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Departments of Physiology (G.P., D.R.S., P.K.L.) and Pediatrics (C.R.F., A.J.D., P.K.L.), University of North Carolina, Chapel, North Carolina 27599-7545; AgResearch, Ruakura Research Center (J.M.O.), Hamilton, New Zealand Private Bag 3213; and the Research Division, Joslin Diabetes Center and Department of Medicine, Harvard Medical School (C.R.K.), Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Dr. Pauline Kay Lund, Ph.D., Department of Cell and Molecular Physiology, CB# 7545, University of North Carolina, Chapel Hill, North Carolina 27599-7545. E-mail empk{at}med.unc.edu
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Abstract |
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Introduction |
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The type 1 IGF receptor (IGF1R) is the primary mediator of the growth-promoting actions of IGF-I (5). The IGF1R is a receptor tyrosine kinase that is related in structure and function to the insulin receptor (IR) (5). Ligand-dependent autophosphorylation of IGF1R or IR leads to binding and tyrosine phosphorylation of a 185-kDa signaling intermediate, insulin receptor substrate-1 (IRS-1) (6). Phosphorylated tyrosines within IRS-1 provide binding motifs for multiple proteins that contain SH2 domains, including the p85 regulatory subunit of phosphatidyl inositol 3-kinase (PI 3-kinase), growth factor receptor bound protein-2, phosphotyrosine phosphatase Syp, and the oncogenic proteins Abl, Crk, Nck, and Fyn (6). These interactions mediate activation of multiple downstream pathways, including PI 3-kinase, extracellular signal-related kinase/mitogen-activated protein kinase, and p70 s6 kinase, and induction of immediate early genes such as c-fos (6).
Mice that are homozygous for targeted deletion of the IRS-1 gene are growth retarded at birth and remain growth retarded to adulthood (7, 8). These IRS-1 null mice show impaired glucose tolerance and a decrease in the magnitude of insulin- or IGF-mediated glucose uptake in vivo, but do not develop type 2 diabetes (7, 8). Insulin-stimulated activation of PI 3-kinase and mitogen-activated protein kinase also is impaired in IRS-1 null mice, but the level of impairment is tissue specific (6, 7, 8, 9). Liver, for example, is affected less than skeletal muscle (9). In liver of IRS-1 null mice, the relative levels of insulin-dependent tyrosine phosphorylation of IRS-2, a molecule that is structurally and functionally related to IRS-1, are greater than those in muscle (9). IRS-2 therefore can substitute for IRS-1 as a mediator of insulin action in a tissue-specific manner (9). Whether different organs or tissues show differential dependence on IRS-1 for normal postnatal growth in vivo has not been examined in detail. To address this question, the present study compared organ growth in mice with targeted deletion of one or both copies of the IRS-1 gene and sex-matched, wild-type (WT) littermates.
Growth retardation in IRS-1 null mice supports the hypothesis that IRS-1 is required for normal growth, but does not define precisely which hormones and associated receptors require IRS-1 as a mediator of their trophic actions. The growth phenotype in IRS-1 null mice could reflect impaired actions of IGF-I or impaired actions of insulin, GH, or other cytokines that activate IRS-1 as an early component of their intracellular signaling pathways (6, 7, 8, 9, 10). The present study aimed to assess the in vivo requirement for IRS-1 as a mediator of IGF-I-dependent body and organ growth. Mice that are heterozygous for targeted deletion of the IRS-1 gene (7) were cross-bred with the transgenic mice that overexpress a metallothionein-1 promoter-driven human IGF-I transgene (2, 3, 4). Subsequent cross-breeding of IRS-1-/+/IGF+ and IRS-1-/+/WT mice generated animals that overexpress IGF-I on a background of zero, one, or two copies of the IRS-1 gene and mice of the same IRS-1 genotype that do not express the IGF-I transgene. Comparisons of IGF-I transgene induced body and organ overgrowth in these mice reveal that absolute IRS-1 deficiency does induce resistance to the growth-promoting actions of IGF-I in vivo, but that these effects are organ specific. Some, but not all, organs can use alternate pathways to mediate IGF-I action.
