Insulin signal transduction in rat small intestine: role of
MAP kinases in expression of mucosal hydrolases
Soheila
Marandi1,
Nadine
De Keyser1,
Alain
Saliez2,
Anne-Sophie
Maernoudt1,
Etienne Marc
Sokal1,
Catherine
Stilmant1,
Mark H.
Rider3, and
Jean-Paul
Buts1
1 Laboratory of Pediatric Gastroenterology and Nutrition,
2 Experimental Surgery, and 3 Physiological
Biochemistry, Christian de Duve Institute of Cellular and Molecular
Pathology, Université Catholique de Louvain, 1200 Brussels,
Belgium
 |
ABSTRACT |
The
postreceptor events regulating the signal of insulin
downstream in rat intestinal cells have not yet been analyzed. Our objectives were to identify the nature of receptor substrates and
phosphorylated proteins involved in the signaling of insulin and to
investigate the mechanism(s) by which insulin enhances intestinal
hydrolases. In response to insulin, the following proteins were rapidly
phosphorylated on tyrosine residues: 1) insulin receptor substrates-1 (IRS-1), -2, and -4; 2) phospholipase
C-isoenzyme-
; 3) the Ras-GTPase-activating protein (GAP)
associated with Rho GAP and p62Src; 4) the
insulin receptor
-subunit; 5) the p85 subunits of
phosphatidylinositol 3-kinase (PI 3-kinase); 6) the Src
homology 2
-collagen protein; 7) protein kinase B;
8) mitogen-activated protein (MAP) kinase-1 and -2; and
9) growth receptor-bound protein-2. Compared with controls,
insulin enhanced the intestinal activity of MAP kinase-2 and protein
kinase B by two- and fivefold, respectively, but did not enhance p70/S6
ribosomal kinase. Administration of an antireceptor antibody or
MAP-kinase inhibitor PD-98059 but not a PI 3-kinase inhibitor
(wortmannin) to sucklings inhibited the effects of insulin on mucosal
mass and enzyme expression. We conclude that normal rat enterocytes
express all of the receptor substrates and mediators involved in
different insulin signaling pathways and that receptor binding
initiates a signal enhancing brush-border membrane hydrolase, which
appears to be regulated by the cascade of MAP kinases but not by PI
3-kinase.
insulin receptor substrates; phosphotyrosine proteins; signal
transduction
 |
INTRODUCTION |
ALTHOUGH THE INTESTINAL
MUCOSA is not a classic target tissue for insulin, accumulating
evidence (2, 3, 6-11) indicates that insulin
determines important physiological effects on intestinal growth, cell
maturation, and enzyme expression in several mammalian species. The
onset of weaning (day 14-17) in the suckling
rat is a critical period during which immature enterocytes exhibit elevated responsiveness to the hormone. At this time, plasma insulin levels rise markedly (3), whereas milk-borne insulin is
still active (10), allowing optimal interaction of the
hormone with intestinal insulin receptors (IR), which are located on
both endoluminal and basolateral membranes of the cell
(8). After weaning, the twofold decrease in IR
concentration is associated with a reduction in responsiveness of
mature enterocytes to the hormone (7, 8). Our recent
studies (9, 11) suggest that the premature induction of
sucrase-isomaltase (SI) is triggered by the binding of the hormone to
the extracellular
-receptor subunit, allowing autophosphorylation of
the tyrosine kinase intrinsic to the juxtamembrane and cytoplasmic
domains of the
-receptor subunit (9). Downstream transduction of the signal into the cell is associated with an increase
in endoluminal polyamine uptake and leads to the final activation of
the SI gene promoter with a dose-dependent accumulation of SI mRNA,
independent of the mitogenic effects of the hormone (11).
In response to IR activation, several receptor substrates [IR
substrate-1 (IRS-1), IRS-2, IRS-4, and Src homology 2
-collagen (Shc)] and tyrosine-phosphorylated proteins associating through Src
homology 2 domains (SH2 domains) have been purified and sequenced from
insulin target cells cultured in vitro (14, 18, 34, 35, 43, 46,
47, 52-54, 56, 57). However, the insulin signal
transduction in the rat small intestine has not been investigated so
far. In addition, the specific pathway by which insulin stimulates brush-border membrane (BBM) hydrolases remains unknown. Using the
intact live animal under conditions relevant to normal insulin responses, the objectives of our study were 1) to analyze
the nature of IRS and phosphorylated proteins involved in insulin signal transmission at the level of the small intestine and
2) and to approach the signal pathway activating BBM hydrolases.
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MATERIALS AND METHODS |
Reagents.
Phenylmethanesulfonic acid, dithiothreitol, HEPES, sodium salt, SDS
(highest purity grade),
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, Triton
X-100, leupeptin, pepstatin, aprotinin, EDTA, wortmannin, PD-98059, and
myelin basic protein were purchased from Sigma Chemical (Bornem,
Belgium). Human recombinant insulin (Actrapid HM) was from Nordisk
(Bagsvaerd, Denmark), and 125I-labeled protein A and
molecular weight standards (CFA 626) were from Amersham Laboratories
(Little Chalfont, UK). Polyvinylidene difluoride (PVDF) membranes were
obtained from Bio-Rad (Nazareth, Belgium), and protein A-Sepharose 4B
was from Pharmacia LKB Biotechnology. Polyclonal antibodies (PY20)
recognizing tyrosine-phosphorylated proteins were purchased from
Transduction Laboratories (Cambridge, UK). Specific polyclonal
antibodies recognizing IRS-1, IRS-2, phospholipase C-isoenzyme-
(PLC-
), Shc, GTPase-activating protein (GAP), protein tyrosine
phosphatase-2 (SYP), growth receptor-bound protein-2 (Grb2), the p85
regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), and
mitogen-activated protein (MAP) kinases were from Upstate Biotechnology
(Lake Placid, NY) and Santa Cruz Biotechnology (Santa Cruz, CA). Anti
IRS-4 polyclonal antibody was produced by repeated rabbit immunizations
with a peptide (31 amino acids) corresponding to the COOH-terminal
sequence of human IRS-4. RPN 538, a monoclonal antibody recognizing
epitopes of the
-extracellular subunit of the IR, was purchased from
Amersham (Brussels, Belgium). 2C4, a monoclonal antibody that
recognizes a 60-kDa receptor substrate associating with GAP in Chinese
hamster ovary cells (CHO) overexpressing IR (CHO-IR) (18)
and immunoprecipitates a docking protein (p62dok)
associated with Ras-GAP in v-abl-transformed murine precursor B cells
(54), was a generous gift of Dr. Richard Roth (Stanford University, Stanford, CA). The anti-protein kinase B (PKB) polyclonal antibody recognizing PKB-
and PKB-
isoforms was raised in rabbits against the COOH-terminal peptide (FPQFSYSASSTA) of rat PKB.
