Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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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 · kg1 · 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
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INTRODUCTION |
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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).
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MATERIALS AND METHODS |
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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 · kg1 · 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.
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 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.
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RESULTS |
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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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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 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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Fructose uptake.
Uptake of 25 mM fructose was similar between treatment groups
(0.74 ± 0.07 nM · min1 · 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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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 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 · kg1 · 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.
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ACKNOWLEDGEMENTS |
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We thank Nancy Sills and Yanira Oneill-Naumann for technical assistance and Dr. Tom Crenshaw for advice and assistance in piglet management.
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FOOTNOTES |
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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.
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