Nutritional state regulates insulin receptor and IRS-1
phosphorylation and expression in chicken
Joëlle
Dupont,
Michel
Derouet,
Jean
Simon, and
Mohammed
Taouis
Endocrinologie de la Croissance et du Métabolisme, Station de
Recherches Avicoles, Institut National de la Recherche Agronomique,
37380 Nouzilly, France
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ABSTRACT |
After insulin
binding, insulin receptors (IR) phosphorylate the insulin receptor
substrate 1 (IRS-1) on specific motifs and thereby initiate insulin
action. The interaction between IR and IRS-1 and their expression were
studied in vivo in two target tissues (muscle and liver) in chickens, a
species that is insulin resistant. To induce extreme changes in plasma
insulin levels, chickens were subjected to three different nutritional
states (ad libitum fed, fasted for 48 h, and refed for 30 min after
48-h fast). Liver membrane IR number was significantly increased in fasted compared with fed chickens. This upregulation of IR number was
concomitant with the an enhanced expression of IR mRNA as determined by
reverse transcription-polymerase chain reaction. In leg muscle, IR mRNA
was not altered by the nutritional state. Using specific antibodies
directed toward human IR, anti-phosphotyrosines, or mouse IRS-1, we
demonstrated that IR and IRS-1 are associated in vivo in liver and
muscles. Tyrosine phosphorylation of liver IR and IRS-1 were
significantly decreased by prolonged fasting and restored by 30-min
refeeding. These alterations were not observed in muscle. Fasting
increased IRS-1 mRNA expression in liver but not in muscle. These
results are the first evidence showing that chicken liver and muscle
express IRS-1. Therefore, the chicken insulin resistance is not
accounted for by the lack of IRS-1. The differences observed for the
regulation of IR and IRS-1 messengers and phosphorylation between liver
and muscle in response to alterations of the nutritional state remain
to be explained.
tyrosine kinase; insulin binding; insulin resistance; insulin
receptor substrate 1
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INTRODUCTION |
INSULIN initiates its metabolic and growth-promoting
effects through a specific transmembrane heterotetrameric tyrosine
kinase receptor, the insulin receptor (IR) (6, 31). The IR tyrosine kinase phosphorylates at least one but more likely several
intracellular substrates. In mammalian species, insulin receptor
substrate 1 (IRS-1) is considered the major substrate for IR. IRS-1
contains motifs that, after tyrosine phosphorylation, are binding sites for proteins containing Src homology 2 (SH2) domains. Such proteins that interact with IRS-1 include phosphatidylinositol 3-kinase, Grb2,
syp (SHPTP2), crk, and nck (5, 14, 18, 24, 25). Furthermore, the
NH2 terminal of IRS-1 contains a
region showing sequence similarity to pleckstrin, which is involved in
protein-protein and phospholipid interactions. Finally, IRS-1 presents
a phosphotyrosine binding domain that interacts with a phosphotyrosine
located in the juxtamembrane NPEY motif of the IR (25). This plethora
of protein-protein interaction motifs confers to IRS-1 the property of
a multisite docking protein, making IRS-1 a key protein in transducing
insulin metabolic and mitogenic effects. These interactions have been
well dissected in vitro in mammalian cells (primary cultured cells and
cell lines).
In the present report, we attempted to demonstrate the presence of
IRS-1, its association with the IR, and its phosphorylation on tyrosine
residues in two chicken target tissues (liver and muscles). Compared
with mammals, this species presents a high plasma glucose level (2 g/l = 11 mM) despite the presence of hyperactive insulin and is largely
resistant to exogenous insulin (20, 23). In the past, our group has
showed that, in several tissues, chicken IR molecular structure and
affinity are very similar to those described for mammalian IR.
Furthermore, the tyrosine kinase activity of the chicken IR (21, 27) is
altered by prolonged fasting or corticosterone treatment in liver but
not in muscle (1, 22, 28).
