IGF-1 Regulates Vertebral Bone Aging Through Sex-Specific and Time-Dependent Mechanisms

Abstract

Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin-like growth factor (IGF-1). Studies have suggested that the reduction in IGF-1 compromises healthspan, whereas others report that loss of IGF-1 is beneficial because it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF-1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF-1 on vertebral bone aging in male and female Igf(f/f) mice. IGF-1 was reduced at multiple specific time points during the mouse lifespan: early in postnatal development (crossing albumin-cyclic recombinase [Cre] mice with Igf(f/f) mice); and in early adulthood and in late adulthood using hepatic-specific viral vectors (AAV8-TBG-Cre). Vertebrae bone structure was analyzed at 27 months of age using micro-computed tomography (μCT) and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age-related reductions in vertebral bone structure. In male mice, reduction of circulating IGF-1 induced at any age did not diminish vertebral bone loss. Interestingly, early-life loss of IGF-1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early-life IGF-1-deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor-activator of NF-κB-ligand (RANKL) levels in circulation. Within 3 months of a loss of IGF-1, there was a 2.2-fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF-1. Together, these data suggest the age-related loss of vertebral bone density in females can be reduced by modifying circulating IGF-1 levels early in life.

Keywords: AGING; BONE HISTOMORPHOMETRY; BONE µCT; IGF-1; OSTEOPROTEGERIN.

Figure 1. Model of IGF-1 deficiency at various timepoints throughout lifespan

(A) Timeline of the onset of IGF-1 deficiency within mice in early post-natal development (LID, onset at 10–15 days, n=7), early adulthood (5m Cre, onset at 5 months, n=6), and late-adulthood (15m Cre, onset at 15 months, n=4). After the time of onset, IGF-1 levels remain low in circulation for the rest of life. Average levels of IGF-1 in circulation in male (B) or female (C) mice early in development (2 months), in adulthood (12 months), and at the end-of-life (27 months). The wild-type denotes data pooled from wild-type igf1^f/f, 5m GFP, and 15m GFP mice. The asterisk indicates a significant difference compared to wild-type controls at that timepoint, while the pound sign indicates a significant difference in the wild-type mice compared to the young 2m time point, *#p<0.05, mean ± SEM.

Figure 2. Aging leads to decreased vertebral bone structure in males

(A) Representative images of the L4 vertebrae in male mice, as observed using µCT. Average bone volume fraction (B), connectivity (1/mm³) (C), structure model index (D), trabecular number (1/mm) (E), trabecular thickness (mm) (F), and trabecular separation (mm) (G) in the various treatment groups. The asterisk indicates a significant difference compared to the aged control mice, *p<0.05, mean ± SEM.

Figure 3. Aging leads to decreased vertebral bone structure in females, which is restored by early-life IGF-1 deficiency

(A) Representative images of the L4 vertebrae in female mice, as observed using µCT. Average bone volume fraction (B), connectivity density (1/mm³) (C), structure model index (D), trabecular number (1/mm) (E), trabecular thickness (mm) (F), and trabecular separation (mm) (G) in the various treatment groups. The asterisk indicates a significant difference compared to the aged control mice, *p<0.05, mean ± SEM.

Figure 4. Osteoprotegerin is elevated in aged, IGF-1 deficient female mice

Average OPG (A), RANKL (B) levels in the sera of the aged female mice. (C) The average ratio of OPG to RANKL in the aged female mice. Average OPG (D), RANKL (E) levels in the sera of the aged male mice. (F) The average ratio of OPG to RANKL in the aged male mice. The asterisk indicates a significant difference between groups, *p<0.05, mean ± SEM, n=4–8.

Figure 5. Loss of circulating IGF-1 does not influence bone acquisition in female mice, but does lead to local upregulation of insulin receptor expression

Average bone volume fraction (A), connectivity density (1/mm³) (B), structure model index (C), trabecular number (1/mm) (D), trabecular thickness (mm) (E), and trabecular separation (mm) (F) in the female igf1^f/f mice three months post injection of AAV8-TBG-Cre or AAV8-TBG-GFP. Average expression of several key anabolic signaling genes in the vertebrae of GFP and Cre-injected igf1^f/f mice, in both females (G) and males (H). The asterisk indicates a significant difference between groups, *p<0.05, mean ± SEM, n=5–6.