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Endocrinology Vol. 140, No. 12 5478-5487
Copyright © 1999 by The Endocrine Society

ARTICLES

Postnatal Growth Responses to Insulin-Like Growth Factor I in Insulin Receptor Substrate-1-Deficient Mice1

Gina Pete, C. Randall Fuller, Jenny M. Oldham, Dani R. Smith, A. Joseph D’Ercole, C. Ronald Kahn and P. Kay Lund

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Organ weight was compared in adult mice with deletion of one (IRS-1-/+) or both (IRS-1-/-) copies of the insulin receptor substrate-1 (IRS-1) gene and IRS-1+/+ littermates. IRS-1-/+ mice showed modest reductions in weight of most organs in proportion to a decrease in body weight. IRS-1-/- mice showed major reductions in weight of heart, liver, and spleen that were directly proportional to a decrease in body weight. In IRS-1-/- mice, kidney and particularly small intestine and brain exhibited proportionately smaller weight reductions, and gastrocnemius muscle showed a proportionately greater weight reduction than the decrease in body weight. Growth deficits in IRS-1-/- mice could reflect impaired actions of multiple hormones or cytokines that activate IRS-1. To assess the requirement for IRS-1 in insulin-like growth factor I (IGF-I)-dependent postnatal growth, IRS-1-/+ mice were cross-bred with mice that widely overexpress a human IGF-I transgene (IGF+) to generate IGF+ and wild-type mice on an IRS-1+/+, IRS-1-/+, and IRS-1-/- background. IGF-I overexpression increased body weight and weight of brain, small intestine, kidney, spleen, heart, and gastrocnemius muscle in IRS-1+/+ mice. IGF-I overexpression could not completely reverse the body growth retardation in IRS-1-/- mice. Absolute or partial IRS-1 deficiency impaired IGF-I-induced body overgrowth more in females than in males. In males and females, IGF-I stimulated similar overgrowth of brain regardless of IRS-1 status, and intestine and spleen showed dose dependence on IRS-1 for IGF-I-induced growth. IGF-I-induced growth of gastrocnemius muscle had an absolute requirement for IRS-1. IGF-I-induced growth of kidney and heart was impaired by IRS-1 deficiency only in females. In vivo, therefore, most organs do not require IRS-1 for IGF-I-induced growth and can use alternate signaling molecules to mediate IGF-I action. Other organs, such as gastrocnemius muscle, require IRS-1 for IGF-I-induced growth in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) plays a key role in regulating body and organ growth during postnatal development (1). Serum levels of IGF-I correlate with postnatal increases in body weight gain and linear growth in humans and rodents (1). Transgenic mice that overexpress a metallothionein promoter-driven human IGF-I transgene show modest increases in plasma levels of IGF-I and exhibit widespread overexpression of the IGF-I transgene in multiple tissues (2, 3, 4). The IGF-I transgenic mice show increased body weight gain from about 2 weeks of age through adulthood and have significant increases in the sizes of a number of organs (2, 3, 4).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice with targeted deletion of the IRS-1 gene
Founder mice heterozygous for targeted disruption of the IRS-1 gene (IRS-1-/+) were created as previously described (7). IRS-1-/+ males and females were bred to derive IRS-1+/+, IRS-1-/+, and IRS-1-/- littermates for initial analyses and for further cross-breeding. The genotype of the mice was identified by PCR of tail DNA. PCR was performed as previously reported using oligomers ML-1 and JB-1 that amplify the WT IRS-1 gene and oligomers Neo and JB-1 that amplify the disrupted IRS-1 gene (7). Tissues from these initial litters were used to establish whether the loss of one or both copies of the IRS-1 gene had selective effects on the growth of different organs and whether growth deficits were associated with changes in plasma IGF-I concentrations or changes in the expression of liver IGF-I messenger RNAs (mRNAs). In this study IRS-1-/- or IRS-1-/+ mice were directly compared with sex-matched IRS-1+/+ littermates to eliminate interlitter variation and to maximize our ability to detect differences.

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body growth and organ-specific growth effects in mice with targeted deletion of the IRS-1 gene
Male and female mice showed essentially the same effect of IRS-1 gene deletion on adult body weight. At 50–60 days of age, body weights in females (expressed as a ratio of weights in sex-matched IRS-1+/+ littermates) were 0.60 ± 0.02 (P < 0.001) in IRS-1-/- mice and 0.90 ± 0.001 (P < 0.001) in IRS-1+/- mice. The corresponding values in males were 0.54 ± 0.01 (P < 0.001) in IRS-1-/- mice and 0.92 ± 0.02 (P < 0.001) in IRS-1+/- mice. The reduction in body weight in IRS-1-/- mice is consistent with previous observations (7). The small, but significant, body weight reduction in IRS-1-/+ mice contrasts with previous reports that the loss of one copy of the IRS-1 gene has no effect on body weight and probably reflects the fact that prior data compared body weights for males and females across litters (7). There were interlitter differences in body weight that would be sufficient to mask the small body weight reduction observed here between age- and sex-matched IRS-1+/- and IRS-1+/+ littermates.