To better understand the mechanisms that control insulin sensitivity in
chicken tissues, we have recently cloned and sequenced the coding
region of chicken IRS-1 gene (cIRS-1) (30). cIRS-1 presents a high
identity with its human, rat, and mouse homologs (30). The most
conserved regions were the pleckstrin homology domain, the IRS-1
homology 2 (IH2), the Shc and IRS-1 NPXY binding domains (SAIN), and
the 12 tyrosine residues that are located in YXXM or YMXM motifs. All
these data suggest that IRS-1 may play an important role in insulin
action in chicken despite the weak hypoglycemic effect observed in
response to exogenous insulin. In the present studies, IR and IRS-1
expressions and their tyrosine phosphorylation were studied in
different and extreme nutritional conditions known to alter plasma
insulin levels: prolonged fasting (48 h), refeeding for 30 min after
48-h fast, and ad libitum feeding. As a main conclusion, IRS-1 is
present in chicken liver and muscle and interacts with the IR. In
liver, but not in muscle, tyrosine phosphorylation of IRS-1 as well as
that of IR is dependent on the nutritional state.
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MATERIALS AND METHODS |
Chemicals.
Monocomponent porcine insulin was from Novo Industrie Pharmaceutique
(Paris, France). 125I was
purchased from Amersham France (Les Ulis, France). Bovine serum albumin
(BSA, fraction V radioimmunoassay grade), phenylmethylsulfonyl fluoride
(PMSF), leupeptin, aprotinin, and protein A-agarose were purchased from
Sigma Chemical (St. Louis, MO). Triton X-100 and sodium dodecyl sulfate
(SDS) were obtained from Bio-Rad Laboratories (Richmond, CA), and
Protogel was from National Diagnostic (Atlanta, GA). The
RNA Insta-Pure system kit for RNA extraction, avian myeloblastosis virus (AMV) reverse transcriptase, and Goldstar
Taq polymerase were obtained from
Eurogentec (Seraing, Belgium). Random hexamers and ribonuclease (RNase)
inhibitor rRNasin were purchased from Promega (Lyon, France), and
dexoynucleotide triphosphate (dNTP) was obtained from Oncor-Appligene
(Illkirch, France). Taq extender kit
was purchased from Stratagene (La Jolla, Ca). Anti-IRS-1 (
IRS-1) and
monoclonal anti-phosphotyrosine (
PY20) antibodies were obtained from
Transduction Laboratories (Lexington, KY). Anti-IR (B10) was a generous
gift from Dr. P. Gorden (National Institute of Diabetes and Digestive
and Kidney Diseases, Bethesda, MD). Primers used for reverse
transcription (RT)-polymerase chain reaction (PCR) were provided by
Genosys (Cambridge, UK).
Animals.
Male chickens (broiler type) were bred in a conventional floor pen, fed
ad libitum a balanced diet (3,100 kcal/kg metabolizable energy and 22%
protein), and exposed to a daily 14-h light period. At 9 wk of age,
animals were separated into three groups:
1) fasted for 48 h,
2) refed for 30 min after 48-h fast,
and 3) fed ad libitum. Liver and leg
muscles were quickly removed, frozen, powdered into liquid nitrogen,
and stored at
80°C.
Determination of plasma glucose and insulin levels.
Plasma glucose levels were measured by the glucose oxidase method using
an automated analyzer (Glucose Beckman Analyzer 2, Palo Alto, CA), and
plasma insulin levels were determined by a radioimmunoassay with a
guinea pig anti-porcine insulin antibody (Ab 27-6, generously
provided by Dr. G. Rosselin, Hôpital Saint-Antoine, Paris) using
chicken insulin as the standard.
Binding of insulin to liver membranes.