Animals.
All procedures were approved by the National and University Animal Care
Committees (Fonds de la Recherche Scientifique Médicale and
Université Catholique de Louvain, Brussels, Belgium). Litters of
Wistar rats, acclimatized to standard conditions of room temperature, light-dark cycles, and feeding schedules, were used. During the nursing
period, pups remained with their mothers in polystyrene cages and had
free access to milk. Suckling rats were killed at days
14-15 postpartum. Adult rats (180 g) were fed a pelleted diet
(AO3 UAR, Villemoisson-sur-Orge, France) ad libitum. To identify receptor substrates, animals were injected under ether anesthesia after
12-24 h of fasting. The femoral vein was exposed after a small
incision, and normal saline (0.9% NaCl) with (10
6 mol/l)
or without insulin was slowly injected (1 ml/min). After infusion, the
animal was killed and the small intestine was excised rapidly, trimmed
of fat and mesentery, and rinsed with ice-cold saline. After being
opened longitudinally, the mucosa was scraped on ice between glass
slides, weighed, and used for protein extraction or stored at
170°C
in liquid nitrogen.
Treatment schedules.
To approach the insulin signaling pathway that activates BBM
hydrolases, antibodies recognizing extracellular epitopes of the IR
(
-subunit) and key enzyme inhibitors (wortmannin and PD-98059) were
injected into suckling pups for 48-72 h. To equalize conditions of
nursing and feeding, dams were reduced to six pups per lactating mother. Human insulin (Actrapid MC, Novo Industries, Brussels, Belgium)
was injected intraperitoneally to sucklings from day 11 to
day 14 postpartum (7:30 AM and 7:30 PM) at a dose of 5 mU · g body wt
1 · day
1 in a
volume of 100 µl. Control groups were treated with equivalent volumes
of 0.9% saline. A monoclonal antibody recognizing epitopes of the
extracellular
-IR subunit (RPN 538, Amersham, Gent, Belgium) was
injected intraperitoneally following the same schedule as insulin at 20 µg protein/dose twice daily. To inhibit PI 3-kinase, wortmannin was
injected intraperitoneally at a dose of 5 µg · g body
wt
1 · day
1 1 h before the
administration of insulin (5 mU/g body wt) from day 11 to
day 14 postpartum. Control animals received insulin alone.
To inhibit MAP kinase kinase and MAP kinases, PD-98059 was injected
intraperitoneally into sucklings at a dose of 2 µg/g body wt twice
daily from day 12 to day 14 postpartum. Insulin
was injected 1 h after administration of the inhibitor at a dose
of 5 mU/g body wt. Control rats received insulin according to the same schedule.
Extraction of phosphotyrosine proteins.
We used the method of Rothenberg et al. (41) with slight
adaptations. Briefly, samples of intestinal mucosa were homogenized for
1 min in a solubilization buffer (1:5 vol/vol) maintained at 100°C in
a water bath (2 min) with an Ultraturax generator (Janke and Kunkel,
Staufen, Germany) operated at maximum speed. The solubilization buffer
was composed of 2% SDS, 100 mmol/l HEPES (pH 7.8 at 22°C), 10 mmol/l
EDTA, 100 mmol/l NaCl, and 50 mmol/l dithiothreitol. The homogenate was
heated further to boiling with gentle stirring for 2 min and then left
to cool to 22°C.
After centrifugation for 2 h at 18°C in a Beckman type 35 rotor ultracentrifuge (143,000 g), the supernatant was
acidified with 100% TCA, added slowly dropwise at 22°C with
vigorous stirring to a final concentration of 10%. The mixture
was then cooled at 4°C overnight. Under these conditions, protein and
nucleic acids form a copious, flocculent, pink precipitate, whereas SDS
remains largely soluble. The precipitate was collected by
centrifugation in a Sorvall RC-5B centrifuge at 4°C for 10 min. The
precipitate was washed once with 25 volumes of 10% TCA at 4°C, and
the TCA was then extracted by six washes, each with 25 volumes of
ethanol and diethyl ether (1:1 vol/vol) at 4°C. The precipitate was
dried in vacuo (Speed Vac) for 12-18 h and pulverized thoroughly
to a fine powder. In this form the extracted proteins could be stored for at least 1 year at
170°C without apparent degradation or significant loss of phosphotyrosine content.
Immunoprecipitation.
For immunoprecipitation of proteins, 0.05 g of dry tissue powder
(±15 mg proteins) was dissolved in 1 ml 0.1 N NaOH with vigorous stirring at 22°C for 5 min. The resulting solution was then
neutralized rapidly to pH 8 with 2 volumes of 100 mmol/l
Tris · HCl, 1 mmol/l EDTA, and a cocktail of protease
inhibitors including 1 mmol/l phenylmethanesulfonic acid, 1 µg/ml
leupeptin, and 1 µg/ml aprotinin. The slightly turbid solution was
clarified with a 0.45-µm pore diameter cellulose/polyvinyl chloride
filter (Millex-HA, Millipore). Protein concentration was measured using
the Lowry assay (31). Poly- or monoclonal antibodies were
added to a final concentration of 1-4 µg IgG/ml and incubated at
4°C for 4 h. Immune complexes were then absorbed to protein
A-Sepharose 4B beads (25 µl of a 50% bead slurry/ml of extract) for
12 h at 4°C with gentle rotation. The immune complexes were
washed twice by resuspension and brief centrifugation in 1 ml of wash
buffer containing 1% Triton X-100, 0.1% SDS, 100 mmol/l NaCl, and 50 mmol/l Tris, pH 7.3, at 22°C and once more in the same buffer lacking
NaCl. After aspirating the excess wash buffer, the immunoprecipitated
proteins were solubilized in 30-50 µl of Laemmli's buffer
(23) and boiled at 100°C for 5 min before being layered
onto gel slots.
Electrophoresis and immunoblotting.