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Materials and Methods |
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Cross-breeding of metallothionein-hIGF-I transgenic mice
(IGF+) and IRS-1-/+ mice
Colonies of mice with germline integration of a human IGF-I
transgene driven by the mouse metallothionein 1 promoter
(IGF+) are established in the laboratory. Postnatal body
and organ growth in these mice have been described in detail (2, 3, 4).
The IGF+ mice were maintained by breeding hemizygous male
transgenics and WT females and were identified by PCR of tail DNA
(2, 3, 4). Initially, IRS-1-/+ females were bred with
IGF+ hemizygous males to generate mice that are
IRS-1-/+ and hemizygous for the IGF-I transgene
(IRS-1-/+/IGF+).
IRS-1-/+/IGF+ males or females were
then bred with IRS-1-/+ mice. This cross-breeding strategy
yielded mice with zero, one, or two copies of the IRS-1 gene that are
either IGF-I transgenic (IGF+) or lack the IGF-I
transgene (WT). Body growth curves and organ weights were compared in
IGF+ and WT mice of each IRS-1 genotype to assess
whether IRS-1 is required for IGF-I-induced growth in vivo
and whether absolute or partial IRS-1 deficiency alters the magnitude
of IGF-I-induced growth. IRS-1 deficiency leads to growth retardation
in utero as well as postnatally (7, 8). It has been
established previously that overexpression of the metallothionein-1
promoter-driven IGF-I transgene influences body and organ growth only
in postnatal life (2, 3, 4). Thus, by analyzing IGF+
and WT mice that have the same IRS-1 genotype, the magnitude of
IGF-I-induced postnatal growth was assessed in animals that shared the
same prenatal growth effects of IRS-1 status. The IGF-I transgene is
constitutively expressed (2, 3, 4), but all mice were given 25
mM zinc sulfate in drinking water at weaning to ensure
maximum expression of the IGF-I transgene in the
IGF+ mice (2, 4). The genotype of the cross-bred
animals was established by PCR of tail DNA (2, 3, 4). Northern blot
hybridization of total RNA from the intestine, a known strong site of
transgene expression (4), was used to confirm IGF-I transgene
expression in IGF+ mice (data not shown).
Body and organ weights
Body weights were measured from day 12 after birth until animals
were killed at 50–75 days of age. Mice were anesthetized using
ketamine hydrochloride (900 µg/g BW) and xylazine hydrochloride (20
µg/g BW). Blood was collected by cardiac puncture. Kidney, brain,
small intestine (from ligament of Treitz to ileocecal valve), spleen,
gastrocnemius muscle, heart, and liver were dissected, and the weights
were recorded. Organs were frozen in liquid nitrogen and stored at -80
C for subsequent analyses.
The animal studies were approved by the institutional animal care and use committee of the University of North Carolina-Chapel Hill. Study protocols were in compliance with the Guide for the Care and Use of Laboratory Animals published by the NIH.
RIA of IGF-I
Plasma samples were extracted with acid-ethanol to remove
IGF-binding proteins (IGFBPs), and IGF-I concentrations were measured
by RIA using an antibody that recognizes human and rodent IGF-I as
previously described (11, 12). Inter- and intraassay variabilities,
assessed as the coefficient of variation for values obtained from
repeated assays of internal control plasma samples, were 19% and 16%,
respectively.
RNA extraction and Northern hybridization
Tissues were homogenized in guanidine thiocyanate, and total RNA
was pelleted over 5.7 M CsCl, then collected by ethanol
precipitation as previously described (13). Liver RNAs were analyzed
for expression of endogenous IGF-I mRNA using a rat IGF-I complementary
RNA (cRNA) probe (14). Intestine RNAs were analyzed for transgene
expression using a human IGF-I cRNA probe (4). The Northern blot
hybridization methods were described previously (4, 14). Blots were
reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cRNA
probe (Ambion, Inc., Austin, TX) to control for RNA
loading. RNA abundance was quantified by densitometry and Image Pro
software.