Table 1Go 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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  		Table 1. The effect of IRS-1 deficiency on organ weight

 
Plasma IGF-I and liver IGF-I mRNAs are unaffected by IRS-1 gene deletion
Plasma IGF-I concentrations were measured to assess whether altered circulating concentrations of IGF-I might contribute to growth deficits in IRS-1-deficient mice. Plasma IGF-I concentrations in IRS-1-/- and IRS-1-/+ mice did not differ significantly from values in IRS-1+/+ littermates, although there was a trend for IRS-1-/- mice to show a small reduction in plasma IGF-I (Table 2Go). 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. 1Go).


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  		Table 2. The effect of IRS-1 deficiency on plasma IGF-I concentrations (nanograms per ml)

 


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  		Figure 1. Abundance of liver IGF-I mRNAs in adult, sex-matched IRS-1-/-, IRS-1-/+, and IRS-1+/+ littermates. A, A representative Northern blot of liver IGF-I mRNAs probed with a 32P-labeled rat IGF-I cRNA and a reprobe of the same blot with GAPDH cRNA. B, A histogram of the abundance (arbitrary units) of two major 7.5- and 1.2-kb IGF-I mRNA variants that differ in length of the 3'-untranslated region (30 ) normalized to the abundance of GAPDH mRNA. Data are the mean ± SE (n = 12 for IRS-1+/+ and IRS-1+/- mice; n = 8 for IRS-1-/- mice).

 
Frequency of genotypes derived from cross-breeding IRS-1-/+/IGF+and IRS-1-/+/WT mice
Cross-breeding of IRS-1-/+/IGF+ and IRS-1-/+/WT mice yielded six different genotypes, and the anticipated frequencies of these six genotypes are compared with actual frequencies in Table 3Go. 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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  		Table 3. Frequency of genotypes from cross-breeding IRS-1+/-/IGF+ and IRS-1+/-/WT mice

 
Growth retardation in IRS-1-deficient mice is not reversed by IGF-I overexpression
Repeated measures ANOVA of body weights measured from 12–50 days of age in IRS-1+/+/IGF+, IRS-1+/+/WT, IRS-1-/+/WT, and IRS-1-/+/IGF+, IRS-1-/-/IGF+, and IRS-1-/-/WT revealed significant main effects of the IGF-I transgene (F1,34 = 54.8; P < 0.001 in males; F1,27 = 58.2; P < 0.001 in females) and IRS-1 gene deletion (F2,34 = 203.1; P < 0.001 in males; F2,27 = 114.4; P < 0.001 in females). There was also a significant interaction between IGF-I transgene and IRS-1 gene deletion in both sexes (F14, 238 = 5.0; P < 0.001 in males; F14,189 = 9.3; P < 0.001 in females), indicating that IRS-1 gene deletion alters IGF-I-induced body growth in both male and female mice.

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. 2AGo). 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. 2BGo).



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  		Figure 2. Postnatal body weights in male (A) or female (B) IRS-1+/+/IGF+, IRS-1+/+/WT, IRS-1+/-/IGF+, IRS-1+/-/WT, IRS-1-/-/IGF+, and IRS-1-/-/WT mice. Data are presented as the mean ± SE, and the number of animals is shown for each genotype. The arrow at day 21 indicates the time when 25 mM zinc sulfate was added to the drinking water. The outcome of post-hoc two-way ANOVA comparisons are shown. Asterisks indicate statistically significant (P < 0.05) main effects of the IGF-I transgene, main effects of IRS-1, or significant transgene x deletion interactions at the postnatal ages indicated. Dashes indicate no significant effect. A significant main effect of IGF-I indicates that IGF-I overexpression affected body growth in one or more of the IGF+ groups studied. Significant main effects of IRS-1 deletion indicate that IRS-1 gene deletion affected body growth in one or more of the groups studied with different IRS-1 genotypes. Significant transgene x deletion interactions indicate that IRS-1 deficiency alters the magnitude of IGF-I-dependent growth at that particular stage in development.

 
Examination of the growth curves for male or female IGF+ and WT mice with two, one, or zero copies of the IRS-1 gene (Fig. 2Go, 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. 2AGo). 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. 2AGo). 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. 2BGo). 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. 2BGo).

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 4Go). 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 2Go and 4Go). Samples from the two groups were assayed at different times, so that interassay variability probably accounts for this difference.