Immunoprecipitated proteins were separated by SDS-PAGE in 7.5%
polyacrylamide gels as described previously (8, 9).
Electrotransfer of proteins to PVDF membranes was performed for 90 min
at 50 V as described by Towbin et al. (50).
Nonspecific protein binding was reduced by preincubating the membrane
overnight at 4°C in blocking buffer containing 5% BSA and 1%
ovalbumin in TN buffer (10 mmol/l Tris, pH 7.2, and 0.9% NaCl). The
membrane was then incubated with appropriate antibodies diluted in
blocking buffer (0.5-2 µg/ml) for 2 h at 22°C and washed
twice for 10 min in TNT buffer (TN buffer plus 0.1% Tween), once for
10 min in TN buffer containing 0.05% Nonidet P-40, and twice for 10 min each in TNT buffer. The blots were then incubated with 50 µCi of
125I-labeled protein A (Amersham) in 10 ml of blocking
buffer for 1 h at 22°C and then washed again as described above.
Bound antiphosphotyrosine or specific antibodies were detected by
autoradiography using 24 × 30 cm Fuji film (St. Nicolas, Belgium)
at
70°C for 72 h as described previously (8, 9).
Band intensities were quantified by optical densitometry using an image
densitometer (CS-690, Bio-Rad). Relative intensity was expressed in
arbitrary optical density units (OD units), whereas relative abundance
was expressed in arbitrary volume units (OD × mm2).
Enzyme assays.
To measure the intestinal activity of PKB, MAP kinase-2, and p70/S6
kinase, mucosal homogenates (1:3 vol/vol) were prepared from
insulin-treated rats and controls under nondenaturating conditions. Enzymes were immunoprecipitated with the corresponding specific antibodies bound to protein A-Sepharose 4B beads in a buffer containing a cocktail of antiproteases. Enzyme activities were determined by the
phosphorylation of three different peptides used as substrates in the
presence of [
-32P]ATP-Mg (sp act >5,000 Ci/mmol,
Amersham). For MAP kinase-2, the substrate was the myelin basic protein
(phosphorylated on serine/threonine residues), for PKB, it was a
synthetic peptide called MR-15 (RPRAATF), and for p70/S6 kinase, it was
the peptide MR-4 (RRLSSLRA). Blanks were handled exactly like samples,
except that the immunoprecipitated enzyme was omitted from the
reaction. The activities were linear for up to 20 min of incubation
time (30°C) and were proportional to the amount of enzyme used in the reaction. Protein kinase activity was expressed in picomoles of substrate phosphorylated per minute per gram of mucosa.
Sucrase, lactase, and maltase activities were assayed in BBM samples by
standard methods (9, 11). Activities were expressed as
micromoles of substrate hydrolyzed per minute per milligram of BBM protein.
Calculations and statistics.
All data are given as means ± SD except enzyme activities, which
are expressed as means ± SE. If not indicated, SD represents <10% of the mean. Differences between controls and insulin-treated animals were tested for statistical significance (P < 0.05) using the nonparametric Mann-Whitney U-test.
 |
RESULTS |
Identification of receptor substrates and insulin-elicited
phosphotyrosine proteins in intestinal mucosal extracts.
To identify IRS and phosphotyrosine proteins, we used the method of
Rothenberg et al. (41) with minor modifications. Because phosphotyrosine proteins are susceptible to rapid phosphatase-mediated dephosphorylation both in vivo (24) and during extraction
procedures (21), we rapidly homogenized intestinal mucosa
from insulin-treated and control rats at 100°C in buffer containing
2% SDS and 50 mmol/l dithiothreitol. The final precipitate yielded
0.1 ± 0.06 g powder/g wet intestinal mucosa
(n = 29) and 0.57 ± 0.09 g protein/g dry powder. We found no difference between the amount of protein extracted from the intestinal mucosa of insulin-treated rats and controls. In
response to intravenous infusion of insulin (10
6 mol/l
for 3 min), direct probing of mucosal extracts with antiphosphotyrosine antibodies (PY20) revealed that nine proteins (p165, p130, p120, p94,
p85, p70, p60, p46, and p42) were rapidly phosphorylated on tyrosine
sites (Fig. 1, left). In
control animals, most of these proteins were undetected, whereas two
(p130 and p60) showed only a weak signal. Densitometric measurements of
each signal revealed that the following seven proteins had emerged by
their signal intensity and relative abundance (in OD units × mm2): p165, p130, p120, p94, p85, p70, and p60 (Fig.
2A). After immunoprecipitation of protein extracts with antiphosphotyrosine antibodies (PY20) and
Western blotting the membrane with the same antibody, p165 and p94 were
detected again as single protein bands in insulin-stimulated rats
without corresponding signal in control rats (Fig. 1,
right). These proteins are most likely IRS-1 and IR
-subunit proteins. The same autoradiography shows a major complex of
several proteins ranging from ~55 to 68 kDa, which were detected in
both insulin-treated and control rats without difference in relative
abundance, indicating the presence of phosphotyrosine proteins
insensitive to insulin and likely the heavy chain of the antibody at 55 kDa. Because we (8) have recently identified in BBM of
suckling rats a 60-kDa protein as a direct substrate of the IR, mucosal
extracts from insulin-treated rats were immunoprecipitated with the
same anti-p60 antibody (clone 2C4). This procedure failed to reveal
this substrate in adult rat mucosal intestinal extracts (Fig. 1,
right). To determine the nature of the p165 phosphotyrosine
protein, protein extracts were immunoprecipitated with an anti-IRS-1
polyclonal antibody raised against the COOH-terminal peptide of rat
liver IRS-1 (YASINFQKQPEDRQ) (46). After
electrotransfer, PVDF membranes were probed with antiphosphotyrosine
antibodies (PY20). Assays performed in adult rats after 3 min of
insulin stimulation revealed a very weak signal (data not shown).
