Involvement of PI 3-kinase in IGF-I stimulation of jejunal Na+-K+-ATPase activity and nutrient absorption

Andrew N. Alexander and Hannah V. Carey

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanisms responsible for increased jejunal transport rates observed in tissues treated with orally administered insulin-like growth factor-I (IGF-I) were studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg · kg-1 · day-1) or control vehicle was given orogastrically for 4 days. Disaccharidase activity, fructose uptake, and Na+-glucose cotransporter SGLT-1 protein abundance were similar between groups. Oral IGF-I produced greater rates of enterocyte Na+-K+-ATPase activity with no significant differences in Na+-K+-ATPase abundance. Cellular mechanisms responsible for transport changes were studied in Ussing chambers. In control tissues, the presence of IGF-I in mucosal solutions increased basal short-circuit current (Isc), potential difference, D-glucose-stimulated Isc, and Na+-K+-ATPase activity; these changes were abolished by preincubation of tissues with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. The results suggest that the effect of IGF-I on jejunal ion and nutrient transport involves activation of PI 3-kinase and stimulation of Na+-K+-ATPase activity in enterocytes.

small intestine; disaccharidase; adenosinetriphosphatase; SGLT-1; neonates


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN-LIKE GROWTH FACTOR-I (IGF-I) is a potent inducer of growth and differentiation in the adult gastrointestinal tract when administered systemically (14-16, 19, 23, 24). Consistent with its presence in breast milk and colostrum, IGF-I also stimulates intestinal growth when administered orally, although significant effects have only been observed in neonates and older animals before weaning (2, 9). Physiological effects of IGF-I on cellular transport processes have been reported in a variety of cell types outside the gut (4, 7, 10, 17, 20, 21). In the intestine, a stimulatory effect of IGF-I on enterocyte transport was shown in adult animals when the peptide was given systemically (3, 16). Recently, our laboratory (1) demonstrated that oral administration of IGF-I increased transepithelial Na+ transport and the absorption of two Na+-coupled nutrients, D-glucose and L-alanine, in neonatal piglet jejunum.

In the present study we extended those findings to determine cellular mechanisms responsible for these effects. We first determined whether the ability of IGF-I to stimulate nutrient transport is specific for Na+-coupled solutes by measuring the transport of fructose, which is not dependent on the Na+ electrochemical gradient into enterocytes. Second, we investigated two potential mechanisms to explain enhanced rates of D-glucose absorption observed in tissues from piglets treated with oral IGF-I: the effect of the peptide on the abundance of the Na+-glucose transporter SGLT-1 and on the activity and abundance of the Na+-K+-ATPase pump. The latter enzyme is responsible for generating the electrochemical gradient that drives transport of Na+ and Na+-coupled nutrients across the enterocyte brush-border membrane. Third, we examined acute effects of IGF-I on jejunal ion and nutrient transport and Na+-K+-ATPase activity in vitro. Finally, we determined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the intracellular signaling cascade initiated by IGF-I that leads to increased jejunal ion and solute transport. This was done because of recent studies demonstrating that IGF-I stimulates transepithelial Na+ absorption in porcine endometrial epithelial cells by stimulating Na+-K+-ATPase activity via a PI 3-kinase-mediated mechanism (7).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and diets. The University of Wisconsin Institutional Animal Care and Use Committee approved the protocol used in this study. Colostrum-deprived neonatal crossbred piglets (1- to 2-kg body wt) were obtained from the University of Wisconsin Swine Teaching and Research herd. To ensure that piglets did not consume mammary gland-derived IGF-I, they were removed from the sow immediately postpartum. All piglets were provided free access to porcine milk replacer (Milk Specialties, Dundee, IL) throughout the study. At birth, piglets were randomly assigned to one of two groups. The first group received recombinant human IGF-I (rhIGF-I; Genentech, South San Francisco, CA) dissolved in 2 ml of milk replacer delivered by orogastric gavage, and the second group received the same volume of milk replacer given orogastrically with no IGF-I added. The dose of IGF-I administered was 3.5 mg · kg-1 · day-1 for 4 days. We chose this dose of oral IGF-I because Burrin et al. (2) showed previously that it stimulated intestinal growth in neonatal piglets. The total daily dose of IGF-I was divided into three equal aliquots administered every 8 h. The final IGF-I dose was given 5 h before euthanasia.

