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Teruyo Kida, Hidehiro Oku, Taeko Horie, Tsunehiko Ikeda; Insulin-induced nitric oxide production under high glucose condition in microvessels of rat retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5835.
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To evaluate insulin-induced nitric oxide (NO) production in retinal microvessels.
Rat retinal microvessels and cultured retinal vascular cells were used in this study. Project (1): Microvascular complexes were freshly isolated from retinas of healthy rats by a “tissue-print” method. Retinal microvessels were incubated in different concentration of glucose (5.5 mM and 25 mM) with or without addition of insulin. Changes of NO production in the retinal microvessels were semiquantitatively determined by the time-lapse recording using DAF, a fluorescein probe for NO, and a laser scanning confocal microscope (LSM 510 Meta, Carl Zeiss, Jena, Germany). Project (2): In addition, we examined cultured retinal endothelial cells and pericytes in different glucose concentrations (5.5 mM and 25 mM), and their DAF intensities were assayed by flow cytometry (EC800 cell analyzer, Sony, Japan). Protein levels of NO synthase (NOS; iNOS and eNOS) were also analyzed by Western blot.
Exposure of microvessels to insulin in 5.5 mM glucose, the fluorescent intensity was significantly increased (P<0.01, ANOVA, post hoc test, Scheffe), while the insulin-induced NO production was significantly suppressed when the vessels were incubated in 25 mM glucose. Expression of NOS was significantly increased in both endothelial cells and pericytes exposed to 25mM glucose (P<0.05, t-test).
Insulin increased production of NO and may contribute to the microcirculation of retina. However, this NO-mediated action of insulin is suppressed under high glucose condition. An immediate intervention of insulin treatment often causes temporary worsening of diabetic retinopathy in untreated diabetic patients. Thus, the balance between activated NOS and NO eliminated by superoxide might be important under high glucose condition.
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