IGFBP mRNA expression in small intestine of rat during postnatal development

C. A. Shoubridge, C.-B. Steeb, and L. C. Read

Child Health Research Institute and Cooperative Research Centre for Tissue Growth and Repair, North Adelaide, South Australia 5006, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In contrast to the adult gut, the immature intestine is refractory to subcutaneously infused insulin-like growth factor I (IGF-I). IGF binding protein (IGFBP) mRNA expression was characterized in intestinal tissues from 6-, 19-, and 90-day-old rats to determine if changes in local expression could account for this age-related change in IGF-I potency. For all age groups, IGFBP-3 to -6, but not IGFBP-1 or -2, were detected by Northern blot analysis. IGFBP-3, -4, and -5 were more intensely expressed in the 6-day-old rat intestine compared with weanling or adult tissue. In contrast, IGFBP-6 expression peaked at the time of weaning. In situ hybridization showed IGFBP-3 to -6 expression was confined to cells of the lamina propria and submucosa and also in the muscularis layer for IGFBP-5. Furthermore, the pattern of IGFBP-5 localization in the intestine changed with development. The findings indicate that the expression of IGFBP-3 to -6 is higher in the immature intestine compared with the adult intestine, suggesting locally produced IGFBPs may inhibit systemically derived IGF-I action in the intestine. Therefore, changes to local IGFBP expression may contribute to the varying response of the rat intestine to IGF-I peptides during postnatal development.

insulin-like growth factor I; Northern blot; growth; in situ hybridization; gastrointestinal tract


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INSULIN-LIKE GROWTH FACTOR (IGF) system plays a major role in cellular growth and development. Ligands of this system are two polypeptide growth factors, IGF-I and IGF-II, which are expressed and secreted by multiple tissues (10, 14-16, 24) and abundant in circulation (9). The biological actions of IGFs are mediated by binding to two specific, high-affinity cell surface receptors (18, 25, 31, 37) and are directly regulated by a family of six IGF binding proteins (IGFBPs).

The gastrointestinal tract is one of the most sensitive target tissues for IGF-I peptides, indicated by the selective growth of the intestine following exogenous IGF-I administration (19, 28), with increases in both wet tissue weight and length of the intestine (34, 35). Although the regulation of IGF-I actions in the gastrointestinal tract remains unclear, in vivo evidence indicates that locally expressed IGFBPs may play a role. Expression of several IGFBP species has been detected in the intestine of adult (1, 2, 32, 33, 39) and fetal rats (26), with all IGFBPs, except IGFBP-1, detected in intestinal tissue of the human fetus (17). Furthermore, expression of IGFBPs has been shown to change in response to several conditions in which gut growth is modified and IGF-I action has been implicated, for example, small bowel resection (3), enterocolitis induced by peptidoglycan-polysaccharide (43), and jejunal atrophy associated with surgical stress and maintenance on total parenteral nutrition (TPN) (41). Most significantly, systemic infusion of the analog long [Arg3]IGF-I (LR3IGF-I; with a reduced affinity to the IGFBPs) has been shown to elicit gut growth in a qualitatively similar manner to infused IGF-I but with at least several fold higher potency (35). There may also be developmental changes in the regulation of IGF action in the intestine by the IGFBPs. This is suggested by our (34, 35) earlier observations that the neonatal rat gut is relatively refractory to IGF-I compared with that in adult rats, whereas the gut is very sensitive to LR3IGF-I in both age groups.

