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Department of Nutritional Sciences (D.M.N., D.J.H., M.B.G., K.R.K., E.M.D.), University of Wisconsin-Madison, Madison, Wisconsin 53706; and Department of Biochemistry (J.L.T., M.L.A.), University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Address all correspondence and requests for reprints to: Denise M. Ney, Ph.D., Department of Nutritional Sciences, University of Wisconsin-Madison, 1415 Linden Drive, Madison, Wisconsin 53706. E-mail: ney{at}nutrisci.wisc.edu
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Abstract |
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Introduction |
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Total parenteral nutrition (TPN) results in marked atrophy of the small intestine in the rat (1, 3) and mucosal hypoplasia in healthy subjects maintained with TPN (9). We have demonstrated that treatment with rhIGF-I or rhGH produce equivalent, significant increases in circulating levels of IGF-I and body weight gain (10), and that IGF-I and GH differentially increase protein synthesis in jejunal mucosa and skeletal muscle, respectively (11). Interestingly, only IGF-I, and not GH, stimulates intestinal growth in parenterally fed rats (10) and orally fed rats subjected to massive intestinal resection (12). In addition, GH does not stimulate crypt cell proliferation in transgenic mice that overexpress GH and show increased circulating levels of IGF-I (13). These model systems suggest that the small intestinal mucosa is resistant to the mitogenic effects of GH-stimulated endogenous IGF-I and/or putative direct mitogenic effects of GH.
The resistance of the small bowel to GH could be due to alterations in IGF-I or insulin receptor binding or postreceptor signaling. Insulin could directly affect mucosal cell proliferation or indirectly modulate IGF-I action. To better understand the selective ability of IGF-I, but not GH, to stimulate intestinal growth, we have compared IGF-I and insulin receptor binding and expression in jejunal membranes from orally fed rats and parenterally fed rats treated with IGF-I, GH or IGF-I plus GH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals were randomized into four TPN groups, using a 2 x 2 factorial treatment design: TPN control, GH, IGF-I and IGF-I + GH. An orally fed, nonsurgical, age-matched group (Oral) was included to provide a "normal" comparison for the clinically relevant TPN model. The Oral group was allowed ad libitum access to a nutritionally complete semipurified diet with a macronutrient composition comparable to the TPN solution (10). The final sample size was 5–6 animals per group.
Jejunal cellularity and morphology
After 4 days of TPN and 3 days of growth factor treatment, rats
were anesthetized with ketamine and then killed by exsanguination. The
jejunum, designated as the intestinal segment from the ligament of
Treitz to 10 cm proximal to the cecum, was rapidly excised and stripped
of vascular and mesenteric connections. The first 10 cm of jejunum
distal to the ligament of Treitz was used to determine the wet weight
of intact jejunum and jejunal mucosa and for analysis of mucosal
protein (bicinchoninic acid protein assay, Pierce Chemical Co., Rockford, IL) and DNA (15) content. The next 2 cm was used
for histology and morphometric measurements as previously described
(10) and for immunohistochemistry to localize the IGF-I receptor. For
half of the animals from each treatment group, the next 15 cm of
jejunum was used for RNA extraction for ribonuclease protection assays
(RPA) and the remaining or distal 30 cm of jejunum was used for
receptor binding studies. This procedure was reversed for the remaining
animals to assure randomization within each treatment group. The
jejunal segments for RPA analysis were flushed with ice-cold saline,
weighed, frozen in liquid nitrogen, and stored at -70 C for later
analysis. Segments to be used for binding assays were flushed with
ice-cold homogenization buffer (see below), weighed, frozen in liquid
nitrogen and stored at -70 C.
