The Regulation of Longitudinal Bone Growth
Wang, Zhou, Bondy
While insulin-like growth factor 1 (IGF1) has long been regarded as the
mediator of growth hormones systemic effects on growth (the somatomedin
hypothesis), evidence has accumulated in recent years suggesting that
growth hormone (GH) may have some IGF1-independent effects on somatic
growth. For example, treating GH-resistant children with IGF1 has not
produced normal growth in stature, as might have been expected if IGF1
could substitute for GH in promoting long bone growth. We hypothesized
that if GH does indeed have IGF1-independent effects on long bone growth,
the linear growth deficit should be more severe in mice with deletion
of the GH receptor (GHR) compared with mice carrying an IGF1 deletion.
Supporting this view, we have recently shown that tibial linear growth
rate is reduced by approximately 35 percent in IGF1 null and by about
65 percent in GHR null mice. The rate of long bone growth is determined
by the rate of epiphysial growth plate chondrocyte generation and the
final size of hypertrophic chondrocytes that form the scaffolding for
bone elongation. Thus, to determine how IGF1 and GHR deletion impairs
long bone growth, we examined these parameters in epiphysial growth plates
from IGF1 and GHR null mice.
The growth plate germinal zone, which gives rise to new chondrocytes,
was significantly enlarged in size and cell number in the IGF1 null but
was severely attenuated in the GHR null growth plate. Given that GH levels
are greatly increased in the IGF1 null (due to lack of negative feedback
from IGF1) while GH effect is abolished in the GHR null mice due to lack
of its receptor, the data support the view that GH stimulates the germinal
zone to generate chondrocyte precursors. In the IGF1 null growth plate
proliferative zone, chondrocyte proliferation and numbers were preserved,
but the rate of proliferation and chondrocyte number were significantly
reduced in the GHR null proliferative zone. Both dwarves had a significant
reduction in chondrocyte hypertrophy. Interestingly, the dimunition in
hypertrophic chondrocyte size was about 35 percent, accounting entirely
for the reduction in long bone growth in IGF1 null mice and suggesting
that IGF1s major contribution to long bone growth is augmentation
of chondrocyte hypertrophy. Looking into the molecular mechanisms underlying
the defect in chondrocyte somatic growth, we found that the expression
of the insulin-sensitive glucose transporter, GLUT4, is significantly
decreased and that the insulin-regulated enzyme, glycogen synthase kinase
3b (GSK3), is hypo-phosphorylated in IGF1 null chondrocytes. Glycogen
levels were significantly decreased, and ribosomal RNA levels were reduced
by almost 75 percent in IGF1 null chondrocytes. The data suggest that
IGF1 promotes chondrocyte hypertrophy through insulin-like
anabolic actions.
Investigation of potential mechanisms whereby the growth plate germinal
zone is expanded and chondrocyte proliferation maintained in the IGF1
null mouse revealed that IGF2 mRNA levels were increased in the germinal
and proliferative zones of the knockout. Given that GH is elevated in
these mice, we hypothesized that GH might stimulate increased growth plate
IGF2 production and thereby germinal zone expansion and chondrocyte proliferation.
Supporting this view, IGF2 mRNA levels were significantly reduced in the
GHR null growth plate. In summary, we have shown for the first time that
loss of GH effect produces a much more severe deficit in long bone growth
than loss of IGF1, indicating that GH truly does have IGF1-independent
effects in promoting growth. In addition, we have provided a mechanistic
explanation for the difference in long bone growth by demonstrating that
both chondrocyte proliferation and hypertrophy are impaired in the GHR
null while only hypertrophy is attenuated in the IGF1 null growth plate.
These observations support dual roles for GH in promoting longitudinal
bone growth: an IGF1-independent role in growth plate chondrocyte generation
(possibly mediated by IGF2) and an IGF1-dependent role in promoting chondrocyte
hypertrophy. The accompanying figure diagrams these effects. The large
and robust differences in long bone growth rate are of obvious biological
and clinical importance and may explain why IGF1 treatment of GH-resistant
children may be significantly less effective than GH in terms of stature.
Figure 1
Diagram of an epiphysial growth plate outlining
our current views on the respective roles of GH, IGF1, and IGF2 in promotion
of longitudinal bone growth. GH stimulates expansion of the germinal zone,
possibly by increasing local IGF2 expression, which enhances chondrocyte
proliferation. GH also stimulates local and systemic IGF1 production,
which promotes chondrocyte hypertrophy.
IGF1 and Brain Development
Cheng, Kelly, Strauss, Tseng, Bondy
The growing brain consumes about 50 percent of the total fuel available
to the organism during early postnatal development. How the brain competes
so successfully with peripheral tissues for energy and substrates has
been unclear. Insulin preferentially enhances the use of glucose by peripheral
tissues, but not by the brain. Based on our observations of cellular patterns
of IGF1 and IGF1 receptor expression in the developing brain, we hypothesized
that endogenous brain IGF1 serves an insulin-like role in promoting neuronal
glucose utilization and hence growth during postnatal development. Supporting
this view, we have shown that brain growth in IGF1-/- mice falls behind
that of wild-type littermates by almost 40 percent during the postnatal
period when brain IGF1 expression is normally most abundant. We have also
demonstrated that brain 2-deoxy-D-[1-14C]glucose uptake (2DGU) parallels
IGF1 expression in wild-type mice and is profoundly reduced in IGF1-/-
mice during early postnatal development, as shown by film autoradiography
in Figure 2..
![Figure 2](images/2_BONDY3.jpg)
Figure 2
We have elucidated the molecular mechanisms whereby IGF1 regulates neuronal
metabolism by demonstrating that the active, phosphorylated form of Akt/PKB
is selectively colocalized with the insulin-sensitive glucose
transporter, GLUT4, in nerve processes of wild-type, IGF1-expressing neurons.
