recombinant human IGF-I was used as the unlabeled standard except that des-IGF-I was used as the unlabeled standard for the des-IGF-I group. Determination
200 -
Resected control IGF-I Des-IGF-I Unoperated control
A-A A-A e--e o-o
g
1951
175--
of IGFBPs in Serum
IGF-binding capacity. Binding capacity was quantified by determining the ability of serum aliquots to bind ‘Z51-IGF-Ias described by McCusker et a1.33This assay measures the quantity of unsaturated IGFBPs in sera. Briefly, 1 pL of serum was incubated for 1 hour with 20,000 cpm of 1251-IGF-I,and the bound ‘2SI-IGF-I was precipitated by centrifugation after adding 0.25% human y-globulin and 12.5% polyethylene glycol (PEG) 8000. Activity is expressed as nanogram equivalents per milliliter. Ligand blotting with ‘251-IGF-I. The proteins of serum were electrophoresed through 12.5% discontinuous sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions as described by McCusker et a1.34The proteins were transferred to 0.05 pm PH 75 nitrocellulose sheets by electroblotting using a Semiphor Semi-dry Transfer Unit (Hoefer Scientific Instrument, San Francisco, CA). Nonspecific binding of the tracer to the nitrocellulose paper was prevented by preincubating the sheets sequentially in 1% Nonidet P-40 (Sigma Chemical Co., St. Louis, MO), 0.1% bovine serum albumin, and 2% Tween20 (Sigma) for 10 minutes, 2 hours and 20 minutes, respectively. To detect IGFBPs, the nitrocellulose sheets were incubated overnight with 100,000 cpm/mL of ‘251-IGF-I. The sheets were then washed, and IGFBPs were visualized by autoradiography. Relative molecular weights were estimated by running prestained molecular weight standards in a parallel lane. The density of each band was measured using a Hoefer 350 program and densitometer. All data were calculated as mean + SEM. Statistically significant differences among group means were determined by Duncan’s New Multiple-Range Test.35
Results Growth curves of animals are sholvn in Figure 1.. All rats initially lost weight; this finding was attributed to the fasting because body weight was depressed even in unoperated controls at day 1. Resected rats infused with IGF-I and des-IGF-I grew faster than resected control rats (P < 0.05). Des-IGF-I was more effective in stimulating the body weight gain of resected rats than IGF-I (P < O.O5).The increased growth rate in the IGF-I- and des-IGF-I-infused rats was not caused by food consumption because food intake did not differ among the three resected groups (data not shown). Body weights were lower in resected rats than in unoperated controls that had been subjected to the same starvation and feeding protocol as resected rats. Food intake of unoperated rats (18 -+ 3 g/day) was higher (P < 0.05) than that of resected controls (15 of:1 g/day). Mucosal DNA, protein, crypt depth, and villus height are shown in Table 1. Both IGF-I and des-IGF-
1251 0
1
2
DAYS
3
AFTER
4
5
6
7
SURGERY
Figure 1. Changes in body weights of animals after 80% jejunoileal resection. Body weights were determined daily beginning the day of surgery. Values represent means + SEM.
I enhanced mucosal weight because of an amplification of duodenojejunal hyperplasia (increased DNA and protein content) above that which normally occurs after 80% small bowel resection. There were no significant differences in duodenojejunal crypt depth among the three resected groups. However, the villus height was significantly higher in rats receiving IGF-I and des-IGF-I than in resected control rats. In the ileum, neither IGF-I nor des-IGF-I affected mucosal wet weight, hyperplasia, or hypertrophy. Crypt depth was greater in the ileal mucosa of rats receiving IGF-I and des-IGF-I than in resected controls. There were no differences in ileal villus height among the three resected groups. In the duodenojejunal mucosa, the specific activities (micromoles per gram protein per minute) of digestive enzymes were not changed by either IGF-I or des-IGF-I except that alkaline phosphatase activity was lower in rats infused with des-IGF-I than in those infused with IGF-I (Table 2). By contrast, when the enzyme activity was expressed as micromoles per centimeter per minute, total sucrase, maltase, alkaline phosphatase, and leucine aminopeptidase activities were higher in IGF-I-infused group, but only maltase and leucine aminopeptidase were significantly elevated by des-IGF-I compared with resected controls. Lactase activity was not affected by IGF infusion. The specific activities (micromoles per gram protein per minute) of sucrase, maltase, and leucine aminopeptidase were higher in the ileal mucosa from rats receiving IGF than in resected control rats. Sucrase and maltase activities in des-IGF-I-injected rats were not statistically different from those of the resected controls. Infusion of these two peptides did not influence lactase or alkaline phosphatase activities in the ileal mucosa (Table 3). Like the effects in
1952VANDERHOOF ET AL.