Because the concentration of IR is higher in the small intestine of
weanling rats than in adult rats (8) and the time of IRS-1
phosphorylation by the IR is very short (41), the
experiment was repeated in weanling rats (day 23) stimulated
for 1 min with insulin (10
6 mol/l). Figure
3, left, shows the detection
in insulin-stimulated rats of two proteins migrating at ~185 and 165 kDa, respectively, whose signals were nearly absent in control rats,
confirming that p165 is a phosphotyrosine protein corresponding to
IRS-1 (41, 46). Because rat IRS-1 and mouse IRS-2 share up
to 40% homology with 455 positive identities on 1,117 amino acid
residues (46, 47) and the anti-rat IRS-1 polyclonal
antibody used recognizes several motifs of the COOH-terminal domain of
IRS-2 (according to the BLAST online search service of the National
Center for Biotechnology Information), it is likely that the antibody
coimmunoprecipitated both IRS-1 and IRS-2 and that the upper signal at
185 kDa corresponded to IRS-2, the reported molecular mass of IRS-2
(47). This was confirmed by immunoprecipitating protein
extracts with an anti-IRS-2 antibody raised against a large fragment of
the COOH-terminal domain of mouse IRS-2 (amino acids 976-1094)
(47). As shown in Fig. 3, right, after 1 min of
insulin stimulation anti-IRS-2 antibodies coimmunoprecipitated the two
IRS (IRS-2 at ~185 kDa and IRS-1 at ~165 kDa), which were not
tyrosine phosphorylated in control animals. Interestingly, IRS-1 and
IRS-2 coimmunoprecipitated with another phosphotyrosine protein
migrating at ~130 kDa, which was not detected in controls. Because
experiments were conducted under strong denaturating conditions, it is
probable that p130 was detected as a protein directly associated with
IRS-1 or IRS-2. Other members of the IRS family (i.e., IRS-4 and IRS-3)
were unlikely candidates because of their molecular mass and relative
mobility (25, 26). Indeed, IRS-3 migrates at 60 kDa
(26) and IRS-4 at 160 kDa in SDS-PAGE (25).
Figure 4, left, clearly shows
the expression of IRS-4, immunoprecipitated with a specific anti IRS-4 antibody from weanling intestinal extracts, as a single
insulin-elicited phosphotyrosine protein migrating at ~160 kDa that
was not phosphorylated in control rats. A likely candidate
associating with IRS-2 in response to insulin could be PLC-
.
Although PLC-
was identified in intestinal protein extracts of
weanling rats as a ~145-kDa phosphotyrosine protein (Fig. 4,
middle), immunoprecipitation of IRS-2 followed by
blotting membranes with anti-PLC-
antibodies failed to confirm
that the p130 associated with IRS-2 was PLC-
(data not
shown). Intestinal cells of weanling rats also expressed PKB, an
important downstream substrate of PI 3-kinase, which binds to
Glut-4-containing vesicles and is believed to activate p70/S6 kinase.
The insulin dependence of PKB, a serine/threonine-phosphorylated kinase of 60 kDa, is shown in Fig. 4, right (relative
abundance for insulin-treated and control rats was 6.29 ± 0.19 and 3.08 ± 0.10 OD units × mm2,
respectively).

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Fig. 1.
Left: autoradiography of phosphotyrosine
proteins detected in intestinal mucosal protein extracts of adult rats
after electrophoretic separation (7.5% SDS-PAGE) and Western blot with
antiphosphotyrosine antibodies (PY20) (see MATERIALS AND
METHODS). After 3 min of intravenous infusion of insulin
(10 6 mol/l), 9 protein bands ranging from ~ 42 to
165 kDa were detected in insulin-stimulated animals (INS+, lane
2), whereas in controls (INS , lane 1) these proteins
were either not or very weakly tyrosine phosphorylated.
Right: mucosal protein extracts from insulin-stimulated
animals (lane 4) or controls (lane 5) were
immunoprecipitated with antiphosphotyrosine antibodies (PY20), and
after electrotransfer, membranes were blotted with the same antibodies,
as described in MATERIALS AND METHODS. Two major
phosphotyrosine proteins of 165 [presumably insulin receptor
substrate-1 (IRS-1)] and 94 kDa (presumably the -subunit of the IR)
were phosphorylated in response to insulin (lane 4) but were
nearly undetected in controls (lane 5). Note a broad band of
several phosphotyrosine proteins (ranging between 55 and 68 kDa) and
most likely the heavy chain of the antibody seen in both
insulin-stimulated (lane 4) and control animals (lane
5). Immunoprecipitation of mucosal extracts with anti-p60 (2C4)
antibodies did not detect p60 phosphotyrosine protein in
insulin-stimulated rats (lane 3).
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Fig. 2.
A: optodensitometric measurements of insulin-elicited
phosphotyrosine proteins detected by Western blot using
antiphosphotyrosine antibodies. (Fig. 1, left). Results are
expressed in relative abundance [arbitrary optical density (OD)
units × mm2]. I, insulin stimulated; C, controls.
B: relative abundance of the p85 subunit of
phosphatidylinositol 3-kinase (PI 3-kinase) and of the p68-associated
protein in mucosal protein extracts of insulin-treated rats and
controls. The autoradiography showing the expression of these
immunoprecipitated proteins is depicted in Fig. 5, top left.
C: relative abundance of the 3 subunits (p66, p52, and p46)
of Src homology 2 -collagen (Shc) in mucosal protein extracts of
insulin-treated rats and controls. The autoradiography showing the
expression of the Shc subunits is depicted in Fig. 5, top
right.
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Fig. 3.
Left: autoradiography of IRS-1 (p165) and a
185-kDa associated protein (presumably IRS-2) in mucosal protein
extracts from weanling rats, treated with insulin intravenously
(10 6 mol/l) for 1 min. After immunoprecipitation with
anti-IRS-1 antibodies, these proteins were detected by Western blot,
using antiphosphotyrosine antibodies (PY20). Both phosphotyrosine
substrates were nearly undetectable in control animals.
Right: autoradiography of IRS-2 (p185) and a 165-kDa
associated protein (presumably IRS-1) in mucosal protein extracts from
weanling rats, treated with insulin intravenously (10 6
mol/l for 1 min). After immunoprecipitation with anti-IRS-2 antibodies,
these proteins were detected by Western blot using antiphosphotyrosine
antibodies (PY20). Both substrates were not detected in control
animals. Note the presence in insulin-treated-animals of a ~130-kDa
associated protein whose nature remains unknown.
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Fig. 4.
