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IGF binding protein-2 complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis
Growth Hormone & IGF Research 2002, 12, 178–183 doi:10.1016/S1096-6374(02)00044-8, available online at http://www.idealibrary.com on
Subcutaneous administration of insulin-like growth factor (IGF)-II/IGF binding protein-2 complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis Cheryl A. Conover,1 Edward W. Johnstone,1 Russell T. Turner,2 Glenda L. Evans,2 F. John Ballard,3 Patrick M. Doran,1 and Sundeep Khosla1 1
Endocrine Research Unit, Division of Endocrinology, Metabolism and Nutrition, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA, Departments of Orthopedics and Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA, 3 GroPep Ltd, Thebarton, SA 5031, Australia 2
Summary Elevated serum levels of insulin-like growth factor binding protein-2 (IGFBP-2) and a precursor form of IGF-II are associated with marked increases in bone formation and skeletal mass in patients with hepatitis Cassociated osteosclerosis. In vitro studies indicate that IGF-II in complex with IGFBP-2 has high affinity for bone matrix and is able to stimulate osteoblast proliferation. The purpose of this study was to determine the ability of the IGF-II/IGFBP-2 complex to increase bone mass in vivo. Osteopenia of the femur was induced by unilateral sciatic neurectomy in rats. At the time of surgery, 14-day osmotic minipumps containing vehicle or 2 lg IGFII + 9 lg IGFBP-2/100 g body weight/day were implanted subcutaneously in the neck. Bone mineral density (BMD) measurements were taken the day of surgery and 14 days later using a PIXImus small animal densitometer. Neurectomy of the right hindlimb resulted in a 9% decrease in right femur BMD (P < 0:05 vs. baseline). This loss in BMD was completely prevented by treatment with IGF-II/IGFBP-2. On the control limb, there was no loss of BMD over the 14 days and IGF-II/IGFBP-2 treatment resulted in a 9% increase in left femur BMD ðP < 0:05Þ. Bone histomorphometry indicated increases in endocortical and cancellous bone formation rates and in trabecular thickness. These results demonstrate that short-term administration of the IGF-II/IGFBP-2 complex can prevent loss of BMD associated with disuse osteoporosis and stimulate bone formation in adult rats. Furthermore, they provide proof of concept for a novel anabolic approach to increasing bone mass in humans with osteoporosis. ª 2002 Elsevier Science Ltd. All rights reserved.
Key words: insulin-like growth factor-II, insulin-like growth factor binding protein-2, bone mineral density, bone histomorphometry, sciatic neurectomy Received 22 April 2002 Revised 23 May 2002 Accepted 3 June 2002 Correspondence to: Cheryl A. Conover, Ph.D., Endocrine Research Unit, Division of Endocrinology, Metabolism and Nutrition, Mayo Clinic and Mayo Foundation, 5-194 Joseph, Rochester, MN 55905, USA. Tel.: +1-507-255-6415; Fax: +1-507-255-4828; E-mail: conover.cheryl@mayo. edu
1096-6374/02/$ - see front matter
INTRODUCTION
Osteoporosis is one of the most important public health problems facing aging Americans.1 Although osteoporosis is more common in women due to its association with estrogen-deficiency following the menopause, men also incur substantial bone loss with aging.2;3 Indeed, as our elderly population grows, the ª 2002 Elsevier Science Ltd. All rights reserved.
Subcutaneous administration of insulin-like growth factor
prevalence of age-related osteoporosis will continue to increase. In addition, there are a number of secondary causes of osteoporosis associated, for example, with hypercortisolism and disuse.4 Therapeutic intervention for osteoporosis requires something safe and relatively specific to the skeleton that not only prevents loss of bone but also reverses the osteoporosis by stimulating the formation of new, functional bone. The recent description by us and others of patients with hepatitis C-associated osteosclerosis (HCAO) has provided clues for a novel approach to increasing bone mass in patients with osteoporosis. To date, only 10 cases (7 men, 3 women) with this unusual disorder have been reported.5–12 The syndrome is characterized by markedly increased osteoblast function and dramatic increases in skeletal mass in adults. Both cancellous and cortical bone mass are increased. Skeletal radiographs have shown increased radiodensity of long bones, hip, and spine, with sparing of the calvarium and facial bones. Histologically, the bone has a normal, lamellar appearance with no evidence of woven bone. Besides the dramatic increase in bone mass, these patients have no major clinical abnormalities. Thus, while HCAO is an extremely rare syndrome, an understanding of the mediators of the increase in skeletal mass could provide clues for developing novel therapeutic approaches to the treatment of osteoporosis. The insulin-like growth factors (IGFs) are critical skeletal growth factors acting through specific receptors identified on various osteoblastic cells.13–15 We previously reported alterations in the circulating IGF system in HCAO.5 Specifically, there were increased levels of an IGF binding protein, IGFBP-2, and a precursor form of IGF-II which circulated together in a complex that can cross the capillary barrier and access target tissues. There was no disruption of the other IGF system components. These clinical observations along with further in vitro studies support a model whereby systemic administration of IGF-II/IGFBP-2 complex would transit the capillary barrier. IGFBP-2, when bound to IGF-II, has enhanced affinity for bone extracellular matrix. Upon binding to matrix, there is an apparent conformational change in IGFBP-2 resulting in the release of IGF-II in the bone microenvironment with a consequent stimulation of osteoblast function and an increase in skeletal mass. Thus, this model suggests a novel approach to potentially match or exceed the efficacy of locally delivered IGF on bone.16–18 The purpose of the present study was to test a predicted outcome of this model by determining the ability of the systemically administered IGF-II/IGFBP-
179
2 complex to increase bone mass in vivo using a rat model of disuse osteoporosis. MATERIALS AND METHODS Materials
Recombinant human IGF-II and bovine IGFBP-2 (receptor-grade) were provided by GroPep Ltd. (Adelaide, Australia). Osmotic minipumps were purchased from Durect Corp. (Cupertino, CA). Unilateral sciatic neurectomy
Female rats at approximately 3 months of age were obtained from Harlan Sprague–Dawley (Madison, WI). Animals were maintained at 24 °C on a 12-h light and 12-h dark cycle with air conditioning in AAALC-accredited and inspected animal facilities. Rats were allowed access to rodent lab chow and water ad libitum. Osteopenia of the femur was induced by unilateral sciatic neurectomy in rats, as previously described.19;20 At the time of surgery, 14-day osmotic minipumps containing vehicle (NaCl/1% BSA, n ¼ 6) or 2 lg IGFII + 9 lg IGFBP-2/100 g body weight/day (n ¼ 5) were implanted at the back of the neck.21 The IGF-II and IGFBP-2 were allowed to complex overnight at room temperature prior to implantation. The stability and bioactivity of the compounds at 37 °C for 14 days was verified in earlier experiments (unpublished data). After surgery, animals were housed in individual cages. At completion of the 14 day infusion period, rat were anesthetized with CO2 before exsanguination by cardiac puncture. This study was approved by Mayo’s Institutional Animal Care and Use Committee. Bone mineral density
Bone mineral density measures of the right and left femurs were taken on the day of surgery and 14 days later using a PIXImus small animal densitometer (Lunar, Madison, WI). Bone histomorphometry
Tetracycline–HCl (Sigma Chemical, St. Louis, MO) at 2 mg/kg body weight was administered as juxta-tail vein injections on day 8 and day 13, and femurs processed for bone histomorphometry as described previously.19–27 The cortical area was measured by tracing the medullar canal and the outer perimeter of the bone using a drawing board (Calcorp digitizer products, Anaheim,