Table 1, Effects of Jejunoileal
Resection
GASTROENTEROLOGY
and IGF-I Treatment
on
Vol. 102, No. 6
MucosafAdaptation Resected
Measurement
Control
Duodenojejunum Mucosal weight (me/cm)
115 + 5”
143 + 5b
149 * 5b
242 + 8’ 13 f 0.5” 120 + 6”
315 + 24b 20 + 1.6b 128 + 7’
313 f lob 18 + 0.4b 130 + go
170 f 5c 10 f o.4c 107 k 7R
838 + 22”
951 It 32b
984 + 22b
662 f 5”
118 481 14 147 578
132 497 16 186 648
130 500 16 193 637
46 209 6 134 260
DNA &g/cm)
Protein (mg/cm) Crypt depth (pm) Villus height @m) Ileum Mucosal weight (mg/cm) DNA @g/cm) Protein (mg/cm) Crypt depth @m) Villus height (pm)
NOTE. Data represent mean + SEM (n = 3-8). “-“Values within rows with different superscripts
IGF-I
+ 5’ T!Z20” + 0.6’ + 8” f 28’
+ 3a + 16’ + 0.3” +. 7b f 24”
+- 7a.b +- 29” + 0.9’ + gb + 15”
Unoperated 80 + 1’
+ t+ * +
4’ 2ob 0.2b 10” 10b
differ (P < 0.05) among different groups.
the duodenojejunum, IGF infusion increased suerase (IGF-I only), maltase, and leucine aminopeptidase total activity (nanomoles per centimeter per minute) in the ileum, but neither affected lactase activity. Serum levels of IGF-I, insulin, glucose, and IGFBPs are shown in Table 4. IGF-I levels were lower in resected control rats than in unoperated rats. As expected, there was an increase in IGF-I levels in rats receiving IGF-I and des-IGF-I compared with resected controls. Insulin levels were lower in resected rats than in unoperated rats and were suppressed further by des-IGF-I infusion but not by IGF-I. IGF-I, but not des-IGF-I, slightly depressed serum glucose levels. The 1Z51-IGF-Ibinding capacity of serum, an indicator of the amount of unsaturated IGFBPs present in
Table 2. Effects of Jejunoileal Resection
Des-IGF-I
serum, was highest in the des-IGF-I-infused group and lowest in the IGF-I-infused group (Table 4). To examine for changes in levels of specific forms of IGFBPs, IGFBPs were quantified by ligand blot analysis (Figure 2). By this analysis, three classes of IGFBPs were detectable in rat sera: (a) a doublet of bands migrating in the 43-39,000 molecular weight range (IGFBP-3), which appeared to increase in intensity following treatment with either IGF-I or des-IGF-I; (b) a 32,000 molecular weight form (IGFBP-2) whose level was elevated by IGF-I only; and (c) a 24,000 molecular weight band (IGFBP4). These and other ligand blots were subjected to scanning densitometry to determine the magnitude of the apparent response (Table 4). Both IGF-I and des-IGF-I treatments increased the serum levels of IGFBP-3 as determined by scanning densitometry of ligand blots. Infusion of
and IGF-I on Duodenojejunal
Mucosal Enzyme Activities Resected
Enzyme activity Sucrase @mol. g protein-’ - min-‘) (nmol - cm-l - min-I)
Maltase @mol. g protein-’ - min-‘) @mol. cm-l . min-‘) Lactase @moI =g protein-’ - min-‘) (nmol - cm-’ *min-‘) Alkaline phosphatase @mol. g protein-’ *min-‘) @moI - cm-’ . min-‘) Leucine aminopeptidase f,umoI - g protein-’ . min-‘) fnmol - cm-’ - min-‘1 NOTE. Data represent mean * SEM (n = 3-8). ‘+Values within rows with different superscripts
Control 54 740 345 4.7 7.0 91
+ + f * f +
3.2’ 62”,’ 13O.b 0.30 1.3” 15O
658 + 40avb 8.9 k 0.7a 39 + 1.6” 521 + 33”
IGF-I 50 1010 323 6.5 6.6 133
+ 2.4’ k 10Sb + 8’ + 0.5b z!z1.2O + 33O
Des-IGF-I
Unoperated
47 841 321 5.8 3.6 65
53 512 388 3.8 7.5 74
f f k + + k
2.8” 50asb 13’ 0.3b 0.9” 15’
681 + 29” 13.8 + 1.5b
563 k 35b 10.2 + 0.7’.b
38 + 1.4’ 758 + 72b
35 * 2.10 636 i: 35b
differ (P < 0.05) among different
groups.
