Alpha-1 anti-trypsin modulates insulin signaling in RPE cells in an vitro model of Diabetic Retinopathy

FERNÁNDEZ ACQUIER, MARIANO; GALLO, JUAN E.; ORTIZ, GUSTAVO A.; PETRIGLIANO, MICAELA; POTILINSKI, MARÍA CONSTANZA

Vancouver

Congreso; ARVO Annual Meeting; 2019

Purpose: Diabetic retinopathy (DR) is associated with persistent inflammation and with damage to the vascular bed. The ophthalmic therapy for this retinal pathology is focused on advanced stages of the disease and there is not a preventive treatment. Previous results obtained by our group showed that Alpha-1 Antitrypsin (A1AT) acts like an anti-inflammatory agent that could play a role on DR therapy. However, it is important to know the effect of A1AT on proteins that are relevant to retinal function and the molecular mechanisms involved. The retinal pigment epithelium (RPE) forms the posterior component of the blood-retinal barrier and it is affected by hyperglycemia. Insulin regulation of blood sugar levels in the body is mainly done through the IR/IRS-1/PI3K/PDK1/AKT/GLUT signaling pathway. Dysfunction of one or more components of this pathway can lead to insulin resistance and the onset or worsening of diabetes, having consequences in the eye. For this reason we evaluated the expression of different components involved in insulin signaling in ARPE-19 cell line.Methods: ARPE-19 cells (ATCC® CRL-2302TM, Manassas, Virginia, USA) were maintained in DMEM/F12 (Invitrogen, Carlsbad, California, USA) containing 2 μM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum. ARPE-19 cells (passages 9 to 12) were incubated 16h with DMEM 5,5 mM glucose (Control), DMEM 5,5 mM glucose + 4.5 mg/ml A1AT (Control + A1AT), DMEM 30 mM glucose (Diabetic), DMEM 30 mM glucose + 4.5 mg/ml A1AT (Diabetic + A1AT). Cells were harvested with RIPA buffer for Western blot assay or fixed for immunohistochemistry. Results: A1AT diminished levels of IRS-1 and AS160 overexpressed in high glucose conditions, A1AT also reduces AKT and pAKT1/2/3 Ser473 expression levels. No changes on GLUT1 expression were observed.Conclusions: Results support the hypothesis that A1AT regulates expression of different components on IR/IRS-1/PI3K/PDK1/AKT/GLUT signaling affecting glucose intake. Moreover, A1AT affects the activation of AKT, a mediator on different signaling pathways essential to the normal function of RPE cells. Taking together with results obtained on inflammatory response, A1AT seems to be a promising molecule to treat DR.