180 C.A. Conover et al.
CA) and Osteomeasure Software (Osteometrics, Inc., Atlanta, GA) at 60 magnification. Cross-sectional area is the entire area within the outer perimeter of the bone. The medullary area is the area within the endosteal surface. The cortical area is the difference between the cross-sectional and medullary areas. Cancellous bone was measured by tracing all trabeculae in the secondary spongiosa at the distal end of the femur at 300 magnification. The area of measurement was 2:28 mm2 . Bone volume is the area that is bone within 1 mm2 of secondary spongiosa. Bone surface is the length of the surface of the trabecular within 1 mm2 of secondary spongiosa. Ultraviolet light microscopy was used to visualize the tetracycline that had been incorporated into newly formed bone at the time of each injection. Fluorescent labels were traced in the cortical and cancellous bone at a 60 and 300 magnification, respectively, using the drawing board and Osteomeasure software, as described previously.27 Bone formation rate (BFR) in cortical and cancellous bone was calculated by division of the ‘‘dual label area’’ by the number of days between tetracycline administration.
ences between limbs, and there was no evidence of an effect of IGF-II/IGFBP-2 treatment to accelerate linear bone growth (IGF-II/IGFBP-2 0:96 0:651 mm/14 days, vehicle 1:58 0:417 mm/14 days).
Blood glucose
Histomorphometry
Blood glucose levels were measured by Mayo’s Immunochemical Core Laboratory using the Hitachi 912 Chemistry Analyzer with hexokinase reagent from Boehringer–Mannheim (Indianapolis, IN).
The results of the bone histomorphometry on the femur are presented in Table 1. There was a 37% increase in endocortical BFR and a doubling of cancellous BFR with IGF-II/IGFBP-2 treatment. These data did not achieve statistical significance, however. There was a significant, 18% increase in trabecular
Statistical analysis
Data are expressed as means SE. Statistical differences were determined by ANOVA followed by twosided t-test, with a P value of 0.05.
Fig. 1 Effect of unilateral sciatic neurectomy and IGF-II/IGFBP-2 treatment on bone mineral density. Rats underwent sciatic nerve resection on the right hindlimb and 14-day osmotic minipumps containing vehicle (n ¼ 6) or IGF-II/IGFBP-2 (II/BP-2, n ¼ 5) were implanted at the back of the neck. BMD measures of the right and left femurs were taken on the day of surgery (baseline, open bars) and 14 days later (solid bars). *P < 0:05 14-day vs baseline following ANOVA.
Table 1 Effect of IGF-II/IGFBP-2 treatment on bone histomorphometry Vehicle
RESULTS BMD
Reduced weight bearing due to motor nerve resection results in osteopenic changes localized to the affected limb.28;29 In this experiment (Fig. 1), sciatic neurectomy of the right hindlimb resulted in a 9% loss in right femur BMD ðP < 0:05Þ that was prevented by treatment with IGF-II/IGFBP-2. The femur of the unoperated left limb experienced no change in BMD over the 14 days. However, IGF-II/IGFBP-2 treatment resulted in a 9% increase in BMD in the left femur ðP < 0:05Þ. As an index of linear growth, tibial length was measured pre- and 14 days post-infusion using the PIXImus scan data. There were no significant differ-
IGF-II/IGFBP-2
Cortical bone Cortical Area (mm) Endocortical BFR
4:56 0:05
4:63 0:01
5:81 0:97
7:94 0:84
19:08 2:19
17:87 1:64
ðmm2 =d 1000Þ Periosteal BFR ðmm2 =d 1000Þ Cancellous bone BV/TV (%)
19:5 1:68
22:9 3:00
Tb Th ðlmÞ
46:1 1:68
54:2 2:04
Tb Sp ðlmÞ
211 20:3
210 25:2
Tb N ðmm1 Þ
4:12 0:30
4:11 0:41
BFR/BS ðlm3 =mm2 =d 100Þ
4:49 2:07
9:22 2:59
Values are means SE: Tb, trabecular; Th, thickness; Sp, separation; N, number; BV, bone volume; TV, total volume; BS, bone surface. * P < 0:05 vs vehicle following one-way ANOVA.
