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Clinical Endocrinology Branch, The National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1758
Address all correspondence and requests for reprints to: Dr. Derek LeRoith, CEB/National Institute of Diabetes and Digestive and Kidney Diseases, Building 10, Room 8D12, National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892-1758. E-mail: Derek{at}helix.nih.gov
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Abstract |
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Introduction |
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Several modes of GH action on postnatal body growth have been proposed (8, 9). GH may act on a major target organ, namely the liver, to stimulate the synthesis and secretion of IGF-I, which reaches its skeletal targets as a true endocrine reagent (the somatomedin hypothesis) (10). GH may stimulate longitudinal bone growth directly through local production of IGF-I (modified somatomedin hypothesis) (11, 12, 13). In addition, GH may have a direct mitogenic action on target tissues such as the chondrocyte precursor cells, or GH action may be mediated by growth factors other than IGF-I (14, 15, 16). For example, GH was found to stimulate synthesis of the bone morphogenetic proteins in the presence of IGF-I antiserum (14). A recent report from our laboratory has demonstrated that liver-derived "endocrine" IGF-I is not essential for postnatal growth and has challenged the classical somatomedin hypothesis (17). In this study, to test the direct effect of GH, we created viable dwarf mice with IGF-I deficiency by the Cre/loxP system and studied the effect of GH administration on their body growth. Our results demonstrate an essential role for IGF-I in GH-induced postnatal body growth in mice and hence support the modified somatomedin hypothesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). At that stage,
Cre-induced recombination of the igf-1 locus had occurred
(18). F2 mice were further intercrossed to generate mice (F3, L/-)
with an allele of igf-1/flox and an allele of
igf-1 null (germ line transmission from F2) which had
segregated the Cre transgene. These L/- mice were assigned to multiple
mating pairs to generate IGF-I null (-/-) mice. Among the three
genotypes generated [L/L, L/-, and -/-] and used in the current
study, L/L and L/- are virtually normal in IGF-I gene expression,
development, and growth and are treated as wild-type mice. On the other
hand, -/- (IGF-I null) mice have no IGF-I expression and demonstrate
a severe defect in intrauterine development and postnatal growth. The
animals were kept in a designated breeding room and were observed
closely throughout the experiment. Genotyping by PCR and Southern blot
analysis has been reported (18). The Animal Care and Use Committee of
the NIDDK, National Institutes of Health (Bethesda, MD) approved all of
the animal manipulations.
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Effect of GH on Jun B gene expression
To study the effect of GH on the expression of immediate early
genes, liver expression of Jun B mRNA was analyzed. Mice of wild-type
and knockout genotypes, at 6 weeks of age, were fasted overnight and
injected with rhGH (3 mg/kg, ip) or diluent. After 30 min, they were
anesthetized and bled. Total RNA was extracted from the liver to
determine the level of Jun B mRNA by RNase protection assay.
RNase protection assay
The expression of IGF-I from various tissues and expression
of Jun B and GH receptor from the liver were studied using the
RNase protection assay (19). 32P-labeled riboprobes were
made from mouse IGF-I exon 4 (18), pTRI-Jun (B)-mouse (Ambion, Inc., Austin, TX), and mouse GH receptor exon 4
(BamHI/AvaI fragment, provided by Dr. John J
Kopchick, Ohio University, Athens, OH) (2).
Hormone assay, serum chemistry, and statistics
The serum concentration of total IGF-I (Diagnostics Systems Laboratories, Inc. Webster, TX), insulin (Linco,
St-Charles, MO), and GH (Amersham Pharmacia Biotech,
Arlington Heights, IL) were determined by RIA kits available
commercially. Glucose was measured with a Glucometer Elite (Bayer Corp., Elkhart, IN). Serum chemistry was performed by the
Pathology Department at the Clinical Center of the NIH. Statistical
significance was determined by the two-tailed t test using
the program SigmaStat 2.03 (Access Softek, Inc., San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Southern blot analysis was performed
on tail DNA to identify the genotype of the offspring (data not shown).