Piglets were killed on day 5. After laparotomy, a 20-cm segment of proximal jejunum beginning ~60 cm from the ligament of Treitz was removed and flushed with chilled Krebs solution (see Acute effect of IGF-I on glucose absorption). A 1-cm segment was fixed for immunohistochemical studies, and the remainder was used to isolate enterocytes.

Enterocyte isolation. Two segments of jejunum, 8-10 cm in length, were ligated at both ends and placed in oxygenated phosphate-buffered saline (in mM: 137 NaCl, 8.2 Na2HPO4, 1.5 KH2PO4, and 3.2 KCl, pH 7.4) at 37°C. The jejunal lumen was filled with 20 ml of buffer A [in mM: 96 NaCl, 5.6 Na2HPO4, 8 KH2PO4, 1.5 KCl, 5 EDTA, 5 EGTA, 0.5 dithiothreitol (DTT), and 185 mannitol with 0.25% BSA, pH 6.8] for 20 min to remove any debris, mucus, and dead cells. Buffer A was replaced with serial washes of buffer B (in mM: 137 NaCl, 8.2 Na2HPO4, 1.5 KH2PO4, 3.2 KCl, 1.5 EDTA, and 1 DTT with 0.1% BSA, pH 7.4) at 5-min intervals to collect enterocytes from the villus tip through the villus base. The first fraction collected was discarded to eliminate damaged cells from the upper villus. Hematoxylin and eosin-stained sections were obtained from small pieces of tissue collected after each wash to monitor the extent and position of dislodged cells along the crypt-villus axis. Enterocytes were pooled from fractions 2-5 representing cells from the upper and midvillus and used to prepare brush-border membranes and whole cell lysates. The former were prepared using a magnesium precipitation technique modified from Kessler et al. (13). For cell lysates, enterocytes were homogenized in buffer B and centrifuged twice at 4°C at 250 g for 5 min to remove nuclei and cellular debris. Protein concentrations were determined with the Pierce BCA kit. Cell lysates and brush-border membranes were stored at -80°C until use.

Enzyme activity. Cell lysates were used to measure specific activities of sucrase and lactase using the Dahlquist technique (6) and of Na+-K+-ATPase using the technique of Forbush (8).

Immunoblotting. Thirty micrograms of either cell lysate or brush-border membranes were separated on a 10% SDS-PAGE gel and electrophoretically transferred to nitrocellulose paper using a wet-transfer apparatus (Bio-Rad, Hercules, CA). Blots were blocked for 1 h in 1% milk in TBS-T (0.15 M Tris base, 10 mM NaCl, and 0.05% Tween-20, pH 7.4). Blots were incubated overnight with either anti-SGLT-1 antibodies (provided by Dr. Ernest Wright, UCLA) or antibodies raised against the alpha 1-subunit of Na+-K+-ATPase (no. 06-167, Upstate Biotechnology). After repeated rinses, blots were incubated with goat-anti-rabbit IgG linked to horseradish peroxidase. Protein bands were visualized using chemiluminescence (KPL, Gaithersburg, MD) and expressed as arbitrary units of optical density.