Subcutaneous administration of insulin-like growth factor
thickness with treatment. There was no apparent effect on cortical area, periosteal BFR, trabecular number or trabecular spacing. Blood glucose
One of the major concerns about systemic administration of IGF is generalized effects on tissues and the risk of hypoglycemia. However, treatment with IGF-II/ IGFBP-2 complex did not lower blood glucose levels in these animals (167 6 mg/dl vehicle, 183 10 mg/ dl IGF-II/IGFBP-2). DISCUSSION
These data provide proof of concept for increasing bone mass in vivo through systemic administration of the IGF-II/IGFBP-2 complex. Treatment of adult rats with IGF-II/IGFBP-2 not only prevented the loss of BMD in the femur effected by sciatic neurectomy, it also significantly increased BMD in the femur of the control limb. The stated purpose of this study was to determine the ability of IGF-II/IGFBP-2 complex to increase bone mass in vivo, and there were no treatment groups consisting of IGF-II or IGFBP-2 alone. Nonetheless, pharmacokinetic profiles indicating generalized distribution,30;31 clinical observations in patients with elevations in either IGF-II or IGFBP-2,5;32 animal studies comparing IGF-I alone and in complex,33 and our in vitro studies5 would predict little or no anabolic effect of systemic administrations of these concentrations of IGF-II or IGFBP-2 alone on bone. Indeed, IGFBP-2 alone might be expected to inhibit bone growth.34 Thus, although we have demonstrated a significant effect of the complex, we cannot draw any conclusion that IGF-II/IGFBP-2 is more effective than IGF-II from these data. This effect of IGF-II/IGFBP-2 to increase BMD appears to be through stimulation of endocortical and cancellous bone formation rates. However, with the small group size powered for the primary outcome variable, BMD, and the inherent variability of bone histomorphometry measures these data did not achieve statistical significance. Nonetheless, trabecular thickness was significantly increased with treatment. If, as these data suggest and as observed in the HCAO patients,5 the IGF-II/IGFBP-2 complex has efficacy at both cortical and cancellous bone sites, then this treatment would have major biomechanical benefit and would be predicted to reduce fracture risk. While our data do not establish the molecular mechanism of IGF-II/IGFBP-2 action in bone, the histomorphometry results are consistent with increased
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osteoblast activity. Bone surface osteoblasts and osteoprogenitor cells in the marrow stroma are induced to proliferate in response to IGFs in vitro and in vivo.13–15;35;36 IGFs also stimulate collagen synthesis, inhibit collagen breakdown through decreased expression of specific skeletal-derived collagenases, and enhance alkaline phosphatase activity.37–39 Osteoblast survival in vitro has been shown to be promoted by IGF.40 In addition, possible effects on bone resorption need to be addressed. Further studies are necessary to determine the mode and target cell of IGF-II/IGFBP-2 action. These preliminary studies have obvious potential implication for the treatment of osteoporosis. The notion of using IGFs to treat osteoporosis is not, however, a novel one and previous human studies using IGF-I treatment have found increases in serum levels of markers of bone formation.41;42 This approach was limited by the multiple systemic effects of IGF-I, including hypoglycemia. Administration of IGFI complexed with IGFBP-3 minimized the risk of hypoglycemia and other side effects, but was not selective for bone.33;43–47 The use of IGF-II to treat osteoporotic patients has not been previously reported. Our observations in HCAO and findings in vitro indicated that IGF-II in complex with IGFBP-2 could be a safe, effective, and relatively selective anabolic for the skeleton. The significant increase in BMD with no effect on blood glucose is consistent with preferential skeletal effects. The relatively low amount of growth factor necessary to elicit the biological response represents a distinct advantage. Indeed, in HCAO it is the precursor form of IGF-II that is elevated and in complex with IGFBP-2 and it remains to be seen if this form of IGF-II is even more effective than mature IGF-II in further preclinical trials. In addition, it will be important to test this treatment in a model of established osteoporosis, such as the ovariectomized rat or mouse.48;49 In summary, short-term administration of IGF-II/ IGFBP-2 prevented the loss of BMD associated with disuse osteoporosis and stimulated bone formation in adult rats. These data support the potential of IGF-II/ IGFBP-2 as a novel anabolic treatment for osteoporosis. REFERENCES 1. National Institutes of Health Consensus Development Conference Statement. Osteoporosis Prevention, Diagnosis, and Therapy. March 27-29, 2000:1–36. 2. Riggs BL, Melton III LJ. Involutional osteoporosis. N Engl J Med 1986; 314: 1676–1686. 3. Orwoll ES, Klein RF. Osteoporosis in men. Endocr Rev 1995; 16: 87–116.
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4. Khosla S, Riggs BL, Melton III LJ. Clinical spectrum. In: Riggs BL, Melton III LJ (eds). Osteoporosis: etiology, diagnosis, and management, second ed. Philadelphia, PA: Lippincott-Raven Publishers; 1995: pp. 205–223. 5. Khosla S, Hassoun AAK, Baker BK, et al. Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis: potential insights into increasing bone mass in adults. J Clin Invest 1998; 101: 2165–2173. 6. Beyer SH, Parfitt AM, Shih MS, Anderson Q, Heath III H. Idiopathic acquired diffuse osteosclerosis in a young women. J Bone Miner Res 1990; 5: 1257–1263. 7. Villareal DT, Murphy WA, Teitelbaum SL, Arens MQ, Whyte MP. Painful diffuse osteosclerosis after intravenous drug abuse. Am J Med 1992; 93: 371–381. 8. Hassoun A, Nippoldt TB, Tiegs RD, Khosla S. Hepatitis C associated osteosclerosis: an unusual syndrome of acquired osteosclerosis in adults. Am J Med 1997; 103: 70–73. 9. Whyte MP, Teitelbaum SL, Reinus WR. Doubling skeletal mass during adult life: the syndrome of diffuse osteosclerosis after intravenous drug abuse. J Bone Miner Res 1996; 11: 554–558. 10. Whyte MP, Reasner CA. Hepatitis C-associated osteosclerosis after blood transfusion. Am J Med 1997; 102: 219–220. 11. Diamond T, Depczynski B. Acquired osteosclerosis associated with intravenous drug use and hepatitis C infection. Bone 1996; 19: 679–683. 12. Shaker JL, Reinus WR, Whyte MP. Hepatitis C-associated osteosclerosis: late onset after blood transfusion in an elderly woman. J Clin Endocrinol Metab 1998; 83: 93–98. 13. Conover CA. The role of insulin-like growth factors and binding proteins in bone cell biology. In: Bilezikian J, Raisz L, Rodan G (eds). Principles of bone biology. San Diego, CA: Academic Press; 1996: pp. 607–618 [chapter 43]. 14. Cohan S, Baylink DJ. IGF system components and their role in bone metabolism. In: Rosenfeld RG, Roberts Jr. CT, (eds). The IGF system: molecular biology, physiology, and clinical applications. Totawa, NJ: Humana Press; 1999: pp. 457–496 [chapter 20]. 15. Canalis E. Insulin like growth factors and the local regulation of bone formation. Bone 1993; 14: 273–276. 16. Tanaka H, Quarto R, Williams S, Barnes J, Liang CT. In vivo and in vitro effects of insulin-like growth factor-I (IGF-I) on femoral mRNA expression in old rats. Bone 1994; 15: 647–653. 17. Wakisaka A, Tanaka H, Barnes J, Liang CT. Effect of locally infused IGF-I on femoral gene expression and bone turnover activity in old rats. J Bone Miner Res 1998; 13: 13–19. 18. Ishibe M, Ishibashi T, Kaneda K, Koda T, Rosier RN, Puzas JE. Stimulation of bone formation in vivo by insulin-like growth factor-II in rats. Calcif Tissue Int 1998; 63: 36–38. 19. Wakley GK, Baum BL, Hannon KS, Turner RT. The effects of tamoxifen on the osteopenia induced by sciatic neurotomy in the rat: a histomorphometric study. Calcif Tissue Int 1988; 43: 383–388. 20. Firling CE, Evans GL, Wakley GK, Sibonga J, Turner RT. Lack of an effect of sodium zeolite A on rat tibia histomorphometry. J Bone Miner Res 1996; 11: 254–263. 21. Dobnig H, Turner RT. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 1996; 136: 3632–3638. 22. Turner RT, Spelsberg TC. Correlation between mRNA levels for bone cell proteins and bone formation in long bones of maturing rats. Am J Physiol 1991; 261: E348–E353.