Jejunal IGF-I receptor immunohistochemistry
Fixed and paraffin-embedded jejunal tissue was cut into 5-µm
sections and processed for localization of the IGF-I receptor as
previously described (16). Briefly, slides were depariffinized,
rehydrated, and treated with 3% hydrogen peroxide in PBS for 10 min to
block endogenous peroxidase activity. Sections were exposed to a 1:100
dilution of the primary antibody (antihuman IGF-I receptor,
-subunit, Upstate Biotechnology, Inc., Lake Placid, NY)
at 4 C overnight, rinsed in PBS and exposed to a 1:500 dilution of the
secondary antibody (biotinylated rabbit anti-chicken IgY, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min at
room temperature. Staining was accomplished by incubating sections in
peroxidase reagent (ABC Reagent, Vectastain Laboratories,
Burlingame, CA), followed by a 3-min incubation in peroxidase substrate
[(0.02% hydrogen peroxide and 0.1% diaminobenzidine
tetrahydrochloride, made in 0.1 M
Tris(hydroxymethyl)-aminomethane (Tris) buffer, pH 7.6].
Stained slides were rinsed and counterstained with a 1:4 dilution of
hematoxylin for 1 min. Control sections were prepared by incubating
with a nonspecific chicken IgY diluted in PBS instead of the primary
antibody. Stained slides were examined under a light microscope to
determine localization and intensity of positive staining.
Serum IGF-I and insulin
Blood was obtained by cardiac puncture, and serum was obtained
by centrifugation at 4 C for 20 min at 1200 x g. Total
serum IGF-I concentrations were determined by RIA after IGF-binding
proteins were removed by HPLC under acid conditions, as previously
described (17). Absence of IGF-binding proteins in the peptide fraction
was verified by Western ligand blot. Serum insulin concentration was
determined using a rat RIA kit (Linco Research, Inc., St.
Charles, MO). Serum samples were assayed in triplicate in a single
assay with intra and inter assay coefficients of variation of 8% and
10%, respectively.
IGF-I and insulin binding assay
The frozen jejunal segments were placed in 5 ml of ice-cold
homogenization buffer (50 mM HEPES, 150 mM
NaCl, 1 mM bacitracin, 1 mM
phenylmethyl-sulfonylfluoride, and 1 trypsin inhibitor unit/ml
aprotinin, pH 7.8) per gram of tissue and immediately homogenized using
a Tekmar Tissumizer (Cincinnati, OH) for 20 sec. The homogenate was
centrifuged at 5000 x g for 10 min at 4 C. The
resulting supernatant was centrifuged at 35,000 x g
for 30 min at 4 C. The supernatant was discarded, and the pellet was
resuspended in homogenization buffer and recentrifuged at 35,000
x g for 30 min at 4 C. The final particulate membrane
pellet was resuspended in homogenization buffer at 2 ml/g initial
jejunal weight. The protein concentration was determined using the
bicinchoninic acid protein assay (Pierce Chemical Co.).
The membranes were stored at -70 C.
The receptor binding assays for both insulin and IGF-I were performed
in triplicate using polyethylene microfuge tubes (8). The assay buffer
contained 100 mM HEPES, 118 mM NaCl, 1.2
mM MgSO4, 8.8 mM dextrose, 5
mM KCl and 1% BSA, pH 8.0. Each tube contained 200 µg
membrane protein and 0.2 ng/ml (3-[125I]
iodotyrosylA14) rhinsulin or (3-[125I]
iodotyrosyl) rhIGF-I (Amersham Pharmacia Biotech,
Arlington Heights, IL). The specific activity of both radioligands was
2000 Ci/mmol. The radioligand binding was competed with 0 to
10-7 M rhIGF-I (Genentech, Inc.)
or 0 to 10-6 M porcine insulin
(Sigma, St. Louis, MO). The final volume of the assay was
320 µl/tube. After incubating for 16 h at 4 C, 400 µl ice-cold
binding buffer was added to each tube and mixed. The bound and free
hormone were separated by centrifugation at 12,500 x g
for 5 min at 4 C. The supernatant was removed and the pellet washed
with 500 µl ice-cold binding buffer. The tubes were recentrifuged at
12,500 x g for 5 min at 4 C. The supernatant was
discarded, and the tips of the tubes containing the pellets were
removed. The 125I in the final pellet was quantitated in a
Wallac, Inc. 
counter (Turku, Finland).