Statistics
All values were expressed as the mean ± SE. In
the initial study to assess whether the body or organ weights in
IRS-1-/- and IRS-1+/- mice differed
significantly from values in IRS-1+/+ littermates, body and
organ weights in individual IRS-1-/- or
IRS-1-/+ mice were expressed as a ratio of the values in
sex-matched IRS-1+/+ littermates. These ratios were
analyzed by the Mann-Whitney U test for independent groups to test for
a significant difference from 1. The organ weight ratios in
IRS-1-/- or IRS-1+/- mice were also compared
with body weight ratios by the Mann Whitney U test to assess whether
IRS-1 gene deletion had differential effects on the weight of a
particular organ relative to the effect on body weight. Plasma IGF-I
levels in IRS-1-/- and IRS-1+/- mice were
compared with values in IRS-1+/+ littermates by Student’s
t test. In the second study of mice derived from
cross-breeding of IGF+/IRS-1+/- and
IRS-1+/-/WT mice, a three-factor mixed model ANOVA was
used to analyze the body weights of IGF+ and WT
mice with different IRS-1 genotypes. In this case IGF-I transgene and
IRS-1 gene deletion were the between-subject factors, and body weight
was the repeated measure. Subsequent analysis of body weights at each
time in development was performed by two-way ANOVA. Organ weights in
the six groups of mice were compared by two-factor ANOVA. Tukey’s test
was used for post-hoc pairwise comparisons of
IGF+ and WT mice of each IRS-1 genotype.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 1
shows organ weights in adult
IRS-1-/- and IRS-1+/- mice expressed as a
ratio of the corresponding organ weights in age- and sex-matched
IRS-1+/+ littermates. All organs examined showed a
significant reduction in weight in IRS-1-/- and
IRS-1+/- mice relative to IRS-1+/+
littermates, but the magnitude of the reduction differed across organs.
In IRS-1-/- mice, gastrocnemius muscle showed the
greatest reduction in weight, and this reduction was proportionately
greater than the decrease in body weight. Heart, liver, and spleen of
IRS-1-/- mice showed major reductions in weight that were
directly proportional to the decrease in body weight. The weights of
brain and small intestine were the least affected in
IRS-1-/- mice, and reductions in weight of brain, small
intestine, and kidney in IRS-1-/- mice were significantly
smaller than reductions in body weight. In IRS-1-/+ mice
there were small, but significant, reductions in the weights of all
organs that were proportional to the small reduction in body
weight.
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).
Liver is a major site of IGF-I synthesis, and the abundance of liver
IGF-I mRNAs is highly dependent on GH (3). GH together with IGF-I are
the major determinants of body growth (3). GH activates IRS-1 (10). The
abundance of IGF-I mRNAs was measured in liver of
IRS-1-/-, IRS-1-/+, and IRS-1+/+
littermates as an indirect measure of whether GH induction of IGF-I
mRNAs was impaired in liver of IRS-1-deficient mice. There were no
significant differences in abundance of the two major 7.5- and 1.2-kb
size classes of liver IGF-I mRNA in IRS-1-/- or
IRS-1-/+ mice compared with IRS-1+/+
littermates (Fig. 1).
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. All
genotypes were represented at approximately the predicted frequency,
except IRS-1-/-/WT and
IRS-1-/-/IGF+ mice, which were
represented at a slightly lower frequency than anticipated. Sex
distribution within each genotype approximated 50% males and females
as would be expected. It should be noted that the relative frequencies
of different genotypes in cross-bred mice did not permit us to limit
comparisons to sex-matched littermates.
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Follow-up two-way ANOVA was performed to assess whether IRS-1 gene
deletion affected IGF-I-induced body growth at particular times in
development. This analysis revealed both age- and sex-specific
differences. Males showed significant transgene x deletion
interactions (P < 0.05) only at times between days 12
and 28 after birth, indicating that IRS-1 gene deletion affects
IGF-I-induced body growth in males only at early times in postnatal
development (Fig. 2A). In contrast,
females showed significant transgene x deletion interactions at
all times in development, indicating that IRS-1 gene deletion affects
IGF-I-induced body growth in females throughout postnatal life (Fig. 2B).
