Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism

Abstract

Serum insulin-like growth factor (IGF) -1 is secreted mainly by the liver and circulates bound to IGF-binding proteins (IGFBPs), either as binary complexes or ternary complexes with IGFBP-3 or IGFBP-5 and an acid-labile subunit (ALS). The purpose of this study was to genetically dissect the role of IGF-1 circulatory complexes in somatic growth, skeletal integrity, and metabolism. Phenotypic comparisons of controls and four mouse lines with genetic IGF-1 deficits-liver-specific IGF-1 deficiency (LID), ALS knockout (ALSKO), IGFBP-3 (BP3) knockout, and a triply deficient LID/ALSKO/BP3 line-produced several novel findings. 1) All deficient strains had decreased serum IGF-1 levels, but this neither predicted growth potential or skeletal integrity nor defined growth hormone secretion or metabolic abnormalities. 2) IGF-1 deficiency affected development of both cortical and trabecular bone differently, effects apparently dependent on the presence of different circulating IGF-1 complexes. 3) IGFBP-3 deficiency resulted in increased linear growth. In summary, each IGF-1 complex constituent appears to play a distinct role in determining skeletal phenotype, with different effects on cortical and trabecular bone compartments.

Figure 1.

Serologic characterization of IGF-1 deficient mice. A) Serum IGF-1. B) GH. C) ALS. D) IGFBP-3. Sera were obtained from 8- to 10-wk-old control, LID, ALSKO, BP3KO, and LAB mice. Data are means ± se; values in parentheses indicate number of mice per group. E) Liver expression of igf-1, als, and igfbp-3 genes was assessed by real-time PCR. *P < 0.05; two-way ANOVA. F) Ternary IGF complex-formation was assessed in sera obtained from control, LID, ALSKO, BP3KO, and LAB mice. Sera were incubated overnight at 22°C with [¹²⁵I]IGF-1 and then cross-linked with disuccinimidyl suberate and fractionated on HiPrep 16/60 Sephacryl S-200 HR columns. Fractions were collected and counted, and Western immunoblots were used to detect the ALS protein in fractions A and B. Data represent mean cpm; 3–5 mice/group.

Figure 2.

IGF-binding proteins and IGF-1 clearance. A) Half-life of [¹²⁵I]IGF-1 in sera obtained from control, LID, ALSKO, and BP3KO mice. Data are means ± se; n >12 mice/group. *P < 0.05 vs. control. B) Ligand blot assay shows IGF-1 binding capacity of serum from control, LID, ALSKO, BP3KO, and LAB mice. Reactive bands corresponding to IGFBP-3 were not detectable in BP3KO and LAB mice and significantly reduced in LID and ALSKO mice. C) Serum IGFBP-2 levels, determined by RIA in sera from 8- to 10-wk-old control, LID, ALSKO, BP3KO, and LAB mice. Data are means ± se (number per group in parentheses). D) Serum levels of IGFBP-5 detected by Western immunoblot and (E) quantified by densitometry, showing twofold increase in BP3KO mice. Data are mean ± se; n = 5/group.

Figure 3.

Tissue expression of igf-1, igfbp-3, and als. PCR analysis of RNA from femoral diaphyses, bone marrow, gonadal fat pad, and muscle. For primers; see Materials and Methods. Each lane represents a single mouse.

Figure 4.

Growth characteristics of IGF-1-deficient mice. A, B) Body weight (A) and body length (B) at 8 wk of age in IGF-1-deficient mice. C) Body compositions determined by MRI, presented as a percentage of fat mass. Data are means ± se; values in parentheses indicate number of mice per group.

Figure 5.

Bone resorption and formation indices. Rankl and opg expression in femoral diaphysis (real-time PCR). For primers, see Materials and Methods. Data are means ± se; n = 5 mice/group.

Figure 6.

A, B) Carbohydrate metabolism indices serum insulin (A) and leptin (B) analyzed in the fed state (RIA). Data are means ± se; n > 10 mice/group. C) Glucose tolerance. Glucose (2 mg/kg) injected intraperitoneally into 6-h fasted mice; blood glucose measured at indicated time points. Data are means ± se; n = 6–8 mice/group.