HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WANG, J. Articles by BONDY, C. A. Search for Related Content PubMed PubMed Citation Articles by WANG, J. Articles by BONDY, C. A.

Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy

Developmental Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA

¹Correspondence: Developmental Endocrinology Branch, NICHD, NIH, Bldg. 10, Rm. 10N262, 10 Center Dr. 1862, Bethesda, MD 20892, USA. E-mail: bondyc{at}exchange.nih.gov

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Longitudinal bone growth, and hence stature, are functions of growth plate chondrocyte proliferation and hypertrophy. Insulin-like growth factor 1 (Igf1) is reputed to augment longitudinal bone growth by stimulating growth plate chondrocyte proliferation. In this study, however, we demonstrate that chondrocyte numbers and proliferation are normal in Igf1 null mice despite a 35% reduction in the rate of long bone growth. Igf1 null hypertrophic chondrocytes differentiate normally in terms of expressing specialized proteins such as collagen X and alkaline phosphatase, but are smaller than wild-type at all levels of the hypertrophic zone. The terminal hypertrophic chondrocytes, which form the scaffold on which long bone growth extends, are reduced in linear dimension by 30% in Igf1 null mice, accounting for most of their decreased longitudinal growth. The expression of the insulin-sensitive glucose transporter, GLUT4, is significantly decreased and the insulin-regulated enzyme glycogen synthase kinase 3ß (GSK3) is hypo-phosphorylated in Igf1 null chondrocytes. Glycogen levels were significantly decreased and ribosomal RNA levels were reduced by almost 75% in Igf1 null chondrocytes. These data suggest that Igf1 promotes longitudinal bone growth by `insulin-like' anabolic actions which augment chondrocyte hypertrophy.—Wang, J., Zhou, J., Bondy, C. A. Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy.

Key Words: glucose transporter • IGF action • insulin • proliferation • epiphysis • short stature

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE RATE OF longitudinal bone growth is approximated by the rate of production of new growth plate chondrocytes times the average size of terminal hypertrophic chondrocytes (1 2 3) . This process is exquisitely sensitive to regulation by growth hormone (GH), which may produce alterations of severalfold in adult stature. Insulin-like growth factor 1 (Igf1, also known as somatomedin C) is an important mediator of GH's effects on longitudinal growth, although its mode of action remains controversial. In the original `somatomedin hypothesis', it was proposed that GH's primary effect was to stimulate Igf1 production by the liver, with Igf1 then responsible for stimulating the longitudinal expansion of growth plates in an endocrine fashion (4) . More recent work has suggested that GH may act directly at the growth plate to amplify the production of chondrocytes from germinal zone precursors and then to induce local Igf1 synthesis, which is thought to stimulate the clonal expansion of chondrocyte columns in an autocrine/paracrine manner (5) . The notion that GH induces chondrocyte Igf1 synthesis has been challenged, however, by the finding that Igf2, rather than Igf1, is synthesized by growth plate chondrocytes in vivo (6 , 7) .

Despite the uncertainty about its mode of action, Igf1 clearly has an important role in longitudinal bone growth, since Igf1 gene deletion results in dwarfism in mice (8 , 9) and extreme short stature in humans (10) . To elucidate Igf1's role in longitudinal bone growth, this study has compared tibial growth and analyzed tibial growth plate characteristics in Igf1 null and littermate wild-type mice.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
The Igf1 deletion mouse line used in this study was generated by Lynn Powell-Braxton at Genentech, Inc., San Francisco (9) , bred for six generations into CD1 strain obtained from Taconic Farms, (Germantown, N.Y.), and maintained in our facility. The mice were used in a protocol approved by the NICHD Animal Use and Care Committee and were killed by CO₂ inhalation between 1:00 and 2:00 PM, 1 h after intraperitoneal ³H-thymidine (2 µCi/g) or BRDU injection (10 µl/g, Calbiochem HCS24). Tibias were dissected, fixed in formalin, decalcified in EDTA, embedded in paraffin, and cut longitudinally into 5 µ sections that were mounted onto poly-L-lysine-coated slides.

