Abstract
Purpose:
To determine whether insulin-like growth factor binding protein 3 (IGFBP-3) is required for Compound 49b to reduce tumor necrosis factor-alpha (TNF-a)-induced insulin resistance in the streptozotocin (STZ)-induced diabetic rat retina.
Methods:
Lewis rats were made diabetic by a 60mg/kg injection of STZ. Compound 49b, a novel b-adrenergic receptor agonist, was initiated once rats became diabetic (glucose >250mg/dl) at 1mM daily/4 drops/ eye for 2 months. Rats were treated with two intravitreal injections of IGFBP-3 siRNA (4 days apart) into both eyes to block IGFBP-3 signaling; empty vector served as a control. Ten days after the initial IGFBP-3 siRNA injection, electroretinogram analyses were done and rats were sacrificed. Whole retinal lysates, blood and splenocytes were collected for functional analyses including Western blot and ELISA of IGFBP-3, TNF-a, suppressor of cytokine signaling 3 (SOCS3), insulin receptor, insulin receptor substrate 1 (IRS-1), and apoptotic markers.
Results:
IGFBP-3 siRNA effectively reduced IGFBP-3 levels in diabetic rats. Compound 49b increased IGFBP-3 levels in diabetic rats and significantly improved their ERG amplitudes, which was not affected by IGFBP-3 siRNA treatment. Compound 49b significantly reduced TNF-a and SOCS3 levels, independent of IGFBP-3 signaling. Since TNF-a and SOCS3 can inhibit insulin receptor signal transduction, we measured insulin receptor phosphorylation of tyrosine 1150/1151. IRTyr1150/1151 was significantly reduced in the diabetic retina, with Compound 49b significantly increasing IRTyr1150/1151, independent of IGFBP-3. The increase in IRTyr1150/1151 phosphorylation actions led to increased phosphorylation of Akt and reduced cleaved caspase 3. We observed systemic reduction of TNF-a levels in plasma, similar to the effects in whole retinal lysates.
Conclusions:
Taken together, these results suggest that Compound 49b can regulate insulin signal transduction independent of IGFBP-3-signaling. We have previously reported that Compound 49b is protective in diabetic retinopathy through both reduced TNF-a and increased IGFBP-3 signaling. This data suggests that while Compound 49b does increase IGFBP-3 in the diabetic retina, this is not required for Compound 49b regulation of insulin signaling.