Acute effect of IGF-I on glucose absorption. Flat sheets of proximal jejunum were stripped of outer muscle layers and mounted in Ussing chambers (1.13 cm2) equipped to measure transepithelial potential difference (PD) and short-circuit current (Isc), a measure of active ion transport. Tetrodotoxin (0.5 µM) was added to the serosal solution to block neurally mediated fluctuations in Isc. Tissue conductance (Gt) was calculated using Ohm's law. The Krebs solution bathing mucosal and serosal sides of tissues contained (in mM) 148.5 Na+, 6.3 K+, 139.7 Cl-, 0.3 H2PO4-, 1.3 HPO42-, 19.6 HCO3-, 3.0 Ca2+, and 0.7 Mg2+. D-Glucose (11.5 mM) or mannitol (11.5 mM) was present in serosal and mucosal solutions, respectively. Tissues were bathed with 10 ml of solution by recirculation from a reservoir maintained at 39°C (porcine core temperature). Solutions were bubbled with a 95% O2-5% CO2 mixture, and solution pH was ~7.4. Ten minutes after mounting, rhIGF-I (0, 10, 60, or 200 µg/l) was added (noncumulative) to the mucosal solution, followed by D-glucose (10 mM) twenty minutes later. In a separate set of experiments, the role of PI 3-kinase in the intracellular signaling cascade evoked by binding of IGF-I to its receptor was investigated using wortmannin, a PI 3-kinase inhibitor. IGF-I-stimulated changes in Isc and PD and D-glucose-stimulated Isc (an indirect measure of D-glucose absorption) were measured in tissues pretreated for 30 min with 1 µM wortmannin added to mucosal and serosal solutions followed by addition of 60 µg/l rhIGF-I.

Acute effect of IGF-I on Na+-K+-ATPase activity. The jejunum was isolated and flushed with ice-cold Krebs solution and bubbled with 95% O2-5% CO2 during tissue preparation. Three segments of jejunum 8-10 cm in length were everted so that the mucosal surface faced outwards, ligated at both ends, and placed in oxygenated Krebs solution. The jejunal serosal lumen was filled with 20 ml of either Krebs solution or Krebs solution containing 1 µM wortmannin. Sleeves were preincubated in warmed (39°C), oxygenated Krebs solution for 30 min and then transferred to incubation solutions containing Krebs solution, Krebs solution containing 60 µg/l rhIGF-I, or Krebs solution containing 60 µg/l rhIGF-I with 1 µM wortmannin. After incubating for 30 min, the mucosa was scraped and immediately flash-frozen in liquid nitrogen. Na+-K+-ATPase enzyme activity was measured using the technique of Forbush (8).

Fructose uptake. Fructose uptake was determined using the everted sleeve technique (18, 22). The jejunum was isolated, flushed with cold Krebs solution, and bubbled with 95% O2-5% CO2 during tissue preparation. Tissues were everted, and 1-cm sleeves were mounted on grooved metal rods (7-mm diameter) suspended in a warmed (39°C), oxygenated Krebs solution over a stir bar rotating at 1,200 rotations/min. Sleeves were preincubated in isotope-free Krebs solution for 5 min and then transferred to incubation solutions containing either 25 mM or 50 mM unlabeled fructose prepared by isosmotic replacement of mannitol to obtain an osmolality of ~290 mosmol/kgH2O. Uptake studies used 4 µCi of [3H]fructose (American Radiolabeled Chemical, St. Louis, MO) added to each incubation solution of cold fructose. After 2-min incubation, sleeves were rinsed in Krebs for 20 s, blotted on filter paper, placed into tared vials, and weighed. Tissue solubilizer (500 µl, Solvable, Packard) was added, and 24 h later 4 ml of aqueous counting scintillant (Ultima Gold, Packard) was added to each vial. Radiotracer counting procedures and data analyses were performed as previously described (12). Uptake rates were corrected for solute present in adherent fluid by addition of tracer amounts of [14C]polyethylene glycol (mol wt 400).

Statistics. Values are expressed as means ± SE. Differences between means were analyzed using Student's t-tests or ANOVA when multiple groups were compared. When differences were identified by ANOVA, the comparison between any two groups was performed using Bonferroni's procedure for multiple comparisons. A probability level of P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weights and rates of weight gain were similar in control and oral IGF-I-treated piglets (data not shown) and were comparable to the results of our previous study (1). A small percentage of animals (5%), which included piglets from both treatment groups, failed to thrive and were subsequently removed from the study.