Crude liver membranes were prepared by differential centrifugation as
previously described (9). Insulin binding to liver membranes was
measured in 0.15 ml of Krebs-Ringer phosphate buffer containing 1% BSA
and 1 mg/ml bacitracin using 0.1 ng/ml
125I-porcine insulin and 0.4 mg/ml
membrane protein (final concn) (13). Tracer binding was inhibited by
increasing concentrations of unlabeled monocomponent porcine insulin
(13-13,000 ng/ml). After 16 h of incubation at 4°C, the
reaction was stopped by centrifugation at 12,000 g for 3 min. The resulting pellets
were washed, and incorporated radioactivity was counted. Nonspecific
binding was determined in the presence of an excess of unlabeled
insulin (13.3 µg/ml) and was found to be 18-25% of total
binding.
Immunoprecipitation.
Powdered tissues (1 g) were homogenized on ice with an Ultraturax
homogenizer in buffer A containing 150 mM NaCl, 10 mM tris(hydroxymethyl)aminomethane (pH 7.4), 1 mM EDTA, 1 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1% Triton X-100, 0.5% NP-40, protease inhibitors (2 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin), and
phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium
pyrophosphate, and 2 mM sodium orthovanadate). Homogenates were
centrifuged at 600 g for 20 min at
4°C, and supernatants were then ultracentrifuged for 45 min at
45,000 rpm. Supernatants were aliquoted, and protein concentrations
were determined using a Bio-Rad kit and stored at
70°C.
Solubilized materials were subjected to immunoprecipitation using
antibodies directed against either phosphotyrosines (
PY20) or IR
(B10).
PY20 and B10 were added to samples at a final dilution of
1:500 and 1:50, respectively, and incubation was carried at 4°C
during 16 h. The immune complexes were precipitated by the addition of
protein A-agarose beads for 1 h at 4°C as previously described (11, 29). After two sequential washes using diluted buffer A (1:2), the resulting pellets
were resuspended in Laemmli buffer containing 80 mM dithiothreitol.
After SDS-polyacrylamide gel electrophoresis (PAGE) and
electrotransfer, proteins containing phosphotyrosines and/or
IRS-1 were detected by sequential incubation with
PY20 or
IRS-1,
respectively, and horseradish peroxidase (HRP)-linked anti-mouse or
anti-rabbit
-globulin (Amersham). Membranes were washed, and
enhanced chemiluminescence (ECL) was performed (29, 30).
In some experiments, liver and muscle extracts (12 µg of protein)
were subjected to SDS-PAGE without previous immunoprecipitation. After
electrotransfer onto nitrocellulose membranes, blots were incubated
with
IRS-1 and subjected to HRP anti-mouse or -rabbit
-globulin.
Finally, bands were revealed by ECL. Band densities were estimated by
using the NIH Image software.
Determination of cIRS-1 expression using RT-PCR.
Total RNAs from liver and muscle of fed, refed, or fasted chickens were
extracted using the RNA Insta-Pure system kit according to
manufacturer's recommendations. Total RNAs were subjected to RT-PCR as
previously described (30). Briefly, 1 µg of total RNA was reverse
transcribed by AMV reverse transcriptase (15 U) in the presence of
random hexamers (1 µg/µl). RT was carried out in the presence of
MgCl2 (25 mM), dNTP mixture (2 mM)
and rRNasin ribonuclease inhibitor. The RT reaction was assessed at
42°C for 60 min followed by an incubation at 95°C for 5 min. RT
products were then subjected to PCR in standard conditions in the
presence of two pairs of primers. The first pair of primers was
specific to cIRS-1: sense, 5'-GCCCGGCCCACGAGGCTG-3' (bases
2630-2648) and antisense, 3'-GTACGCTTGTCCGTAACG-5'
(bases 3120-3102) flanking a 490-bp region. The second pair of
primers was specific to chicken IR tyrosine kinase domain cDNA (19):
sense, TTGGGATGGGTTTATGAGGG and antisense, TAATAGTCGGTTTCATATAGAT
flanking a 470-bp region. As an internal control of RT and PCR, we have
used an amplification of 18S RNA using two primers flanking a 515-bp
fragment (sense, 5'-CTGCCCTATCAACTTTCG-3' and antisense,
5'-CATTATTCCTAGCTGCGG-3').