Expression of IRS-4, phospholipase C-isoenzyme-
(PLC- ), and protein kinase B (PKB) in protein extracts from weanling
rats (day 25), treated with insulin (10 6 mol/l
iv for 3 min) or its vehicle. Left: intestinal proteins were
immunoprecipitated (IP) with an anti-IRS-4 polyclonal antibody, and
after electrotransfer, samples were immunoblotted (Western blot, WB)
with antiphosphotyrosine antibodies (PY20). IRS-4 was detected by
autoradiography as a single phosphotyrosine substrate of ~160 kDa in
insulin-treated rats but not in controls. Middle: PLC-
was detected by the same method as a 145-kDa phosphotyrosine protein,
present only in insulin-treated rats. Right: PKB was
evidenced in insulin-treated rats as a single 60-kDa protein using a
polyclonal anti-PKB antibody for both the immunoprecipitation and
immunoblotting procedures (dilution 1:10,000). HC, heavy chain of the
antibody.
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In concordance, Table 1 shows that in
response to insulin, the activity of PKB measured in immunoprecipitated
samples was enhanced five times over the activity measured in control
rats. Interestingly, the basal activity of p70/S6 kinase, although
present, was found to be unresponsive to insulin in the small
intestine.
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Table 1.
Activity of protein kinases in immunoprecipitated mucosal samples
from insulin-treated rats and controls
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The 85-kDa phosphotyrosine protein detected by probing protein extracts
with antiphosphotyrosine antibodies (Fig. 1, left) was
identified as the p85 subunit of PI 3-kinase, a heterodimer containing
a regulatory subunit (p85) and a catalytic subunit (p110). Using a
polyclonal antibody recognizing a large sequence (corresponding to
amino acids 333-428 of the NH2-terminal SH2 domain) of
human p85 subunit of PI 3-kinase, two isoforms, the
- and
-p85
subunits, were identified in the small intestine (Fig.
5, top left). The
predominant isoform was
-p85. This adaptor molecule associates in
other cell lines with the IR at tyrosine 1322 (44) and
activates tyrosine kinase substrates (IRS-1, Grb2, SYP, and IRS-3)
through SH2 domains and thus serves as a link between PI 3-kinase and
other ligand-activated substrates (27, 35, 44, 55). The
-p85, a protein whose function remains unknown, was much less
abundant. The same autoradiography shows a broad and intense signal
extending from 50 to 55 kDa detected in both controls and
insulin-treated rats. This suggests that, in addition to
-p85, two
other isoforms, p55 and p50, previously evidenced in rat brain, muscle,
and liver (19), are also expressed in the small intestine,
especially because our antibody was raised against the entire
NH2-SH2 domain of
-p85, which is common to the three
isoforms
-p85,
-p55, and
-p50 (19).
Figure 5, top left, also demonstrates a ~68-kDa protein
that coimmunoprecipitated with the
- and
-p85, p55, and p50
isoforms of PI 3-kinase. Relative abundance of this protein (Fig.
2B) was greater in insulin-stimulated rats (3.84 ± 0.07 OD units × mm2) than in controls (2.43 ± 0.06 OD units × mm2). Its relative molecular mass was
estimated by imaging densitometry to be 68 kDa in controls and 70 kDa
in stimulated rats, the decrease in mobility likely being due to
phosphorylation of residues in response to insulin. Attempts to
identify the nature of this protein by probing immunoprecipitated p85
material with several antibodies against SH2 domain containing proteins
remained unsuccessful. A potential candidate could be SYP (68 kDa),
which contains two NH2-terminal SH2 domains interacting
directly with the SH2 domain of p85 via its motif
EY380GVM even under denaturating conditions
(29, 55, 57).

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Fig. 5.
Top left: autoradiography showing
the expression of the PI 3-kinase subunits ( - and -p85,
p50-55) in mucosal protein extracts from adult rats treated with
insulin (10 6 mol/l for 3 min) or its vehicle. The p55 and
p50 subunits form a large complex that was not separated by the
electrophoretic conditions. Protein extracts were immunoprecipitated
with a polyclonal antibody raised against the p85 subunit of PI
3-kinase, and the immunoprecipitated proteins were detected with the
same antibody in Western blot. Note that the PI 3-kinase subunits
coimmunoprecipitated with a 68-kDa protein, whose abundance was
increased in insulin-treated rats. Although the nature of this protein
is unknown, it could presumably be the protein tyrosine phosphatase-2.
Top right: expression of the 3 subunits (p66, p52, p46) of
Shc in mucosal extracts prepared from insulin-treated rats
(10 6 mol/l for 3 min) and controls immunoprecipitated
with anti-Shc antibodies. Note that abundance of the p46 subunit is
enhanced in insulin-treated rats. Immunoprecipitated proteins were
detected with the same antibody in Western blot. Bottom
middle: expression of the growth receptor-bound protein-2 (Grb2,
p24) in intestinal extracts from rats treated with insulin
(10 6 mol/l for 3 min) or its vehicle. Grb2 was probed
directly by anti-Grb2 antibodies on polyvinylidene difluoride
membranes. The protein was clearly present in insulin-treated rats but
not in controls. LC, the end of the gel.
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Intestinal extracts from adult rats stimulated with insulin
(10
6 mol/l for 3 min) and controls were
immunoprecipitated and Western blotted with a polyclonal antibody
reacting with a COOH-terminal peptide of the human Shc protein (amino
acids 359-473 of the SH2 domain). This allowed the detection of
three Shc proteins of ~66, 52, and 46 kDa, respectively (Fig. 5,
top right). Densitometric measurements (Fig. 2C)
showed that p46 was increased in relative abundance in response to
insulin (insulin vs. controls, 3.16 ± 0.08 vs. 2.15 ± 0.03 OD units × mm2, respectively), whereas p66 and p52
were equally abundant in insulin-treated and control rats, which could
be explained by the presence of the heavy chain of the antibody.
The Shc family of proteins are believed to be important substrates of
growth factor receptors, including the IR, and associate with Grb2/sem
5 gene products that link receptor tyrosine kinases and p21 Ras,
suggesting a role for Shc in the Ras-GAP pathway (17, 20).
We also identified in intestinal extracts of insulin-treated rats Grb2
as a ~24-kDa tyrosine phosphorylated protein, which was undetectable
in control rats (Fig. 5, bottom middle). Grb2 is a small
cytoplasmic protein containing two SH3 domains and one SH2 domain
(52), which interacts in response to IR activation with
mammalian son of sevenless (mSOS), IRS-1, and Shc via its SH2 domain
(17, 36). Like in target cell lines stimulated in vitro
with insulin or with other growth factors (epidermal growth factor,
platelet-derived growth factor), we have detected in intestinal
extracts from weanling rats (day 23) a ~120-kDa protein
identified as GAP (the mean GTPase-activating protein of the normal
form of p21 Ras). In animals injected with insulin (10
6
mol/l, 1 min) immunoprecipitation and Western blotting of protein extracts with an anti-GAP polyclonal antibody, reacting with a large
protein fragment of human GAP (corresponding to amino acids 171-443, which include 2 adjacent SH2 domains and 1 SH3 domain), revealed a major signal of ~120 kDa (p120 GAP) and two
phosphotyrosine proteins of ~190 (Rho GAP) and ~62 kDa
(p62Src), respectively (Fig.