23. Westerlind KC, Wakley GK, Evans GL, Turner RT. Estrogen does not increase bone formation in growing rats. Endocrinology 1993; 133: 2924–2934. 24. Cavolina JM, Evans GL, Harris SA, Zhang M, Westerlind K, Turner RT. The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signalling peptides in ovariectomized growing rats. Endocrinology 1997; 138: 1567–1576. 25. Kidder LS, Schmidt IU, Evans GL, Turner RT. Effects of growth hormone and low dose estrogen on bone growth and turnover in long bones of hypophysectomized rats. Calcif Tissue Int 1997; 61: 327–335. 26. Turner RT, Vandersteenhoven JJ, Bell NH. The effects of ovariectomy and 17b-estradiol on cortical bone histomorphometry in growth rats. J Bone Miner Res 1987; 2: 115–122. 27. Turner RT, Wakley GK, Hannon KS, Bell NH. Tamoxifen inhibits osteoclast-mediated bone resorption of trabecular bone in ovarian hormone-deficient rats. Endocrinology 1988; 122: 1146–1150. 28. Gillespie JA. The nature of the bone changes associated with nerve injuries and disuse. J Bone Joint Surg 1954; 36B: 464–473. 29. Landry M, Fleisch H. The influence of immobilization on bone formation as evaluated by osseous incorporation of tetracyclines. J Bone Joint Surg 1964; 46B: 764–771. 30. Ballard FJ, Knowles SE, Walton PE, et al. Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(1–3)IGF-I in rats. J Endocrinol 1990; 128: 197–204. 31. Young SCJ, Miles MV, Clemmons DR. Determination of the pharmacokinetic profiles of insulin-like growth factor binding proteins-1 and -2 in rats. Endocrinology 1992; 131: 1867–1873. € nschoff B, Blum WF, Wingen A-M, Mehls O, and the European 32. To Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood. Serum insulin-like growth factors IGFs and IGF binding proteins1,2, and3 in children with chronic renal failure:relationship to height and glomerular filtration rate. J Clin Endocrinol Metab 1995;80:2684–2691. 33. Bagi CM, Brommage R, Deleon L, Adams S, Rosen D, Sommer A. Benefit of systemically administered rhIGF-I and rhIGF-I/ IGFBP-3 on cancellous bone in ovariectomized rats. J Bone Miner Res 1994; 9: 1301–1312. 34. Hoeflich A, Wu M, Mohan S, et al. Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain. Endocrinology 1999; 140: 5488–5496. 35. Machwate M, Zerath E, Holy X, Pastoureau P, Marie PJ. Insulin-like growth factor-I increases trabecular bone formation and osteoblastic cell proliferation in unloaded rats. Endocrinology 1994; 134: 1031–1038. 36. Thomas T, Gori F, Spelsberg TC, Khosla S, Riggs BL, Conover CA. Response of bipotential human marrow stromal cells to insulin-like growth factors: effect on binding protein production, proliferation, and commitment to osteoblasts and adipocytes. Endocrinology 1999; 140: 5036–5044. 37. McCarthy TL, Centrella M, Canalis E. Regulatory effects of IGF-I and IGF-II on bone collagen synthesis in rat calvarial cultures. Endocrinology 1989; 124: 301–309. 38. Canalis E. Insulin like growth factors and the local regulation of bone formation. Bone 1993; 14: 273–276.
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39. Richman C, Baylink DJ, Lang K, Dony C, Mohan S. Recombinant human insulin-like growth factor-binding protein-5 stimulates bone formation parameters in vitro and in vivo. Endocrinology 1999; 140: 4699–4705. 40. Hill PA, Tumber A, Meikle MC. Multiple extracellular signals promote osteoblast survival and apoptosis. Endocrinology 1997; 138: 3849–3858. 41. Rosen CJ, Donahue LR. Insulin-like growth factors: potential therapeutic options for osteoporosis. Trends Endocrinol Metab 1995; 6: 235–241. 42. Johansson AG, Rosen CJ. The IGFs as potential therapy for metabolic bone diseases. In: Bilezikian J, Raisz L, Rodan G (eds). Principles of bone biology. San Diego, CA: Academic Press; 1996: pp. 1099–1109 [chapter 79]. 43. Hamon GA, Hunt TK, Spencer EM. In vivo effects of systemic insulin-like growth factor-I alone and complexed with insulinlike growth factor binding protein-3 on corticosteroid suppressed wounds. Growth Regul 1993; 3: 53–56. 44. Narusawa K, Nakamura T, Suzuki K, et al. The effects of recombinant human insulin-like growth factor rhIGF-I and rhIGF-I/IGF binding protein-3 administration on rat osteopenia
45.
46.
47.
48.
49.
183
induced by ovariectomy with concomitant bilateral sciatic neurectomy. J Bone Miner Res 1995; 10: 1853–1864. Bagi CM, DeLeon E, Brommage R, Rosen D, Sommer A. Treatment of ovariectomized rats with the complex of rhIGF-I/ IGFBP-3 increases cortical and cancellous bone mass and improves structure in the femoral neck. Calcif Tissue Int 1995; 57: 40–46. Sandra A, Boes M, Dake BL, Stokes JB, Bar RS. Infused IGF-I/ IGFBP-3 complex causes glomerular localization of IGF-I in the rat kidney. Am J Physiol 1998; 275: E32–E37. Svanberg E, Ohlsson C, Kimball SR, Lundholm K. rhIGF-I/IGFBP3 complex, but not free rhIGF-I, supports muscle protein biosynthesis in rats during semistarvation. Eur J Clin Invest 2000; 30: 438–446. Geddes AD. Animal models of bone disease. In: Bilezikian JP, Raisz LG, Rodan GA (eds). Principles of bone biology. New York, NY: Academic Press; 1996: pp. 1343–1354. Alexander JM, Bab I, Fish S, et al. Human parathyroid hormone 1–34 reverses bone loss in ovariectomized mice. J Bone Miner Res 2001; 16: 1665–1673.