Morphometry
The longitudinal dimensions of the tibia and of the proximal tibial growth plate were measured on photomicrographs of anatomically matched, midsagittal sections taken at 5 and 200x. For the growth plate dimensions, three different measurements, clustered about the midline, were taken on each of two sections for each animal; the data were meaned for each animal, then pooled and meaned for each group. Terminal chondrocyte longitudinal diameter was measured using micrographs taken at 400x. Measurements were made only on cells with intact superior and inferior transverse septa. The photographs were coded and masked prior to analysis so that the observer did not know to which group the samples belonged.

Histochemistry
Immunohistochemistry was performed by the avidin-biotin-immunoperoxidase method as described previously (11) . Tissue sections were deparaffinized, rehydrated, and digested in 0.5% Trypsin (Sigma, St. Louis, Mo.) at 37°C for 30 min prior to immunoreaction. A polyclonal antibody reactive against the unique carboxyl terminal domain of collagen X was a gift from Dr. Haronen (12) . The polyclonal antibodies used to detect BRDU, GLUT 4, and the serine-phosphorylated form of GSK-3ß (9) were obtained from Chemicon (Temecula, Calif.). The monoclonal antibody detecting both active and inactive forms of GSK-3ß was obtained from Transduction Laboratories (Lexington, Ky.). All primary antibodies were used at a dilution of 1:200. Sections were incubated with peroxidase-conjugated secondary antibody (1:200) for 30 min at room temperature. The signal was detected and amplified using the ABC peroxidase method (Vector, Burlingame, Calif.) and visualized with 3,3'-diaminobenzidine. Sections were counterstained with methyl green. Parallel incubations omitting the primary antibody were used to establish the level of nonspecific background staining.

Deparaffinized sections were dipped in Kodak NTB2 nuclear emulsion and exposed at 4°C for 3 wk, developed, fixed, and lightly counterstained with hematoxylin for detection of nuclear ³H-thymidine incorporation. Positive cells had 5 or more grains/nucleus. Slides were masked and coded to conceal group designations prior to analysis.

Histological staining of glycogen was performed by the periodic acid-Schiff (PAS) reaction. Sections were first deparaffinized in xylene, rehydrated, and oxidized in 0.1% periodic acid for 5 min. After washing in water, slides were stained in 0.5% Schiff's solution (Sigma) for 15 min and counterstained with hematoxylin. For negative control, parallel sections were treated with 0.5% -amylase (Sigma) at 37°C for 90 min before PAS staining. PAS `positivity' was determined by the presence of fuchsia-colored staining.

In situ hybridization was performed as described previously (7) using ³⁵S-labeled antisense RNA probes synthesized from DNA fragments corresponding to nucleotides 4400–4515 of the 28S- and nucleotides 715–794 of the 18S ribosomal RNA genes (Ambion, Austin, Tex.). After exposure to Kodak NTB2 emulsion for 3 days, hybridized slides were developed, fixed, and counterstained with hematoxylin and eosin. RNA was quantified by silver grain counting under oil at 630x.

Data analysis
Data for each group (n=4–8) are expressed as means with standard errors. Differences between groups were analyzed by analysis of variance using Stat View 4.1 (Abacus Systems), followed by Fisher's least significant difference tests. A P value <0.05 was taken as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Growth plate morphometry and chondrocyte proliferation
To determine the cause of reduced long bone growth in the Igf1 null mouse, we compared chondrocyte proliferation and hypertrophy in tibial growth plates from wild-type (WT) and Igf1 null mice at postnatal day 20 (P20, Fig. 1 , Table 1 ). P20 was chosen because this is when the GH/IGF1-dependent growth spurt starts. Furthermore, the epiphysial growth plate is most active in terms of proliferation at this time (13) . The cells of the epiphysial growth plate demonstrate synchronized functional and morphological changes, which are manifested in characteristic horizontal zones. The germinal zone (GZ) consists of a thin layer of small, undifferentiated cells immediately adjacent to epiphysial bone giving rise to proliferative chondrocytes. This zone is significantly increased in width and cell number in the Igf1 null growth plate (Fig. 1 , Table 1 ). This expansion of the GZ is likely due to enhanced GH activity in the Igf1 null mouse, since circulating Igf1 normally inhibits GH secretion in a negative feedback loop.