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, A and B) illustrates these differences. As
previously reported, a major difference in body weight between male
IRS-1+/+/IGF+ and
IRS-1+/+/WT mice becomes evident between 21 and 28 days of
age immediately after zinc induction of maximum IGF transgene
expression (2, 3, 4). IRS-1+/+/IGF+
males then remain significantly larger than IRS-1+/+/WT
mice throughout adulthood. Similarly,
IRS-1+/-/IGF+ males showed dramatic
increases in body growth between 21 and 50 days of age, and growth
curves for IRS-1+/-/IGF+ and
IRS-1+/+/IGF+ males were almost
identical during this period (Fig. 2A). These data indicate that the
loss of one copy of the IRS-1 gene is without effect on IGF-I-induced
body overgrowth. IRS-1-/-/IGF+
males also showed higher mean body weight than
IRS-1-/-/WT from day 28 up to 50 days of age. The
acceleration of growth in
IRS-1-/-/IGF+ males between days 21
and 28 was, however, not as dramatic as that in
IRS-1+/+/IGF+ or
IRS-1+/-/IGF+ mice. Later in
development, the rate of growth of
IRS-1-/-/IGF+ males more
closely approximated that of
IRS-1+/+/IGF+ and
IRS-1-/+/IGF+ mice, and statistical
analyses revealed no significant transgene x deletion interaction
after day 28 (Fig. 2A). These data support the conclusion that absolute
IRS-1 deficiency attenuates IGF-I-dependent body overgrowth in males,
particularly at early times in postnatal development before
adulthood.
Female IRS-1+/+/IGF+ mice, like
males, showed a major acceleration in body growth relative to
IRS-1+/+/WT animals between days 21 and 28 days of age and
after zinc induction of maximum transgene expression and remained
heavier than IRS-1+/+/WT mice throughout the
period studied (Fig. 2B). Even though IRS-1+/-/WT and
IRS-1+/+/WT females had virtually superimposable
growth curves, body weight in
IRS-1+/-/IGF+ females was
significantly lower than that in
IRS-1+/+/IGF+ females from 28–50
days of age. Thus, the loss of one copy of the IRS-1 gene greatly
attenuated IGF-I-induced body overgrowth in females. Impaired IGF-I
action was even more pronounced in females with absolute IRS-1
deficiency. Post-hoc Tukey’s analysis after two-way ANOVA
revealed no statistically significant increase in body weight in
IRS-1-/-/IGF+ females compared
with IRS-1-/-/WT females at any stage in development, and
mean body weight in IRS-1-/-/IGF+
females became only slightly higher than that in
IRS-1-/-/WT by 50 days after birth (Fig. 2B).
Neither male nor female IRS-1-/-/IGF+ mice achieved the same body weight as IRS-1+/+/WT by 50 days of age, demonstrating that IGF-I overexpression in the period between birth and maturity cannot elicit catch-up growth to compensate for the growth retardation that results from absolute IRS-1 deficiency.
IGF-I transgene expression elevates plasma IGF-I in all IRS-1
genotypes
Two-way ANOVA revealed significant main effects of the IGF-I
transgene (F1,44 = 97.6; P < 0.001)
and IRS-1 gene deletion (F2,44 = 8.0;
P = 0.001) on plasma IGF-I concentrations and a
significant transgene x deletion interaction
(F2,44 = 7.8; P < 0.001).