Subcutaneous administration of insulin-like growth factor (IGF)-II/IGF binding protein-2 complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis Cheryl A. Conover,1 Edward W. Johnstone,1 Russell T. Turner,2 Glenda L. Evans,2 F. John Ballard,3 Patrick M. Doran,1 and Sundeep Khosla1 1
Endocrine Research Unit, Division of Endocrinology, Metabolism and Nutrition, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA, Departments of Orthopedics and Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA, 3 GroPep Ltd, Thebarton, SA 5031, Australia 2
Summary Elevated serum levels of insulin-like growth factor binding protein-2 (IGFBP-2) and a precursor form of IGF-II are associated with marked increases in bone formation and skeletal mass in patients with hepatitis Cassociated osteosclerosis. In vitro studies indicate that IGF-II in complex with IGFBP-2 has high affinity for bone matrix and is able to stimulate osteoblast proliferation. The purpose of this study was to determine the ability of the IGF-II/IGFBP-2 complex to increase bone mass in vivo. Osteopenia of the femur was induced by unilateral sciatic neurectomy in rats. At the time of surgery, 14-day osmotic minipumps containing vehicle or 2 lg IGFII + 9 lg IGFBP-2/100 g body weight/day were implanted subcutaneously in the neck. Bone mineral density (BMD) measurements were taken the day of surgery and 14 days later using a PIXImus small animal densitometer. Neurectomy of the right hindlimb resulted in a 9% decrease in right femur BMD (P < 0:05 vs. baseline). This loss in BMD was completely prevented by treatment with IGF-II/IGFBP-2. On the control limb, there was no loss of BMD over the 14 days and IGF-II/IGFBP-2 treatment resulted in a 9% increase in left femur BMD ðP < 0:05Þ. Bone histomorphometry indicated increases in endocortical and cancellous bone formation rates and in trabecular thickness. These results demonstrate that short-term administration of the IGF-II/IGFBP-2 complex can prevent loss of BMD associated with disuse osteoporosis and stimulate bone formation in adult rats. Furthermore, they provide proof of concept for a novel anabolic approach to increasing bone mass in humans with osteoporosis. ª 2002 Elsevier Science Ltd. All rights reserved.
Key words: insulin-like growth factor-II, insulin-like growth factor binding protein-2, bone mineral density, bone histomorphometry, sciatic neurectomy Received 22 April 2002 Revised 23 May 2002 Accepted 3 June 2002 Correspondence to: Cheryl A. Conover, Ph.D., Endocrine Research Unit, Division of Endocrinology, Metabolism and Nutrition, Mayo Clinic and Mayo Foundation, 5-194 Joseph, Rochester, MN 55905, USA. Tel.: +1-507-255-6415; Fax: +1-507-255-4828; E-mail: conover.cheryl@mayo. edu
1096-6374/02/$ - see front matter
INTRODUCTION
Osteoporosis is one of the most important public health problems facing aging Americans.1 Although osteoporosis is more common in women due to its association with estrogen-deficiency following the menopause, men also incur substantial bone loss with aging.2;3 Indeed, as our elderly population grows, the ª 2002 Elsevier Science Ltd. All rights reserved.
Subcutaneous administration of insulin-like growth factor
prevalence of age-related osteoporosis will continue to increase. In addition, there are a number of secondary causes of osteoporosis associated, for example, with hypercortisolism and disuse.4 Therapeutic intervention for osteoporosis requires something safe and relatively specific to the skeleton that not only prevents loss of bone but also reverses the osteoporosis by stimulating the formation of new, functional bone. The recent description by us and others of patients with hepatitis C-associated osteosclerosis (HCAO) has provided clues for a novel approach to increasing bone mass in patients with osteoporosis. To date, only 10 cases (7 men, 3 women) with this unusual disorder have been reported.5–12 The syndrome is characterized by markedly increased osteoblast function and dramatic increases in skeletal mass in adults. Both cancellous and cortical bone mass are increased. Skeletal radiographs have shown increased radiodensity of long bones, hip, and spine, with sparing of the calvarium and facial bones. Histologically, the bone has a normal, lamellar appearance with no evidence of woven bone. Besides the dramatic increase in bone mass, these patients have no major clinical abnormalities. Thus, while HCAO is an extremely rare syndrome, an understanding of the mediators of the increase in skeletal mass could provide clues for developing novel therapeutic approaches to the treatment of osteoporosis. The insulin-like growth factors (IGFs) are critical skeletal growth factors acting through specific receptors identified on various osteoblastic cells.13–15 We previously reported alterations in the circulating IGF system in HCAO.5 Specifically, there were increased levels of an IGF binding protein, IGFBP-2, and a precursor form of IGF-II which circulated together in a complex that can cross the capillary barrier and access target tissues. There was no disruption of the other IGF system components. These clinical observations along with further in vitro studies support a model whereby systemic administration of IGF-II/IGFBP-2 complex would transit the capillary barrier. IGFBP-2, when bound to IGF-II, has enhanced affinity for bone extracellular matrix. Upon binding to matrix, there is an apparent conformational change in IGFBP-2 resulting in the release of IGF-II in the bone microenvironment with a consequent stimulation of osteoblast function and an increase in skeletal mass. Thus, this model suggests a novel approach to potentially match or exceed the efficacy of locally delivered IGF on bone.16–18 The purpose of the present study was to test a predicted outcome of this model by determining the ability of the systemically administered IGF-II/IGFBP-
179
2 complex to increase bone mass in vivo using a rat model of disuse osteoporosis. MATERIALS AND METHODS Materials
Recombinant human IGF-II and bovine IGFBP-2 (receptor-grade) were provided by GroPep Ltd. (Adelaide, Australia). Osmotic minipumps were purchased from Durect Corp. (Cupertino, CA). Unilateral sciatic neurectomy
Female rats at approximately 3 months of age were obtained from Harlan Sprague–Dawley (Madison, WI). Animals were maintained at 24 °C on a 12-h light and 12-h dark cycle with air conditioning in AAALC-accredited and inspected animal facilities. Rats were allowed access to rodent lab chow and water ad libitum. Osteopenia of the femur was induced by unilateral sciatic neurectomy in rats, as previously described.19;20 At the time of surgery, 14-day osmotic minipumps containing vehicle (NaCl/1% BSA, n ¼ 6) or 2 lg IGFII + 9 lg IGFBP-2/100 g body weight/day (n ¼ 5) were implanted at the back of the neck.21 The IGF-II and IGFBP-2 were allowed to complex overnight at room temperature prior to implantation. The stability and bioactivity of the compounds at 37 °C for 14 days was verified in earlier experiments (unpublished data). After surgery, animals were housed in individual cages. At completion of the 14 day infusion period, rat were anesthetized with CO2 before exsanguination by cardiac puncture. This study was approved by Mayo’s Institutional Animal Care and Use Committee. Bone mineral density
Bone mineral density measures of the right and left femurs were taken on the day of surgery and 14 days later using a PIXImus small animal densitometer (Lunar, Madison, WI). Bone histomorphometry
Tetracycline–HCl (Sigma Chemical, St. Louis, MO) at 2 mg/kg body weight was administered as juxta-tail vein injections on day 8 and day 13, and femurs processed for bone histomorphometry as described previously.19–27 The cortical area was measured by tracing the medullar canal and the outer perimeter of the bone using a drawing board (Calcorp digitizer products, Anaheim,