In the IGF1-/- brain, however, Akt phosphorylation is barely detectable
and GLUT4 is concentrated in neuronal perikarya, implicating IGF1-induced
Akt phosphorylation in translocation of GLUT4 to the neuronal membrane.
Moreover, we find that the phosphorylated form of GSK3beta and glycogen
stores are abundant in wild-type, IGF1-expressing neurons. In IGF1-/-
neurons, however, phospho-GSK3beta and glycogen are barely detectable,
suggesting that IGF1 normally augments glucose uptake and storage by familiar,
insulin-like pathways. We have proposed that IGF1s primary physiological
role in normal brain development is to promote the growth of projection
neurons. IGF1 expression is most abundant in neurons (e.g., Purkinje cells)
destined to be the largest and most complex in the brain, implicating
IGF1s insulin-like, anabolic effects in this extraordinary growth.
IGF1-expressing neurons grow large perikarya, long axons, and extraordinarily
prolific dendritic arbors, which are severely hypoplastic in the IGF1
null brain (see figure below). Homozygous IGF1 deletion results in mental
retardation, showing that this anabolic peptides effects on neuronal
metabolism and growth have important effects on cognitive function. Current
work in our laboratory is aimed at discovering factors regulating neuronal
IGF1 expression with a view to therapeutic manipulation to increase the
potential for cognitive development and successful responses to brain
injury.
Estrogen Effects in the Primate Neocortex
Cheng, Bondy
Estrogen stimulates neurite growth and neurotransmitter synthesis and
protects against diverse types of neural injury in vitro. In vivo, estrogen
treatment is reputed to protect against Alzheimers disease in menopausal
women. To investigate the molecular basis for estrogens neuroprotective
effects in a model system relevant to humans, we evaluated the expression
of glucose transporters and IGF1 in the prefrontal cortex of estradiol-
versus placebo-treated ovariectomized rhesus monkeys. We investigated
the expression of facilitative glucose transporters (Gluts) 1, 3, and
4 by using in situ hybridization, immunohistochemistry, and immunoblot
analysis. Gluts 3 and 4 were concentrated in cortical neurons while Glut1
was localized in capillaries and glial cells. Estradiol (E2) treatment
induced two- to four-fold increases in Glut 3 and Glut 4 mRNA and protein
levels. E2 treatment also induced an increase in glial Glut1 mRNA levels
but did not appreciably affect vascular Glut1 gene expression. IGF1 mRNA
was concentrated in cortical neurons together with Gluts 3 and 4 and was
significantly increased in E2-treated animals. These novel data suggest
that the up-regulation of Gluts 3 and 4 and IGF1 expression contribute
to estrogens salutary effects on neural tissue. In support of this
view is the finding by other researchers that glucose transport and transporter
expression are reduced in subjects with Alzheimers disease, suggesting
one way in which estrogen may address the Alzheimers neuropathology.
Current studies are aimed at profiling the entire spectrum of genes regulated
by estrogen effect in the nonhuman primate neocortex.
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PUBLICATIONS
- Bondy CA. Insulin-like growth factors in the nervous system. In: Smith
BH, Adelson G, eds. Encyclopedia of neuroscience, in press.
- Bondy CA, Cheng C. IGF1 and brain development. In: LeRoith D, Baxter
R, eds. Insulin-like growth factors. Landes Bioscience, in press.
- Cheng C, Bondy CA. IGF1 and brain glucose utilization. In: Dwyer D,
ed. Brain glucose metabolism. Int Rev Neurosci, Academic Press, in press.
- Cheng
C, Cohen M, Tseng V, Bondy C. Endogenous IGF1 enhances cell survival
in the postnatal dentate gyrus. J Neurosci Res 2001;64:341-347.
- Cheng, C, Cohen M, Wang J, Bondy CA. Estrogen augments glucose transporter
and IGF1 expression in primate cerebral cortex. FASEB J 2001;15:907-915.
- Cheng
C, Reinhardt RR, Lee W-H, Patel SC, Bondy CA. IGF1 regulates developing
brain glucose metabolism. Proc Natl Acad Sci USA 2000;97:10236-10241.
- Kadakia
R, Arraztoa J, Bondy C, Zhou J. Granulosa cell proliferation is
impaired in the Igf1 null ovary. Growth Horm IGF Res, in press.
- Le
Roith D, Bondy C, Yakar S, Liu JL, Butler A. The somatomedin hypothesis:
2001. Endocr Rev 2001;22:53-74.
- Monget
P, Bondy C. Importance of the IGF system in early folliculogenesis.
Mol Cell Endocrinol 2000;163:89-93.
- Valencia
JC, Matsui K, Bondy C, Zhou J, Rasmussen A, Cullen K, Yu ZX, Moss J,
Ferrans VJ. Distribution and mRA expression of insulin-like growth
factor system in pulmonary lymphangioleiomyomatosis. J Investig Med
2001;49:421-433.
- Wang J, Zhou J, Cheng C, Kopchick JJ, Bondy CA. Evidence that GH has
IGF1-independent effects on somatic growth. Endocrinol, in press.
- Zhou
J, Anderson K, Bievre M, Ng S, Bondy CA. The primate mammary gland
insulin-like growth factor system: cellular localization and regulation
by sex steroids. J Invest Med 2001;49:47-55.
- Zhou
J, Bievre M, Bondy CA. Reduced GLUT1 expression in Igf1-/- null
oocytes and follicles. Growth Horm IGF Res 2000;10:111-117.
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