Post-hoc comparisons revealed that plasma IGF-I levels were
significantly elevated in all animals expressing the IGF-I transgene
relative to those in mice of the corresponding IRS-1 genotype that lack
the transgene (Table 4). Plasma IGF-I
levels were, in fact, significantly higher in
IRS-1-/+/IGF+ and
IRS-1-/-/IGF+ mice than in
IRS-1+/+/IGF+ mice (P
< 0.002). Reduced circulating concentrations of IGF-I therefore cannot
account for the reduced growth rates in IGF+ mice
that lack one or both copies of the IRS-1 gene. We have no evidence to
indicate that sex-specific differences in plasma IGF-I concentrations
in IRS-1-/-/IGF+ or
IRS-1-/+/IGF+ mice account for the
more pronounced effect of IRS-1 deficiency on IGF-I-induced body
overgrowth in females than males. Plasma IGF-I concentrations in male
IRS-1-/+/IGF+ mice (511 ± 31
ng/ml; n = 5) did not differ significantly from values in female
IRS-1-/+/IGF+ mice (564 ± 42
ng/ml; n = 5). Due to sampling difficulties in
IRS-1-/- mice we do not have sufficient numbers of
IRS-1-/-/IGF+ mice to make
meaningful statistical comparisons between plasma levels of IGF-I
in males vs. females, but the available data suggest that
plasma levels of IGF-I are similar in both sexes. We note that plasma
IGF-I levels in IRS-1-/-/WT and
IRS-1+/+/WT mice from the cross-bred colony were somewhat
lower than those in mice of the same genotype derived from the initial
IRS-1-/+ founders (compare Tables 2 and 4). Samples from
the two groups were assayed at different times, so that interassay
variability probably accounts for this difference.
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and 6 and
Fig. 3, A and B. Tables 5 and 6 show the
mean organ weights in male and female mice of each genotype. The tables
include the results of post-hoc pairwise comparisons to
assess whether IGF+ mice showed significant
increases in organ weight relative to WT mice of the same IRS-1
genotype and thereby establish whether the loss of one or both copies
of the IRS-1 gene prevents IGF-I-induced organ overgrowth. Figure 3, A
and B, shows the increase in mean weight of each organ in
IGF+ mice with two, one, or zero copies of the
IGF-I gene, expressed as the percent increase relative to mean organ
weight in WT mice of the same IRS-1 genotype. Figure 3, A and B, serve
to illustrate that loss of one or both copies of the IGF-I gene alters
the magnitude of IGF-I-induced growth in some, but not all, organs
examined and includes the outcome of two-way ANOVA tests for
transgene x deletion interactions that provide statistical
validation of these effects.
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and Fig. 3A). The weights of these organs in
IRS-1-/-/IGF+ males were similar to
or greater than organ weights in IRS-1+/+/WT (Table 5).
Together, these data suggest that in males, IRS-1 is not required for
IGF-I-induced growth of kidney, brain, small intestine, and spleen and
that IGF-I overexpression can completely correct the growth deficit in
these organs that results from absolute IRS-1 deficiency. In kidney,
IRS-1 gene copy number had no effect on the magnitude of IGF-I-induced
overgrowth, indicating that in males, the normal trophic effects of
IGF-I on kidney are unaffected by partial or absolute IRS-1 deficiency
(Table 5 and Fig. 3A). In, small intestine and spleen there was a
reduction in the magnitude of IGF-I-induced growth in mice with one
copy of the IRS-1 gene and a further reduction in mice with zero copies
of the IRS-1 gene. In these organs, therefore, although IRS-1 is not
required for IGF-I-induced organ growth, there is some dose dependence
on IRS-1 for maximum growth effects of IGF-I. Gastrocnemius muscle
showed a different response, in that IGF-I overexpression induced
significant muscle growth in males with two copies of the IRS-1 gene,
but the growth effect was greatly attenuated in mice that lacked one or
both copies of the IRS-1 gene (Table 5). Muscle weight did not differ
significantly between IRS-1+/-/IGF+
and IGF+/-/WT or between
IRS-1-/-/IGF+ and
IRS-1-/-/WT males. Muscle weight was lower in
IRS-1-/-/IGF+ than in
IRS-1+/+/WT males (Table 5). These data provide evidence
that IRS-1 is required for IGF-I-induced growth of gastrocnemius
muscle. IGF-I overexpression induced significant increases in heart
weight in male mice with two copies or one copy of the IRS-1 gene. The
mean heart weight in IRS-1-/-/IGF+
males was higher than that in IRS-1-/-/WT (Table 5), but
this did not achieve statistical significance (P =
0.188). In males, the similar magnitude of IGF-I-induced growth of
heart regardless of IRS-1 status and the lack of a significant
transgene x deletion interaction (Fig. 3A) provide evidence that
IGF-I-induced heart growth does not require IRS-1 or show dose
dependence on IRS-1 in males. Analyses of liver weights in
IGF+ and WT mice with different IRS-1 gene copy
numbers indicate that IGF-I has a modest effect on liver growth
regardless of IRS-1 status (Table 5 and Fig. 3A).