180 C.A. Conover et al.
CA) and Osteomeasure Software (Osteometrics, Inc., Atlanta, GA) at 60 magnification. Cross-sectional area is the entire area within the outer perimeter of the bone. The medullary area is the area within the endosteal surface. The cortical area is the difference between the cross-sectional and medullary areas. Cancellous bone was measured by tracing all trabeculae in the secondary spongiosa at the distal end of the femur at 300 magnification. The area of measurement was 2:28 mm2 . Bone volume is the area that is bone within 1 mm2 of secondary spongiosa. Bone surface is the length of the surface of the trabecular within 1 mm2 of secondary spongiosa. Ultraviolet light microscopy was used to visualize the tetracycline that had been incorporated into newly formed bone at the time of each injection. Fluorescent labels were traced in the cortical and cancellous bone at a 60 and 300 magnification, respectively, using the drawing board and Osteomeasure software, as described previously.27 Bone formation rate (BFR) in cortical and cancellous bone was calculated by division of the ‘‘dual label area’’ by the number of days between tetracycline administration.
ences between limbs, and there was no evidence of an effect of IGF-II/IGFBP-2 treatment to accelerate linear bone growth (IGF-II/IGFBP-2 0:96 0:651 mm/14 days, vehicle 1:58 0:417 mm/14 days).
Blood glucose
Histomorphometry
Blood glucose levels were measured by Mayo’s Immunochemical Core Laboratory using the Hitachi 912 Chemistry Analyzer with hexokinase reagent from Boehringer–Mannheim (Indianapolis, IN).
The results of the bone histomorphometry on the femur are presented in Table 1. There was a 37% increase in endocortical BFR and a doubling of cancellous BFR with IGF-II/IGFBP-2 treatment. These data did not achieve statistical significance, however. There was a significant, 18% increase in trabecular
Statistical analysis
Data are expressed as means SE. Statistical differences were determined by ANOVA followed by twosided t-test, with a P value of 0.05.
Fig. 1 Effect of unilateral sciatic neurectomy and IGF-II/IGFBP-2 treatment on bone mineral density. Rats underwent sciatic nerve resection on the right hindlimb and 14-day osmotic minipumps containing vehicle (n ¼ 6) or IGF-II/IGFBP-2 (II/BP-2, n ¼ 5) were implanted at the back of the neck. BMD measures of the right and left femurs were taken on the day of surgery (baseline, open bars) and 14 days later (solid bars). *P < 0:05 14-day vs baseline following ANOVA.
Table 1 Effect of IGF-II/IGFBP-2 treatment on bone histomorphometry Vehicle
RESULTS BMD
Reduced weight bearing due to motor nerve resection results in osteopenic changes localized to the affected limb.28;29 In this experiment (Fig. 1), sciatic neurectomy of the right hindlimb resulted in a 9% loss in right femur BMD ðP < 0:05Þ that was prevented by treatment with IGF-II/IGFBP-2. The femur of the unoperated left limb experienced no change in BMD over the 14 days. However, IGF-II/IGFBP-2 treatment resulted in a 9% increase in BMD in the left femur ðP < 0:05Þ. As an index of linear growth, tibial length was measured pre- and 14 days post-infusion using the PIXImus scan data. There were no significant differ-
IGF-II/IGFBP-2
Cortical bone Cortical Area (mm) Endocortical BFR
4:56 0:05
4:63 0:01
5:81 0:97
7:94 0:84
19:08 2:19
17:87 1:64
ðmm2 =d 1000Þ Periosteal BFR ðmm2 =d 1000Þ Cancellous bone BV/TV (%)
19:5 1:68
22:9 3:00
Tb Th ðlmÞ
46:1 1:68
54:2 2:04
Tb Sp ðlmÞ
211 20:3
210 25:2
Tb N ðmm1 Þ
4:12 0:30
4:11 0:41
BFR/BS ðlm3 =mm2 =d 100Þ
4:49 2:07
9:22 2:59
Values are means SE: Tb, trabecular; Th, thickness; Sp, separation; N, number; BV, bone volume; TV, total volume; BS, bone surface. * P < 0:05 vs vehicle following one-way ANOVA.
