Download Insulin-Like Growth Factor-I: Compartmentalization

Insulin-Like Growth Factor-I: Compartmentalization
Within the Somatotropic Axis?
Andrew A. Butler,1 Shoshana Yakar,2 and Derek LeRoith2
1
The Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201; and 1Clinical Endocrinology Branch, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1758
Insulin-like growth factor-I (IGF-I) is essential for normal growth; igf-1 gene mutations are associated
with extreme growth retardation in mice and, very rarely, in humans. The relative contributions of
tissue vs. endocrine (hepatic) IGF-I to the regulation of growth has been a fundamental question. New gene targeting technologies are providing answers for these questions.
he growth of an organism is dependent on the integration
of internal signals, which ultimately stem from the program
driven by the genome, with environmental cues. Nutrition is
an obvious environmental factor over which the organism has
no influence, but the organism must be able to respond to it to
produce a viable adult. Temperature is another important variable, with the increased metabolic demands of low temperatures leading to changes in the size of certain organs. The
organism must also determine when to stop growing and commit resources to reproduction (i.e., puberty). Flexibility of the
genetic program is therefore a requirement, with growth resulting from the integration of the constraints imparted by the environment with the genetic program.
It has been known for over half a century that the primary
factor controlling growth rate is growth hormone (GH) (8). GH
was originally believed to stimulate growth via the stimulation
of an intermediary, somatomedin, which is secreted by the
liver and acts as a circulating growth factor (4). The isolation
and sequencing of the genes and cDNAs of the insulin-like
growth factors (IGF) led to the discovery that, although the livers of adult mice had by far the highest level of expression,
IGF-I mRNA expression was essentially ubiquitous. IGF-I
mRNA was also found to be expressed in the fetus, a stage in
development at which GH does not appear to play a significant role in regulating growth (3). The question thus raised was:
what are the respective roles of endocrine IGF-I, primarily
hepatic in origin, and IGF-I synthesized in the peripheral tissues and presumably acting in an autocrine/paracrine manner?
This review will provide a brief introduction to the somatotropic axis and will discuss some of the recent developments
made possible by the use of modern genetic techniques.
Overview of the somatotropic axis
GH is secreted from somatotrophs, specialized cells present
in the anterior pituitary, and represents the leading candidate
for the apical “growth regulator.” Modern genetic analysis has
shown that dwarfism in humans can result from mutations
leading to loss of GH secretion, such as, for example, mutations in the GH-releasing hormone (GHRH) receptor, in the
GH gene itself, in transcription factors necessary for GH gene
activity (e.g., Pit-1), or in the GH receptor (GHR, “Laron
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Dwarfism”) (8). However, the observation of rare cases in
which normal growth is observed to be associated with normal
serum IGF-I levels in the absence of pituitary GH suggests that
our understanding of growth regulation is not complete.
GH activity is regulated at several levels, with both the magnitude and pattern of GH secretion influencing the biological
response (8). The secretion of GH from pituitary stores has long
been known to be regulated by the interplay of two hypothalamic neuropeptides. GH release is stimulated by GHRH and is
inhibited by somatostatin. The pattern of GH secretion is an
important determinant of growth rate, with a discrete pulsatile
secretory pattern stimulating growth more efficiently than continuous infusion. The pattern of GH release is sexually dimorphic. Males exhibit discrete pulses with low interpeak concentration, whereas females tend toward a high basal level of
GH and less evidence of pulsatility. GH activity can be regulated at the level of the GHR, with malnutrition in particular
associated with a state of GH resistance.
