Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
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ABSTRACT |
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The insulin receptor substrates (IRSs) function in insulin signaling. Four members of the family, IRS-1 through IRS-4, are known. Previously, mice with targeted disruption of the genes for IRS-1, -2, and -3 have been characterized. To examine the physiological role of IRS-4, we have generated and characterized mice lacking IRS-4. Male IRS-4-null mice were ~10% smaller in size than wild-type male mice at 9 wk of age and beyond, whereas the female null mice were of normal size. Breeding pairs of IRS-4-null mice reproduced less well than wild-type mice. IRS-4-null mice exhibited slightly lower blood glucose concentration than the wild-type mice in both the fasted and fed states, but the plasma insulin concentrations of the IRS-4-null mice in the fasted and fed states were normal. IRS-4-null mice also showed a slightly impaired response in the oral glucose tolerance test. Thus the absence of IRS-4 caused mild defects in growth, reproduction, and glucose homeostasis.
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INTRODUCTION |
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THE INSULIN RECEPTOR is a tyrosine kinase. Signaling from it proceeds by the phosphorylation of specific substrate proteins by the activated receptor. Among these substrate proteins are a group of four now referred to as insulin receptor substrates 1-4 (IRS-1, -2, -3, and -4) (reviewed in Ref. 21). The IRSs share a common architecture, consisting of a COOH-terminal pleckstrin homology domain followed by a phosphotyrosine binding domain followed by a large COOH-terminal region containing multiple sites of tyrosine phosphorylation. The pleckstrin homology and phosphotyrosine binding domains are required for the efficient tyrosine phosphorylation of the IRSs by the insulin receptor. The sites of tyrosine phosphorylation lie in short motifs that bind to the SH2 domains of specific signaling proteins. As a consequence, the tyrosine-phosphorylated IRSs act as adaptor/effector proteins for various SH2 domain-containing signaling proteins. The most important of these appears to be phosphatidylinositol 3-kinase (PI 3-kinase). Each of the IRSs has been found to associate with PI 3-kinase; in the case of IRS-1, this association has been shown to activate PI 3-kinase (9, 21). Many of the metabolic effects of insulin are signaled through pathways that lie downstream of activated PI 3-kinase (reviewed in Refs. 1 and 17). Besides insulin, a number of other hormones and cytokines, acting through receptors complexed with the JAK tyrosine kinases, also elicit the tyrosine phosphorylation of various IRSs, and consequently the IRSs may also be key intermediates in signaling from other receptors (21).
The occurrence of four IRSs raises the question of the physiological
role of each. Despite their similarity, the IRSs have sufficiently
different structures so that differences in their tyrosine
phosphorylation by the insulin and other receptors and differences in
their association with various SH2 domain-containing proteins were
expected and are being found (5, 8, 14-16). Moreover, differences
exist in the intracellular locations of the IRSs, with IRS-1 and -2 associated with intracellular structures and IRS-3 and -4 associated
with the plasma membrane (2, 7). Finally, the IRSs differ in their
sites and levels of expression. IRS-1 and IRS-2 show widespread
tissue expression at relatively high levels, whereas IRS-3 and
IRS-4 exhibit more limited tissue expression at relatively lower levels
(summarized in Ref. 6; also see DISCUSSION for tissue
distribution of IRS-4). These considerations indicate that the IRSs are
likely to have different physiological roles. The results of targeted
disruptions of the genes for IRS-1, -2 and -3 have shown that this is
the case. Mice lacking IRS-1 are ~60% of normal size. They are
mildly insulin resistant; they exhibit elevated plasma insulin
levels and impaired glucose and insulin tolerance tests. However, their
blood glucose levels are normal in the fasted and ad libidum-fed states
(3, 19). In contrast, mice lacking IRS-2 are only slightly
smaller than normal but develop diabetes because of a combination
of insulin resistance and impaired proliferation of the -cells of
the pancreas (22). Mice lacking IRS-3 show no defects in growth or
glucose homeostasis (11). Thus IRS-1 plays an important role in
growth; IRS-2 plays an important role in
-cell proliferation,
and both play roles in the insulin responsiveness of major tissues. On
the other hand, IRS-3 appears not to have a major role in any of these processes.