Subcutaneous administration of insulin-like growth factor
thickness with treatment. There was no apparent effect on cortical area, periosteal BFR, trabecular number or trabecular spacing. Blood glucose
One of the major concerns about systemic administration of IGF is generalized effects on tissues and the risk of hypoglycemia. However, treatment with IGF-II/ IGFBP-2 complex did not lower blood glucose levels in these animals (167 6 mg/dl vehicle, 183 10 mg/ dl IGF-II/IGFBP-2). DISCUSSION
These data provide proof of concept for increasing bone mass in vivo through systemic administration of the IGF-II/IGFBP-2 complex. Treatment of adult rats with IGF-II/IGFBP-2 not only prevented the loss of BMD in the femur effected by sciatic neurectomy, it also significantly increased BMD in the femur of the control limb. The stated purpose of this study was to determine the ability of IGF-II/IGFBP-2 complex to increase bone mass in vivo, and there were no treatment groups consisting of IGF-II or IGFBP-2 alone. Nonetheless, pharmacokinetic profiles indicating generalized distribution,30;31 clinical observations in patients with elevations in either IGF-II or IGFBP-2,5;32 animal studies comparing IGF-I alone and in complex,33 and our in vitro studies5 would predict little or no anabolic effect of systemic administrations of these concentrations of IGF-II or IGFBP-2 alone on bone. Indeed, IGFBP-2 alone might be expected to inhibit bone growth.34 Thus, although we have demonstrated a significant effect of the complex, we cannot draw any conclusion that IGF-II/IGFBP-2 is more effective than IGF-II from these data. This effect of IGF-II/IGFBP-2 to increase BMD appears to be through stimulation of endocortical and cancellous bone formation rates. However, with the small group size powered for the primary outcome variable, BMD, and the inherent variability of bone histomorphometry measures these data did not achieve statistical significance. Nonetheless, trabecular thickness was significantly increased with treatment. If, as these data suggest and as observed in the HCAO patients,5 the IGF-II/IGFBP-2 complex has efficacy at both cortical and cancellous bone sites, then this treatment would have major biomechanical benefit and would be predicted to reduce fracture risk. While our data do not establish the molecular mechanism of IGF-II/IGFBP-2 action in bone, the histomorphometry results are consistent with increased
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osteoblast activity. Bone surface osteoblasts and osteoprogenitor cells in the marrow stroma are induced to proliferate in response to IGFs in vitro and in vivo.13–15;35;36 IGFs also stimulate collagen synthesis, inhibit collagen breakdown through decreased expression of specific skeletal-derived collagenases, and enhance alkaline phosphatase activity.37–39 Osteoblast survival in vitro has been shown to be promoted by IGF.40 In addition, possible effects on bone resorption need to be addressed. Further studies are necessary to determine the mode and target cell of IGF-II/IGFBP-2 action. These preliminary studies have obvious potential implication for the treatment of osteoporosis. The notion of using IGFs to treat osteoporosis is not, however, a novel one and previous human studies using IGF-I treatment have found increases in serum levels of markers of bone formation.41;42 This approach was limited by the multiple systemic effects of IGF-I, including hypoglycemia. Administration of IGFI complexed with IGFBP-3 minimized the risk of hypoglycemia and other side effects, but was not selective for bone.33;43–47 The use of IGF-II to treat osteoporotic patients has not been previously reported. Our observations in HCAO and findings in vitro indicated that IGF-II in complex with IGFBP-2 could be a safe, effective, and relatively selective anabolic for the skeleton. The significant increase in BMD with no effect on blood glucose is consistent with preferential skeletal effects. The relatively low amount of growth factor necessary to elicit the biological response represents a distinct advantage. Indeed, in HCAO it is the precursor form of IGF-II that is elevated and in complex with IGFBP-2 and it remains to be seen if this form of IGF-II is even more effective than mature IGF-II in further preclinical trials. In addition, it will be important to test this treatment in a model of established osteoporosis, such as the ovariectomized rat or mouse.48;49 In summary, short-term administration of IGF-II/ IGFBP-2 prevented the loss of BMD associated with disuse osteoporosis and stimulated bone formation in adult rats. These data support the potential of IGF-II/ IGFBP-2 as a novel anabolic treatment for osteoporosis. REFERENCES 1. National Institutes of Health Consensus Development Conference Statement. Osteoporosis Prevention, Diagnosis, and Therapy. March 27-29, 2000:1–36. 2. Riggs BL, Melton III LJ. Involutional osteoporosis. N Engl J Med 1986; 314: 1676–1686. 3. Orwoll ES, Klein RF. Osteoporosis in men. Endocr Rev 1995; 16: 87–116.
182 C.A. Conover et al.
4. Khosla S, Riggs BL, Melton III LJ. Clinical spectrum. In: Riggs BL, Melton III LJ (eds). Osteoporosis: etiology, diagnosis, and management, second ed. Philadelphia, PA: Lippincott-Raven Publishers; 1995: pp. 205–223. 5. Khosla S, Hassoun AAK, Baker BK, et al. Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis: potential insights into increasing bone mass in adults. J Clin Invest 1998; 101: 2165–2173. 6. Beyer SH, Parfitt AM, Shih MS, Anderson Q, Heath III H. Idiopathic acquired diffuse osteosclerosis in a young women. J Bone Miner Res 1990; 5: 1257–1263. 7. Villareal DT, Murphy WA, Teitelbaum SL, Arens MQ, Whyte MP. Painful diffuse osteosclerosis after intravenous drug abuse. Am J Med 1992; 93: 371–381. 8. Hassoun A, Nippoldt TB, Tiegs RD, Khosla S. Hepatitis C associated osteosclerosis: an unusual syndrome of acquired osteosclerosis in adults. Am J Med 1997; 103: 70–73. 9. Whyte MP, Teitelbaum SL, Reinus WR. Doubling skeletal mass during adult life: the syndrome of diffuse osteosclerosis after intravenous drug abuse. J Bone Miner Res 1996; 11: 554–558. 10. Whyte MP, Reasner CA. Hepatitis C-associated osteosclerosis after blood transfusion. Am J Med 1997; 102: 219–220. 11. Diamond T, Depczynski B. Acquired osteosclerosis associated with intravenous drug use and hepatitis C infection. Bone 1996; 19: 679–683. 12. Shaker JL, Reinus WR, Whyte MP. Hepatitis C-associated osteosclerosis: late onset after blood transfusion in an elderly woman. J Clin Endocrinol Metab 1998; 83: 93–98. 13. Conover CA. The role of insulin-like growth factors and binding proteins in bone cell biology. In: Bilezikian J, Raisz L, Rodan G (eds). Principles of bone biology. San Diego, CA: Academic Press; 1996: pp. 607–618 [chapter 43]. 14. Cohan S, Baylink DJ. IGF system components and their role in bone metabolism. In: Rosenfeld RG, Roberts Jr. CT, (eds). The IGF system: molecular biology, physiology, and clinical applications. Totawa, NJ: Humana Press; 1999: pp. 457–496 [chapter 20]. 15. Canalis E. Insulin like growth factors and the local regulation of bone formation. Bone 1993; 14: 273–276. 16. Tanaka H, Quarto R, Williams S, Barnes J, Liang CT. In vivo and in vitro effects of insulin-like growth factor-I (IGF-I) on femoral mRNA expression in old rats. Bone 1994; 15: 647–653. 17. Wakisaka A, Tanaka H, Barnes J, Liang CT. Effect of locally infused IGF-I on femoral gene expression and bone turnover activity in old rats. J Bone Miner Res 1998; 13: 13–19. 18. Ishibe M, Ishibashi T, Kaneda K, Koda T, Rosier RN, Puzas JE. Stimulation of bone formation in vivo by insulin-like growth factor-II in rats. Calcif Tissue Int 1998; 63: 36–38. 19. Wakley GK, Baum BL, Hannon KS, Turner RT. The effects of tamoxifen on the osteopenia induced by sciatic neurotomy in the rat: a histomorphometric study. Calcif Tissue Int 1988; 43: 383–388. 20. Firling CE, Evans GL, Wakley GK, Sibonga J, Turner RT. Lack of an effect of sodium zeolite A on rat tibia histomorphometry. J Bone Miner Res 1996; 11: 254–263. 21. Dobnig H, Turner RT. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 1996; 136: 3632–3638. 22. Turner RT, Spelsberg TC. Correlation between mRNA levels for bone cell proteins and bone formation in long bones of maturing rats. Am J Physiol 1991; 261: E348–E353.