IGF-I is also essential for normal growth, with the original
gene targeting studies in the mouse indicating that IGF-I is
important for pre- and postnatal growth (1, 5). Confirmation of
the significance of IGF-I in human physiology was obtained by
the discovery of a patient with intrauterine growth retardation
and postnatal growth failure associated with a mutation in the
igf-1 gene (11). Mice lacking a functional igf-1 gene exhibit
growth retardation both in utero and postnatally, whereas mice
lacking a functional igf-2 gene exhibit intrauterine growth
retardation but have normal growth velocity postnatally. This is
consistent with the developmental profile of IGF-I and IGF-II
mRNA expression in the mouse and rat, in which IGF-II mRNA
expression dramatically declines in the first 10 days postpartum. However, an important caveat is that the profile of IGF
expression varies considerably between species. In other
mammalian species, including humans, IGF-II expression and
presence in the circulation persists into adulthood. The function of IGF-II in the adult of nonrodent mammals remains an
enigma, because there have been no reports of naturally
occurring igf-2 mutants.
IGF-I and its receptor (IGF-IR) belong to a family of peptides
related in structure to insulin and its receptor (Fig. 1). Two IGF
peptides have been isolated that exhibit distinct development
and tissue-specific patterns of expression. Both IGF-I and IGF0886-1714/02 5.00 © 2002 Int. Union Physiol. Sci./Am. Physiol. Soc.
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T
II activate IGF-IR, and IGF-II is also an agonist of a splice variant of the insulin receptor that is expressed at high levels in
fetal and neoplastic tissues. A third receptor, the mannose-6phosphate/IGF-IIR, binds IGF-II with high affinity and appears
primarily to function as a “biological sink” for IGF-II, increasing clearance and degradation.
The liver is the primary source of circulating IGF-I
Interaction between GH and IGF-I in regulating growth
Given the level of interest in the field concerning the respective roles of GH and IGF-I in growth regulation, it is perhaps
surprising that the first study of ghr
igf-1 knockout mice has
only recently been published (7). This study has confirmed that
GH and IGF-I have independent and common functions. Double ghr
igf-1 nullizygotes exhibit a more severe phenotype
than either the igf-1 or ghr knockout. This study provides an
interesting comparison of the respective roles of GH, signaling
through the GHR, and IGF-I in murine growth, which is summarized in Table 1. The study confirms several previous reports
that GH does not appear to significantly affect growth until late
in the second week postpartum, with the most dramatic differences in growth velocity between GH-sufficient and GH-deficient mice occurring between postnatal days 20 and 40.
Significantly, the growth rates of double ghr
igf-1 nullizygotes are not significantly different from those of igf-1 nullizygotes until 2 wk postpartum, at which time the double mutants
began to exhibit a more significant growth retardation. Ultimately, adult ghr
igf-1 nullizygotes weigh ~17% of normal,
FIGURE 1. Schematic of the insulin and insulin-like growth factor (IGF) systems. The majority of IGF-I and IGF-II circulate bound to IGF binding proteins
(IGFBPs). Both IGF-I and IGF-II act through the IGF-I receptor (IGF-IR). IGF-II
and insulin (INS) can act through one or both forms of the insulin receptor (IRA, IR-B).
whereas igf-1 nullizygotes weigh ~30% of normal. Thus ~83%
of the final weight of a mouse is due to the growth-stimulating
actions of the somatotropic axis. The authors use the growth
curves of the single and double knockouts to estimate the contribution of GH and IGF-I separately and in synergy with the
83% of growth due to the somatotropic axis. They suggest that
~35% of the final weight of a mouse is due to IGF-I action,
14% is due to GH acting independently of IGF-I, and the GHIGF-I interaction contributes 34%. Note that these figures represent approximations for the contribution of GH and IGF-I in
the background of the mouse strains used for the study.
Tissue-specific knockouts to examine the role of hepatic
IGF-I
Recently there has been an advance in in vivo gene targeting studies allowing the study of the function of genes in either
a tissue- or temporal-specific manner in the whole animal.