* + + k + k
5.3a 58’ 7b 0.2” 1.5O 15a
950 f 25’ 9.2 f 0.4avb 25 + 0.7b 239 _+ 3c
IGF-I AND MUCOSAL ADAPTATION
Tune 1992
Table 3. Effects of Jejunoileal
Resection
1953
and IGF-I on Ileal Mucosal Enzyme Activities Resected
Enzyme activity
IGF-I
Control 14 206 147 2.1 3.7 51
Sucrase @moI . g protein-’ *min?) (nmol . cm-’ - min-‘) Maltase @mol. g protein-’ - min-‘) @mol. cm-’ . min-‘) Lactase @mol. g protein-’ - min-‘) (nmol . cm-’ - min-‘) Alkaline phosphatase @mol. g protein-‘. min-‘) (nmol - cm-‘. min-‘) Leucine aminopeptidase @mol. g protein-’ . min-‘) (nmol . cm-’ . min-‘) NOTE. Data represent mean f SEM (n = 3-8). “-“Values within rows with different superscripts
f f i + k f
2.3” 38’ 18’,’ 0.3O 0.5” 7O.b
26 403 213 3.3 4.4 68
+ f f + t f
2.8a.b 41“,b 20USb 0.3b 0.5” 6’
5 27 96 0.6 5.2 32
+_ 1.8’ + 10” + 22” + 0.1” +_ 0.5” k 4b
55 + 7.6’ 860 k 114O
43 * 4.5O 694 ?I 99”
45 f 7.0” 268 k 38’
13 f 2.0° 187 + 33”,’
22 iz 2.8b 343 + 41b
25 f 3.5b 334 f 49b
13 + 2.9” 77 f 16”
differ (P < 0.05) among different groups.
IGF-I may be potential therapeutic agents for short bowel patients. A probable role of IGF-I in controlling intestinal growth was shown when northern blot and in situ hybridization analyses revealed the presence of messenger RNA for both IGF-I and IGF-II in rat and human intestine.3”,37 Furthermore, mouse intestinal explants release IGF-I in vitro3’ and rat intestinal epithelium contains both IGF-I and IGF-II receptors.3 In addition, IGF-I is a potent stimulator of intestinal epithelial cell (IEC-6 cell) proliferation.’ We have also observed that fibroblasts isolated from the rat small intestine and IEC-6 cells produce IGF-like peptides and IGFBPs (unpublished data). This evidence suggests that IGFs and IGFBPs are physiologically important in the intestinal mucosa. In the present study, we have shown that IGF-I enhanced mucosal hyperplasia after small bowel resection in vivo. Unoperated rats subjected to the same fasting and refeeding protocol as the resected rats were included
Discussion An important finding of the present study is that IGF-I and des-IGF-I significantly enhance mucosal adaptation that normally occurs after massive small bowel resection. These changes were observed morphometrically by increased villus height in the duodenojejunal segment and increased crypt depth in the ileum. Furthermore, biochemical measures of intestinal hyperplasia (DNA and protein content) were increased significantly by IGF-I and des-IGF-I in the duodenojejunal segment of the remaining intestine. Cellular differentiation, as reflected by total disaccharidase activity within duodenojejunal and ileal segments, was also stimulated significantly by both IGF-I and des-IGF-I. The implications of these results are significant because both IGF-I and des-
Resection
19 281 205 3.1 4.1 63
Unoperated
50 f 2.5” 724 + 53’
IGF-I but not des-IGF-I increased the serum levels of IGFBP-2. Neither IGF treatments changed serum levels of the 24,000 molecular weight IGFBP-4.