View larger version (127K): [in this window] [in a new window]   Figure 1. Representative proximal tibia sections from P20 WT (A, B) and Igf1 null (C, D) mice. The tissues were stained with the Masson trichrome technique in which nuclei are black, collagen is blue, and calcified matrix is red. The gross anatomical features of the bone are similar but the growth plate functional zones are significantly different in WT and null mice (B, D). The germinal zone (GZ) is expanded whereas the hypertrophic zone (HZ) is attenuated in the Igf1 null growth plate compared with WT (see Table 1 ). Bar = 200 µ for (A, B) and 50 µ for (C, D).

The proliferative zone (PZ) is formed of columns of rapidly dividing chondrocytes resembling stacks of coins. The length of the PZ and the number of chondrocytes per column are equal in WT and Igf1 null mice (Fig. 1 and Table 1 ). The proliferation rate, assessed by DNA labeling index (Table 1) , is also equal. Growth plate chondrocytes undergo terminal differentiation as hypertrophic chondrocytes, ultimately increasing in height in the direction of growth by four- to fivefold (2 , 3) before they are incorporated into bone at the metaphysial end of the growth plate. The length of the Igf1 null hypertrophic zone (HZ) is reduced by ~35%, although the number of hypertrophic cells per column is equal to WT (Fig. 1 , Table 1 ). Igf1 null chondrocytes are obviously smaller at all levels of the HZ, with the mean longitudinal diameter of terminal hypertrophic cells reduced by ~30% (Table 1) . This degree of impairment in chondrocyte hypertrophy correlates with the decreased rate of longitudinal bone growth observed in Igf1 null mice. The GH-dependent postnatal growth spurt occurs between P20–40. During this period, the WT tibia grows at a rate of ~290 µ/day (from 13.3±0.5 to 19.1±0.8 mm), whereas the Igf1 null grows ~185 µ/day (from 10.5±0.6 to 14.2±0.4 mm). This represents an ~35% reduction in longitudinal growth rate for the Igf1 null mouse. These data suggest that impaired chondrocyte hypertrophy is the major cause of short stature in Igf1 deficiency.

Chondrocyte hypertrophy
The differentiation of proliferative into hypertrophic chondrocytes is marked by the appearance of collagen X expression (14 , 15) . Collagen X immunoreactivity is qualitatively normal, although somewhat less abundant in the Igf1 null growth plate (Fig. 2 ). A similar pattern was found for the expression of alkaline phosphatase and bone sialoprotein, other `markers' of hypertrophic differentiation (data not shown). Thus, it appears that these cells differentiate normally, but fail to attain full somatic growth. Given the marked homology between insulin and Igf1 and between their cognate receptors (16) , we hypothesized that Igf1 may act in insulin-like anabolic fashion to promote chondrocyte hypertrophy. Thus, we examined the expression of well established targets of insulin's anabolic actions. Among the facilitative glucose transporters, GLUT4 expression is known to be highly dependent on insulin (17) . GLUT4 immunoreactivity was significantly reduced in Igf1 null hypertrophic chondrocytes, both in terms of the number of positively stained cells and in the degree of intensity demonstrated by positive cells (Fig. 3 A, B, Table 2 ).

View larger version (166K): [in this window] [in a new window]   Figure 2. Collagen X immunostaining in WT (A) and Igf1 null (B) hypertrophic chondrocytes. Collagen X immunoreactivity is first detected in the cytoplasm and plasma membrane of cells in the early hypertrophic zone and becomes more abundant as the hypertrophy progresses. In the largest hypertrophic cells, collagen X immunoreactivity is extracellular, concentrated in the space between the plasma membrane and the pericellular matrix (pc). These large cells are particularly susceptible to shrinkage and plasmalemmal rupture during fixation. The rupture of the cells and loss of extracellular proteoglycans during conventional tissue processing leave clearly demarcated lacunae, which outline the spaces occupied by healthy hypertrophic chondrocytes in vivo. Some collagen X immunostaining is found in the longitudinal septa of the extracellular matrix (mx). Bar = 25 µ.

View larger version (105K): [in this window] [in a new window]   Figure 3. Reduced metabolic function in Igf1 null chondrocytes. Representative wild-type sections are shown on the left (A, C, E) and Igf1 null on the right (B, D, F). A, B) Immunostaining of the insulin-regulated GLUT4. Little GLUT4 is detected in Igf1 null chondrocytes although it is abundant in some bone marrow cells in these mice (B). C, D) Immunodetection of the ser9-phosphoform of GSK3b; E, F) PAS staining of glycogen deposits.