Compared with males, female mice that have two copies of the IRS-1 gene
showed a similar magnitude of IGF-I-induced overgrowth of all organs
studied except heart, where the effect was more pronounced than in
males (Table 6 and Fig. 3B). This is important because it suggests that
any differential effect of IRS-1 deficiency on IGF-I-induced growth
reflects a differential dependence on IRS-1 as a mediator of IGF-I
action rather than sex-specific differences in the trophic effects of
IGF-I. In females, effects of partial or absolute IRS-1 deficiency on
IGF-I-induced growth of brain, small intestine, and spleen were similar
to those observed in males. IGF-I overexpression induced major
increases in growth of these organs regardless of IRS-1 status, but
showed dose dependence on IRS-1 to elicit maximum growth effects (Table 6 and Fig. 3B). Females differed from males in that kidney and heart
showed clear dose dependence on IRS-1 for IGF-I-induced growth (Table 6
and Fig. 3B). As in males, the data in females provide evidence that
IGF-I-induced growth of gastrocnemius muscle has an absolute
requirement for IRS-1 (Table 6 and Fig. 3A), although, surprisingly,
there were not significant transgene x deletion interactions
(P = 0.178) for muscle in females. Nonetheless, the
similar muscle weights in
IRS-1-/-/IGF+ and
IRS-1-/-/WT mice (Table 6) support the conclusion that
IRS-1 is required for IGF-I-induced muscle growth in females. In
females, there was an increase in liver weight in
IRS-1+/+/IGF+ females that did not
reach statistical significance (P < 0.076), suggesting
that IGF-I may have a minor effect on liver growth in females. A lesser
effect of IGF-I overexpression on liver weight in mice with one or zero
copies of the IRS-1 gene also suggests that IGF-I action in liver may
show modest dependence on IRS-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address whether complete or partial IRS-1 deficiency alters IGF-I-dependent postnatal body growth or growth of brain, skeletal muscle, and other organs in vivo, the current study developed mice that overexpress IGF-I in postnatal life on an IRS-1+/+, IRS-1+/-, or IRS-1-/- background. Our findings that IRS-1-/-/IGF+ mice remain smaller than IRS-1+/+/WT mice up to adulthood demonstrate that IGF-I overexpression cannot correct the body growth deficit that results from absolute IRS-1 deficiency. Observations that male and female mice with zero copies of the IRS-1 gene show impaired ability of IGF-I to induce postnatal body overgrowth provide conclusive evidence that absolute IRS-1 deficiency causes resistance to IGF-I action in vivo. IGF-I-induced body overgrowth was, however, impaired more in IRS-1 null females than in males, and loss of one copy of the IRS-1 gene reduced IGF-I-induced body overgrowth in postweaning females, but not in males. These findings are of interest in light of recent evidence that estrogen induces IRS-1 expression and enhances IGF-I-dependent IRS-1 tyrosine phosphorylation in breast (18) and uterus (19) and that this contributes to the synergistic growth effects of estrogen and IGF-I. The present findings indicate that IRS-1 may have more widespread relevance as a mediator of estrogen/IGF interactions to regulate normal body or organ growth in vivo.