Nonspecific binding was measured in the presence of excess unlabeled hormone (10-6 M porcine insulin or 10-7 M rhIGF-I). Total binding was measured in the absence of cold competitor. Specific binding was calculated by subtracting nonspecific binding from total binding. Nonspecific binding was not significantly different between treatment groups for either IGF-I or insulin binding. In preliminary experiments, the specific binding of insulin and IGF-I was linear from 100–400 µg of membrane protein/tube.
The specificity of binding to the IGF-I receptor was confirmed by competing radiolabeled IGF-I with unlabeled porcine insulin. The unlabeled insulin was about 300-fold less effective at competing off the IGF-I tracer than unlabeled IGF-I. Half-maximal competition of [125I]-IGF-I binding occurred at a concentration of 6 x 10-10 M unlabeled IGF-I. The specificity of binding to the insulin receptor was determined by competing off radiolabeled insulin with unlabeled rhIGF-I. The unlabeled rhIGF-I was about 300-fold less effective at competing off the insulin tracer than unlabeled insulin. Half-maximal competition of [125I]-insulin binding occurred at a concentration of 3 x 10-10 M unlabeled insulin. Neither IGF-I nor insulin binding was affected by the proximity of the jejunal segments to the ligament of Treitz.
The LIGAND iterative curve fitting program (Biosoft,Ferguson, MO) was used to generate and analyze Scatchard plots to determine receptor number (Ro) and affinity (Kd). The IGF-I binding data produced linear Scatchard plots that were best described by a single site model. The insulin binding data displayed curvilinear Scatchard plots for four of the five treatment groups. These curves indicate insulin receptor negative cooperativity and were best fit using a two-site model to generate total receptor capacity. Dr. Robert McCusker (University of Illinois, Urbana, IL) generated affinity constants for the empty (Ke) and filled (Kf) insulin receptor occupancy states using a custom computer program. In contrast to the other four treatment groups, rats treated with GH showed insulin binding Scatchard plots that were linear and were best fit with a one-site model.
Jejunal IGF-I and insulin receptor messenger RNA (mRNA)
Ribonuclease protection assay (RPA) was used to measure IGF-I
and insulin receptor mRNA in full thickness jejunum. A description of
the probes follows. A pGEM 3 vector containing 265 bp of rat IGF-I
receptor complementary DNA (cDNA) sequence (18) was kindly provided by
Drs. Werner, LeRoith, and Roberts, Jr. The vector was linearized with
EcoRI, and Sp6 RNA polymerase was used to synthesize an
antisense RNA probe that contained 40 b of vector sequence and
265 b complementary to the rat IGF-I receptor mRNA sequence. Thus, a
protected band of 265 b was obtained in RPA. A pGEM 4 vector containing
747 bp of rat insulin receptor genomic sequence was kindly provided by
Dr. C. T. Roberts, Jr. of Oregon Health Science University
(Portland, OR). The vector was linearized with EcoRI and T7
RNA polymerase was used to synthesize an antisense RNA. The probe
included 478 b complementary to the insulin receptor mature mRNA
sequence reported in (19); thus, a 478-b protected band was obtained in
RPA reflecting mature insulin receptor mRNA. Antisense RNA probes were
synthesized using 32P-UTP, 800 Ci/mmol (NEN Life Science Products, Boston, MA) and reagents from the Maxi Script
kit (Ambion, Inc. Austin, TX). After the labeling
reaction, the DNA template was digested and probes were extracted and
precipitated (20).