23. Westerlind KC, Wakley GK, Evans GL, Turner RT. Estrogen does not increase bone formation in growing rats. Endocrinology 1993; 133: 2924–2934. 24. Cavolina JM, Evans GL, Harris SA, Zhang M, Westerlind K, Turner RT. The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signalling peptides in ovariectomized growing rats. Endocrinology 1997; 138: 1567–1576. 25. Kidder LS, Schmidt IU, Evans GL, Turner RT. Effects of growth hormone and low dose estrogen on bone growth and turnover in long bones of hypophysectomized rats. Calcif Tissue Int 1997; 61: 327–335. 26. Turner RT, Vandersteenhoven JJ, Bell NH. The effects of ovariectomy and 17b-estradiol on cortical bone histomorphometry in growth rats. J Bone Miner Res 1987; 2: 115–122. 27. Turner RT, Wakley GK, Hannon KS, Bell NH. Tamoxifen inhibits osteoclast-mediated bone resorption of trabecular bone in ovarian hormone-deficient rats. Endocrinology 1988; 122: 1146–1150. 28. Gillespie JA. The nature of the bone changes associated with nerve injuries and disuse. J Bone Joint Surg 1954; 36B: 464–473. 29. Landry M, Fleisch H. The influence of immobilization on bone formation as evaluated by osseous incorporation of tetracyclines. J Bone Joint Surg 1964; 46B: 764–771. 30. Ballard FJ, Knowles SE, Walton PE, et al. Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(1–3)IGF-I in rats. J Endocrinol 1990; 128: 197–204. 31. Young SCJ, Miles MV, Clemmons DR. Determination of the pharmacokinetic profiles of insulin-like growth factor binding proteins-1 and -2 in rats. Endocrinology 1992; 131: 1867–1873. € nschoff B, Blum WF, Wingen A-M, Mehls O, and the European 32. To Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood. Serum insulin-like growth factors IGFs and IGF binding proteins1,2, and3 in children with chronic renal failure:relationship to height and glomerular filtration rate. J Clin Endocrinol Metab 1995;80:2684–2691. 33. Bagi CM, Brommage R, Deleon L, Adams S, Rosen D, Sommer A. Benefit of systemically administered rhIGF-I and rhIGF-I/ IGFBP-3 on cancellous bone in ovariectomized rats. J Bone Miner Res 1994; 9: 1301–1312. 34. Hoeflich A, Wu M, Mohan S, et al. Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain. Endocrinology 1999; 140: 5488–5496. 35. Machwate M, Zerath E, Holy X, Pastoureau P, Marie PJ. Insulin-like growth factor-I increases trabecular bone formation and osteoblastic cell proliferation in unloaded rats. Endocrinology 1994; 134: 1031–1038. 36. Thomas T, Gori F, Spelsberg TC, Khosla S, Riggs BL, Conover CA. Response of bipotential human marrow stromal cells to insulin-like growth factors: effect on binding protein production, proliferation, and commitment to osteoblasts and adipocytes. Endocrinology 1999; 140: 5036–5044. 37. McCarthy TL, Centrella M, Canalis E. Regulatory effects of IGF-I and IGF-II on bone collagen synthesis in rat calvarial cultures. Endocrinology 1989; 124: 301–309. 38. Canalis E. Insulin like growth factors and the local regulation of bone formation. Bone 1993; 14: 273–276.
Subcutaneous administration of insulin-like growth factor
39. Richman C, Baylink DJ, Lang K, Dony C, Mohan S. Recombinant human insulin-like growth factor-binding protein-5 stimulates bone formation parameters in vitro and in vivo. Endocrinology 1999; 140: 4699–4705. 40. Hill PA, Tumber A, Meikle MC. Multiple extracellular signals promote osteoblast survival and apoptosis. Endocrinology 1997; 138: 3849–3858. 41. Rosen CJ, Donahue LR. Insulin-like growth factors: potential therapeutic options for osteoporosis. Trends Endocrinol Metab 1995; 6: 235–241. 42. Johansson AG, Rosen CJ. The IGFs as potential therapy for metabolic bone diseases. In: Bilezikian J, Raisz L, Rodan G (eds). Principles of bone biology. San Diego, CA: Academic Press; 1996: pp. 1099–1109 [chapter 79]. 43. Hamon GA, Hunt TK, Spencer EM. In vivo effects of systemic insulin-like growth factor-I alone and complexed with insulinlike growth factor binding protein-3 on corticosteroid suppressed wounds. Growth Regul 1993; 3: 53–56. 44. Narusawa K, Nakamura T, Suzuki K, et al. The effects of recombinant human insulin-like growth factor rhIGF-I and rhIGF-I/IGF binding protein-3 administration on rat osteopenia
45.
46.
47.
48.
49.
183
induced by ovariectomy with concomitant bilateral sciatic neurectomy. J Bone Miner Res 1995; 10: 1853–1864. Bagi CM, DeLeon E, Brommage R, Rosen D, Sommer A. Treatment of ovariectomized rats with the complex of rhIGF-I/ IGFBP-3 increases cortical and cancellous bone mass and improves structure in the femoral neck. Calcif Tissue Int 1995; 57: 40–46. Sandra A, Boes M, Dake BL, Stokes JB, Bar RS. Infused IGF-I/ IGFBP-3 complex causes glomerular localization of IGF-I in the rat kidney. Am J Physiol 1998; 275: E32–E37. Svanberg E, Ohlsson C, Kimball SR, Lundholm K. rhIGF-I/IGFBP3 complex, but not free rhIGF-I, supports muscle protein biosynthesis in rats during semistarvation. Eur J Clin Invest 2000; 30: 438–446. Geddes AD. Animal models of bone disease. In: Bilezikian JP, Raisz LG, Rodan GA (eds). Principles of bone biology. New York, NY: Academic Press; 1996: pp. 1343–1354. Alexander JM, Bab I, Fish S, et al. Human parathyroid hormone 1–34 reverses bone loss in ovariectomized mice. J Bone Miner Res 2001; 16: 1665–1673.