6, left). The p120 GAP was
also present in control rats (relative abundance: 4.07 ± 0.20 OD
units × mm2), but its abundance was stimulated
threefold (n = 4) in insulin-treated rats (11.59 ± 0.31 OD units × mm2). Interestingly, p190 Rho-GAP
and p62Src protein were not detected in controls but
coimmunoprecipitated with p120-GAP only in response to insulin,
indicating that their interactions with p120 GAP are physiologically
relevant and likely representing a direct binding between these
proteins. Likewise, in cell lines stimulated in vitro with growth
factors, Ras-GAP is phophorylated on both tyrosine and serine residues
and forms complexes with two phosphorylated proteins, p190 Rho GAP and
p62Src. These associations inhibit the GTPase activity of
Ras-GAP (44, 55).

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|
Fig. 6.
Left: mucosal protein extracts of weanling
rats (day 25) untreated or treated with insulin
(10 6 mol/l, 3 min) were immunoprecipitated with
anti-GTPase-activating protein (GAP) antibodies. The immunoprecipitated
proteins were detected by Western blot using the same antibody. The
resultant autoradiography shows 3 proteins, p190 (Rho-GAP), p120 (GAP),
and p62 GAP-associated protein, whose abundance was markedly enhanced
in insulin-stimulated animals. In controls the relative abundance of
GAP (p120) was decreased while Rho-GAP and
p62Src-GAP-associated protein were nearly absent. Because
of a slight deviation in the run, the figure of the autoradiography has
been cut between 62 and 120 kDa to perfectly align the signals in the
lanes of insulin-treated rats and controls. Right:
expression of mitogen-activated protein kinases (MAP kinases) in
mucosal extracts of adult rats treated with insulin (10 6
M for 3 min) or with its vehicle. MAP kinase-1 (p44) and -2 (p42) were
immunoprecipitated using a polyclonal anti-MAP kinase-1 antibody and
detected by Western blot using antiphosphotyrosine antibodies (PY20).
Both kinases were not detected in control animals.
|
|
MAP kinases [or extracellular signal-activated kinases (ERK)] are
cytoplasmic and nuclear protein kinases acting as intermediates between
the insulin-stimulated phosphorylated cascade (Ras-GAP pathway) and the response of eukaryotic cells to extracellular signals (32). To identify MAP kinase-1 and -2 in rat small
intestine, we immunoprecipitated protein extracts from adult rats
stimulated with insulin (10
6 mol/l for 3 min) or saline
because MAP kinase expression increases during development
(4). The polyclonal antibody used was raised against a
36-amino-acid peptide (PFTFDMELDDLPKSERLKELIFQETARFQPGAPEAP) corresponding to the COOH terminus of rat MAP kinase-1. As shown in
Fig. 6, right, both MAP kinase-1 (p44) and MAP kinase-2
(p42) were tyrosine phosphorylated in response to insulin and were not detected in control rats. The antibody used also recognized MAP kinase-2 because the rat immunogenic sequence of MAP kinase-1 has 90%
identity with mouse or human MAP kinase-2 (BLAST search). The
corresponding signals shown in Fig. 6, right, are not
abundant after probing the immunoprecipitates with antiphosphotyrosine antibodies (PY20), because these proteins are serine/threonine kinases
(4, 32) and are phosphorylated in response to insulin only
on one tyrosine site (Tyr205) and one threonine site
(Thr203) (4, 32). In concordance, the activity
of MAP kinase-2 measured in immunoprecipitated samples was two times
higher (P < 0.01, n = 7) in
insulin-treated rats than in controls (n = 5) (Table 1).
Response of rat immature intestinal mucosa to in vivo
administration of inhibitors.
The effects of the IP administration of a monoclonal anti-IR antibody
(RPN 538) recognizing epitopes of the
-extracellular subunit on the
specific activity of BBM hydrolases are detailed in Table
2. Sucklings treated with two doses of 20 µg/day of the antibody from day 11 to day 14 postpartum had growth rates similar to saline controls. However,
mucosal weight expressed per centimeter of gut length was significantly
lower in the monoclonal-treated group with depression by day
14 of the specific activity of SI and maltase. There was no change
in the activity of lactase between the two groups. Corticosteronemia
was equivalent between the three groups of rats (mean: 1.34 µg/dl,
saline controls; 1.54 µg/dl, anti-IR treated group; and 1.39 µg/dl,
insulin-treated group). These hormone levels are negligible compared
with the active circulating levels measured in 18-day-old weanling rats
(17.6 µg/dl) (7, 9). The identification in rat small
intestine of the regulatory subunits (
-p85,
-p85,
-p55, and
-p50) of PI 3-kinase by immunoprecipitation and Western blot
prompted us to determine whether this key enzyme could regulate a
specific pathway stimulating BBM enzymes. Wortmannin, an irreversible
inhibitor of PI 3-kinase, was administered at low doses of 5 µg/g
body wt (median lethal dose = 500 µg/g) to sucklings for 72 h,
1 h before the administration of insulin. The results are
presented in Table 3. Growth rates were
similar in the wortmannin-treated group and controls. There was no
change in BBM protein concentration or in mucosal mass expressed per unit of length. Surprisingly, compared with insulin-treated controls, wortmannin enhanced SI activity fivefold, maltase activity 2.7-fold, and lactase activity 1.7-fold. Measurements of plasma insulin levels
revealed a marked increase in insulinemia in the wortmannin-treated group (wortmannin vs. controls, 67 ± 0.5 vs. 10 ± 0.1 µU/ml, respectively).