RNase protection assay on several tissues was employed to assess the
level of IGF-I gene expression and serum IGF-I level was determined by
RIA. As shown in Fig. 2, wild-type mice
have varying amounts of IGF-I mRNA in the liver, heart, and kidney,
while IGF-I null mice have undetectable IGF-I mRNA. Their circulating
IGF-I concentration was below the detection limit (Table 1). As such, the gene deletion was
confirmed at the level of tissue DNA, mRNA, and circulating
peptide.
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). Consistent with GH hypersecretion, adult
IGF-I null mice show specific and significant increases in the relative
weight of the following organs: brain (+113%), liver (+41%), heart
(+37%), and kidney (+21%). No change was found in the weight of the
spleen (Table 2).
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). This
finding is similar to a report on severe IGF-I deficient (midi) mice
created by an insertional mutation (20). In the standard serum
chemistry assay, there was a dramatic 58% reduction in alkaline
phosphatase (Table 1), associated with the reduction in bone
growth.
Postnatal survival and growth rates
While the overall phenotype of the IGF-I null mice generated in
this study remains similar to those previously reported (4, 7),
demonstrating severe defects in prenatal development and postnatal
growth, our IGF-I null mice demonstrate improved postnatal survival
rate, which enables a more detailed postnatal study. At birth, viable
IGF-I null pups weigh 65% of their wild-type litter mates, which
decreased to 50% after 2 weeks, and then the growth curve flattened
and body weight decreased to approximately 30% of wild-type litter
mates as adults (Fig. 3). Thus, while
IGF-I is essential for both intrauterine and postnatal development, the
growth defect becomes more profound postnatally.
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). This
rate is relatively high but within the reported range (5% in C57BL/6J,
10% in 129/sv, 16% in C57BL/6J and 129/sv mix, and 68% in MF1 and
129/sv mix) (4, 7).
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). From 2 weeks on, the
IGF-I null mice grow more slowly and have delayed onset of the
prepubertal growth and do not undergo pubertal changes. Their final
body weight becomes approximately one third and body length about two
thirds of their wild-type litter mates as adults. As shown in Fig. 4
and Table 2, continuous injection of rhGH did not affect their growth
rate at all, when measured both by weight and length. In contrast,
wild-type mice receiving the same doses of rhGH exhibited accelerated
growth starting at P21 and continuing to P56, where they showed a 29%
increase in body weight and 12% in body length in comparison to those
treated with diluent alone.
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). As stated earlier, IGF-I null mice have enlarged
brain, liver, heart, and kidney and GH injection caused a further
increase of 16% in liver weight (P < 0.05) (Table 2).
Therefore, GH induced liver enlargement despite the IGF-I
deficiency.
GH receptor expression in the liver
To exclude the possibility that the lack of growth responsiveness
to GH is due to a decrease in GH receptor expression in the face of
IGF-I deficiency, we performed RNase protection assays from mouse liver
at the end of the period of GH injections. As shown in Fig. 5, hepatic GH receptor mRNA level was
affected neither by IGF-I deficiency nor by GH treatment.
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). Thus, GH actions (on Jun B expression
and liver enlargement) are not affected by IGF-I deficiency. It further
demonstrates that GH was functional in IGF-I null mice although it
failed to increase body growth.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to the previously reported phenotype of pre- and postnatal growth retardation, perinatal lethality, and infertility, in our IGF-I-deficient mice, we propose a 30% prenatal lethality, which suggests that some IGF-I null fetuses did not survive due to developmental retardation. We further demonstrate, in these IGF-I null mice, GH-resistant dwarfism and marked serum GH elevation. In addition, there is enlargement of multiple organs (brain, liver, kidney, and heart). Finally, IGF-I null mice have normal liver GH receptor expression and liver Jun B response to GH.