Total RNA from intact jejunal segments was prepared using the Ultraspec Reagent (Biotecx, Houston, TX). RNA concentration was determined by measuring the absorbance at 260 nm and the integrity and quantification were confirmed using agarose/formaldehyde gel electrophoresis (20). RPAs were performed on 10 or 20 µg of total RNA using the reagents and protocols supplied in the RPA II kit from Ambion, Inc.) with the following modifications (20). Two-hundred thousand cpm of labeled probe was precipitated with 10 or 20 µg of RNA. After the hybridization and RNase digestion steps, the reaction mixtures were extracted and precipitated. Alternatively, the protocol described in (20) was used for the entire RPA procedure. Protected hybrids were resolved on urea/PAGE gels. Autoradiography was performed, followed by phosphorimage analysis to quantify mRNA abundance (Molecular Dynamics, Sunnyvale, CA). The data are expressed as a fold of the mean phosphorimage units obtained for the orally fed reference rats or TPN control rats.
Insulin receptor immunoblotting
Jejunal particulate membranes prepared for the receptor binding
assays were also used for immunoblotting to measure insulin receptor
protein content. Samples containing 400 µg of protein were diluted in
an equal volume of buffer containing 125 mM Tris-HCl, pH
6.8, 4% SDS, 20% (vol/vol) glycerol, 100 mM
dithiothreitol and 0.05% (wt/vol) bromophenol blue and then incubated
in a boiling water bath for four minutes. The solubilized jejunal
proteins were fractionated by 7% SDS-PAGE and transferred to
nitrocellulose (Schleicher & Schuell, Inc., Keene, NH) at
100 V for 1 h using buffer containing 25 mM Tris, 192
mM glycine, 0.05% SDS and 20% (vol/vol) methanol. The
nitrocellulose blots were blocked for 1 h at room temperature in a
solution made up of 5% nonfat milk, 20 mM Tris, pH 7.6,
137 mM NaCl, 10 mM Na azide and 0.1% Tween-20.
The blots were washed for 10 min at room temperature in TTBS (20
mM Tris, pH 7.6, 137 mM NaCl and 0.1%
Tween-20) followed by incubation in the appropriate primary antibody
diluted in TTBS. The blots were then washed three times for 10 min each
in TTBS at room temperature. Subsequently, the blots were incubated for
1 h at room temperature in a 1:5000 TTBS dilution of donkey
antirabbit horseradish peroxidase conjugated secondary antibody
(Amersham Pharmacia Biotech). Finally, the nitrocellulose
sheets were washed three times for 10 min each in TTBS at room
temperature. Binding of the secondary antibody was detected by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech).
Immunoblots were probed initially with an antibody generated in rabbits against an 11 amino acid sequence from the carboxy terminus of the rat insulin receptor ß subunit between residues 1328–1338 (generously provided by Dr. R. Smith, Joslin Diabetes Center, Boston, MA). This purified antibody was diluted to 5.4 µg/ml and incubated with the jejunal membrane blots for 16 h at 4 C, which resulted in a single band at MW 95,000. Insulin receptor protein abundance was standardized by stripping the blots of all previous antibodies and reprobing with a 1:1000 dilution of rabbit antirat actin antibody (Sigma) for 2 h at room temperature. This antibody recognizes cytoskeletal actin and produced a doublet at about 40,000 MW that served to validate equal protein loading per lane.
Autoradiographs were analyzed with a PDI scanning densitometer, model DNA 35, using Quantity One software (Huntington Station, NY). To control for variation in band intensity in multiple blots due to differences in film exposure time, the most intense band on each blot was set to an arbitrary value of 1.0. All remaining bands on the blot were then expressed as a percentage of that darkest band. Two samples from each treatment group were included on every blot. A ratio of insulin receptor protein abundance/actin protein abundance was calculated for each animal and used in the statistical analysis.
Statistical analysis
All groups were compared using one-way ANOVA (21). The four TPN
groups were also compared in a two-way ANOVA that included IGF-I and GH
main effects. Group means were considered significantly different at
P < 0.05, as determined by the protected least
significant difference technique. Data are presented as mean ±
SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IGF-I main effects,
P < 0.04; GH main effects, P < 0.07,
no significant interaction).