On the basis of these observations, we predicted that the developmental responsiveness of the gut to IGF-I reflected either a change in circulating IGFBPs and therefore delivery to the gut or the expression of local IGFBPs in the gut tissue, which would affect the potency once the IGF-I peptide was delivered. Because there is little information on the developmental changes in local IGFBP expression in the gut, we have undertaken in this study to characterize IGFBP mRNA expression in the small intestine of the rat during postnatal development and identify any developmental trends that correlate with the changes in the biological potency of IGF-I in the small intestine.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Female and male hooded Wistar rats were obtained from the University of Adelaide breeding colony. All experimental procedures in this study were approved by the Animal Care and Ethics Committees of the Women's and Children's Hospital and the University of Adelaide. Animal handling and experimentation followed the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Sample collection. To minimize differences in the fed vs. fasting state of experimental animals, all adult rats had ad libitum access to water and rat chow up until time of death, with the suckling pups remaining with the dam until death. Because rats are predominately nocturnal feeders, they were killed for collection of tissue samples first thing in the morning so that they had already fed. This was confirmed by the stomachs of adults and pups containing foodstuffs at the time of collection. The study examined three distinct developmental time points with tissue and blood samples taken from a minimum of six animals at days 6, 19, and 90 postpartum to represent the early suckling, weaning, and early adult periods, respectively. For each time point, animals were stunned and killed by decapitation. Trunk blood was collected into heparinized tubes and centrifuged (for 5 min at 2,000 g at 4°C), and plasma was stored at -20°C for Western blot analysis. Immediately thereafter, a midline incision was made and the most proximal portion of the small intestine was collected in 10% (vol/vol) buffered formalin for standard histological processing and paraffin embedding. The remaining proximal small intestine was collected rapidly, snap frozen in liquid nitrogen, and stored at -70°C until required for mRNA extraction. Kidney samples were collected for use as positive control tissue for IGFBP mRNA expression studies.

RNA preparation. Total RNA was extracted from 0.5 to 1 g of proximal small intestine and kidney samples by the single-step acid guanidine-phenol-chloroform method of Chomczynski and Sacchi (6). Final RNA concentrations were determined spectrophotometrically at 260 nm.

Northern blot analysis. Total RNA (10 µg/lane) from the small intestine of all age groups and from adult kidney was electrophoresed on a 1% agarose gel containing 6% formaldehyde after denaturation. The separated RNA was transferred to a Zetaprobe GT nylon membrane (Bio-Rad, Hercules, CA) in a LKB Vacugene vacuum-blotting apparatus at 40 cmH2O vacuum pressure for 2 h. The RNA was then fixed to the membrane using a Stratalinker ultraviolet crosslinker (Stratagene, La Jolla, CA) at 1,200 mJ. RNA integrity was confirmed by ribosomal 18S and 28S RNA ethidium bromide staining. The membranes were hybridized with 32P-labeled cDNA probes at 1 × 106 cpm/ml at 42°C for 40-48 h. They were then exposed to a phosphor screen followed by scanning on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) for quantitative analysis. IGFBP in each age group were analyzed from two replicate membranes.

Analysis of mRNA expression. The abundance of mRNA for each of the IGFBP was quantified from the Northern blots using ImageQuant (Molecular Dynamics) image analysis software attached to the PhosphorImager. To correct for minor variations in sample loading and confirm the integrity of the RNA in each lane, the membranes were finally hybridized with a 18S rRNA DECAprobe (Ambion).

In situ hybridisation. Tissue samples from the proximal small intestine were fixed in 10% (vol/vol) buffered formalin for 12 h, rinsed, and stored in 70% ethanol until routine paraffin processing. Sections (5 µm) were cut and transferred to slides coated with 3-aminopropyltriethoxy-silane (Sigma, St. Louis, MO). In situ hybridization using sense and antisense rRNA probes was carried out as described by Powell and Rogers (28a) for IGFBP-3 to IGFBP-6, with exposure times of up to 24 days at 4°C.

Probe preparation. The cDNA probes for rat IGFBP-1 to -6 were generously provided by P. K. Lund (Department of Physiology, University of North Carolina, Chapel Hill, NC). The probes for rat IGFBP-1, -2, -3, and -5 were used with the kind permission of A. J. D'Ercole (Department of Pediatrics, University of North Carolina). The probes for rat IGFBP-4 and -6 were used with the kind permission of S. Shimasaki (The Scripps Research Institute, La Jolla, CA).