The ability to inactivate genes in a tissue- and time-regulated
manner represents an incredibly powerful new tool for the
molecular biologist. Genes may be excised from the genome
by using a site-specific recombination technology adapted
from bacteriophage. Bacteriophage P1 Cre recombinase, a
38-kDa protein, recognizes 34-bp DNA sequences called
locus of crossover P1 (loxP) sites (2, 9). When the loxP sites
are in tandem and flanking an essential exon of the gene of
interest, Cre induces an intramolecular recombination and
excision of the intervening DNA, resulting in deletion of the
exon. By placing the Cre recombinase under the control of tissue-specific or inducible promoters, the function of a gene in
a certain tissue(s) can be examined in the absence of potenNews Physiol Sci • Vol. 17 • April 2002 • www.nips.org
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Several observations supported the contention that IGF-I in
the circulation is primarily hepatic in origin. In rodents, IGF-I
mRNA expression increases dramatically in the liver during the
first 50 days of life. The increase in expression ensures that, in
the young adult, IGF-I mRNA is approximately two orders of
magnitude more abundant in liver than other tissues. The rise
in hepatic production coincides with an increase in IGF-I in
the circulation, while hepatic IGF-I mRNA exhibits the most
dramatic response to GH depletion and replenishment.
A recent publication reports that serum IGF-I levels in mice
lacking a functional GHR could be as low as 0.2% of normal
levels (7). A common finding in mutant mice in which either
GH or the GHR genes are compromised is the dramatic
decline in IGF-I mRNA in liver, accompanied by smaller
declines in some tissues (e.g., kidney, ovary, muscle) and some
tissues in which IGF-I expression is not affected (e.g., lung,
heart, testis, brain). Indeed, this has led Lupu and colleagues
(7) to suggest that circulating IGF-I is exclusively hepatic in origin and that the residual levels observed in mice in which the
igf-1 gene has been targeted for excision by the Cre-loxP system, as discussed below, is perhaps due to incomplete recombination. However, it could also be argued that IGF-I mRNA
expression is not necessarily correlated with translation. Measurements of IGF-I protein in the tissues of the GHR knockout
in which IGF-I mRNA expression is not reduced will be
needed to confirm that posttranscriptional steps of IGF-I synthesis have not been affected.
TABLE 1. Body weight comparison for igf-1, ghr, and double-knockout
mice at various stages of development
0
10 days
IGF-I
Period/Predominance
2
3 wk
3
7 wk
IGF-I
GH and IGF-I
Igf-1 knockout
60
40
30
Ghr knockout
Normal
Minor reduction
50
60
40
17
Double knockout
Values are % of normal. Note that mice lacking a functional ghr gene
have normal body weight at birth and do not show signs of growth retardation until 3 wk of age. In contrast, mice lacking a functional igf-1 gene
exhibit growth retardation throughout all stages of development.
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Insulin resistance in muscle of liver-specific igf-1 knockouts
The increase in circulating GH consistently observed in
liver-specific igf-1 knockouts could potentially have important
physiological consequences (10, 13). Insulin resistance is a
FIGURE 2. Deletion of the liver IGF-I gene results in elevated serum growth
hormone (GH) levels due to the reduced circulating IGF-I levels. This is associated with insulin resistance, primarily in the skeletal muscle, and with secondary hyperinsulinemia, which may also be caused by GH-stimulated islet
cell hyperplasia.
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tially critical developmental abnormalities. Mice with a gene
flanked by loxP sites therefore represent an extremely valuable resource.
By using homologous recombination, a mouse line has been
established in which two loxP sites in tandem flank the fourth
exon of the IGF-I gene (6, 10, 13). Exon 4 encodes amino acid
residues 26
70 of the IGF-I peptide, including part of the B
domain and the entire C, A, and D domains. This region of the
peptide is solely responsible for IGF-I binding to its cognate
receptor (IGF-IR) and has been previously targeted by others to
create a total IGF-I knockout for developmental studies (1). To
create mice with targeted inactivation of the IGF-I gene in the
liver, transgenic mice that express Cre recombinase exclusively
in the liver by expressing Cre under the control of the albumin
promoter were generated (13). The albumin gene is activated
in hepatocytes during fetal development, with transcripts
becoming easily detectable at ~10 days postnatally. Crossbreeding of the loxP-flanked IGF-I mice and the albumin-Creexpressing mice resulted in deletion of the IGF-I gene in the
liver. As assayed by Southern blot analysis, the IGF-I gene deletion effect in liver was ~95%. In other tissues, there was no
evidence of recombination. Analysis of IGF-I mRNA expression by solution hybridization RNAse protection assays confirmed that the targeting had worked, with liver IGF-I mRNA
levels reduced to <1% of wild-type expression levels. IGF-I
mRNA levels measured in nonhepatic tissues of the liver-specific knockouts such as heart, muscle, fat, spleen, and kidney
were similar to levels in control animals.