Another well-known target of insulin action is the enzyme glycogen synthase kinase 3ß (GSK3ß). Activation of a signaling cascade proceeding through PI3K and protein kinase B/Akt results in the serine phosphorylation and inhibition of GSK3ß activity (reviewed in ref 18 ). This in turn relieves GSK3ß-induced inhibition of glycogen synthase, thus promoting glycogen synthesis. Glycogen normally is accumulated by proliferative and early hypertrophic chondrocytes and depleted during the late stages of chondrocyte hypertrophy (19) . GSK3ß immunoreactivity was abundant in proliferative and hypertrophic chondrocytes in both WT and Igf1 null growth plates (Table 2) . An antibody specific for serine-phosphorylated GSK3ß (9) , however, demonstrated greatly reduced immunostaining for the inactive form of the enzyme in Igf1 null chondrocytes (Fig. 3C, D , Table 2 ). As predicted from the reduced level of serine-phosphorylated GSK3ß, PAS histochemical staining showed significantly reduced glycogen stores in the Igf1 null growth plate (Fig. 3E, F , Table 2 ).

Reduced glucose transport and intracellular glycogen stores may result in reduction of intracellular ATP supply and substrates for protein synthesis, particularly in cells dependent on glycolysis for energy production, as are hypertrophic chondrocytes (20) . Since energy and/or substrate deficit may lead to down-regulation of ribosomal RNA (rRNA) synthesis (21) , we compared cellular levels of rRNA in WT and Igf1 null growth plates using in situ hybridization. This analysis showed a drastic, ~70% reduction in rRNA content in Igf1 null hypertrophic chondrocytes and a lesser reduction in proliferative chondrocytes (Fig. 4 and Table 3 ). It is noteworthy that rRNA is more abundant in hypertrophic than proliferative chondrocytes, consistent with observations that mitochondria, Golgi, and endoplasmic reticulum are all more abundant in hypertrophic than in proliferative chondrocytes (22) .

View larger version (118K): [in this window] [in a new window]   Figure 4. Ribosomal RNA in WT (A, B) and Igf1 null (C, D) growth plates, as shown in paired dark- and bright-field photomicrographs. In situ hybridization using an 35S-labeled RNA probe complementary to the 28S ribosomal RNA shows that steady-state RNA levels are greater in hypertrophic compared with proliferative chondrocytes, and markedly greater in WT than Igf1 null chondrocytes in both zones (see Table 3 ). Bar = 200 µ.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study of longitudinal bone growth in the Igf1 null mouse has shown that growth plate chondrocyte proliferation and cell numbers are preserved despite an ~35% reduction in the rate of long bone growth. The growth defect due to Igf1 deletion is traced to an attenuation of chondrocyte hypertrophy, which is associated with reduced glucose transporter expression and glycogen synthesis and with reduced ribosomal RNA levels. These findings suggest that Igf1's mechanism of action in promoting statural growth involves `insulin-like' anabolic effects, supporting the extraordinary biosynthetic activity, somatic growth, and matrix production that characterize hypertrophic chondrocytes.

The present study supports the view that GH expands the pool of chondrocyte progenitors through direct effects on the growth plate germinal zone (23 , 24) , since the increased cellularity of the Igf1 null germinal zone may be attributed to increased GH secretion in the absence of negative feedback from Igf1. Igf1's role in long bone growth revealed in this study, however, refutes the prevailing view that this peptide is responsible for stimulating the clonal proliferation of growth plate chondrocytes (reviewed in ref 5 ). We have previously questioned this hypothesis, based on the finding that Igf2 rather than Igf1 is expressed by proliferating chondrocytes (6 , 7) . The present data demonstrate that Igf1's major effect on the growth plate is amplification of chondrocyte size, not proliferation. This is not to say that Igf1 normally has no role in chondrocyte proliferation, since enhanced GH action in the Igf1 null growth plate may compensate for some loss of Igf1 stimulation of chondrocyte proliferation. Also, the present data do not dispute the fact that exogenous Igf1 supplied in vivo or added to growth plate or chondrocyte cultures in vitro may enhance proliferation, since the Igf1 receptor is expressed by all types of chondrocytes (7) . We would argue, however, that chondrocyte proliferation in vivo is stimulated primarily by Igf2 in an autocrine/paracrine fashion, explaining why no major deficit is seen with Igf1 deletion. In contrast, the ~30% reduction in linear growth of hypertrophic chondrocytes and corresponding reduction in longitudinal bone growth seen in Igf1 null mice demonstrate that augmentation of chondrocyte somatic growth is Igf1's unique and essential role in amplifying statural growth.

