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 insulin-like 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 (DeltaIGF-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 DeltaIGF-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, DeltaIGF-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 DeltaIGF-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.
Highlights
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
These findings provide the first ing the osteoblasts) were pooled together, centrifuged, washed unequivocal evidence for direct actions of insulin on osteo- with ␣MEM containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, blasts and suggest that the strength of insulin generated signals and plated overnight at 37 °C in a humidified incubator supin this cell type is tempered through interactions with insulinlike growth factor type 1 receptor (IGF-1R). plied with 5% CO2
insulin receptor (IR)/IGF-1R hybrid receptor were clearly detected in cells expressing both IR and IGF-1R but were greatly diminished in ⌬IGF-1R osteoblasts (Fig. 1, F and G)
Summary
Materials—Cell culture medium, ␣-minimal essential with adenovirus encoding Cre recombinase (Ad-Cre) (Vector medium (␣MEM), was obtained from Cellgro-Mediatech (Her- Biolabs, Philadelphia, PA) at a titer of 100 multiplicity of infecdon, VA), and fetal bovine serum (FBS) was from Invitrogen. tion (MOI). The cells were serum-starved in 0.05% FBS for 48 h and blasts were replated on 100-mm tissue culture plates for restimulated with 100 nM insulin for 24 h. For signaling experiments, ⌬IGF-1R and control cells were cul- and subsequently purified as per TrueLabeling-AMPTM prototured in 10% FBS ␣MEM to 90% confluence and serum- col recommended by manufacturer (SuperArray Bioscience starved in 0.1% bovine serum albumin for 24 h to reduce cellular Corp., Frederick, MD). Alkaline Phosphatase and von Kossa Staining—The ⌬IGF-1R and control osteoblasts were cultured in 6-well plates with 2.0 ϫ 105 cells/well in ␣MEM with 10% FBS for 4 days until they were confluent. [3H]2-Deoxyglucose (2-DOG) Uptake Assay—The ⌬IGF-1R and control osteoblasts were cultured in 12-well plates till confluent. The values are expressed as the means Ϯ S.E
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