Circulating IGF-I levels in mice lacking a functional IGF-I
gene in liver were markedly reduced at 6 wk of age, being only
25% of that in wild-type animals. This was associated with an
approximately fourfold increase in circulating GH levels, presumably due to the decrease in negative feedback control by
circulating IGF-I on GH secretion by the pituitary. At 6 wk of
age, mice were killed and body length (nose to anus) was
measured, femoral length was measured by x-ray, and the
weights of individual organs were recorded. No significant differences in any of these parameters were observed between
liver-specific IGF-I knockout animals and their wild-type littermates. The only exception was a reduction in spleen size in the
knockout animals, perhaps as a result of chronically reduced
levels of circulating IGF-I. Sexual maturation was normal, as
demonstrated by normal fertility, normal-size litters, and normal lactation and weaning. In a second model, we produced
a liver-specific deletion of the IGF-I gene by using an inducible
interferon promoter to drive the expression of Cre. Once again,
the circulating levels of IGF-I were reduced by ~75% but postnatal growth and development were considered normal (10).
Because the circulating (endocrine form) of IGF-I was
markedly reduced and GH levels were correspondingly
increased in these animals, we also considered the possibility
that the normal growth and development of these mice was the
result of compensation from nonhepatic tissues. Using the
solution hybridization/RNase protection assay, which provides
high specificity and sensitivity, IGF-I mRNA levels were measured in various tissues. In all tissues examined, including heart,
muscle, fat, spleen, and kidney, IGF-I mRNA levels were not
different from wild-type levels. From these results, we have
concluded that these tissues do not show compensation,
although we cannot exclude the possibility that compensation
could occur at the level of IGF-I translation.
At this stage it is still unclear whether the normal growth and
development in these mice is due entirely to local IGF-I production or whether the free circulating IGF-I levels are sufficient to maintain this function. This latter possibility could
explain why when IGF-IR mRNA levels were measured in various tissues they were not different from those in wild-type
mice. Alternatively, free IGF-I levels are normal (10) and could
be sufficient to maintain endocrine IGF-I-induced growth.
However, it is clear that no compensation is seen with IGF-II,
because IGF-II mRNA levels in tissue and IGF-II protein levels
in serum were both undetectable in the IGF-I gene-deleted
mice.
Conclusions
Recent studies suggest that the majority of growth (83%) in
the mouse is the result of the somatotropic axis. The contribution of the various components of the axis (GH, IGF-I) varies
considerably between developmental stages. IGF-I stimulates
growth throughout development, whereas GHR-dependent
growth does not develop until the second week of life. The
growth-promoting effects of IGF-II, which have not been discussed in this review, are limited to growth in utero in the
mouse. The concept of separate endocrine vs. paracrine compartments of IGF-I is supported by the observation that GHR
mutants have no measurable IGF-I in the circulation but have
normal expression of IGF-I mRNA in peripheral tissues. Confirmation that IGF-I production is truly GH independent in
peripheral tissues will require the measurement of IGF-I protein in these tissues in the GHR knockout and also in models
such as the liver-specific IGF-I knockout. Studies examining
the function and regulation of IGF-I will be greatly aided by the
development of tissue-specific knockouts of the IGF-I and IGFIR genes.
References
1. Baker J, Liu JP, Robertson EJ, and Efstratiadis A. Role of insulin-like growth
factors in embryonic and postnatal growth. Cell 75: 73
82, 1993.
2. Gu H, Marth JD, Orban PC, Mossmann H, and Rajewsky K. Deletion of a
DNA polymerase beta gene segment in T cells using cell type-specific
gene targeting. Science 265: 103
106, 1994.