It is noteworthy that IRS-1+/-/IGF+ and IRS-1-/-/IGF+ mice had higher levels of plasma IGF-I than IRS-1+/+/IGF+ mice. This demonstrates that impaired body growth in IRS-1+/-/IGF+ and IRS-1-/-/IGF+ mice does not reflect reduced plasma IGF-I but, rather, impaired IGF-I action. We considered the possibility that elevated plasma IGF-I levels in IRS-1+/-/IGF+ and IRS-1-/-/IGF+ mice could reflect some change in circulating IGFBPs that can alter the half-life of IGF-I in the circulation (7, 8, 20). Preliminary radioligand blot analyses provide no indication that IGFBP levels differ in IRS-1+/- and IRS-1-/- mice relative to those in IRS-1+/+ mice regardless of whether the mice express the IGF-I transgene or are WT (our unpublished observations). Definitive analyses of the effect of IRS-1 deficiency on circulating or tissue IGFBPs will require Western immunoblot or more quantitative analyses by specific RIAs, which are ongoing in our laboratories.
Analyses of organ weights indicate organ-specific differences in the role of IRS-1 as a mediator of IGF-I action. IGF-I-dependent growth of gastrocnemius muscle appeared the most impaired of all organs studied, providing direct support for the hypothesis that IRS-1 is a primary mediator of IGF-I-induced growth of skeletal muscle. In both males and females, gastrocnemius muscle appears to require IRS-1 for IGF-I-induced growth, because there was little or no growth effect of IGF-I on gastrocnemius muscle in IRS-1 null mice. At present, the cellular and molecular bases for the effects of IRS-1 deficiency to impair muscle growth and to specifically impair IGF-I-induced growth are not defined. IGF-I and IGF1R null mice show hypoplasia of skeletal muscle and reduced cell size (16, 17). IGF-I and IGF-II are expressed in satellite cells and proliferating myoblasts in developing or regenerating muscle and are important mediators of myoblast proliferation and differentiation (21). Differentiative effects are associated with induction of the myogenin gene (21). IRS-1 deficiency thus may impair IGF-I-dependent myoblast proliferation, protein synthesis, or myogenin gene expression. The cross-bred mice developed in the present study provide useful models for future studies to define the components of IGF-I-dependent skeletal muscle growth that require IRS-1. IRS-2 is a major alternative substrate for the IGF1R (6), and prior studies have shown that insulin-stimulated tyrosine phosphorylation of IRS-2 is enhanced in muscle of IRS-1 null mice (9, 22). The present observations indicate, however, that this enhanced IRS-2 activation is not sufficient to mediate normal IGF-I-dependent growth of skeletal muscle in vivo. These observations in skeletal muscle are consistent with recent observations in skin fibroblasts from IRS-1 null mice, in which impaired mitogenic responses to IGF-I cannot be normalized by overexpression IRS-2 (23).
In both male and female IRS-1 null mice IGF-I overexpression induced dramatic overgrowth of brain, small intestine, and spleen and completely corrected the growth deficit due to absolute IRS-1 deficiency. In these organs, therefore, IRS-1 is not required for IGF-I action, and IGF-I must be able to activate signaling molecules other than IRS-1 to elicit organ growth. In brain, small intestine, and spleen enhanced expression or activation of IRS-2 or other signaling molecules, such as Shc, may permit IGF-I-induced growth. In this regard it is of interest that one report demonstrates high level expression of IRS-2 relative to IRS-1 mRNA in brain (24), the organ whose growth was shown here to be the least affected by IRS-1 deficiency. Recent studies indicate that Shc expression and activation are increased in chick hepatoma cells made deficient in IRS-1 by antisense strategies (25). Other studies in CHO cells demonstrate that the levels of IRS-1 expression alter downstream signaling, such that IRS-1 overexpression decreases the levels of Shc associated with growth factor receptor bound protein-2 (26). It will be of interest to establish whether organ-specific differences in dependence on IRS-1 for IGF-I-induced growth in vivo correlate with organ-specific differences in the levels of expression or IGF-dependent activation of IRS-2, Shc, or other signaling molecules.
Although IRS-1 is not required for IGF-I-induced growth of small intestine and spleen, there was a dose-dependent decrease in the magnitude of IGF-I-dependent growth in mice with two, one, or zero copies of the IRS-1 gene. Thus, IRS-1 deficiency compromises the maximum growth effects of IGF-I in these organs. This suggests that even though the IGF-1R can use signaling molecules other than IRS-1 to elicit growth of small intestine and spleen, a component of IGF-I-induced growth requires normal levels of IRS-1. Dose dependence could exist because only particular cell populations within an organ show impaired IGF-I-induced growth as a result of partial or absolute IRS-1 deficiency or because levels of IRS-1 are rate limiting for IGF1R coupling to IRS-1.
