Protein kinase C-mediated insulin receptor phosphorylation in diabetic rat retina
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Diabetic retinopathy (DR) involves a proliferation of vascular endothelial cells and loss of pericytes. There is a link among the action of protein kinase C (PKC) and insulin signaling. Thus, we investigated the differences between these cells in insulin receptor (IR) phosphorylation in DR.
Retinas were removed from streptozotocin-induced diabetic or healthy rats, and IR expression levels were compared by immunoblot and immunohistochemistry. In vitro assays also were performed in order to determine the expressions of phosphorylated IR in both cells cultured under 5.5 or 25 mM glucose by immunoblot. Cell viability was determined in both cells cultured under different concentrations of phorbol myristate acetate (PMA), a PKC activator. To determine the involvement of the PI3 kinase pathway of IR, PMA with or without wortmannin-induced changes in Akt was also analyzed.
Immunoreactivity to the IR was decreased in diabetic retina. High glucose (25 mM) increased phosphorylated IR levels in endothelial cells but not in pericytes. PMA (1 nM or higher) induced death of pericytes, while endothelial cells were increased. PMA increased phosphorylated Akt in endothelial cells and decreased in pericytes. Wortmannin suppressed the PMA-induced phosphorylation of Akt in endothelial cells.
The different responses to 25 mM glucose and PMA were observed between retinal endothelial cells and pericytes. Thus, IR phosphorylation is likely important for retinal vascular cells to survive in diabetic retina.
KeywordsInsulin receptor PMA (phorbol myristate acetate) Retinal vascular endothelial cell Pericyte Akt
Grant-in-Aid for Scientific Research (C) (Researcher No. 90610105) from the Japan Society for the Promotion of Science (Tokyo, Japan), and the Osaka Eye Bank Association Fund (Osaka, Japan) provided financial support. The sponsor had no role in the design or conduct of this research.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval (animal experiments)
All applicable guidelines of ARVO statement for the care and use of animals were followed. Our experimental protocols conformed to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines.
All procedures performed in studies involving animals were in accordance with the ethical standards of the Osaka Medical College Committee on the Use and Care of Animals (approval number: 27114) at which the studies were conducted.
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