Abstract
Purpose :
Thyroid Eye Disease (TED) is thought to occur due to an immune response to autoantigens expressed on thyrocytes and orbital fibroblasts (OFs), the thyroid stimulating hormone receptor (TSHR) and the insulin-like growth factor-1 receptor (IGF-1R), which induces a series of inflammatory cytokine cascades causing soft tissue expansion through fibrosis and adipogenesis. Single nuclear RNA sequencing (snRNAseq) has revealed that OFs undergoing adipogenesis upregulate the expression of both IGF-1, and IGF-1R, early in the course of adipogenesis. To test the function of the IGF-1R signaling pathway in adipogenesis, we used the small molecule IGF-1R antagonist linsitinib to inhibit IGF-1R function.
Methods :
OFs were induced to undergo adipogenesis as previously described. Linsitinib was added to the adipogenic media at the concentration indicated in the first week only. On days 0, 5, and 9, cellular protein was collected and prepared for immunoblotting according to standard technique. For quantification of adipocytes, oil red O staining was performed on fixed cells to visualize adipocytes. Cells were imaged by a masked study team member using a Keyence BZ-X710 microscope. Four high-power field images were obtained per replicate. Cell counts were performed by a masked study team member using ImageJ. Each time point was analyzed in triplicate.
Results :
Treatment of OFs undergoing adipogenesis with linsitinib decreases phosphorylation of the IGF-1R in a dose-dependent manner(Figure 1). Treatment of OFs with linsitinib decreases adipogenesis in a dose-dependent manner using standard adipogenic media (Figure 2A) and insulin-free media (Figure 2B).
Conclusions :
Loss of IGF-1R function decreases phosphorylation of IGF1-R in OFs undergoing adipogenesis and decreases adipogenesis of OFs. This may contribute to the mechanism of action of teprotumumab, the anti-IGF-1R that is currently approved for use in TED.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.