The rate of growth retardation of the IGF-I null mice started at 35% at birth and worsened to approximately 65% as adults, in terms of body weight. This may indicate that the fetal development of the IGF-I null mice is partially compensated by IGF-I produced from the placenta or possibly the presence of IGF-II and insulin. The placenta produces a large number of hormones, including GH, IGF-I, and IGF-II, that may reach the fetus (25, 26). Deficiency in either IGF-II or insulin contributes to intrauterine growth retardation. Birth weights of IGF-II knockout mice are 60% and IGF-I/II double knockout are only 30% of their wild-type litter mates (27, 28), while insulin-1 and -2 double knockouts also induce 15–20% growth retardation in mouse embryos (29). After birth, a further growth retardation was observed, since the pups were no longer influenced by placental IGF-I, mouse IGF-II level diminishes quickly, and the role of insulin on postnatal growth is apparently very limited (29).
According to the somatomedin hypothesis, production of IGF-I from the liver and other tissues is regulated by GH release from the pituitary. Like many other physiological systems, overproduction of IGF-I sends a signal to inhibit GH production via a long loop (involving GHRH and somatostatin) and a short loop (directly on somatotrophs) negative feedback mechanism (30, 31, 32). It is well established that exogenous IGF-I, when administered in vivo, suppresses GH secretion, while IGF-I deficiency is accompanied by GH hypersecretion (20, 23, 33). In a separate study, when IGF-I production was abolished exclusively from the liver (using albumin-Cre/loxP system), we effectively decreased serum IGF-I level by 75% without affecting the growth and development of extrahepatic tissues. Serum GH increased 6.4-fold in these mice, suggesting that circulating IGF-I affects GH secretion (17). The current study abolished both endocrine and paracrine IGF-I production and thereby increased serum GH level even further to 12.4-fold. The fold increase in serum GH concentration correlates well with serum IGF-I levels under these circumstances. Therefore, our studies demonstrate the presence of a potent negative feedback on GH secretion by IGF-I.
A central issue in the somatomedin hypothesis is that GH acts through IGF-I production from the liver or local tissues in stimulating body growth. There have been suggestions that GH exerts some direct effects on target tissues that are not mediated by IGF-I (15, 16, 25, 34). The current study demonstrates that IGF-I null mice are selectively resistant to GH in terms of body growth, directly confirming that GH works exclusively via IGF-I production in this aspect. This is consistent with an earlier report on the ineffectiveness of GH on weight gain of IGF-I null mice (35). As for the role of IGF-I in the circulation or as a paracrine growth factor, our study on liver-specific IGF-I gene deletion demonstrated that liver-derived endocrine IGF-I is not essential for postnatal growth (17, 36). These studies, as well as those by other investigators (37), demonstrate that IGF-I, most probably produced by nonhepatic tissues, is essential for GH-stimulated postnatal growth.
One exception to the above conclusion is that the profound defect in development and metabolism caused by IGF-I deficiency may have impaired the ability of GH and its other growth mediators to promote body growth. To address this question, we have demonstrated that IGF-I null mice have normal levels of GH receptor mRNA in the liver and that rhGH caused a significant increase in liver size. Furthermore, in an acute experiment, we tested the effect of rhGH on liver Jun B expression. Early response genes encode for transcription factors including Fos, Jun, Jun B, and Myc, which can be induced by GH and are involved in activating DNA synthesis, downstream gene expression, and cell proliferation (21, 22). As expected, Jun B expression was activated within 30 min of an in vivo rhGH injection in wild-type mice. The response in IGF-I null mice was virtually identical, further demonstrating the presence of a functional GH signaling system. The current study therefore does not support direct GH action on growth and indicates that those IGF-I-independent mechanisms of GH at least in mice, are not involved in body growth regulation.