Enzyme activity. Enterocyte Na+-K+-ATPase activity was significantly greater in piglets treated with oral IGF-I (Table 1). This was not caused by a general effect of oral IGF-I on all membrane-bound enzymes because activities of sucrase and lactase were unchanged.

                              
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Table 1.   Jejunal enterocyte enzyme activities

Na+-K+-ATPase and SGLT-1 protein abundance. These experiments tested the hypothesis that increased numbers of SGLT-1 transporters in brush-border membranes and/or increased numbers of Na+-K+-ATPase pumps in basolateral membranes are responsible for the effect of oral IGF-I on nutrient and ion transport in piglet jejunum. Treatment with oral IGF-I did not alter the protein abundance of the alpha 1-subunit of Na+-K+-ATPase in cell lysates (Fig. 1). Likewise, SGLT-1 protein abundance in brush-border membranes was similar between treatment groups (Fig. 2).


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Fig. 1.   Effect of oral insulin-like growth factor I (IGF-I) on Na+-K+-ATPase protein abundance. Values are means ± SE from Western blots derived from 11 control and 9 IGF-I-treated piglets. Inset, representative blots from each group. Each lane contains 30 µg of enterocyte protein. Arrow indicates the 112-kDa alpha 1-subunit of Na+-K+-ATPase.



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Fig. 2.   Effect of oral IGF-I on Na+-glucose cotransporter SGLT-1 protein abundance. Values are means ± SE from Western blots derived from 7 control and 7 IGF-I-treated piglets. Inset, representative blots from each group. Each lane contains 30 µg of enterocyte brush-border membrane protein. Arrow indicates the 70-kDa SGLT-1 protein band.

Fructose uptake. Uptake of 25 mM fructose was similar between treatment groups (0.74 ± 0.07 nM · min-1 · mg-1 in 4 IGF-I piglets vs. 0.76 ± 0.10 nM · min-1 · mg-1 in 4 control piglets; P > 0.05). Likewise, fructose uptake at 50 mM did not differ between groups (data not shown). As we (1) observed previously, oral IGF-I had no effect on jejunal tissue mass, because the wet weights of 1-cm jejunal sleeves were similar between four IGF-I-treated and four control piglets (106.8 ± 5.1 mg vs. 118.9 ± 6.4 mg; P > 0.05).

Acute effect of IGF-I and role of PI 3-kinase. We (1) showed previously that oral administration of IGF-I enhanced jejunal glucose absorption in piglets. In these experiments, we determined whether IGF-I added directly to mucosal solutions bathing jejunal tissues mounted in Ussing chambers would also influence Na+-coupled glucose transport. If such an effect were measurable, this protocol would allow us to examine the effect of IGF-I on cellular mechanisms more directly. We used a range of IGF-I concentrations that enhanced glucose-stimulated Isc in jejunal tissues from rats maintained on total parenteral nutrition (TPN) when glucose was added to serosal solutions (16).

In control piglets, incubation of tissues for 30 min with the two highest IGF-I concentrations (60 and 200 µg/l) significantly increased basal Isc and PD but not Gt (Table 2). This effect was not observed in tissues from IGF-I-treated piglets (Table 2). In control tissues, mucosal addition of 60 and 200 µg/l IGF-I significantly increased the change in Isc evoked by subsequent addition of D-glucose (an indirect measure of D-glucose absorption) (Fig. 3) compared with tissues not exposed acutely to the peptide. These IGF-I concentrations also increased D-glucose absorption in piglets treated with oral IGF-I, but the magnitude of the effect was greater in control piglets (Fig. 3). For example, preincubation of tissues with 60 µg/l IGF-I enhanced D-glucose absorption by 40% in control piglets and only 28% in IGF-I-treated piglets compared with tissues incubated in Krebs alone.

