Effects of intravitreal insulin and insulin signaling cascade inhibitors on emmetropization in the chick

Abstract

Purpose: Intravitreal insulin has been shown to be a powerful stimulator of myopia in chickens, in particular if the retinal image is degraded or defocused. In most tissues, the insulin receptor activates two main signaling pathways: a) the mitogen-activated protein kinase (MAPK) cascade (e.g., mitogen-activated protein kinasem kinase [MEK] and extracellular regulated kinase [ERK]) and b) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In the current study, insulin was injected, and these pathways were separately inhibited to determine which is activated when the retinal image is defocused by spectacle lenses.

Methods: Chicks were treated with either +7 D, -7 D, or no lenses. They were intravitreally injected with insulin, the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or a combination of insulin and one of the inhibitors. Refractions and ocular dimension were measured at the beginning and after four days of treatment. The retinal proteins of the chicks were measured with western blots after 2 h and four days of treatment. Incubation occurred with anti-Akt1, anti-Erk1/2, anti-phospho-Akt(Thr308), and anti-phospho-Erk1/2((Thr202/Tyr204)) antibodies, and the ratio between the relative intensity of the phospho-form and the total-form was calculated.

Results: Chicks wearing positive lenses and injected with saline and with PI3K inhibitor compensated for the imposed defocus and became hyperopic. Insulin injections and insulin plus PI3K inhibitor injections prevented lens-induced hyperopia, whereas the MEK inhibitor alone and insulin plus MEK inhibitor had no effect. Obviously, the MEK inhibitor suppressed the effect of insulin on eye growth in the plus lens-treated animals. Chicks treated with negative lenses and injected with insulin, or with insulin plus MEK inhibitor, overcompensated for the imposed defocus. This effect of insulin was not detected in eyes injected with PI3K inhibitor plus insulin, suggesting that the PI3K inhibitor suppressed the effects of insulin in minus lens-treated animals. Insulin increased the ratio of phospho-Akt/total-Akt in animals with normal visual exposure but even more so in chicks wearing plus or minus lenses. The increase was blocked by simultaneous PI3K inhibitor injections in control eyes but not in lens-treated eyes. Insulin also increased the ratio of phospho-ERK/total-ERK in animals with normal visual exposure and in animals wearing positive lenses, compared to U0126- and Ly294002-injected eyes. In contrast, no significant activation of the MEK/ERK pathway was observed in the negative lens-treated animals.

Conclusions: Intravitreal insulin promoted axial eye growth and stimulated both signaling pathways. The PI3K/Akt pathway was activated in control and plus and minus lens-treated eyes, but the MEK/ERK pathway was activated only with positive lenses or no lenses. With negative lenses, insulin did not stimulate the MEK/ERK signaling cascade. Independent of the pathway stimulated after insulin binding, the effect on insulin was always the same: an increase in eye growth.

Figure 1

Effects of intravitreal saline, insulin, mitogen-activated protein kinase kinase inhibitor U0126, and phosphatidylinositol 3-kinase inhibitor Ly294002 injections on the development of the refractive state in chicks without any lenses, and after bilateral treatment with +7 D: lenses and −7 D: lenses. The large squares represent the means of the changes in refractive state±SEM from the beginning of the experiment and four days later. The small squares denote data from individual eyes. Six animals per group were used. Insulin and insulin + PI3K inhibitor prevented hyperopia development in positive lens–wearing animals, whereas the MEK inhibitor blocked the insulin effect. Animals wearing negative lenses and injected with insulin and insulin+ MEK inhibitor overcompensated the imposed defocus. In contrast, with the same lens treatment, the PI3K inhibitor plus insulin–injected eyes had similar refractions as insulin-injected eyes. Statistically significant differences, as determined by one-way ANOVA, are denoted in the graph (* p<0.05, ** p<0.01).

Figure 2

Effects of intravitreal saline, insulin, mitogen-activated protein kinase kinase inhibitor U0126, and phosphatidylinositol 3-kinase inhibitor Ly294002 injections on the ocular compartments in chicks without any lenses. The large squares represent the means of the changes±SEM from the beginning of the experiment and four days later. The small squares denote data from individual eyes. Six animals per group were used. Animals injected with PI3K inhibitor had deeper vitreous chambers compared with all the other types of injections. No significant changes were observed in anterior chamber depth, lens thickness, and axial length. Statistically significant differences, as determined with one-way ANOVA, are denoted in the graph (* p<0.05, ** p<0.01).

Figure 3

Effects of intravitreal saline, insulin, mitogen-activated protein kinase kinase inhibitor U0126, and phosphatidylinositol 3-kinase inhibitor Ly294002 injections on the ocular compartments in chicks treated with positive lenses during four days. The large squares represent the means of the changes±SEM from the beginning of the experiment and four days later. The small squares denote data from individual eyes. Six animals per group were used. Statistically significant differences, as determined with one-way ANOVA, are denoted in the graph (* p<0.05, ** p<0.01).

Figure 4

Effects of intravitreal saline, insulin, mitogen-activated protein kinase kinase inhibitor U0126, and phosphatidylinositol 3-kinase inhibitor Ly294002 injections on the ocular compartments in chicks treated with negative lenses during four days. The large squares represent the means of the changes±SEM from the beginning of the experiment and four days later. The small squares denote data from individual eyes. Six animals per group were used. Statistically significant differences, as determined with one-way ANOVA, are denoted in the graph (* p<0.05, ** p<0.01).

Figure 5

Short time effects (2 h) of intravitreal saline, insulin, mitogen-activated protein kinase kinase inhibitor U0126, U0126 plus insulin, phosphatidylinositol 3-kinase inhibitor Ly294002, or Ly294002 plus insulin injections on phospho-Akt/total-Akt and phospho-Erk/total-Erk levels in chicks not wearing any lenses, and after bilateral treatment with +7 D: lenses and −7 D: lenses. The same independent control sample was analyzed on each blot and used for normalization of the data. Band intensities were converted to percentages of the control sample value. The ratio of phospho-form/total-form was then calculated using the normalized values. The bars represent the mean ratio of relative intensity (mean±SEM) between the phospho-forms and non-phosphorylated proteins in percent. Each group included six animals. In control, positive and negative lens–wearing animals (Figure 5A) insulin injections highly increased the retinal ratio of phospho-Akt/total-Akt. In control and animals wearing positive lenses, insulin injections led to an increase in phospho-Erk/total-Erk levels, compared to either the MEK inhibitor or the PI3K inhibitor (Figure 5A). The PI3K inhibitor blocked the stimulatory effect of insulin in control animals without lenses but not in positive and negative lens–treated animals (Figure 5B). The MEK inhibitor significantly blocked the effect of insulin on ERK phosphorylation (Figure 5B). Significant differences were determined with one-way ANOVA and are denoted in the graph (* p<0.05, ** p<0.01, and *** p<0.001).