For Northern blot analysis, IGFBP-1 to -6 rat cDNA fragments and 18S rRNA cDNA DECAprobe were labeled with [32P]deoxy-CTP using a megaprime labeling system (Amersham Australia, Castle Hill, New South Wales, Australia). For in situ hybridization, sense and antisense probes were generated from linearized cDNA probes for IGFBP-3 to -6. The probes were labeled with [33P]UTP using a riboprobe in vitro transcription kit (Promega). In brief, 200 µg of linearized DNA were incubated at 37°C for 2 h in the presence of 3,000 Ci/mM of [33P]UTP and the appropriate polymerase T3 or T7 under the transcription reaction conditions outlined in the manufacturer's instructions. Removal of template DNA by incubation of the completed transcription reaction with 0.3 U/reaction of RNase-free DNase preceded the removal of unincorporated nucleotides by passing the diluted reaction volume, in the presence of an RNase inhibitor, through a ProbeQuant G50 MicroSpin column (Pharmacia Biotech). Before use in the in situ hybridization procedure, the integrity of each labeled probe was confirmed by denaturing gel electrophoresis and visualization by autoradiography.

Western ligand blot. For each age group, the circulating IGFBP protein profile was determined using pooled plasma (2 µl/lane) subjected to SDS-PAGE and ligand blotting, as described previously (5), with exposure to X-ray film at -80°C for 6 days.

Statistical analysis. All values are reported as means ± SE. Statistical comparisons between IGFBP mRNA levels in the three developmental age groups were by one-way ANOVA with significance set at P < 0.05. Where significance was reached, the Student-Newman-Keuls post hoc test for all pair-wise comparisons was used to analyze variance in mRNA levels between age groups. All statistical analyses were performed using StatView software (Jandel Scientific).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Circulating IGFBP profile. To confirm the circulating profile of the IGFBPs in the different age groups, plasma was subjected to SDS-PAGE and Western ligand blotting. With the exception of a minor 22-kDa band, the levels of circulating IGFBPs appeared to be markedly lower in the suckling and weaning periods than in the adult circulation (Fig. 1). In the early suckling period, the predominant species detected was a 30-kDa protein, presumably rat IGFBP-2 and/or -1. Low levels of the 22-kDa species were also present, most likely representing IGFBP-4. The profile of circulating binding proteins in the weanling animals was similar to that observed for the suckling rats, with the addition of low levels of the doublet migrating at 40 kDa, characteristic of IGFBP-3. The circulating IGFBP profile in the adult rats at day 90 was similar to that seen in the older adult rat plasma controls and typically showed the IGFBP-3 doublet, plus a well-defined 30-kDa band, representing IGFBP-1 and/or -2. Overall, these results are in agreement with previous reports (11, 29) for IGFBP levels in serum from suckling and adult rats.


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Fig. 1.   The circulating insulin-like growth factor binding protein (IGFBP) profile in rat plasma detected by Western ligand blot analysis changes markedly during postnatal development. Lanes contained 2 µl of pooled plasma from rats at day 6 (lanes 1-3), day 19 (lanes 4-6), and day 90 (lanes 7-9) and control adult rats (lanes 10 and 11). 14C-labeled molecular weight markers were run in the lane at left (M).

IGFBP mRNA expression in rat small intestine. Preliminary Northern blots detected all six IGFBP mRNA species in samples from the rat kidney, at the expected migration sizes (23, 30) (results not shown). Expression of IGFBP-3 to -6 mRNA was apparent in the proximal small intestine in all postnatal age groups examined. However, hybridization to IGFBP-1 and to IGFBP-2 mRNA in the small bowel was below detectable limits by Northern blot analysis in all cases (results not shown).

To quantitate changes in IGFBP expression during postnatal development, Northern blots were repeated with four animals per age group included on each membrane. Figure 2 shows representative blots probed for IGFBP-3, -4, -5, and -6 mRNA and corresponding 18S rRNA for each sample. Ribosomal 18S RNA was measured to normalize expression data of the IGFBPs by correcting for any minor variations in sample loading per lane. The expression of rat ribosomal 18S RNA has been described as one of the least variable of the housekeeping genes (40), and in this study there were no significant changes in 18S rRNA expression with developmental age (Fig. 2). Expression of mRNA in the proximal small intestine varied markedly between individual binding proteins. IGFBP-5 and -3 mRNA displayed the strongest signals for all IGFBPs examined, requiring overnight exposures. A 6-kb transcript was detected for IGFBP-5. This species was strongly expressed in the adult kidney and proximal small intestine for all age groups. IGFBP-3 mRNA was expressed as a 2.6-kb transcript in the proximal small intestine and adult kidney. IGFBP-4 and -6 mRNA levels were moderate to weak in expression, with clear hybridization signals requiring exposure times of between 2 and 4 days. A 2.6-kb transcript of IGFBP-4 and a smaller 1.3-kb transcript of IGFBP-6 were expressed in the adult kidney and the proximal small intestine of all age groups examined (Fig. 2).