                              
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Table 2.   Effect of mucosal IGF-I on electrical parameters in jejunal tissues



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Fig. 3.   Effect of mucosal IGF-I in vitro in tissues from control and IGF-I-treated piglets. Changes in short-circuit current (Delta Isc) after mucosal addition of 10 mM D-glucose in the presence of increasing concentrations of mucosal IGF-I in piglet jejunum are shown. Data represent means ± SE for 2 tissues per animal for each IGF-I concentration from 8 control (filled bars) and 8 IGF-I-treated (open bars) piglets. a, Significantly different from tissues exposed to 0 µg/l mucosal IGF-I (P < 0.001) in respective treatment groups; b, significantly different from tissues exposed to 10 µg/l mucosal IGF-I (P < 0.001).

To determine whether the effect of mucosal IGF-I on glucose absorption and basal electrical parameters involved a PI 3-kinase-mediated pathway, we preincubated tissues from control piglets with 1 µM wortmannin 30 min before mucosal addition of 60 µg/l IGF-I or vehicle. As expected, IGF-I alone increased Isc, PD (Table 3), and glucose absorption (Fig. 4). Wortmannin alone did not alter basal electrical parameters (data not shown). Pretreatment of tissues with wortmannin inhibited the IGF-I-induced increases in Isc, PD (Table 3), and glucose absorption observed in tissues exposed acutely to IGF-I (Fig. 4). In a separate set of experiments, incubation of tissues from six control piglets for 30 min in 60 µg/l IGF-I increased Na+-K+-ATPase activity compared with tissues not exposed to the peptide (4.2 ± 0.5 vs. 2.0 ± 0.2 µmol Pi · h-1 · mg protein-1; P < 0.01). This effect was abolished in six tissues that were pretreated for 30 min with wortmannin, resulting in Na+-K+-ATPase activities that were not significantly different from control tissues not exposed to IGF-I (2.5 ± 0.5 µmol Pi · h-1 · mg protein-1; P < 0.05).

                              
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Table 3.   Effect of wortmannin on IGF-I-induced changes in basal electrical parameters in control piglet jejunum



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Fig. 4.   Effect of 60 µg/l mucosal IGF-I on D-glucose absorption in tissues from control piglets pretreated with wortmannin in vitro. Values are means ± SE for 2 jejunal tissues per piglet from 7 control piglets per treatment group. D-Glucose absorption as indicated by Delta Isc after mucosal addition of 10 mM D-glucose is shown. **Significantly different from control tissues (P < 0.01); #significantly different from IGF-I tissues (P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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We (1) showed previously that oral administration of IGF-I to neonatal piglets increases small intestinal Na+ and Cl- absorption and Na+-coupled glucose and alanine absorption. These effects were not caused by alterations in jejunal mucosal mass, villus or crypt lengths, or brush-border microvillar dimensions (1). We therefore hypothesized that oral IGF-I may exert its effects on epithelial transport through a physiological rather than a proliferative effect and that it might occur via an increase in the electrochemical gradient for Na+ entry across the brush-border membrane. The current study provides several lines of evidence to support this idea. First, we found that oral IGF-I has no effect on intestinal fructose absorption, which is mediated by the facilitated glucose transporter GLUT5 and therefore is not dependent on the brush-border Na+ electrochemical gradient. Second, we showed that the enhanced Na+-coupled glucose absorption induced by oral IGF-I (1) is not accompanied by an increase in the abundance of SGLT-1 protein in the brush-border membrane, which suggests that the peptide probably influenced the activity of existing cotransporters without increasing their number. The lack of effect of oral IGF-I on the specific activity of two brush-border disaccharidases, lactase and sucrase, further supports the idea that IGF-I did not exert a general effect on enterocyte maturation.