Terminal hypertrophic chondrocytes are the quantal units for transformation of cartilage into bone. If the number of chondrocytes coming through the system per day is equal, then the size of the terminal units determines the longitudinal bone growth rate (2 , 3 , 22 , 25) . The present observations that the number of cells in the chondrocyte columns and chondrocyte DNA labeling indices are equal in the two groups suggest that the chondrocyte proliferation rate is equal in Igf1 null and WT mice. The differentiation of proliferative into hypertrophic chondrocytes also appears to occur normally, as evidenced by typical collagen X, bone sialoprotein and alkaline phosphatase expression in the Igf1 null growth plate. Furthermore, the fact that the proportion of cells in proliferative and hypertrophic zones is equal in both groups argues that progress in differentiation from one stage to another proceeds normally in Igf1 null growth plates. Thus, the only feature of growth plate expansion that is clearly disturbed in Igf1 null mice is the extent of chondrocyte hypertrophy, and the degree of attenuation of this process corresponds to the degree of reduction in longitudinal growth.

The rapid expansion of hypertrophic chondrocyte size is extraordinary, but the factors regulating this dramatic process have been unknown. The present data suggest that Igf1 enhances chondrocyte hypertrophy by insulin-like actions such as boosting glucose and amino acid uptake and utilization. Supporting this view, the Igf1 receptor, which is structurally and functionally homologous to the insulin receptor, is known to mediate such anabolic effects (16) , including regulation of GLUT4 (26) . We have shown that GLUT4 expression and GSK3ß serine phosphorylation are significantly diminished in Igf1 null hypertrophic chondrocytes, resulting in reduced glycogen in these cells. Glycogen stores are normally accumulated by proliferative and early hypertrophic chondrocytes and depleted during maturation of the hypertrophic chondrocytes (19 , 27) . Hypertrophic chondrocytes are highly active metabolically and are dependent on glycolysis to fuel their expansive biosynthetic activity (20) . The decrease in rRNA in Igf1 null hypertrophic chondrocytes may reflect cellular `starvation' for fuel and building blocks for protein synthesis (21) . The present data suggest that Igf1 acts in a truly insulin-like manner to enhance chondrocyte glucose uptake and glycogen and protein synthesis, thus promoting maximal somatic growth and matrix production and enhancing the rate of linear bone growth by ~30%.

	ACKNOWLEDGMENTS

 
We are grateful to Lynn Powell-Braxton and Genentech for making the Igf1 deletion line available to us, to Rita Haronen and Bjorn Olsen for providing the collagen X antibody, and to Ricardo Dreyfuss for expert photomicrography.