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Fig. 2.   Representative Northern blot showing changes in IGFBP mRNA expression in the rat proximal small intestine during postnatal development. Each lane represents 10 µg total RNA extracted from the proximal small intestine of individual rats aged 6, 19, or 90 days (n = 4/age group). Membranes were sequentially hybridized for expression of IGFBP-5, -3, -4, or -6. The membranes were also probed for 18S rRNA expression, used as a control to normalize any sample or loading variability. The control lane at right contained 10 µg of total RNA from rat kidney (K) as a positive hybridization control for each of the IGFBPs.

Changes to IGFBP mRNA expression in small intestine during postnatal development. The pattern of mRNA expression for the four IGFBPs present in the proximal small intestine across postnatal development is shown in Fig. 3. The IGFBP-3 mRNA level in the proximal small intestine was significantly higher in tissue from the early suckling rats compared with tissue from the older age groups (Fig. 3A). IGFBP-4 mRNA expression was significantly higher during the suckling and weaning periods compared with adult animals. Although there were no significant differences in the expression levels of IGFBP-5 mRNA across developmental age, the highest level of mRNA was observed in tissues from the early suckling period with a 1.4- and 2.4-fold decrease in the weanling and adult gut, respectively. This trend indicates IGFBP-5 has a similar expression pattern to that observed for IGFBP-3 and -4 (Fig. 3C). IGFBP-6 showed a different pattern, with mRNA expression peaking during the weaning period (Fig. 3D). We conclude that, notwithstanding differences in IGFBP expression during the suckling to weaning periods, there is a clear developmental trend toward lower levels of total IGFBP expression in the small intestine of adult rats compared with the developing small bowel.


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Fig. 3.   Northern blot analysis of IGFBP mRNA expression in the rat proximal small intestine at days 6, 19, and 90 postpartum. The intensity of hybridization signal for IGFBP-3 (A), IGFBP-4 (B), IGFBP-5 (C), and IGFBP-6 (D) mRNA in each age group was normalized for variability in sample loading and expressed as % of intensity of the signal in the kidney. Data are means ± SE from 4 animals per age group, from 2 replicate membranes. All group comparison was achieved by ANOVA. * P < 0.05 vs. day 6; dagger  P < 0.05 vs. day 19.

Tissue localization of IGFBP mRNA expression in proximal small intestine. The cellular localization of the four IGFBPs detected in the proximal small intestine was characterized by in situ hybridization. Tissue sections from the proximal small intestine of three individual animals per age group were hybridized with either IGFBP-3, -4, -5, or -6 sense and antisense probes. In agreement with the Northern blots, IGFBP-5 and -3 were strongly expressed, with moderate levels of IGFBP-4 and -6 mRNA, indicated by longer exposure times required to gain clear hybridization signals. The level of nonspecific hybridization in intestinal tissues was negligible, measured by the absence of specific hybridization signal with the sense probe for each of the IGFBP examined (data not shown).

The pattern of expression for IGFBP-3, -4, and -6 mRNA did not change during postnatal development despite the age-related differences in the total level of expression. Localization patterns for each of the IGFBPs are shown in weanling animals in the representative photomicrographs (Fig. 4). IGFBP-3 mRNA was localized to cells in discreet clusters in the lamina propria of the mucosal layer and in cells around the crypt enterocytes in the pericryptal region (Fig. 4A). Expression of IGFBP-4 mRNA was relatively low in all age groups examined and displayed a diffuse signal in cells of the lamina propria and submucosa (Fig. 4B). The hybridization signal for IGFBP-6 mRNA was also localized to discreet cell clusters within the lamina propria along the entire length of villi and to cells in the submucosal region (Fig. 4D). A strong hybridzation signal for IGFBP-5 mRNA was observed in cells within the pericryptal region and lamina propria in the weanling rat intestine (Fig. 4C). However, in contrast to the other IGFBPs, IGFBP-5 mRNA displayed a distinct developmental change in cellular localization within the proximal small intestine. In young suckling rats, IGFBP-5 expression was localized to cells in the muscularis externa in addition to the expression in the mucosal layer observed in the weanling and adult intestine (Fig. 5).