To complete the analysis of the physiological roles of the IRSs by targeted gene disruption, we have now generated and characterized mice lacking IRS-4. These exhibit slight defects in growth, reproduction, and glucose homeostasis.
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EXPERIMENTAL PROCEDURES |
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Generation of IRS-4-null mice.
We have previously described the isolation and restriction mapping of
the mouse IRS-4 gene and shown that the coding region of the
IRS-4 gene contains no introns (6). On the basis of this
information, the targeting vector for disruption of the IRS-4 gene was assembled in pBluescriptSK+ (Stratagene) as follows (see Fig.
1A). A 3.3-kb Avr
II-Xho I fragment of IRS-4 containing the entire coding
sequence except for 457 bp at its 3' end was replaced by a 1.8-kb
fragment containing the neomycin-resistance gene (neo) in
opposite orientation. The neo gene was flanked by a 2.9-kb Nhe I-Avr II fragment and a 4.8-kb Xho
I-Sal I fragment derived from the IRS-4 regions
5' and 3' to the deleted coding region. A 2.6-kb thymidine
kinase gene was placed at the 3' end of the targeting vector.
Both the neo gene and the thymidine kinase gene included the
phosphoglycerate kinase promoter to drive expression. The targeting
vector was linearized with Not I and electroporated into J1
embryonic stem (ES) cells. Clones that were resistant to G418 (Life
Technologies) and gancyclovir (Syntex) were picked and expanded as
described (10). These were screened for homologous recombination by
Southern blotting of BamH I-digested genomic DNA. Out of 200 ES
clones screened, two showed the 5.4-kb fragment expected for the
IRS-4-null allele (see Fig. 1B). These two ES clones
were injected into blastocysts derived from C57BL/6 and BALB/c mice in
the facility of Dr. Arlene Sharpe, Department of Pathology, Brigham and
Women's Hospital (Boston, MA). Both clones yielded highly chimeric
male mice.
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Analysis for IRS-4 mRNA by reverse transcription PCR.
Tissues were removed from mice, rinsed in cold phosphate-buffered
saline, frozen in liquid nitrogen, and stored at70°C until use. Total RNA was isolated from the various tissues using the TRIzol
reagent according to the manufacturer's instructions (Life Technologies). To reduce genomic DNA contaminating the RNA, the RNA was
treated with deoxyribonuclease, followed by phenol-chloroform extraction and ethanol precipitation. In addition, before reverse transcription, RNA samples were subjected to a second deoxyribonuclease I treatment, according to the procedure in the Life Technologies manual
for preparation of an RNA sample for reverse transcription PCR. Reverse
transcription was performed with 1 µg of total RNA and a primer
corresponding to nucleotides 3926-3945 of the IRS-4 gene
(6) with SuperScriptII according to the manufacturer's instructions
(Life Technologies). In control reactions to test for the presence of
genomic DNA, water replaced the reverse transcriptase. Aliquots of the
reverse transcription and control reactions were amplified using
primers corresponding to nucleotides 3153-3174 and 3788-3807
of the IRS-4 gene with AmpliTaq Gold (Perkin-Elmer) as
described previously (6).
Blood glucose and plasma insulin. In all cases, blood samples were collected from tails cut at the tip. Mice were either fed ad libitum, and samples taken between 7 and 8 AM, or they were fasted from 6 AM to 9 PM and samples then taken. Blood glucose concentration was measured with the PrecisionG glucose testing system (Medisense). For measurement of insulin, plasma was obtained from the blood by the addition of heparin at 10 U/ml to the blood, followed by centrifugation at 8,000 rpm in a microfuge for 10 min at 4°C. The concentration of insulin in the plasma was measured by radioimmune assay (Linco Research, catalog numbers RI and SRI 13K).