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  		Table 4. Plasma IGF-I concentrations (nanograms per ml) in offspring of cross-bred IRS-1+/-/IGF+ and IRS-1+/-/WT mice

 
Excess IGF-I induces selective organomegaly in IRS-1-deficient mice
A major goal in cross-breeding IGF-I transgenic mice and mice with deletion of the IRS-1 gene was to assess whether IRS-1 is required for IGF-I-induced organ growth in vivo and to determine whether partial or absolute IRS-1 deficiency altered the magnitude of IGF-I-induced organ overgrowth. Two-way ANOVA revealed significant effects of IRS-1 gene deletion (P < 0.01) in all organs examined in both sexes. Significant effects of the IGF-I transgene (P < 0.01) were observed in all organs examined in males and in all organs examined (P < 0.01), except liver (P = 0.103), in females. Transgene x deletion interactions were observed in some, but not all, organs examined, and there were some differences between the sexes, suggesting that IRS-1 gene deletion has organ- and sex-specific effects on IGF-I-induced growth. These effects are described in more detail below and are illustrated in Tables 5Go and 6Go and Fig. 3Go, A and B. Tables 5Go and 6Go 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 3Go, 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 3Go, 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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  		Table 5. The effect of IRS-1 deficiency on IGF-I-induced organ growth in males

 

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  		Table 6. The effect of IRS-1 deficiency on IGF-I-induced organ growth in females

 


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  		Figure 3. IGF-I-dependent increases in organ growth in male (A) or female (B) IGF+/IRS-1+/-, IGF+/IRS-1+/-, and IGF+/IRS-1-/- mice. Values are the increase in mean organ weight in IGF+ mice expressed as a percentage of the mean organ weight in WT mice with the same IRS-1 genotype. a, P < 0.1 (significant transgene x deletion interactions). F and P values for transgene x deletion interactions were as follows: in males: kidney, F2,36 = 0.838; P = 0.441; brain, F2,42 = 5.2; P = 0.009; small intestine (S. I.), F2,42 = 2.985; P = 0.061; spleen, F2,36 = 6.569; P = 0.004; muscle, F2,27 = 4.058; P = 0.029; liver, F2,40 = 2.225; P = 0.121; heart, F2,36 = 1.024; P = 0.369; in females: kidney, F2,23 = 3.472; P = 0.048; brain, F2,30 = 7.862; P = 0.002; small intestine (S. I.), F2,29 = 3.960; P = 0.030; spleen, F2,24 = 4.903; P = 0.016; muscle, F2,22 = 1.870; P = 0.178; liver, F2,29 = 1.648; P = 0.210; heart, F2,24 = 7.497; P = 0.003.

 
In males, IGF-I overexpression induced robust overgrowth of kidney, brain, small intestine, and spleen regardless of IRS-1 genotype (Table 5Go and Fig. 3AGo). The weights of these organs in IRS-1-/-/IGF+ males were similar to or greater than organ weights in IRS-1+/+/WT (Table 5Go). 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 5Go and Fig. 3AGo). 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 5Go). 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 5Go). 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 5Go), 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. 3AGo) 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 5Go and Fig. 3AGo).

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 6Go and Fig. 3BGo). 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 6Go and Fig. 3BGo). Females differed from males in that kidney and heart showed clear dose dependence on IRS-1 for IGF-I-induced growth (Table 6Go and Fig. 3BGo). 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 6Go and Fig. 3AGo), 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 6Go) 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.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Deletion of both copies of the IRS-1 gene induces dramatic prenatal growth retardation that persists throughout postnatal life (7, 8). The present study provides evidence that the requirement for IRS-1 as a mediator of growth in vivo is organ specific. Our findings in IRS-1-/- and IRS-1+/+ littermates indicate that gastrocnemius muscle growth is the most affected by absolute IRS-1 deficiency, whereas the small intestine and particularly the brain are the least affected. IGF-I is a major mediator of normal pre- and postnatal body and organ growth (1, 2, 3, 4), and IRS-1 is considered a major mediator of IGF-I action (5, 6, 7, 8). Deletion of both copies of the IGF-I gene results in a more severe phenotype than IRS-1 gene deletion and is accompanied by significant perinatal lethality that reflects in part impaired growth of skeletal muscle (15, 16). Brain growth is also severely affected in IGF-I null mice (15, 16, 17). These and the present findings in IRS-1-/- and IRS-1+/+ littermates support the hypothesis that normal growth of both brain and skeletal muscle in vivo requires IGF-I, but that brain may use signaling molecules other than IRS-1 to mediate IGF-I-induced growth, whereas muscle may not. Comparisons between IRS-1 null and IGF-I null mice cannot, however, provide definitive evidence in support of this hypothesis, because body and organ growth retardation in IRS-1 null mice could reflect the impaired actions of other hormones or cytokines that activate IRS-1, including GH or insulin (5, 6, 10).

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.


   Acknowledgments
 
The authors thank Drs. Jens Bruning and Eiichi Araki for provision of IRS-1 null heterozygote breeding pairs and advice on breeding and genotyping. Drs. Judson Van Wyk and Neil Cox are gratefully acknowledged for useful discussions. The authors thank Mr. Chris DaCosta for technical help, Ms. Evonne Bruton for assistance with RIAs, and Ms. Eileen Hoyt and Ms. Deborah L. Carver for assistance with the preparation of the manuscript. The transgenic mouse and biostatistics cores of the Center for Gastrointestinal Biology and Disease facilitated these studies.


   Footnotes
 
1 This work was supported by NIH Grants DK-40247 (to P.K.L.), DK-33201 (to C.R.K.), AG09973 (to D.S. and P.K.L.), and HD-08299 (to A.J.D.) and the New Zealand/USA Cooperative Science Program of the ISAT Linkages Fund (to J.M.O.). Back

Received March 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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