Because MAP-kinases I and II are known to be critical enzymes
regulating the mitogenic effects of insulin, PD-98059, a specific inhibitor of MAP kinase kinase, was administered intraperitoneally to
sucklings at low doses (2 µg/g body wt twice daily) (median lethal
dose = 200 µg/g) for 48 h, 1 h before the administration of
insulin. As detailed in Table 4,
sucklings treated with PD-98059 and insulin gained weight as did the
control group without change in final intestinal length. However,
mucosal mass expressed per centimeter of length was significantly lower
in the experimental group compared with the control group. Sucrase
activity was 3.7 times lower in the experimental group than in the
insulin-treated group, suggesting that PD-98059, by inhibiting MAP
kinases, prevented the ontogenic induction of the enzyme. In
concordance, maltase and lactase activities decreased 42% and
44%, respectively, compared with the activities measured in controls.
 |
DISCUSSION |
Our studies had two complementary goals: first to identify in the
intestinal cells of intact living animals, under physiological conditions, the receptor substrates and phosphorylated proteins involved in the signal transmission of insulin and second to approach the signal pathway activating BBM hydrolases. To accomplish these objectives, we adapted to the small intestinal mucosa the method of
Rothenberg et al. (41) used to extract phosphotyrosine
proteins from rat liver. Our results demonstrate that the SDS
denaturation method allows a direct assessment in vivo of
insulin-elicited tyrosine phosphorylation of endogenous substrates of
physiological significance. There is so far no information regarding
the mechanism(s) by which the signal of insulin is transduced from the
IR downstream into the intestinal cell. The main reason is probably
that intestinal cells are not considered to be typical target cells for
insulin, although the hormone is essential for intestinal growth and
cell maturation. Using the denaturating method of protein extraction, coupled with immunoprecipitation and immunoblotting proteins with antiphosphotyrosine antibodies, two proteins migrating at ~165 and 94 kDa (corresponding to IRS-1 and the
-subunit of the IR) were
tyrosine phosphorylated in the small intestine of insulin-treated rats
but not in controls (Fig. 1, right). A major complex of
several phosphotyrosine proteins ranging from ~55 to 68 kDa was also
immunoprecipitated and detected without difference between
insulin-stimulated and control rats. However, when immunoblots of
protein extracts were probed directly with antiphosphotyrosine
antibodies (PY20), we detected in insulin-treated rats at least nine
individual phosphotyrosine proteins, ranging from ~165 to 42 kDa,
whose signals were either nearly absent or very weak in controls (Fig.
1, left). These findings suggest that during the
immunoprecipitation process, a majority of phosphotyrosine proteins
escape detection because of extremely low abundance (i.e.,
p62Src-GAP associated protein) or rapid dephosphorylation
(i.e., IRS-2) or weak binding to antiphosphotyrosine antibodies (i.e.,
MAP kinases). Specific immunoprecipitations of protein extracts allowed
the detection of both IRS-1 (p165) and IRS-2 (p185) in intestinal cells
of insulin-stimulated rats. As shown in Fig. 1, IRS-1 remained phosphorylated after 3 min of insulin infusion, a time at which IRS-2
was already dephosphorylated. However, after 1 min of insulin infusion
both IRS-1 and IRS-2 were phosphorylated (Fig. 3), indicating that
IRS-2 is dephosphorylated more rapidly than IRS-1. A similar observation has been published by Ogihara et al. (39), who
found in skeletal muscle cells that IRS-2 was dephosphorylated after 3 min of insulin stimulation, whereas IRS-1 remained phosphorylated for
60 min, most likely because of differences in the associations of these
substrates with PI 3-kinase (39). This finding has raised
the speculation that IRS-1 would transmit continuous signals from the
IR, whereas IRS-2 would mediate transient signals activating PI
3-kinase more transiently.
After insulin binds to the
-subunit (extramembranous) of its
receptor, the
-subunit becomes autophosphorylated and rapidly phosphorylates Shc and IRS-1 on multiple tyrosine residues. IRS-1 in
turn recognizes and binds directly to SH2 domain-containing proteins
(SH2 proteins), including the regulatory subunits of PI 3-kinase,
-p85 (35),
-p55 (40),
-p50
(19), Grb2 (30), SYP or SHPTP-2
(22), and Nck (28). Consequently, IRS-1
mediates activation of PI 3-kinase, PKB, Grb2, mSOS, p21 Ras-GAP, the
MAP kinase cascade, and finally the nuclear translocation of a family of cytoplasmic transcription factors called STATS (for signal transducer and activator of transcription) (43). This
cascade of activations results in the promotion of glucose uptake,
glycogen synthesis, mitogenis, or gene expression according to the
specificity of the target tissue. In the present study, most of these
receptor substrates, SH2 proteins, and phosphotyrosine and
serine/threonine kinases have been identified in the small intestine of
intact animals in response to insulin stimulation. In addition, the
activity of MAP kinase-2 and PKB, two key enzymes regulating different pathways of the insulin signal, was enhanced in response to insulin.
Studies (1, 49) using IRS-1-deficient mice derived from
targeted gene disruption have demonstrated that IRS-2 functions as an
alternative substrate for the IR and can activate PI 3-kinase (47). Furthermore, two other IRS candidates have been
sequenced: IRS-3 in adipocytes (26) and IRS-4 in human
embryonic kidney cells (25). In the present study, IRS-4
was also detected in rat intestinal cells. In contrast to IRS-2, IRS-4
was still phosphorylated after 3 min of insulin stimulation. The
aligned structure of all of the members of the IRS family is relatively
similar and includes from the NH2 terminus a highly
conserved pleckstrin homology domain (PH domain), followed by a highly
conserved protein tyrosine-binding domain (PTB domain), a non-PTB
domain referred to as the SAIN domain (IRS-1 and IRS-2), and a second
COOH-terminal domain (IRS-2) containing multiple tyrosine
phosphorylation sites that can bind to various SH2 proteins (13,
25). Although the PH and PTB domains are highly conserved in all
IRS, there is little homology between the COOH-terminal domains in
IRS-1 and IRS-2 and the corresponding regions in IRS-3 and in IRS-4
(25, 26). Binding specificity to SH2 proteins is
determined by the amino acid sequence motif around the phosphotyrosine
residue (52, 57). For instance, upstream both the non-SH2
PTB domains of IRS-1 and IRS-2 interact with phosphorylated NPXY motifs
in the receptors for insulin, IGF-1, and interleukin-4 (12,
16), whereas downstream IRS-1 interacts with at least four sites
of the SH2 domains of
-p85: Y608MPM,
Y939MPM, Y987MTM, and Y460ICM
(45). PI 3-kinase plays a pivotal role in signal
transduction. In this study in the rat small intestine, we have
detected four isoforms of the regulatory subunits of PI 3-kinase:
-
and
-p85, p55, and p50 (fusioned). Similar findings have been
published by Inukai et al. (19) in the rat liver.