Briefly, 2 µl of RT products were amplified by PCR in a final volume
of 50 µl containing 5 µl of Taq
10× buffer, 3 µl of MgCl2 (1.5 mM final concn), 2 µl of dNTP (10 mM), 1 µl of sense and antisense primers, and 1 U of Goldstar
Taq polymerase. Forty cycles were
performed, each cycle consisting of denaturation (94°C, 1 min),
annealing (58°C, 1 min), and elongation (72°C, 1 min) except for the first cycle, in which denaturation was for 4 min, and the last
cycle, in which the extension time was for 10 min. RT-PCR products were
analyzed in an agarose gel (1.5%) stained with ethidium bromide (1 mg/ml).
Statistical analysis.
Statistical analysis was performed using analysis of variance to detect
significant intergroup differences (ad libitum fed, fasted for 48 h,
and refed for 30 min after fast period). Data from multiple
immunoblotting and RT-PCR experiments were expressed in arbitrary
densitometry units normalized against the mean of ad libitum-fed
control values within each experiment. Comparisons between ad
libitum-fed (control) and fasted (48 h) chickens or between control and
refed chickens (30 min after 48-h fast) were made using the Student's
t-test. Data are expressed as means ± SE, and P < 0.05 was
considered statistically significant.
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RESULTS |
Glucose and insulin plasma levels.
Insulin and glucose plasma levels were determined in the different
nutritional conditions: ad libitum fed, fasted for 48 h, and refed for
30 min after 48-h fast (Table 1). Fasting
reduced plasma insulin levels (P < 0.05) compared with fed or refed states (Table 1), whereas plasma
glucose levels were not significantly affected by the nutritional state
(Table 1).
Insulin binding to chicken liver membranes.
Insulin binding to crude liver membranes was measured in the various
nutritional states. As previously shown (1), such direct measurement
cannot be performed with muscle membranes, since, in this tissue, the
proportion of nonspecific binding is too high. Liver membranes were
incubated in the presence of
125I-porcine insulin and
increasing concentrations of unlabeled insulin. Specific tracer insulin
binding (B0), as a percentage of total radioactivity (T)
added, was significantly increased in fasted chickens
(B0 / T = 15.2 ± 0.7%, mean ± SE,
n = 4) compared with fed chickens
(B0 / T = 10.3 ± 0.7%,
P < 0.05; Fig.
1,
inset). Specific insulin binding
level in refed chicken liver was intermediate. Figure 1 shows that the
nutritional states did not affect IR affinity as estimated by the
concentration of unlabeled insulin inhibiting tracer binding by 50%:
57 ± 3 (fed ad libitum), 62 ± 3 (fasted for 48 h), and 61 ± 3 (refed for 30 min after 48-h fast) ng insulin/ml, i.e., minor
differences (P < 0.05).

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Fig. 1.
Competitive inhibition of
125I-labeled porcine insulin
binding to chicken liver membranes in various nutritional states. Liver
membranes were prepared from chickens that were either fasted for 48 h,
ad libitum fed, or refed for 30 min after a 48-h fast. Crude membranes
were incubated in presence of labeled insulin (0.1 ng/ml) for 16 h at
4°C in absence or presence of increasing concentrations of
unlabeled porcine insulin. Percentage of maximal specific binding
(B / B0) of
125I-insulin was determined and
plotted against different unlabeled insulin concentrations.
Inset: specific binding of labeled
insulin (B) was expressed as percentage of total radioactivity added
(T). Data are means ± SE; n = 4.
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Chicken liver and muscle express IRS-1.
Liver and muscle of fasted (48 h), fed (ad libitum), and refed (30- min
refed after 48-h fast) chickens were solubilized and subjected to
SDS-PAGE as described in MATERIALS AND
METHODS. After SDS-PAGE under reducing conditions and
electrotransfer onto nitrocellulose membranes, blots from liver and
muscle were probed with
IRS-1. After addition of secondary antibody
coupled to HRP (anti-mouse-HRP or anti-rabbit-HRP), bands were revealed
by ECL as previously described (11, 29). In the three nutritional
states,
IRS-1 revealed a band with a high molecular mass
(180-185 kDa) in both liver (Fig.