Glucose and insulin tolerance tests. For the glucose tolerance test, mice were fasted from 6 PM to 9 AM; then an oral glucose dose of 1 g D-glucose/kg body weight, in a solution of 0.25 g/ml, was administered with a feeding needle. Glucose levels were determined in blood drops from the tail taken immediately before and 10, 20, 30, 60, 90, and 120 min after the administration of glucose. The insulin tolerance test was performed at 2-3 PM on mice fed ad libitum. Insulin (Humulin, 100 U/ml, from Eli Lilly) at 0.75 U/kg body weight was administered by intraperitoneal injection of a 0.25 U/ml solution in 0.9% NaCl. Blood glucose concentration was measured in blood drops obtained from the tail immediately before the injection and at 10-min intervals for the hour after injection.
Statistical analysis. For each experiment, the unpaired two-tailed t-test was used to compare the mean values for the wild-type and IRS-4-null mice. Differences between means were considered significant for P values <0.05. Data are presented as the means ± SE.
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RESULTS |
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Generation of IRS-4-null mice.
The generation of the IRS-4-null mice is described in
EXPERIMENTAL PROCEDURES. The genotyping of these (Fig. 1,
B and C) shows that the IRS-4 gene was
disrupted. As further evidence for this conclusion, we examined the
expression of the IRS-4 mRNA in brain, skeletal muscle, liver, and
kidney by reverse transcription PCR. We have previously found that
IRS-4 mRNA is expressed in these tissues in wild-type mice (6). The
data in Fig. 2 show that the IRS-4 mRNA is,
as expected, absent from the null mice and present in the wild-type
mice.
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Growth and development of IRS-4-null mice.
The IRS-4 gene is located on the X chromosome. Consequently, as
described in EXPERIMENTAL PROCEDURES, the breeding scheme to obtain IRS-4-null male and female mice differed from the one for genes on other chromosomes. IRS-4+/ females,
obtained from the mating of chimeric male mice with wild-type females,
were mated with wild-type males. From 14 breeding pairs, a total of 167 pups with an average litter size of 6.7 were obtained and genotyped at
~3 wk of age. The proportions of the male wild-type, male null,
female wild-type, and female heterozygous mice were 26:23:27:24. The
IRS-4-null males were then mated with the heterozygous females.
From 12 breeding pairs a total of 85 pups with an average litter size
of 5.8 were obtained. The proportions of male wild-type, male null,
female heterozygous, and female null mice at 3 wk of age were
27:24:26.5:22.5. Because the phenotypic distribution in both of these
breedings is very close to the one of 25:25:25:25 expected in the
absence of any perturbation, there was little or no embyronic lethality
or early death associated with the IRS-4-null phenotype.
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Reproduction of IRS-4-null mice. Preliminary observations suggested that the IRS-4-null mice were not reproducing as well as the wild-type mice. To examine this issue, eight breeding pairs of null mice and seven of wild-type mice, matched in age at 8-12 wk, were followed over a 3-mo period. The IRS-4-null mice produced an average of 1.8 ± 0.4 litters per pair (range 0-3 litters) over this period, whereas the wild-type mice produced an average of 2.9 ± 0.1 litters per pair (range 2-3 litters) over this period. This difference in the number of litters per pair between the null and wild-type mice is significant (P = 0.03).