	FOOTNOTES

 
Received for publication March 1, 1999. Revised for publication May 24, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Kember, N. F., Walker, K. V. R. (1971) Control of bone growth. Nature (London) 229,428-429[Medline]
2. Hunziker, E. B., Schenk, R. K. (1989) Physiological mechanisms adopted by chondrocytes in regulating longitudinal bone growth in rats. J. Physiol. (London) 414,55-71[Abstract/Free Full Text]
3. Breur, G. J., VanEnkevort, B. A., Farnum, C. E., Wilsman, N. J. (1991) Linear relationship between the volume of hypertrophic chondrocytes and the rate of longitudinal bone growth in growth plates. J. Orth. Res. 9,348-359
4. Daughaday, W. H., Hall, K., Raben, M. S., Salmon, W. D., Jr, den Brande, J. L., van Wyk, J. J. (1972) Somatomedin: proposed designation for sulphation factor. Nature (London) 235,107[Medline]
5. Ohlsson, C., Bengtsson, B. A., Isaksson, O. G., Andreassen, T. T., Slootweg, M. C. (1998) Growth hormone and bone. Endocr. Rev. 19,55-79[Abstract/Free Full Text]
6. Shinar, D. M., Endo, N., Halperin, D., Rodan, G. A., Weinreb, M. (1993) Differential expression of insulin-like growth factor-I (IGF-I) and IGF- II messenger ribonucleic acid in growing rat bone. Endocrinology 132,1158-1167[Abstract/Free Full Text]
7. Wang, E., Wang, J., Chin, E., Zhou, J., Bondy, C. A. (1995) Cellular patterns of insulin-like growth factor system gene expression in murine chondrogenesis and osteogenesis. Endocrinology 136,2741-2751[Abstract]
8. Liu, J. P., Baker, J., Perkins, A. S., Robertson, E. J., Efstratiadis, A. (1993) 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[Medline]
9. Powell-Braxton, L., Hollingshead, P., Warburton, C., Dowd, M., Pitts-Meek, S., Dalton, D., Gillett, N., Stewart, T. A. (1993) IGF-I is required for normal embryonic growth in mice. Genes Dev 7,2609-2617[Abstract/Free Full Text]
10. Woods, K. A., Camacho-Hubner, C., Savage, M. O., Clark, A. J. (1996) 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[Free Full Text]
11. Cheng, C. M., Joncas, G., Reinhardt, R. R., Farrer, R., Quarles, R., Janssen, J., McDonald, M. P., Crawley, J. N., Powell-Braxton, L., Bondy, C. A. (1998) Biochemical and morphometric analyses show that myelination in the insulin-like growth factor 1 null brain is proportionate to its neuronal composition. J. Neurosci. 18,5673-5681[Abstract/Free Full Text]
12. Kwan, K. M., Pang, M. K., Zhou, S., Cowan, S. K., Kong, R. Y., Pfordte, T., Olsen, B. R., Sillence, D. O., Tam, P. P., Cheah, K. S. (1997) Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: implications for function. J. Cell Biol. 136,459-471[Abstract/Free Full Text]
13. Walker, K. V. R., Kember, N. F. (1972) Cell kinetics of growth cartilage in the rat tibia: measurements during aging. Cell Tissue Kinet 5,409-415[Medline]
14. Grant, W. T., Sussman, M. D., Balian, G. (1985) A disulfide-bonded short chain collagen synthesized by degenerative and calcifying zones of bovine growth plate cartilage. J. Biol. Chem. 260,3798-3803[Abstract/Free Full Text]
15. Gerstenfeld, L. C., Landis, W. J. (1991) Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system. J. Cell Biol. 12,501-513
16. De Meyts, P., Wallach, B., Christoffersen, C. T., Urso, B., Gronskov, K., Latus, L. J., Yakushiji, F., Ilondo, M. M., Shymko, R. M. (1994) The insulin-like growth factor-I receptor structure, ligand-binding mechanism and signal transduction. Horm. Res. 42,152-169[Medline]
17. Mueckler, M (1994) Facilitative glucose transporters. Eur. J. Biochem. 219,713-725[Medline]
18. Cohen, P., Alessi, D. R., Cross, D. A. (1997) PDK1, one of the missing links in insulin signal transduction?. FEBS Lett 410,3-10[Medline]
19. Brighton, C. T., Ray, R. D., Soble, L. W. (1969) In vitro epiphysial plate growth in various oxygen tensions. J. Bone Joint Surg. 1A,1383-1396
20. Brighton, C. T., Lackman, R. D., Cuckler, J. M. (1983) Absence of the glycerol phosphate shuttle in the various zones of the growth plate. J. Bone Joint Surg. 65A,663-666[Abstract/Free Full Text]
21. Roberts, J. (1997) Control of the supply line. Science 278,2073-2074[Free Full Text]
22. Hunziker, E. B., Schenk, R. K., Cruz-Orive, L. M. (1987) Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J. Bone Joint Surg. 69A,162-173[Abstract/Free Full Text]
23. Ohlsson, C., Nilsson, A., Isaksson, O., Lindahl, A. (1992) Growth hormone induces multiplication of the slowly cycling germinal cells of the rat tibial growth plate. Proc. Natl. Acad. Sci. USA 89,9826-9830[Abstract/Free Full Text]
24. Hunziker, E. B., Wagner, J., Zapf, J. (1994) Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo. J. Clin. Invest. 93,1078-1086
25. Breur, G. J., Lapierre, M. D., Kazmierczak, K., Stechuchak, K. M., McCabe, G. P. (1997) The domain of hypertrophic chondrocytes in growth plates growing at different rates. Calcif. Tissue Int. 61,418-425[Medline]
26. Wilson, C. M., Mitsumoto, Y., Maher, F., Klip, A. (1995) Regulation of cell surface GLUT1, GLUT3, and GLUT4 by insulin and IGF-I in L6 myotubes. FEBS Lett 368,19-22[Medline]
27. Pritcherd, J. J. (1952) A cytological and histochemical study of bone and cartilage formation. J. Anat. 8,259-277