2A) and
muscle (Fig. 2B). The high identity
found between mouse and chicken IRS-1 coding regions (87%; Ref. 30) and the cross-reactivity observed with mouse
IRS-1 led us to conclude that this 180- to 185-kDa band is the chicken homolog of
IRS-1.

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Fig. 2.
Insulin receptor substrate 1 (IRS-1) expression in chicken liver and
muscle. Livers (A) and muscles
(B) from ad libitum-fed, fasted for
48 h, and refed (for 30 min after 48-h fast) chickens were isolated and
solubilized as described in MATERIALS AND
METHODS. Aliquots were resolved on 7.5% sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel (12 µg of
protein) and transferred to nitrocellulose filter by electroblotting.
Blot was probed with an anti-IRS-1 antibody, and bands were revealed by
enhanced chemiluminescence (ECL).
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In vivo association of IRS-1 with IR in chicken liver and muscle.
Supernatants from solubilized liver materials were subjected to
immunoprecipitation using an anti-IR antibody (B10). After SDS-PAGE and
electrotransfer, nitrocellulose membranes were incubated in the
presence of
IRS-1. ECL revelation showed one band of high molecular
mass (180-185 kDa) corresponding to IRS-1 (Fig.
3A), which was present independent of the nutritional status. The
coimmunoprecipitation of IRS-1 and IR strongly indicates that, in vivo,
these two proteins are associated. The intensities of the bands were
measured using NIH Image software. The association of IR and
IRS-1 was slightly but consistently reduced in fasting state despite
the increase in IR number induced by fasting. This suggests that liver
IR and IRS-1 interaction is regulated by the nutritional state. IR and IRS-1 coimmunoprecipitation was also demonstrated in muscle from fed,
fasted, and refed chickens (Fig.
3B). However, in muscle, the
IRS-1-IR association does not appear to be qualitatively influenced by
nutritional states.

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Fig. 3.
Association of IRS-1 with insulin receptor (IR) in chicken liver and
muscle. Livers (A) and muscles
(B) from ad libitum-fed, fasted for
48 h, and refed chickens were solubilized. Solubilized samples were
adjusted to 5 mg of protein, and IRs were immunoprecipitated (IP) with
anti-IR antibody (B10). Immunoprecipitates were analyzed on SDS-PAGE
(7.5%) followed by electroblotting. Blot was probed with either
IRS-1 or PY20, and bands were detected by ECL. Results shown are
representative of at least 2 independent experiments repeated twice.
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Regulation of IR and IRS-1 phosphorylation by nutritional state.
To determine the level of tyrosine phosphorylation of IR and IRS-1 in
response to nutritional states, supernatants from liver or muscle
lysates were immunoprecipitated with
PY20. After SDS-PAGE and
electrotransfer, nitrocellulose membranes were incubated with either
PY20 or
IRS-1. In liver, after immunoprecipitation and blotting
with
PY20, ECL detection revealed two major bands with apparent
molecular masses of 95 and 185 kDa, corresponding to the IR
-subunit
and to IRS-1, respectively (Fig. 4). As
shown in Fig. 4A, the level of
tyrosine phosphorylation of IR
-subunit and IRS-1 were reduced in
fasted state compared with fed and refed states. The level of tyrosine
phosphorylation was quantified using NIH Image software, and the mean
results are represented in Fig. 5,
A and
B. Results (arbitrary units) were
expressed as percentage of the level measured in fed state. Fasting
significantly reduced IRS-1 phosphorylation (by 21 ± 4%,
P < 0.05, Fig.