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Fig. 4.   In situ hybridization of IGFBP-3 (A), IGFBP-4 (B), IGFBP-5 (C), and IGFBP-6 (D) expression in tissue of sections from the proximal small intestine of weanling rats. Representative darkfield micrographs (×100) show all 4 IGFBPs are expressed in the mucosal layer, specifically in cells of the lamina propria (LP), with only IGFBP-5 expression observed in the muscularis externa (ME).



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Fig. 5.   In situ hybridization shows that the localization of IGFBP-5 mRNA in the small intestine changes with developmental age in the rat. IGFBP-5 expression is seen in rats aged 6 (A), 19 (B), and 90 (C) days. Expression is predominantly in the ME during early postnatal life and in the LP of the mucosal layer with increasing developmental age.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We predicted that the responsiveness of the gut to IGF-I may be due, at least in part, to developmental changes in IGFBPs produced in the gut tissues. Hence, IGFBP mRNA expression was measured in tissue samples from the small intestine of rats during three important developmental stages to assess local production of these diverse proteins. Here we have shown, with the exception of IGFBP-6, the levels of IGFBP mRNA expression decrease in the postnatal intestine during development.

At the local tissue level, IGFBPs can have either stimulatory or inhibitory effects on IGF-I action. For example, IGFBP-3 and -5 have been shown (20, 23) to bind IGF-I with high affinity when found in extracellular fluid, thereby inhibiting IGF-I action by sequestration of the peptide away from the receptor. However, the same IGFBPs also bind to proteins on cell surfaces and in the extracellular matrix, reducing their affinity for IGFs and enhancing bioavailability of the peptide (22, 23). Not all IGFBPs are attributed with dual roles in regulation of IGF-I action; indeed, IGFBP-4 has always been shown (23, 38) to inhibit the biological effects of IGF-I in both in vitro and in vivo settings. Therefore, the developmental change in responsiveness of the gastrointestinal tract to subcutaneously administered IGF-I could potentially reflect a decrease in the local expression of inhibitory IGFBPs or an increase of stimulatory IGFBPs from neonatal to adult life. Nevertheless, the results from this study support an overall inhibitory role for IGFBP-3, -4, and -5 expressed by intestinal tissues in modulating systemically derived IGF-I action on the growth of the rodent gastrointestinal tract.

The peaking of IGFBP-6 mRNA expression at weaning, however, would argue against IGFBP-6 having a major role in regulating the bioavailability of systemically derived IGF-I. In both early suckling and weanling rats in the previous infusion trials (34, 35), the gut was relatively refractory to the native peptide. From this study, the significance of the developmental expression pattern of IGFBP-6 remains unclear but may reflect the marked affinity of IGFBP-6 for IGF-II, which is present in high levels during fetal and early postnatal life. Conversely, changes to IGFBP-6 may relate not to its modulation of IGF actions but to its emerging role in relation to the biological actions of retinoids (8, 36).

Although this study has focused on local intestinal expression of the IGFBPs, contributions to the developmental responsiveness of the intestine by other components of the IGF system cannot be ruled out. Indeed, changes to expression of IGFBPs at the tissue level may reflect mechanisms to regulate the normally high levels of IGF-II present early in postnatal life. Additionally, regulation of IGF action by tissue-derived IGFBPs may be required to compensate for the low levels of regulatory IGFBPs in circulation during the early postnatal period. Our results confirmed that the concentrations of all forms of the IGFBPs in the plasma were low during early postnatal life and increased markedly in adult life, in agreement with the literature (11, 29). Despite evidence (4, 5, 13, 42) that plasma IGFBPs slow the clearance of IGFs from the circulation and delivery to tissues, the impact of changing circulating IGFBP levels during postnatal development on the delivery of IGFs to the intestine requires further study to be fully understood.