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In association with continuous infusion of TPN solution containing 18% dextrose, serum insulin concentration was increased by approximately 80% in TPN control compared with orally fed animals, P < 0.0001. Serum concentrations of insulin were reduced by 50–75% in parenterally fed animals treated with IGF-I or IGF-I plus GH compared with treatment with GH or TPN alone, P < 0.0001.
Jejunal cellularity, morphology and immunohistochemistry for the
IGF-I receptor
Four days of TPN induced marked jejunal atrophy. TPN induced a
significant (32–36%) decrease in jejunal wet weight and significant
decreases in the concentrations of protein and DNA in jejunal mucosa
compared with orally fed rats, Table 2.
Treatment with IGF-I alone or in combination with GH significantly
attenuated the jejunal atrophy induced by TPN, as evidenced by a
32–35% increase in jejunal wet weight and parallel increases in the
concentrations of protein and DNA in jejunal mucosa from IGF-I treated
compared with TPN control rats. The greater jejunal mass of animals
treated with IGF-I can be attributed to cellular hyperplasia, as
indicated by parallel increases in protein and DNA content. Consistent
with our previous findings, GH did not attenuate the jejunal atrophy
induced by TPN (10, 22).
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). IGF-I, but not
GH treatment, significantly increased villus height (IGF-I main
effects, P < 0.009).
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). There
appeared to be greater intensity of staining for the IGF-I receptor in
the TPN control compared with the orally fed group, as shown in Fig. 2.
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. Treatment of parenterally
fed rats with IGF-I or IGF-I + GH significantly increased serum IGF-I
concentrations and significantly reduced IGF-I specific binding by
approximately 25% compared with treatment with GH or TPN alone (IGF-I
main effects, P < 0.0015). Scatchard analysis of IGF-I
binding data indicated that the apparent dissociation constants
(Kd) were not significantly different among the five
treatment groups and ranged from 4.9 ± 0.5 to 7.9 ±
1.3 x 10-10 M. TPN increased IGF-I
receptor binding capacity (Ro) by approximately 70%
compared with oral feeding, P < 0.01. In contrast,
treatment of parenterally fed rats with IGF-I or IGF-I + GH reduced
Ro by approximately 50% compared with treatment with
GH or TPN alone (IGF-I main effects, P < 0.0007).
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and 4. IGF-I receptor mRNA levels in the
jejunum of rats treated with IGF-I were reduced by approximately
11–13%, which was not significantly different compared with TPN
alone, whereas receptor number was significantly decreased by
approximately 50%, Figs. 3 and 5.
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. Treatment of parenterally
fed rats with GH alone significantly reduced insulin-specific binding
by approximately 35% compared with TPN control. Insulin-specific
binding was not significantly different among the TPN control and IGF-I
treated groups.
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. Despite greater insulin-specific
binding with TPN, there was no significant difference in the affinity
parameters for insulin receptors in the TPN control compared with the
oral group. In contrast, significant differences in insulin receptor
affinity were noted, in association with similar insulin-specific
binding, with IGF-I treatment compared with the TPN control group.
Treatment with IGF-I alone showed a significantly greater insulin
receptor Kf compared with TPN control, whereas simultaneous
treatment with IGF-I and GH showed significantly greater insulin
receptor Ke and Kf compared with TPN
control.
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To determine whether the changes in insulin receptor binding were
accompanied by changes in receptor mRNA levels, RPAs for insulin
receptor mRNA were conducted. Insulin receptor mRNA levels in the
jejunum of TPN control rats were not significantly different compared
with orally fed rats, Fig. 7. Insulin
receptor mRNA levels in the jejunum of rats treated with GH or IGF-I
alone were reduced by approximately 24 and 21%, respectively compared
with TPN alone (P < 0.08), Fig. 8.
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.