Table 4. Effects of Jejunoileal
+ 3.3b + 5ob + 23b + 0.3b -Irl.oa + 14O.b
Des-IGF-I
and IGF-I Treatment
on Serum IGF-I and IGFBP Levels Resected
Measurement IGF-I (ng/mL) Insulin (pILJ/mL) Glucose (mg/lOO mL)
IGF-binding capacity (ngEq/mL) IGFBP-3 (43-39,000 mol wt)d IGFBP-2 (32,000 mol wt)d IGFBP-4 (24,000 mol wt)d
Control
IGF-I
328 f 23”
521 zk 23b 20 I? 2.5” 139 + 2b
20 k 1.3” 146 + 2’ 177 +_ 5” 100 -t 7O
100 f 7” 100 + 15”
NOTE. Data represent mean + SEM (n = 3-8). “-“Values within rows with different superscripts differ (P < 0.05). dValues for IGFBP-3, -2, and -4 are expressed in scanning densitometry
155 176 181 125
-+ 5b + 17b.C F 28b k 26a.b
Des-IGF-I 570 15 143 680 150 119 112
k 27b -t l.lb xk 2”.b + 120” f 13b t 21a.b rf: 12”
units relative to levels in resected
control rat sera.
Unoperated 515 f 8b 29 f 3.3c 153 f 4” 178 f 5”
209 t 6” 143 + 32n,b 141 + 23b
1954
VANDERHOOF ET AL.
Cntl
IGF-I
GASTROENTEROLOGY Vol. 102, No. 6
;;e,s_; Unop
Resected Figure 2. Ligand blot of serum with ‘Z51-IGF-I. Rat sera were electrophoresed through 12.5% sodium dodecyl sulfate-polyacrylamide gels, and Western blots were prepared. Ligand blot analysis was used to determine the size and relative amounts of the various IGFBPs present as described in Materials and Methods. A representative set (n = 3-6) is shown. Sizes (molecular weight X 10e3) of the IGFBPs were estimated relative to the mobility of molecular weight standards. The 43-39,660 molecular weight IGFBPs are glycoprotein variants of the same growth hormone-dependent form, IGFBP-3.’ IGFBP-2 was confirmed as such by immunoblotting the Western blots with antisera specific to this form of IGFBP (data not shown). Rat and human IGFBP-4 has recently been cloned and named,6 and the 24,000 molecular weight IGFBP in sera has an N-terminal sequence identical to this recently characterized protein.‘5
in this study. Although these animals are not the ideal control group for the resected animals, results from these unoperated animals provide some interesting information. Serum levels of IGF-I, IGFBP-3, and IGFBP-4 decreased in resected control rats compared with unoperated rats. This decrease may be caused partially by the slight decrease in either food intake or absorption in resected rats, which would explain the decrease in serum insulin and glucose levels. Other studies have indicated that circulating levels of IGF-I were reduced in fasted rats,3Q by ligand blot analysis the levels of 4%39,000 molecular weight IGFBP-3 and 24,000 molecular weight IGFBP4 are markedly reduced in fasted animals, and IGFBP-2 is not consistently depressed compared with contro1s.34 In addition, serum concentrations of IGFBP-3 in rats have been shown to be IGF-I dependent.40*41Thus, the reduced serum levels of IGFBPs in resected rats in this study may reflect serum levels of IGF-I. Levels of IGFBP-3 were returned toward the levels measured in the sera of unoperated controls by IGF infusion (IGF-I and des-IGF-I) (Table 4), confirming the hypothesis. Des-IGF-I did not significantly change either IGFBP-2 or IGFBP-4 levels although IGFBP-2 levels were elevated in IGF-I treated rats compared with resected controls (Table 4).