In contrast to these findings, oral IGF-I significantly increased enterocyte Na+-K+-ATPase activity. This stimulation occurred without a measurable increase in abundance of Na+-K+-ATPase alpha 1-subunit protein, suggesting a physiological action of the peptide on enzyme activity. The mucosal addition of IGF-I in vitro to tissues of control piglets increased basal Isc and PD, enhanced glucose-stimulated Na+ absorption, and stimulated Na+-K+-ATPase enzyme activity within 20-30 min. Together, these findings suggest that IGF-I can affect brush-border transport processes and Na+-K+-ATPase activity within a relatively short time period and without synthesis of new brush-border transporters or Na+-K+-ATPase pumps. However, because Na+-K+-ATPase enzyme activity and protein abundance were measured in whole cell lysates, not in isolated basolateral membranes, we cannot rule out the possibility that IGF-I stimulated Na+-K+-ATPase enzyme activity by a redistribution of Na+-K+-ATPase pumps from intracellular to basolateral pools. This would not be detectable from Western blot experiments using enterocyte lysates but might be detected as an increase in enzyme activity, if one assumes that the activity of pumps in the intracellular pool is not measurable using the assay we used.

This is the first study to demonstrate an effect of IGF-I on Na+-K+-ATPase activity in intestinal epithelial cells. However, there is evidence from other cell types that, like the related peptide insulin, IGF-I can stimulate Na+-K+-ATPase activity (7, 21). In porcine endometrial epithelial cells this effect was responsible for the ability of IGF-I to stimulate transepithelial Na+ absorption (7). Although our evidence is indirect, we speculate that the greater enterocyte Na+-K+-ATPase activity resulting from IGF-I action is responsible for the enhanced ion and nutrient transport we observed in piglets treated in vivo with the peptide. This effect most likely occurs through a lowering of intracellular Na+ concentration that subsequently increases the electrochemical driving force for transport of Na+ and Na+-coupled nutrients into enterocytes.

The lack of effect of oral IGF-I on jejunal sucrase and lactase activities in our study contrasts with results of other work that showed an effect of oral IGF-I on enterocyte disaccharidase activity. For example, Houle et al. (9) found that 7- and 14-day-old piglets consuming formula containing 200 µg · kg-1 · day-1 of IGF-I for 6 days had greater small intestinal sucrase activity in the jejunum and lactase activity in the jejunum and ileum compared with controls. The discrepancy among these studies may reflect differences in the dose, duration, and delivery method of IGF-I as well as piglet age and thus the subsequent maturity of the enterocytes that were studied.

In our previous study (1), treatment of neonatal piglets with oral IGF-I increased the maximal rate of jejunal D-glucose uptake with no effect on the affinity constant for SGLT-1, which resulted in greater carrier-mediated D-glucose absorption (1). An acute effect of IGF-I on carrier-mediated glucose absorption was observed by Peterson et al. (16), who reported that serosal administration of IGF-I in vitro to jejunal tissues of rats maintained on TPN enhanced D-glucose-stimulated Na+ absorption (as indicated by enhanced change in Isc). Thus the functional effects of IGF-I on jejunal sugar transport are apparent when the peptide is given both in vivo and in vitro. In the present study, we confirmed these results by showing that tissues from control piglets that were incubated with mucosal IGF-I for 20 min demonstrated increased basal Isc, PD, and D-glucose-stimulated Na+ absorption. Although the maximal change in Isc evoked by mucosal glucose at all IGF-I concentrations was greater in piglets that previously received the peptide orally, the ability of mucosal IGF-I to acutely enhance D-glucose absorption was proportionately greater in tissues from control piglets. Thus administration of oral IGF-I appeared to dampen the maximal effect the peptide exerts on glucose absorption when it is given acutely to control tissues. Similarly, acute addition of IGF-I failed to stimulate Isc and PD in IGF-I-treated piglets, whereas it did in control animals.