5B), which was restored after 30 min
of food intake (refed). Similarly, IR
-subunit phosphorylation was reduced after 48 h of fasting period (18 ± 2%,
P < 0.05) and restored after 30 min
of refeeding (Fig. 5A). The decrease
in IRS-1 phosphorylation in response to prolonged fasting was also
confirmed when
IRS-1 was used for immunoblotting after
PY20
immunoprecipitation (Fig. 4B). In
muscle, immunoprecipitation and immunoblotting using
PY20 showed
that IRS-1 and IR
-subunit were tyrosine phosphorylated (Fig.
6A). In
contrast to liver, the levels of muscle IRS-1 and IR
-subunit
tyrosine phosphorylations were not clearly decreased by prolonged
fasting, and this was confirmed by the quantification of band densities
(Fig. 6B).

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Fig. 4.
IRS-1 and IR -subunit tyrosine phosphorylation in chicken liver in
various nutritional states. Livers from fed, refed (after 48 h of
fast), and fasted chickens were extracted and solubilized. Solubilized
proteins were equalized to 5 mg and immunoprecipitated using
anti-phosphotyrosine antibody ( PY20). Immunoprecipitates were
analyzed on SDS-PAGE (12%) followed by electroblotting. Blots were
probed with either PY20 (A) or an
anti-IRS-1 antibody ( IRS-1; B),
and bands were detected by ECL.
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Fig. 5.
Quantification of liver IR and IRS-1 phosphorylation. Band densities
from Fig. 4 were quantified by scanning densitometry and expressed as
percent arbitrary units (nutritional state/fed state × 100). IR
and IRS-1 phosphorylation levels are represented in
A and
B, respectively. Values are means ± SE; n = 6. * P < 0.05.
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Fig. 6.
IRS-1 and IR -subunit tyrosine phosphorylation in chicken muscle in
various nutritional states. Muscles extracted from fed, refed, and
fasted chicken were solubilized. Solubilized proteins (5 mg) were
immunoprecipitated using anti-phosphotyrosine antibody ( PY20).
Immunoprecipitates were resolved on SDS-PAGE (12%) followed by
electroblotting. Blot was probed with PY20, and bands were detected
by ECL (A). This result is
representative of 6 and 2 independent experiments repeated twice for
IRS-1 and IR -subunit phosphorylations, respectively
(B).
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Regulation of in vivo IRS-1 and IR mRNA expression by nutritional
state.
To determine the consequence of the nutritional status on IRS-1 and IR
mRNA expression, total RNAs from chicken liver and muscle were
subjected to RT-PCR using specific primers to amplify cIRS-1 and cIR
cDNAs. RT and PCR were primed with random hexamers and two pairs of
primers, respectively. For cIRS-1, specific primers were chosen
flanking a 490-bp region as indicated in MATERIALS AND
METHODS. For cIR, primers were selected in the tyrosine
kinase domain and were specific to IR without overlapping with
insulin-like growth factor I receptor cDNA as previously reported by
Scavo et al. (19). They were flanking a fragment of 470 bp. As RNA expression control, we have subjected the same RT products to PCR using
18S RNA specific primers flanking a 515-bp region. After RT-PCR,
ethidium bromide-stained agarose gel showed that IRS-1 and IR
expressions were increased in liver after 48-h fast compared with the
fed state (Fig. 7,
A, lane
7 vs. 5, and
B, lane
4 vs. 2), whereas
the expression of 18S RNA was not influenced by the nutritional states
in either liver or muscle (Fig. 7C).
Gel density quantification using NIH Image software showed that liver
cIRS-1 and cIR expression increased by about threefold in fasting
chicken compared with fed chicken (Fig. 8).
In muscle, IRS-1 expression was not significantly affected by the
nutritional state, whereas IR expression was slightly increased in
fasted state (Figs. 7 and 8). These data were confirmed by Northern
blot analysis (data not shown).

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Fig. 7.