3. Han VK, D’Ercole AJ, and Lund PK. Cellular localization of somatomedin
(insulin-like growth factor) messenger RNA in the human fetus. Science
236: 193
197, 1987.
4. Le Roith D, Bondy C, Yakar S, Liu JL, and Butler A. The somatomedin
hypothesis: 2001. Endocr Rev 22: 53
74, 2001.
5. Liu JP, Baker J, Perkins AS, Robertson EJ, and Efstratiadis A. Mice carrying
null mutations of the genes encoding insulin-like growth factor I (Igf-1) and
type 1 IGF receptor (Igf1r). Cell 75: 59
72, 1993.
6. Liu JL, Grinberg A, Westphal H, Sauer B, Accili D, Karas M, and LeRoith
D. Insulin-like growth factor-I affects perinatal lethality and postnatal
development in a gene dosage-dependent manner: manipulation using
the Cre/loxP system in transgenic mice. Mol Endocrinol 12: 1452
1462,
1998.
7. Lupu F, Terwilliger JD, Lee K, Segre GV, and Efstratiadis A. Roles of growth
hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev
Biol 229: 141
162, 2001.
8. Reiter EO and Rosenfeld RG. Normal and aberrant growth. In: Williams
Textbook of Endocrinology, edited by Wilson JD, Foster DW, Kronenberg
HM, and Larsen PR. Philadelphia: W. B. Saunders, 1998, p. 1427
1507.
9. Sauer B. Manipulation of transgenes by site-specific recombination: use of
Cre recombinase. Methods Enzymol 225: 890
900, 1993.
10. Sjogren K, Liu JL, Blad K, Skrtic S, Vidal O, Wallenius V, LeRoith D, Tornell
J, Isaksson OG, Jansson JO, and Ohlsson C. Liver-derived insulin-like
growth factor I (IGF-I) is the principal source of IGF-I in blood but is not
required for postnatal body growth in mice. Proc Natl Acad Sci USA 96:
7088
7092, 1999.
11. Woods KA, Camacho-Hubner C, Savage MO, and Clark AJ. Intrauterine
growth retardation and postnatal growth failure associated with deletion of
the insulin-like growth factor I gene. N Engl J Med 335: 1363
1367, 1996.
12. Yakar S, Liu JL, Fernandez AM, Wu Y, Schally AV, Frystyk J, Chernausek SD,
Mejia W, and LeRoith D. Liver-specific igf-1 gene deletion leads to muscle
insulin-insensitivity. Diabetes 50: 1110
1118, 2001
13. Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, and LeRoith D.
Normal growth and development in the absence of hepatic insulin-like
growth factor I. Proc Natl Acad Sci USA 96: 7324
7329, 1999.
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common feature of states in which GH levels are chronically
elevated, and a recent study has confirmed that a state of
insulin insensitivity exists in liver-specific igf-1 knockouts (12).
Liver-specific igf-1 knockouts are mildly hyperinsulinemic and
are resistant to the glucose-lowering effects of exogenous
insulin, but they clear a glucose load normally. Insulin-induced
phosphorylation of the insulin receptor and insulin receptor
substrate-1 is absent in muscle but is normal in liver and white
adipose tissue. Chronic treatment with IGF-I reduced circulating GH to normal levels and increased insulin sensitivity. Treatment with a GHRH antagonist, which reduced GH levels, also
increased insulin sensitivity.
This study also showed that the levels of “free” IGF-I were
normal in liver-specific igf-1 knockouts. The insulin insensitivity thus appears to be a deficit associated with mild hypersomatotropism. An interesting question yet to be answered is
why GH levels are elevated despite unbound IGF-I, which presumably represents the biologically active fraction of IGF-I in
the circulation, being normal. Nevertheless, it appears that a
major role of IGF-I in the circulation is to promote insulin sensitivity in muscle, perhaps indirectly via a feedback effect on
GH secretion (Fig. 2).