A potential complication in understanding the modulation of IGF action in the intestine by locally derived IGFBPs arises due to the complex structure of the small intestine, comprising a range of different cell types in distinct functional layers. Previous studies (34, 35) have demonstrated that in all age groups examined, IGF-I stimulates the proportional growth of all tissue layers of the small intestine. Nevertheless, it is feasible that different IGFBPs may influence IGF-I action within these separate layers. Therefore, in addition to measuring the overall expression levels, IGFBP mRNA was localized to cellular sites within the different tissues layers of the rodent small intestine. The results of this study showed that although there was no expression of IGFBPs mRNA in the mucosal epithelial layer, all four IGFBPs expressed in the intestine during postnatal development were found in cells of the lamina propria and submucosal regions. In agreement, other studies (27, 39) have localized IGFBP-3 mRNA expression to cells of the lamina propria in the adult rodent intestine and shown an absence of expression in the mucosal epithelial cells. In our study, the detection of IGFBPs in the lamina propria and submucosa suggests they have the potential to influence IGF-I actions on crypt epithelial proliferation and therefore mucosal growth. It is difficult to identify the cell types expressing the various IGFBPs in the lamina propria from the tissue sections used for in situ hybridization analysis. However, in vitro studies (7, 21) have shown that cultured intestinal fibroblasts, myofibroblasts, smooth muscle cells, and a range of immune cell types express several of the IGFBPs, including IGFBP-3, -4, and -5. Our study, along with others (39, 43), indicates the IGFBPs are localized to cells of the lamina propria, and the smooth muscle cell layer of the small intestine in the case of IGFBP-5, suggesting that mesenchymal cells are a source of IGFBPs in the rat small intestine. The IGFBPs derived from the mesenchyme may act in a paracrine manner to modulate IGF-I action on nearby epithelial cells or in an autocrine manner on cells within the mesenchymal layer itself. In support, IGFBP-3, which is predominantly localized in the lamina propria of the rat small intestine, displays a reduced level of expression in conditions of mucosal hyperplasia following resection (3) and fasting and refeeding (39), and these changes may contribute to the bioavailability of IGF-I at the tissue level.

Changes in the expression of IGFBP-3, -4, and -6 mRNA by in situ hybridization were commensurate with the developmental changes measured by Northern blot. Interestingly, the pattern of IGFBP-5 mRNA localization in the intestine changed with postnatal development. In early postnatal life, IGFBP-5 mRNA was localized predominantly in the muscularis layer (the only IGFBP expressed in this layer), with the greatest intensity of mRNA signal in the older age groups localized to the lamina propria and submucosa. This raises the possibility that locally produced IGFBP-5 may enhance the bioavailability of infused IGF-I in the mucosal layer of the small intestine but have an inhibitory effect on systemically delivered IGF-I activity in the muscularis layer, especially during early life. Reports of cellular localization of the IGFBPs in the postnatal intestine are quite limited, although IGFBP-5 mRNA localization has been reported (43) in the adult rat cecum. This report (43) showed that IGFBP-5 mRNA was confined to the smooth muscle layer in normal adult rat cecum but was expressed in cells of the submucosa and serosa of inflamed cecum after enterocolitis induced by peptidoglycan-polysaccharide. Similarly, changes to IGFBP-5 expression in the jejunal mucosa of rats surgically stressed and maintained on TPN were suggested to contribute to the ability of IGF-I but not growth hormone in attenuating the jejunal atrophy caused by the surgical intervention (41). On closer examination, when rats undergoing TPN were treated with IGF-I, IGFBP-5 expression was induced in the lamina propia in addition to the normal expression in the muscularis layer (27). Therefore, changes in the expression pattern of the IGFBPs at the level of different cells and tissue layers in vivo may have important physiological consequences in modulation of IGF-I actions.

In summary, the IGFBPs expressed in the proximal small intestine of the rat display distinct developmental patterns during postnatal life. These patterns of expression may help to explain the potency of systemically derived LR3IGF-I in the intestine over that observed with IGF-I infusion, especially during early postnatal life.


    FOOTNOTES

Address for reprint requests and other correspondence: C. Shoubridge, Child Health Research Institute, North Adelaide, South Australia 5006, Australia (E-mail: cheryl.shoubridge{at}adelaide.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 March 2001; accepted in final form 28 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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