The content of insulin receptor protein was increased by 40% in TPN
control compared with oral feeding, an effect that was not
significantly different at P > 0.05. Treatment with GH
alone significantly reduced insulin receptor protein content by 59%
compared with TPN control. There was a significant 115% increase in
insulin receptor protein content due to treatment with IGF-I plus GH
compared with TPN control.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I receptor binding in jejunum was increased as the result of parenteral feeding and was decreased when parenterally fed rats were treated with IGF-I. The most likely explanation is that the effects on IGF-I receptor binding were secondary to the changes in serum concentrations of IGF-I, as IGF-I is known to regulate IGF-I binding (25, 26). In the current study, animals maintained with TPN alone showed significantly lower circulating levels of IGF-I and significantly greater IGF-I receptor binding capacity (Ro) and abundance of IGF-I receptor mRNA in jejunum compared with orally fed animals. Winesett et al. also noted that parenteral compared with enteral infusion of an elemental diet increased the abundance of IGF-I receptor mRNA in jejunum (24). In contrast, coinfusion of IGF-I with TPN solution elevated circulating levels of IGF-I by 2–3 fold, stimulated mucosal hyperplasia, and reduced IGF-I receptor Ro by approximately 50%.
Fasting decreases the circulating concentration of IGF-I and increases IGF-I receptor binding in a tissue-specific manner (26). The stomach, lung, and kidney showed significantly greater IGF-I binding and IGF-I receptor number in conjunction with greater IGF-I receptor mRNA levels, whereas brain and testes showed no change in IGF-I binding or level of IGF-I receptor mRNA in response to fasting. Unlike the increased IGF-I receptor binding capacity in jejunum noted with TPN in the current study, fasting does not increase IGF-I receptor binding in jejunum in association with dramatically reduced circulating levels of IGF-I (8, 24). One possible explanation is that the systemic nutrition provided by TPN, in contrast to fasting, signals the body that nutrients are available to support tissue turnover and growth, a process that is supported by the presence of IGF-I receptors.
Research has demonstrated that IGF-I mRNA is expressed at very low levels in the jejunum and is localized to the lamina propria (22, 24). Evidence also suggests that changes in locally produced IGF-I mRNA cannot account for the jejunal atrophy induced by TPN (22, 24) or the stimulation of growth induced by exogenous IGF-I in rats maintained with TPN (22). Winesett et al. (24) compared parenteral and enteral administration of an elemental diet and noted similar expression of IGF-I mRNA in rat jejunum although jejunal mass was significantly reduced with parenteral compared with enteral feeding. We noted similar expression of IGF-I mRNA in rat jejunal mucosa using RPA, as well as in intact rat jejunum using in situ analysis, during altered jejunal growth due to oral feeding, TPN, or TPN with growth factor treatment in separate experiments (22). Moreover, we assessed the abundance of IGF-I mRNA by RPA in intact jejunum in the current study (data not shown) and confirmed that there is no significant difference in local IGF-I expression among TPN control, TPN + GH, or TPN + IGF-I groups. In contrast, changes in abundance of jejunal IGF-I mRNA correlate with changes in jejunal mass and circulating levels of IGF-I induced by fasting and oral refeeding (24). Maintenance of local expression of IGF-I by systemic nutrition is not sufficient to maintain gut mass during TPN. Regulation of jejunal growth appears to be dependent on both systemic nutrition and the presence of luminal nutrients.
Physiologically, the changes in IGF-I receptor binding, both receptor number and affinity, cannot explain the jejunal growth responses and suggest that the IGF-I receptor is not limiting for gut growth. Changes in IGF-I receptor mRNA qualitatively correlated with changes in IGF-I binding. Moreover, there was more IGF-I receptor protein in the TPN animals, as assessed by immunohistochemistry. Thus, at least a portion of the regulation of IGF-I receptor occurred at the level of either transcriptional control or mRNA stability. However, a significant portion of the effect may have occurred at the level of translation, protein trafficking, protein stability and/or inherent binding activity.