These litter numbers include litters that died completely within a few days of birth, and in fact a substantial percentage (36%) of the litters of the IRS-4-null mice was completely dead within a few days of birth. For some litters, we observed that the pups were alive at birth. For other litters, which were not examined until a day after birth and were dead at that time, it is possible that the pups were stillborn. In contrast, only 5% of the litters of the wild-type mice were completely lost within a few days of birth. Expression of these results as number of litters completely lost per mating pair gives values of 0.6 ± 0.3 for the IRS-4-null mice and 0.1 ± 0.1 for the wild-type mice; the P value for this difference is 0.15. The average size of the surviving litters from the IRS-4-null mice was slightly less than that of the wild-type litters [6.5 ± 0.8 pups (n = 9 litters) vs. 7.8 ± 0.5 pups (n = 19); P = 0.18 for the difference]. Similar results were obtained in a comparison of mice of the 129 × BALB/c background. In this case, five breeding pairs of null mice and eight of wild-type mice, matched in age at 8-12 wk, were followed over a 3-mo period. The IRS-4-null mice produced an average of 2.0 ± 0.5 litters per pair (range 0-3), whereas the wild-type mice produced an average of 3.1 ± 0.1 litters per pair (range 3-4); this difference is statistically significant, with P = 0.03. A high percentage (50%) of the null litters was completely dead within a few days of birth, whereas only 8% of the wild-type litters was lost. These values correspond to 1.0 ± 0.3 and 0.3 ± 0.2 litters lost per mating pair for the null and wild-type mice; this difference is statistically significant, with P = 0.04. The average sizes of the surviving litters were almost the same for the null and wild-type mice [7.0 ± 1.3 (n = 5 litters) vs. 6.8 ± 0.5 (n = 23)]. As described above, there was no embryonic lethality or early death associated with the IRS-4-null phenotype. Consequently, the lower number of total litters from the IRS-4-null mice indicates that the null pairs were less fertile than the wild type. Although we have not systematically examined the IRS-4-null male and female mice separately to determine which sex is responsible for this difference from the wild type, we did not observe a reduced frequency of total or surviving litters upon mating the male IRS-4-null mice with the heterozygous females (see Growth and development of IRS-4-null mice). Consequently, most likely it is the IRS-4-null female, rather than the male, that is less fertile. In addition, the lower number of surviving litters from the IRS-4-null pairs indicates that the null females are less nurturing of their pups than are the wild-type females.Blood glucose and plasma insulin levels in IRS-4-null mice.
To determine whether glucose homeostasis was affected by the absence of
IRS-4, the concentrations of blood glucose and plasma insulin in the
fed and fasted states were compared between IRS-4-null and
wild-type mice at ages 6-10 wk and again at age 6 mo (Table 1). In the case of the males at 6-10
wk of age, the null mice exhibited a slightly lower blood glucose
concentration in both the fasted and fed states, and the difference
with the wild-type mice was statistically significant. The
IRS-4-null females at 6-10 wk showed the same effect, but
the difference with the wild type was not statistically significant. In
the case of the 6-mo-old mice, both the IRS-4-null males and
females had slightly lower blood glucose in the fed state, and in the
case of the males, this was also true for the fasted state. However,
these differences were not statistically significant. In the case of
the plasma insulin concentration, neither male nor female
IRS-4-null mice at either age showed any difference from the
corresponding wild-type mice.
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Glucose and insulin tolerance tests.
To further examine glucose homeostasis in the IRS-4-null mice,
glucose tolerance tests were carried out. Male and female null mice of
6-10 wk of age exhibited a slightly impaired tolerance compared
with wild-type mice (Fig. 4A). For
the males, the differences at 90 and 120 min were statistically
significant, but in the case of the females, the differences with the
wild type at the various times were not significant. To determine
whether the impairment in glucose tolerance became more pronounced with
age, the test was also performed on mice of 6 mo of age (Fig.
4B). The results were similar to those for the 6- to 10-wk-old
mice. The male IRS-4-null mice showed a slightly impaired
tolerance compared with the wild type, which was statistically
significant at the 20-min time point; the female mice exhibited only a
very slightly impaired tolerance, which was significantly different
from the wild type at 10 min but not at the other times. We also
performed the oral glucose tolerance test on male IRS-4-null
and wild-type mice of the 129 × BALB/c background (8 mice of each
type). The null mice showed an impaired tolerance that was
statistically significant at 60, 90, and 120 min (data not shown).
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DISCUSSION |
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This study has revealed that IRS-4-null mice show some modest differences from wild-type mice. The males were slightly smaller beyond 6 wk of age. Pairs of mice reproduced less well. The null mice had slightly lower blood glucose levels in the fasted and fed states and exhibited a slightly impaired oral glucose tolerance test.