This article has been cited by other articles:

				A. Giustina, G. Mazziotti, and E. Canalis Growth Hormone, Insulin-Like Growth Factors, and the Skeleton Endocr. Rev., August 1, 2008; 29(5): 535 - 559. [Abstract] [Full Text] [PDF]

				S. A. Kaplan and P. Cohen REVIEW: The Somatomedin Hypothesis 2007: 50 Years Later J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4529 - 4535. [Abstract] [Full Text] [PDF]

				K. E. Govoni, S. K. Lee, Y.-S. Chung, R. R. Behringer, J. E. Wergedal, D. J. Baylink, and S. Mohan Disruption of insulin-like growth factor-I expression in type II{alpha}I collagen-expressing cells reduces bone length and width in mice Physiol Genomics, August 20, 2007; 30(3): 354 - 362. [Abstract] [Full Text] [PDF]

				M. R. Hutchison, M. H. Bassett, and P. C. White Insulin-Like Growth Factor-I and Fibroblast Growth Factor, But Not Growth Hormone, Affect Growth Plate Chondrocyte Proliferation Endocrinology, July 1, 2007; 148(7): 3122 - 3130. [Abstract] [Full Text] [PDF]

				S. Ciarmatori, D. Kiepe, A. Haarmann, U. Huegel, and B. Tonshoff Signaling mechanisms leading to regulation of proliferation and differentiation of the mesenchymal chondrogenic cell line RCJ3.1C5.18 in response to IGF-I J. Mol. Endocrinol., April 1, 2007; 38(4): 493 - 508. [Abstract] [Full Text] [PDF]

				K. Frazier, R. Thomas, M. Scicchitano, R. Mirabile, R. Boyce, D. Zimmerman, E. Grygielko, J. Nold, A.-C. DeGouville, S. Huet, et al. Inhibition of ALK5 Signaling Induces Physeal Dysplasia in Rats Toxicol Pathol, February 1, 2007; 35(2): 284 - 295. [Abstract] [Full Text] [PDF]

				N. Ben Lagha, D. Seurin, Y. Le Bouc, M. Binoux, A. Berdal, P. Menuelle, and S. Babajko Insulin-Like Growth Factor Binding Protein (IGFBP-1) Involvement in Intrauterine Growth Retardation: Study on IGFBP-1 Overexpressing Transgenic Mice Endocrinology, October 1, 2006; 147(10): 4730 - 4737. [Abstract] [Full Text] [PDF]

				D. Kiepe, S. Ciarmatori, A. Haarmann, and B. Tonshoff Differential expression of IGF system components in proliferating vs. differentiating growth plate chondrocytes: the functional role of IGFBP-5 Am J Physiol Endocrinol Metab, February 1, 2006; 290(2): E363 - E371. [Abstract] [Full Text] [PDF]

				D. Kiepe, S. Ciarmatori, A. Hoeflich, E. Wolf, and B. Tonshoff Insulin-Like Growth Factor (IGF)-I Stimulates Cell Proliferation and Induces IGF Binding Protein (IGFBP)-3 and IGFBP-5 Gene Expression in Cultured Growth Plate Chondrocytes via Distinct Signaling Pathways Endocrinology, July 1, 2005; 146(7): 3096 - 3104. [Abstract] [Full Text] [PDF]

				D. A. M. Salih, S. Mohan, Y. Kasukawa, G. Tripathi, F. A. Lovett, N. F. Anderson, E. J. Carter, J. E. Wergedal, D. J. Baylink, and J. M. Pell Insulin-Like Growth Factor-Binding Protein-5 Induces a Gender-Related Decrease in Bone Mineral Density in Transgenic Mice Endocrinology, February 1, 2005; 146(2): 931 - 940. [Abstract] [Full Text] [PDF]