Kidney and heart showed sex-specific differences in the requirement for IRS-1 as a mediator of IGF-I action. In males, IGF-I-induced growth of these tissues was not compromised by partial or absolute IRS-1 deficiency, indicating that in males, these tissues share the ability of brain, small intestine, and spleen to use signaling molecules other than IRS-1 to mediate IGF-I-dependent growth. In females, however, there was clear dose dependence of heart and kidney on IRS-1 for IGF-I-mediated growth. This together with a lack of significant growth of these organs in IRS-1 null females suggests that heart and kidney have impaired ability to use alternate signaling molecules to mediate IGF-I-dependent growth in females. It seems possible that IGF-I may interact with estrogen to regulate kidney and heart growth in an IRS-1-dependent manner, as observed in uterus and breast (18, 19). These findings in kidney and heart indicate that the role of IRS-1 as an important mediator of estrogen/IGF interactions to regulate growth of particular organs females in vivo warrants further investigation.
GH interacts with IGF-I to regulate postnatal growth (1, 3, 10). GH is a primary regulator of IGF-I synthesis in the liver and of the circulating concentration of IGF-I (1, 4). Impaired GH action or GH induction of hepatic IGF-I synthesis in liver could contribute to the growth retardation in IRS-1 null mice. Our observations that liver IGF-I mRNAs and plasma levels of IGF-I are normal in IRS-1 null mice indicate that reduced hepatic IGF-I synthesis or reduced plasma IGF-I do not contribute significantly to the growth deficit in IRS-1 null mice and provide indirect evidence that GH action to induce IGF-I synthesis in liver is not impaired by IRS-1 deficiency. At present, we have no evidence about the effects of IRS-1 deficiency on IGF-I synthesis in nonhepatic tissues.
The present findings that liver weight is reduced in IRS-1 null mice indicate that normal growth of liver is dependent on IRS-1. IGF-I is not generally considered a major mediator of normal liver growth, especially in the postnatal or adult liver, where there are few IGF1R (27, 28). This concept is supported by the current findings that IGF-I overexpression had little effect on liver size regardless of IRS-1 status. Reduced liver weight in IRS-1 null mice may reflect impaired actions of hormones other than IGF-I. GH is known to stimulate hepatic growth (29). Observations that plasma IGF-I and hepatic IGF-I mRNA are normal in IRS-1 null mice provide indirect evidence that GH action is not impaired in liver of IRS-1 null mice. Preliminary data indicate that mice overexpressing a bovine GH transgene on an IRS-1 null background show similar liver overgrowth as GH transgenics with two copies of the IRS-1 gene (Lund, P. K., unpublished observations). Together, these observations indicate that hepatic growth effects of GH are normal in mice with complete IRS-1 deficiency. At present, therefore, the hepatic growth deficiency in IRS-1 null mice cannot be attributed to defects in signaling by a particular ligand or receptor.
In summary, our findings demonstrate that normal IGF-I-dependent increases in body weight postnatally require IRS-1, and the dependence on IRS-1 is greater in females than in males. In vivo, normal IGF-I-induced growth of skeletal muscle requires IRS-1 in both males and females. In brain, small intestine, and spleen of both sexes, major IGF-I-dependent increases in organ size occur even on a background of complete IRS-1 deficiency, and IGF-I overexpression can completely reverse the in vivo growth deficits that occur due to IRS-1 deficiency. Thus, these organs must use signaling molecules other than IRS-1 to mediate IGF-I action. Other organs, such as kidney and heart, show sexually dimorphic effects of IRS-1 deficiency, such that IGF-I-dependent growth is affected more in IRS-1-deficient females than in males. The mouse models developed here will prove useful in future studies to elucidate the organ-specific, IRS-1-dependent and -independent pathways that mediate IGF-I-induced growth of various organs in vivo.
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Received March 11, 1999.
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