Defects in GH receptor or downstream targets (such as STAT5b, HNF-1
,
and IGF-I) cause GH insensitivity (GHIS) or Laron Syndrome. Patients
with GH receptor gene mutations show normal prenatal development,
severe postnatal growth retardation, high serum GH, and low levels of
serum IGF-I that cannot be corrected by GH injection (38). A second
class of Laron type dwarfism, induced by HNF-1 deficiency in mice,
demonstrates normal GH receptor level, elevated serum GH, and
diminished IGF-I and insulin concentrations (23). These HNF-1 null
mice are viable and sterile and develop non-insulin-dependent diabetes
mellitus. Deficiency in STAT5b (signal transducer and activator of
transcription 5b), another transcription factor downstream of GH, also
causes Laron type of dwarfism with virtually identical features to
HNF-1 (hepatocyte nuclear factor 1) null mice (39).
Defects in IGF-I production are rare, probably due to intrauterine
lethality. The only case of IGF-I deficiency reported features growth
failure before and after birth, high GH level, and resistance to GH
treatment (6). In agreement with the report on patients, our
data demonstrate a dramatic increase in serum GH level and the
inability of GH to promote body growth in the face of IGF-I deficiency.
It clearly defines a new class of GH-resistant dwarfism (similar to
GHIS) and proves that IGF-I is essential for GH-stimulated postnatal
growth. This mouse model provides a valuable tool with which to explore
the relationship of the GH-IGF-I axis and to provide therapeutic
modalities to treat such dwarfism.
The finding of multiple organ enlargement (relative weight) in IGF-I null mice is an extension of previously observed weight increases of the brain and the liver and may be the result of a sustained GH hypersecretion (7, 40). To exclude the influence of an overall change in body weight, we expressed the organ weight as per body weight and found that IGF-I null mice have a 21–113% increase in the weight of the kidney, heart, liver, and brain. GH further induced changes in both wild-type and IGF-I null mice. In wild-type mice, chronic rhGH administration increased the weight of the liver by 27% but decreased the relative weights of the brain and the kidney. In IGF-I null mice, a 15% increase in liver weight, caused by GH treatment, and no change in other organs were observed.
The relationship of GH with organ growth has been well studied. For example, prolonged excessive secretion of GH by GH3 tumor cells in rats induced widespread visceromegaly affecting the liver, kidney, spleen, heart, and adrenals that was associated with an increase in DNA synthesis (41); transgenic mice overexpressing GH have selective splanchnomegaly coupled with glomerular sclerosis and hepatomegaly (42); pulsatile administration of GH in broiler pullets induced hepatomegaly due largely to cellular hypertrophy (43); and GH-transgenic mice have accelerated liver, kidney, and skeletal growth (44). Because the IGF-I null mice have no IGF-I production and a marked increase in GH secretion, we propose an IGF-I-independent, direct GH action on hepatocyte growth and proliferation under conditions of both IGF-I-deficient status and when GH was exogenously administered.
Analysis of a standard serum chemistry profile revealed a marked decrease in alkaline phosphatase and glucose concentrations among IGF-I null mice. Alkaline phosphatase from the bone and other tissues is essential for normal skeletal mineralization and growth (45, 46, 47). IGF-I has been found to influence alkaline phosphatase activity as part of the mechanism of stimulating bone formation. For example, locally infused IGF-I into mouse femurs can stimulate expression of alkaline phosphatase and bone formation; the age-dependent decline in bone formation can be attributed in part to a decline in local IGF-I expression and response (48); and IGF-I stimulates alkaline phosphatase activity and type I procollagen mRNA expression in osteoblastic cells in vitro (49). The current study demonstrates severe hypophosphatasia due to IGF-I deficiency and suggests that alkaline phosphatase activity may be an important downstream target of IGF-I action on postnatal skeletal growth.
In conclusion, using the Cre/loxP system, we have generated IGF-I null mice that have a 42% postnatal survival rate and enable an in-depth postnatal study. These mice exhibited multiple organ enlargement accompanied by decreased serum alkaline phosphatase and glucose concentrations. The increase in serum GH concentration and failure of GH in stimulating body growth in these IGF-I null mice clearly demonstrate GH resistance (with respect to body growth) and the role of IGF-I in feedback control of GH secretion.
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Acknowledgments |
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Footnotes |
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Received June 10, 1999.
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7) and IGF-I null
(n 