Additional experiments were performed in control piglets to determine whether the effects of IGF-I on intestinal transport involve activation of PI 3-kinase, which occurs after autophosphorylation of the IGF-I receptor (11). Incubation of jejunal tissues in vitro with the PI 3-kinase inhibitor wortmannin inhibited the IGF-I-stimulated increases in Isc, PD, and glucose absorption observed in tissues exposed to 60 µM mucosal IGF-I. These results suggest that PI 3-kinase is involved in the IGF-I receptor signaling cascade that culminates in enhanced Na+ and Na+-coupled nutrient transport. This is consistent with the observation that wortmannin also abolished IGF-I-stimulated increases in Na+-K+-ATPase activity. Our choice of the 1 µM concentration of wortmannin was based on the study by Deachapunya et al. (7), who reported that this concentration abolished the stimulatory effects of insulin on Na+ transport in porcine endometrial cells, an effect that is mediated by enhanced activity of the Na+-K+-ATPase pump and is similar to the actions of IGF-I on those cells. Wortmannin at concentrations >= 100 nM has been reported to inhibit other enzymes besides PI 3-kinase (5); thus we cannot rule out the possibility that the effects of wortmannin on jejunal transport and Na+-K+-ATPase activity in our study may be mediated by inhibition of other enzymes in addition to PI 3-kinase. Nevertheless, the results presented here support a model in which activation of PI 3-kinase secondary to binding of IGF-I to its receptor leads to an increase in Na+-K+-ATPase activity. This results in enhanced Na+ and Na+-coupled nutrient absorption via a steepening of the Na+ electrochemical gradient into enterocytes. This mechanism likely accounts for the acute effects of IGF-I on jejunal ion and nutrient transport observed in Ussing chambers. However, it is possible that in piglets treated orally with IGF-I for 4 days an additional effect of the peptide on brush-border Na+ conductance, leading to a further enhancement of Na+-K+-ATPase pump activity, may contribute to the increase in jejunal ion and nutrient transport observed in vivo. For example, whereas short-term treatment of porcine endometrial cells with insulin had no effect on apical Na+ conductance, treatment of cells for 4 days led to a doubling of apical Na+ conductance and an additional increase in Na+-K+-ATPase activity (7).

In conclusion, our findings provide evidence that administration of oral IGF-I to neonatal piglets stimulates jejunal Na+-K+-ATPase activity through a mechanism that involves PI 3-kinase and results in enhanced rates of Na+ and Na+-coupled nutrient absorption. The potential use of oral IGF-I as a proabsorptive agent for neonates, particularly those with compromised mucosal function as in short bowel syndrome, deserves further study.


    ACKNOWLEDGEMENTS

We thank Nancy Sills and Yanira Oneill-Naumann for technical assistance and Dr. Tom Crenshaw for advice and assistance in piglet management.


    FOOTNOTES

This study was supported by US Department of Agriculture Grant 93-37206-9222 (to H. V. Carey), National Institute of Diabetes and Digestive and Kidney Diseases Grant F32-DK-09629 (to A. N. Alexander), and grants from the University of Wisconsin-Madison Graduate School and the University of Wisconsin-Madison School of Veterinary Medicine.

Address for reprint requests and other correspondence: H. V. Carey, Dept. of Comparative Biosciences, Univ. of Wisconsin, 2015 Linden Dr. West, Madison, WI 53706 (E-mail: careyh{at}svm.vetmed.wisc.edu).

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 12 June 2000; accepted in final form 8 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alexander, AN, and Carey HV. Oral IGF-I enhances nutrient and electrolyte absorption in neonatal piglet intestine. Am J Physiol Gastrointest Liver Physiol 277: G619-G625, 1999[Abstract/Free Full Text].

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5.   Cross, MJ, Stewart A, Hodgkin MN, Kerr DJ, and Wakelam MJ. Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem 270: 25352-25355, 1995[Abstract/Free Full Text].

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13.   Kessler, M, Acuto O, Storelli C, Murer H, Muller M, and Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membrane. Their use in investigating some properties of D-glucose and choline transport systems. Biochim Biophys Acta 506: 136-154, 1978[ISI][Medline].

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Am J Physiol Gastrointest Liver Physiol 280(2):G222-G228
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