Treatment with IGF-I or IGF-I and GH significantly reduced serum concentrations of insulin and increased apparent affinity of the insulin receptor compared with TPN control. Stimulation of mucosal hyperplasia by treatment with IGF-I or IGF-I and GH resulted in significantly greater insulin-specific binding and greater insulin receptor protein content, as assessed by immunoblotting, compared with treatment with GH, which did not stimulate mucosal growth. When mucosal hyperplasia due to IGF-I treatment is taken into account, the greater content of insulin receptor protein per unit length of jejunum with IGF-I treatment is even more dramatic.
Treatment with GH alone dramatically altered insulin receptor binding kinetics compared with oral or parenteral feeding as evidenced by a linear Scatchard plot best fit with a one-site model. All other groups showed the expected curvilinear Scatchard plot consistent with the concept of insulin receptor negative cooperativity (23). Treatment with GH is known to induce insulin resistance in skeletal muscle and adipose tissue in vivo, an effect that appears to occur at the postreceptor level (27, 28, 29, 30). The current results suggest that GH may reduce insulin action in the jejunum during parenteral nutrition by decreasing insulin receptor binding. It is also of interest that the changes in insulin receptor binding do not correlate systematically with the changes in insulin receptor mRNA, either qualitatively or quantitatively. This suggests that GH alters insulin receptor binding principally by altering mRNA translation, receptor protein turnover, or inherent binding activity.
We conclude that factors associated with stimulation of jejunal growth during TPN include increased serum concentration of IGF-I, decreased IGF-I receptor binding, and reduced serum concentration of insulin. The role of the insulin receptor in stimulation of jejunal growth is unclear. It is possible that the 2- to 3-fold increase in circulating concentration of IGF-I in IGF-I treated rats stimulated jejunal growth by binding and activation of the insulin receptor or the IGF-I receptor. However, increased insulin binding without increased serum IGF-I concentration was not sufficient for jejunal growth because TPN control and IGF-I treated groups showed similar insulin-specific binding.
The absence of luminal nutrients due to TPN in the current study or fasting (8) appears to increase jejunal insulin receptor binding. However, unlike the reduced serum insulin concentrations and increased insulin binding due to fasting, consistent associations between circulating concentrations of insulin and insulin binding in jejunum were not observed in the complex TPN model, which is known to alter insulin dynamics (31). Given the greater jejunal insulin and IGF-I binding with TPN compared with oral feeding, it appears that the number and affinity of either receptor cannot account for the intestinal atrophy induced by TPN.
This study provides new information about the role of IGF-I and insulin receptors in the regulatory actions of luminal and circulating nutrients, as well as exogenous IGF-I and GH, in mediating small bowel growth. The data support the concept that jejunal IGF-I receptor number is determined by ligand-mediated down-regulation that occurs in part at the level of gene expression. Stimulation of jejunal growth during TPN was associated with elevated serum concentration of IGF-I and decreased IGF-I receptor binding in jejunum, presumably as a reflection of increased IGF-I "action." We cannot rule out the possibility that IGF-I action is being signaled in part through the insulin receptor. Given the clinical interest in utilization of IGF-I and GH to promote anabolism in critically ill patients who often require TPN (32), the current study provides relevant insights into the mechanisms of action of IGF-I and GH in the small bowel.
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Footnotes |
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Received February 18, 1999.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
; TPN control, 
; TPN + GH, 
; TPN + IGF-I, •; TPN + IGF-I +
GH = 
. IGF-I-specific binding was increased in the TPN control
and TPN + GH groups compared with the oral and IGF-I groups,
P < 0.003. There were no significant differences
in receptor binding affinity (Kd or slope). IGF-I receptor
number (Ro) was increased in TPN control compared with oral feeding and
decreased by IGF-I treatment compared with TPN control,
P < 0.01. Means ± SEM, n =
5–6. Values with different letter designations are
significantly different.