Expression of IRS-1 and IR in chicken liver and muscle. Total RNA from
liver and muscle of fed, refed, and fasted chickens was prepared from
~100 mg of tissues as indicated in MATERIALS AND
METHODS. A: primer,
IRS-1 sense, 5'-GCCCGGCCCACGAGGCTG-3' (bases
2630-2648) and antisense, 3'-GTACGCTTGTCCGTAACG-5'
(bases 3103-3120). B: IR sense
primer, TTTGGATGGGTTTATGAGGG and antisense primer,
TAATAGTCGGTTTCATATAGAT. C: 18S RNA
sense primer, 5'-CTGCCCTATCAACTTTCG-3' and antisense
primer, 5'CATTATTCCTAGCTGCGG-3'. In
A, muscle and liver transcripts were
deposited in lanes 2-4 and
5-7, respectively. In
B and
C, liver and muscle transcripts were
deposited in lanes 2-4 and
5-7, respectively.
Lanes 2 and
5, 3 and 6, and
4 and
7 represent nutritional states,
respectively: ad libitum fed, refed for 30 min after 48-h fast, and
fasted for 48 h. Lane 1 indicates
migration of marker molecules (100 bp). Products of amplification were
run on a 1.5% agarose gel and visualized by staining with ethidium
bromide. Expected size of amplified producted is 490, 470, and 515 bp
for IRS-1, IR, and 18S, respectively, and is indicated on
right.
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Fig. 8.
Relative levels of IRS-1 and IR mRNA in liver and muscle from fasted
chickens compared with fed chickens. IRS-1 and IR mRNA in liver and
muscle from fasted and fed chickens were determined by reverse
transcription-polymerase chain reaction, and band densities were
quantified using NIH software. Values shown represent relative IRS-1
and IR mRNA levels compared with fed state. In each case, level of
IRS-1 and IR mRNA as adjusted to 1.0, and level in fasted state was
normalized accordingly. Value for IRS-1 mRNA represents means of 4 separate assays, each done on RNA preparations prepared from different
animals (n = 4). Value for IR mRNA
represents mean of 2 separate assays.
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DISCUSSION |
IRS-1 has been identified as a major substrate for the IR tyrosine
kinase. IRS-1 gene has been cloned and sequenced in several species:
mouse (2), rat (26), human (3),
Xenopus (12), and chicken (30). It
showed a high conservation during evolution (91% of identity for amino
acid sequence between human and chicken IRS-1). The high conservation
of cIRS-1 in chicken raised the question about its role in this
species, which exhibits a large resistance to exogenous insulin and a
high blood glucose level despite the presence of an hyperactive
endogenous insulin (20, 23). The mechanisms of this insulin resistance
are still unclear. To address this question, we first characterized
cIRS-1 in vivo in two insulin target tissues, liver and muscle. We then
studied the interaction between IRS-1 and IR, and their levels of
phosphorylation and expression in vivo in different nutritional
situations.
Three different nutritional states were chosen to exaggerate the
differences in circulating insulin levels and reveal any modulation of
IRS-1 expression and tyrosine phosphorylation: fasting for 48 h,
refeeding for 30 min after 48-h fast, and ad libitum feeding (control
state). The insulinemia was significantly decreased in fasted chickens;
this decrease was accompanied by an upregulation of liver IR number. In
response to 30-min refeeding, plasma insulin levels increased and liver
IR number started to decrease, recovering from the upregulation state
induced by prolonged fasting. In previous experiments in which the
refeeding period was extended to 24 h, liver and kidney IR number (4,
22) decreased under the level observed in the fed state. As mentioned
in the introduction, technical problems do not permit such
determinations in muscle tissues. The degree of IR tyrosine
phosphorylation was then determined in liver and muscle. In liver, IR
phosphorylation was reduced by 48-h fast and restored by 30-min
refeeding. The inhibitory effect of prolonged fasting observed for IR
phosphorylation is in good agreement with previous studies showing that
fasting reduced the intrinsic tyrosine kinase activity of solubilized
and lectin-purified chicken liver IR (22). In contrast, in the present
study, muscle IR phosphorylation was not altered by prolonged fasting.
