Circulating levels of IGF-1 directly regulate bone growth and density

Abstract

IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. Liver IGF-1-deficient (LID) mice and ALS knockout (ALSKO) mice exhibited relatively normal growth and development, despite having 75% and 65% reductions in serum IGF-1 levels, respectively. Double gene disrupted mice were generated by crossing LID+ALSKO mice. These mice exhibited further reductions in serum IGF-1 levels and a significant reduction in linear growth. The proximal growth plates of the tibiae of LID+ALSKO mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, the double gene disruption LID+ALSKO mouse model demonstrates that a threshold concentration of circulating IGF-1 is necessary for normal bone growth and suggests that IGF-1, IGFBP-3, and ALS play a prominent role in the pathophysiology of osteoporosis.

Figure 1

Serum levels of IGF-1, IGFBPs, GH, and insulin in control, ALSKO, LID, and LID+ALSKO mice. (a) Serum was treated with acid/ethanol to remove IGFBPs. Total IGF-1 was determined by RIA (provided by the National Hormone and Pituitary Program)in single serum samples from control (Ctl) mice (n = 23), ALSKO mice (n = 36), LID mice (n = 21), and LID+ALSKO mice (n = 61). *P < 0.01 compared with control mice. ^†P < 0.01 compared with LID or ALSKO mice. (b) GH levels were determined in the serum of control mice (n = 9), ALSKO mice (n = 11), LID mice (n = 10), and LID+ALSKO mice (n = 28). (c) Serum insulin levels, measured in the fed state, were determined in the serum of control mice (n = 12), ALSKO mice (n = 10), LID mice (n = 17), and LID+ALSKO mice (n = 35) (d) Upper panel shows a representative ligand blot assay performed on sera from control, ALSKO, LID, and LID+ALSKO mice. Serum proteins were separated by 4–20% SDS-PAGE and IGFBPs were detected by incubating the blots with ¹²⁵I–IGF-1, as described in Methods. The graph shows quantification of ¹²⁵I–IGF-1 binding to IGFBP-3 in samples from control (n = 11), ALSKO (n = 9), LID (n = 14), and LID+ALSKO (n = 17) mice. (e) IGFBP-3 mRNA expression does not differ significantly between control, ALSKO, LID, and LID+ALSKO mice. Upper panel shows a representative Northern blot assay. The graph shows quantification of IGFBP-3 mRNA detected by Northern blot analysis (n = 6 per group).

Figure 2

Serum levels of free IGF-1 and clearance studies. (a) Levels of total IGF-1 and free IGF-1 were measured by RIA in the same serum samples. Graph shows free IGF-1, expressed as a percentage of total IGF-1, in control (n = 6 samples, drawn from a tube containing 23 pooled samples), LID (n = 5 from 19 pooled samples), ALSKO (n = 9 from 35 pooled samples), and LID+ALSKO mice (n = 9 from 64 pooled samples). (b) Serum clearance of ¹²⁵I–IGF-1 in the various genotypes. Serum was obtained from the retro-orbital vein at the intervals shown after ¹²⁵I–IGF-1 was injected into control (n = 3), LID (n = 3), ALSKO (n = 3), and LID+ALSKO (n = 3) mice. The percentage of TCA-precipitable ¹²⁵I is expressed as the ratio of TCA-precipitable ¹²⁵I at an indicated time divided by TCA-precipitable ¹²⁵I at 0 time point. Inset shows in vitro degradation of ¹²⁵I–IGF-1 protein, expressed as the ratio of TCA-precipitable ¹²⁵I divided by total count.

Figure 3

Postnatal growth in LID+ALSKO mice. (a) Representative examples of body, femur, and tibia size in control, LID, ALSKO, and LID+ALSKO mice at postnatal day 28. (b) Body length was measured from nose to anus at weekly intervals (n = 20–30 mice per group). (c) Femur length (acetabulum to trochanter) was measured from radiographs taken after the mice were euthanized (n = 10–20 mice per group). (d) Body weight was measured at weekly intervals from birth to the age of 8 weeks (n = 30–60 mice per group).

Figure 4

LID+ALSKO mice exhibit alterations in growth plate height. Representative proximal tibia sections from control (a), LID (b), ALSKO (c), and LID+ALSKO (d) mice at postnatal day 28. Sections were visualized with the Masson trichrome technique, which stains nuclei black, stains collagen blue, and stains calcified matrix red. In LID+ALSKO mice, the total height of the growth plate is significantly reduced (bar, 200 μm). Inserts (bar = 50 μm) show the germinal zone (GZ), proliferating zone (PZ), and hypertrophic zone (HZ) of chondrocytes.

Figure 5

Four weeks of IGF-1 treatment results in increased serum levels of IGF-1 and IGFBP-3 and an increase in body and femoral lengths. (a) Serum levels of IGF-1 following 4 weeks of IGF-1 injections were determined by RIA as described above. (b) Serum levels of IGFBP-3 were determined by ligand blot assay. The graph shows quantification of ¹²⁵I–IGF-1 binding to IGFBP-3 in samples from control, LID, ALSKO, and LID+ALSKO mice (n = 8 per group). (c) Body length of control, LID, ALSKO, and LID+ALSKO mice at 8 weeks of age, with and without IGF-1 treatment. (d) Length of femurs of ALSKO and LID+ALSKO mice at 8 weeks of age, with and without IGF-1 treatment.