Interestingly, p50 exhibited a markedly higher capacity for activation
of associated PI 3-kinase via insulin stimulation and had a higher
affinity for tyrosine-phosphorylated IRS-1 than the other isoforms
(19). Each isoform has a different tissue distribution and
may have specific functions in various tissues. The regulatory subunits
(p85) of PI 3-kinase activate the serine/threonine kinase PKB
(5), which in turn activates in some tissues p70/S6
ribosomal kinase (51), an enzyme critical for cell cycle
progression through G1. In the present study, intestinal PKB activity was markedly enhanced by insulin, whereas the basal activity of p70/S6 kinase remained unresponsive to the hormone.
Besides the phosphorylation of IRS-1 and IRS-2, activation of the IR
phosphorylates another cellular substrate, Shc. As shown in Fig. 4, Shc
is expressed in the rat small intestine as three proteins, p46, p52,
and p66, which differed in relative abundance. Each Shc protein
contains a NH2-terminal PTB domain, a central glycine/proline rich sequence, and a COOH-terminal SH2 domain (17, 20, 56). The three Shc proteins are phosphorylated by
activated growth factor receptors (e.g., insulin, EGF, IGF-1) and
oncogen tyrosine kinases to form complexes with Grb2. We also clearly
demonstrated in intestinal protein extracts of insulin-stimulated rats
an increase in abundance of the p120-Ras-GAP and the presence of two
associated proteins, p190 (Rho GAP) and p62Src. Rho GAP is
a phosphotyrosine protein tightly bound to p120 GAP in nearly
stoichiometric amounts. The virtual absence of Rho-GAP and
p62Src in intestinal extracts from control rats indicates
that the associations between these molecules and p120 GAP likely
represent a direct binding. This is further attested to by the fact
that Rho GAP exhibits three domains that share strong homology with
GTP-binding proteins, (Rho) GAP-like molecules, and the putative
glucocorticoid gene repressor (42). When the cell is
stimulated, Rho-GAP appears to be an effector acting via GAP to
transduce signals from p21 Ras to the nucleus, because ~25% of the
immunoprecipitated p190 is detected in the nuclear compartment
(42). The p62Src GAP-associated protein could
be another effector of the p21 Ras pathway, because it contains DNA-
and RNA-binding domains and exhibits similarities to nuclear
ribonucleoproteins (38, 53).
The mechanism(s) by which insulin stimulates enzyme expression in rat
immature enterocytes has been little investigated. Besides receptor
activation, a direct effect of insulin on target cells, unrelated to
the receptor status, has been postulated. Experimental evidence
(33) has shown that insulin can exert mitogenic effects in
Xenopus laevis oocytes by direct contact with the nuclei in the absence of membrane receptors. These observations prompted us to
clarify whether IR binding is necessary for inducing BBM enzyme
expression. The monoclonal antibody used here recognizes extracellular
epitopes of the
-subunit of the human IR, whose molecular structure
closely resembles that of the rat. When administered at low doses to
suckling pups, anti-IR antibodies clearly prevented the action of
endogenous insulin by inhibiting mucosal growth and the ontogenic
expression of sucrase and maltase compared with age-matched controls.
These data are in agreement with our previous study on insulin
B-ASP10 (11) receptor binding and provide
unequivocal evidence that endogenous insulin plays a physiological role
in the ontogenic expression of BBM enzymes. As a result, the downstream
signal is at least in part triggered by binding of the hormone to the extramembranous sites of the IR. Similar effects of anti-IR antibodies on the growth of chicken embryos have been observed by Girbau et al.
(15). In the study (15), it was found that
administration of 200 and 400 µg protein/day resulted in a
dose-related inhibition of growth with marked depression of total body
DNA, RNA, and protein contents of the embryos, resulting in a mortality
of 20-40%, proportional to the dose of antibody given. The
inhibition of PI 3-kinase by its irreversible inhibitor wortmannin not
only failed to block the enzymatic response to insulin but
significantly stimulated the BBM enzyme activities by an overproduction
of insulin. This insulin overproduction was apparently induced by
wortmannin itself, because the control and experimental groups received
the same doses of insulin. A similar observation has recently been
published by Nunoi et al. (37). Perfusion of freshly
isolated rat pancreas islets by wortmannin (10
4 to
10
8 M) markedly enhanced insulin secretion by the
inhibition of phosphodiesterase, resulting in increased cAMP content.
Despite inhibition of PI 3-kinase, the enzymatic response to insulin
was not inhibited, suggesting that PI 3-kinase is not critical for the
transduction of the signal.
The detection of MAP kinase-1 and -2 in the rat small intestine and the
stimulation of their activity by insulin (Fig. 6) prompted us to
inhibit their activation, especially because these key enzymes
downregulate the mitogenic effects of insulin. PD-98059 is a specific
inhibitor of MAP kinase kinase that in turn activates MAP kinase-1 and
-2. The administration of PD-98059 to suckling pups at low doses
clearly inhibited both mucosal mass and the expression of BBM
hydrolases after 48 h of treatment (Table 4). Together, these
results suggest that after receptor binding the signal of insulin that
produces mitogenic effects and BBM enzyme stimulation is transduced via
the pathway (Grb2, SOS, Raf, Ras-GAP ?) that activates MAP kinase-1 and
-2. Because our (11) previous studies have shown that the
stimulation of gene transcription by insulin is independent of the
mitogenic effects of the hormone, further studies are warranted to
clarify the role of this signaling pathway in the expression of BBM enzymes.
 |
ACKNOWLEDGEMENTS |
We thank Dominique Vermeulen for assistance in redaction and typing
the manuscript.
 |
FOOTNOTES |
This work was supported by Fonds de la Recherche Scientifique
Médicale Grant 3.4546.95 and Fonds de Développement
Scientifique (Université Catholique de Louvain, Brussels,
Belgium) Grant 629205.
Address for reprint requests and other correspondence: J.-P.
Buts, Cliniques Universitaires St. Luc, 10 Ave. Hippocrate, 1200 Brussels, Belgium (E-mail: buts{at}gype.ucl.ac.be).
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 6 December 1999; accepted in final form 30 August 2000.
 |
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