Disruption of the Insulin-like Growth Factor Type 1 Receptor in Osteoblasts Enhances Insulin Signaling and Action

Abstract

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulinlike growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (⌬IGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In ⌬IGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, ⌬IGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-3 H]-deoxy-D-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of ⌬IGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.Insulin and insulin-like growth factor 1 (IGF-1) 2 are related signaling molecules that evolved from a common ancestor pathway originally involved in sensing and integrating signals arising from nutrient and growth factor availability. During evolution, this primitive receptor pathway diverged into two distinct hormonal systems that in mammals serve different but overlapping developmental and metabolic functions (1, 2). These receptors belong to the family of ligand-activated receptor kinases and are abundantly expressed in osteoblasts (3, 4). Unlike other receptor tyrosine kinases, these proteins exist at the cell surface as homodimers composed of two identical ␣/␤ (␣ 2 /␤ 2 ) monomers or as heterodimers (discussed below) composed of two different receptor monomers (e.g. IR ␣ ␤/IGF-1R ␣ ␤). Upon ligand binding, they undergo a conformational change that facilitates binding to ATP and autophosphorylation (5, 6). Autophosphorylation increases the kinase activity of IR-type receptors by 3 orders of magnitude, enabling them to phosphorylate a number of substrate proteins and engender growth or metabolic responses (7). IRS proteins act as mediators of insulin, IGF, and cytokine signaling in a variety of cell types. These adaptor molecules recruit and activate downstream signaling cascades su...

Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2

“…In accordance with our proposed mechanism for insulin resistance in human VSMCs (Fig. 8), disruption of IGF1R in breast cancer cells, osteoblast, and rat VSMC is followed by an increased fraction of insulin holoreceptors and reduced insulin resistance (Fulzele et al 2007, Zhang et al 2007, Engberding et al 2009). …”

Human microvascular endothelial cells are sensitive to IGF-I but resistant to insulin at the receptor level

“…In osteoblasts, a cell type with both IGF-IR and IR, and containing insulin/IGF-I hybrid receptors, disruption of the IGF-IR was found to enhance insulin signalling and action indicating that the insulin-generated signal was tempered through interactions with the IGF-IR (Fulzele et al, 2007). Correspondingly, sequestration of insulin receptors into insulin/IGF-I hybrid receptors in HMVEC probably makes the cells insulin resistant.…”

IGF-1 regulation of key signaling pathways in bone

“…The osteocalcin promoter is active at around e17dpc in mice and has been shown to be specifically expressed in mature osteoblasts. 41 The mice with the loss of IGF1R have decreased bone formation at 6 weeks of age and although they have a sufficient number of osteoblasts these mice have very low bone formation rates, suggesting the work of osteoblasts is impaired. One of the interesting observations from the knockout of IGF-1 is the requirement of IGF-1 for parathyroid hormone (PTH) action on bone.…”