Because the male IRS-4-null mice were only slightly smaller in size, IRS-4 does not play a major role in the control of growth. Moreover, IRS-4 does not play a large role in the insulin responsiveness of muscle and liver, major tissues controlling blood glucose levels. If it did, the absence of IRS-4 would have resulted in insulin resistance in both male and female mice, manifested by elevated plasma insulin in the fed state and markedly impaired glucose and insulin tolerance tests. In contrast, as described in the introduction, the phenotype of mice lacking IRS-1 and IRS-2 shows that IRS-1 has a major role in growth and indicates that both IRS-1 and -2 are required for normal insulin responsiveness of muscle and liver. This difference between IRS-4 and IRS-1 and/or -2 can be explained by the greater abundance of IRS-1 and -2. Although IRS-4 mRNA is present in mouse skeletal muscle, heart, liver, brain, and kidney, IRS-4 protein could not be detected in these tissues by immunoprecipitation and immunoblotting with sensitive antibodies (6). In contrast, the IRS-1 and -2 proteins are readily detectable in these tissues (18).
We note that our results cannot rigorously exclude the possibility that IRS-4 normally plays a significant role in growth and/or glucose homeostasis and that, in the IRS-4-null mice, one or more of the other IRSs functions in place of IRS-4. Because each of the IRS genes is located on a separate chromosome (6), it will be possible through appropriate breeding of mice lacking IRS-4 with mice lacking another IRS to examine the effects of the combined deletion of IRS-4 and each of the other IRSs. Characterization of such mice may reveal whether another IRS compensates for the lack of IRS-4.
In this investigation of IRS-4-null mice and the corresponding control mice, it would have been desirable to examine the activation of components of insulin signaling cascades, such as PI 3-kinase, in a tissue where insulin-elicited tyrosine phosphorylation of IRS-4 could be detected. This is the approach that has been taken in characterization of mice lacking IRS-1, -2, and -3 (3, 11, 19, 22). However, despite considerable effort to date by use of sensitive antibodies against IRS-4, we have not been able to detect the protein in any mouse tissue (6). We have examined whether the absence of IRS-4 affected the levels of expression of IRS-1 and IRS-2 by immunoblotting SDS lysates of liver, white fat, skeletal muscle, heart, and brain from IRS-4-null and wild-type mice for these two proteins. With the possible exception of skeletal muscle, where myosin interfered with the detection of the IRSs, there was no difference in the amounts of IRS-1 and IRS-2 (data not shown).
Very recently a report has appeared (12) that describes the distribution of the mRNA for IRS-4, as well as that for IRS-1 and IRS-2, in the rat brain as determined by in situ hybridization. Remarkably, IRS-4 mRNA is concentrated in the hypothalamus and in a few regions of the telencephalon and mesencephalon and is relatively strongly expressed in these regions. The hypothalamus regulates eating, mating, and nurturing behaviors, growth, and counterregulatory responses to lowered blood glucose (4, 13). This region of the brain contains insulin receptors, as well as other receptors that could potentially require IRS-4 for signal transduction (13, 20). Thus the differences between the IRS-4-null and the wild-type mice found in this study could arise from altered hypothalamic functions due to the absence of IRS-4. In the future, it may be possible to detect and characterize IRS-4 protein in the hypothalamus and other regions of the brain where its mRNA is highly expressed. Moreover, it will be of interest to compare the structures and outputs of these regions of the brain in IRS-4-null and wild-type mice. The investigation of IRS-4 in the brain should lead to a clearer understanding of the cellular and physiological roles of IRS-4.
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ACKNOWLEDGEMENTS |
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We are deeply indebted to Dr. Nancy Speck for advice and encouragement in the generation of the mice with disruption of the IRS-4 gene. We thank Drs. Suzanne Numan and David Russell for allowing us to include their information about the location of IRS-4 in the brain.
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FOOTNOTES |
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This research was supported by a grant (DK-48216) from the National Institute of Diabetes and Digestive and Kidney Diseases.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. E. Lienhard, Dept. of Biochemistry, Vail Bldg., Dartmouth Medical School, Hanover, NH 03755 (Email: gustav.e.lienhard{at}dartmouth.edu).
Received 30 June 1999; accepted in final form 24 September 1999.
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