Again, this result is in good agreement with previous results showing that the tyrosine kinase activity of lectin purified muscle IR was
unchanged after a 48-h fast (1). The absence of regulation of IR
tyrosine kinase activity in response to prolonged fasting in muscle and
its presence in liver has also been described for rat (8, 17). To
further analyze the associations between IR and IRS-1 in liver and
muscle of intact animals, coimmunoprecipitation experiments were
performed after solubilization using various antibodies as mentioned in
RESULTS. The coimmunoprecipitation of
IR and IRS-1 in both tissues indicates the existence of a functional complex between IR and IRS-1 in intact animals as previously described in several cell lines (7, 10, 15, 16, 26). Futhermore, this association
is regulated most likely in response to the ambient insulinemia: a
slight increase in the association between IR and IRS-1 was suggested
in the liver of refed chickens. Prolonged fasting also induced changes
in the IR and IRS-1 mRNA levels in chicken liver. IR mRNA was
overexpressed and thus should contribute to the development of the IR
upregulation observed after fasting in this tissue as discussed
earlier. Liver IRS-1 mRNA was also overexpressed in the fasted state
without, however, clear changes in the protein level. Differences in
the turnover of these two proteins may be involved. This discrepancy
may also be explained by the techniques used to quantify IR and IRS-1:
IR was quantified using
125I-insulin binding, whereas
IRS-1 was estimated by Western blotting and densitometry. The latter
techniques are less sensitive than the former. As observed for liver IR
tyrosine phosphorylation, liver IRS-1 tyrosine phosphorylation was
significantly decreased in fasted chickens and restored by 30-min
refeeding. These data suggest that liver IRS-1 phosphorylation is
coordinately regulated by the IR phosphorylation state, most likely
through changes in circulating insulin levels. In contrast, muscle
IRS-1 phosphorylation and, as discussed earlier, IR phosphorylation
were not significantly affected by nutritional states. The origin and
the physiological significance of the lack of any regulation of the
phosphorylation state of both IR and IRS-1 in muscle despite large
changes in plasma insulin concentrations are surprising and remain
unexplained.
In conclusion, this is the first characterization of IRS-1 in chicken
tissues. IRS-1 is present in liver and muscle; it associates with the
IR and is phosphorylated on tyrosine residues. In liver, IRS-1
expression is increased and its tyrosine phosphorylation is decreased
by prolonged fasting despite an increase in IR number. This change,
which is most likely accounted for by a decrease in the IR tyrosine
phosphorylation (and IR kinase activity), may lead to insulin
resistance in the liver. Refeeding rapidly restores the phosphorylation
of IR and IRS-1. To our knowledge, this is the first evidence showing
that IRS-1 expression and phosphorylation are dependent on the
nutritional state and, therefore, on the ambient circulating insulin
levels in intact animals. In muscle, IRS-1 expression and tyrosine
phosphorylation were not altered by the nutritional state, suggesting
the presence of a tissue-specific regulation that remains unexplained.
Although some peculiarities are likely to be present somewhere on the
insulin signaling pathway to account for the natural resistance state
found in chickens compared with mammals, the present studies
demonstrate the presence of IRS-1 in chicken and suggest that IRS-1
also exerts a regulatory role in the control of metabolism and growth
in this species. This role has now to be further characterized using
different experimental models in which sensitivity is known to be
altered.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Phillip Gorden (Diabetes Branch) for
providing insulin receptor antibody (B10), Dr. G. Rosselin for providing guinea pig anti-porcine insulin antibody and L. Ruffier and
Dr. M. Duclos for providing insulin tracer.
 |
FOOTNOTES |
This study was supported by the Association Française contre les
Myopathies.
Address for reprint requests: M. Taouis, Endocrinologie de la
Croissance et du Métabolisme, Station de Recherches Avicoles,
INRA, 37380 Nouzilly, France.
Received 9 June 1997; accepted in final form 24 October 1997.
 |
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