Despite similar immunoassayable serum IGF-I levels in IGF-I- and des-IGF-I-infused rats and a lower total induction of serum IGFBP levels by des-IGF-I, the ‘251-IGF-I-binding capacity of sera was greatest in des-IGF-I-infused rats (Table 4). This finding indicates that the infused des-IGF-I either is free or dissociates from the IGFBPs during the binding capacity assay because of its low affinity for IGFBPs. Thus, with unaltered IGFBP levels the degree of unsaturation of circulating IGFBPs appears to be elevated in des-IGF-I-infused animals. Dissociation of des-IGF-I appears to be a plausible explanation because the clearance of des-IGF-I from rat serum is approximately 2 hours42 due to des-IGF-I association with the l50- and SO-kilodalton IGFBP complexes in sera. Dissociation of des-IGF-I from the IGFBPs during measurement of IGF distribution in sera may explain the apparent lack of IGFBP binding of des-IGF-I that has been published previously.43 Differential distribution of des-IGF-I vs. IGF-I between the various forms of IGFBPs present in serum and differences in tissue availability would be expected to alter the bioactivity of des-IGF-I compared with IGF-I. Such differences were found. In the present study, des-IGF-I was a better stimulant of somatic growth (body weight) than IGF-I (Figure 1). These results are consistent with those of Lemmey et a1.,44 who compared these two analogues at the dosage of 0.96 mg/kg per day in the same animal model. In their studies, des-IGF-I was also more potent in stimulating increases in duodenal weight, kidney weight, and body length than IGF-I.44 In contrast, we found that des-IGF-I was either lower or equipotent to IGF-I in stimulating duodenojejunal mucosal adaptation and ileal mucosal enzyme activities (Tables 2 and 3). It is possible that the dose of both IGFs (1.5 mg/kgper day) used in the present studies is high enough to produce the maximal responses in the intestine, thus masking some of the possible differences in activity between the two forms. These results also suggest a degree of tissue specificity in the effects of des-IGF-I vs. IGF-I. The precise role of IGFBPs in controlling differential growth of various tissues is unclear at this time. Future studies will be needed to determine the role of IGF induction of serum or locally secreted IGFBPs in controlling these effects. In the duodenojejunum, infusion of IGF-I and desIGF-I resulted in increased DNA, protein, and villus height but no change in crypt depth. In the ileum, these growth factors increased crypt depth but did not affect DNA, protein, or villus height. The magnitude of increases in DNA, protein, and villus height in resected controls compared with unoperated controls was much greater in the ileum (greater than twofold in each case) than in the duodenojejunum (1.26-1.42-fold increase) (Table 1). One can speculate
June 1992
that the adaptive responses to resection were already maximal in the ileum so that exogenous IGF-I could not further enhance these responses. Alternatively, the increase in crypt depth in the ileum of IGF-infused animals suggests that the adaptation process in the ileum was still in progress 7 days after resection. If the rats had been killed later, the differences in crypt depth may have disappeared and an increase in villus height may have become apparent. In the duodenojejunum, infusion of IGF-I and desIGF-I increased DNA and protein contents without altering enzyme activities per unit protein. It is possible that the enzyme activities were already so high in the proximal intestine that they could not be increased further. By contrast, the activities of maltase, sucrase, and leucine aminopeptidase in the ileum were increased significantly by IGF infusion without changes in mucosal DNA and protein contents. One possible explanation for this result is that IGF infusion stimulated nutrient transport in the enterocytes of the ileum, which then induced these digestive enzymes. In fact, alkaline phosphatase and lactase, which are of minimal significance for nutrient digestion and absorption in adult rats, were not induced by infusion of either IGF-I or des-IGF-I. In conclusion, continuous infusion of IGF-I or desIGF-I enhanced mucosal adaptation after small bowel resection in rats. These results suggest that both IGF-I and des-IGF-I have potential as therapeutic agents for patients with short bowel syndrome. It remains to be determined through dose-response studies whether des-IGF-I or IGF-I is the more efficacious stimulant of mucosal adaptation. References 1.
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IGF-I AND MUCOSAL ADAPTATION
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Received July 17,199l. Accepted November 121991. Address requests for reprints to: Jung H. Y. Park, Ph.D., Swanson Hall Room 3049, 502 South 44th Street, Omaha, Nebraska 68105-1065. Supported by a grant from Caremark, Inc., Deerfield, Illinois. The authors are grateful to Drs. L. E. Underwood and J. J. Van Wyk (University of North Carolina, Chapel Hill, North Carolina) and the National Hormone and Pituitary Program (Baltimore, Maryland) for the provision of rabbit insulinlike growth factor I antiserum. They thank Dr. L. E. Underwood for the “‘I-insulinlike growth factor I used for ligand blot analysis.