				C. W. E. Meyer, D. Korthaus, W. Jagla, E. Cornali, J. Grosse, H. Fuchs, M. Klingenspor, S. Roemheld, M. Tschop, G. Heldmaier, et al. A Novel Missense Mutation in the Mouse Growth Hormone Gene Causes Semidominant Dwarfism, Hyperghrelinemia, and Obesity Endocrinology, May 1, 2004; 145(5): 2531 - 2541. [Abstract] [Full Text] [PDF]

				T. Mushtaq, P. Bijman, S. F. Ahmed, and C. Farquharson Insulin-Like Growth Factor-I Augments Chondrocyte Hypertrophy and Reverses Glucocorticoid-Mediated Growth Retardation in Fetal Mice Metatarsal Cultures Endocrinology, May 1, 2004; 145(5): 2478 - 2486. [Abstract] [Full Text] [PDF]

				B. C. J. van der Eerden, M. Karperien, and J. M. Wit Systemic and Local Regulation of the Growth Plate Endocr. Rev., December 1, 2003; 24(6): 782 - 801. [Abstract] [Full Text] [PDF]

				C. W. Brown, L. Li, D. E. Houston-Hawkins, and M. M. Matzuk Activins Are Critical Modulators of Growth and Survival Mol. Endocrinol., December 1, 2003; 17(12): 2404 - 2417. [Abstract] [Full Text] [PDF]

				L. Longobardi, M. Torello, C. Buckway, L. O'Rear, W. A. Horton, V. Hwa, C. T. Roberts Jr., F. Chiarelli, R. G. Rosenfeld, and A. Spagnoli A Novel Insulin-Like Growth Factor (IGF)-Independent Role for IGF Binding Protein-3 in Mesenchymal Chondroprogenitor Cell Apoptosis Endocrinology, May 1, 2003; 144(5): 1695 - 1702. [Abstract] [Full Text] [PDF]

				S. Mohan, C. Richman, R. Guo, Y. Amaar, L. R. Donahue, J. Wergedal, and D. J. Baylink Insulin-Like Growth Factor Regulates Peak Bone Mineral Density in Mice by Both Growth Hormone-Dependent and -Independent Mechanisms Endocrinology, March 1, 2003; 144(3): 929 - 936. [Abstract] [Full Text] [PDF]

				W. M. Naranjo, S. Yakar, M. Sanchez-Gomez, A. U. Perez, J. Setser, and D. LERoith Protein Calorie Restriction Affects Nonhepatic IGF-I Production and the Lymphoid System: Studies Using the Liver-Specific IGF-I Gene-Deleted Mouse Model Endocrinology, June 1, 2002; 143(6): 2233 - 2241. [Abstract] [Full Text] [PDF]

				G. Camarero, C. Avendano, C. Fernandez-Moreno, A. Villar, J. Contreras, F. de Pablo, J. G. Pichel, and I. Varela-Nieto Delayed Inner Ear Maturation and Neuronal Loss in Postnatal Igf-1-Deficient Mice J. Neurosci., October 1, 2001; 21(19): 7630 - 7641. [Abstract] [Full Text] [PDF]

				D. Le Roith, C. Bondy, S. Yakar, J.-L. Liu, and A. Butler The Somatomedin Hypothesis: 2001 Endocr. Rev., February 1, 2001; 22(1): 53 - 74. [Abstract] [Full Text]

				C. M. Cheng, R. R. Reinhardt, W.-H. Lee, G. Joncas, S. C. Patel, and C. A. Bondy Insulin-like growth factor 1 regulates developing brain glucose metabolism PNAS, August 17, 2000; (2000) 170008497. [Abstract] [Full Text]

				C. M. Cheng, R. R. Reinhardt, W.-H. Lee, G. Joncas, S. C. Patel, and C. A. Bondy Insulin-like growth factor 1 regulates developing brain glucose metabolism PNAS, August 29, 2000; 97(18): 10236 - 10241. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WANG, J. Articles by BONDY, C. A. Search for Related Content PubMed PubMed Citation Articles by WANG, J. Articles by BONDY, C. A.