Insulin Acts as a Powerful Stimulator of Axial Myopia in Chicks

Abstract

purpose. In animal models, it has been shown that the retina can use the defocus of the projected image to control emmetropization. Glucagon may be involved in the sign of defocus detection, at least in chickens. Since glucagon and insulin often have opposite effects in metabolic pathways, the effect of insulin on eye growth was investigated.

methods. Chicks were treated with either positive or negative spectacle lenses and intravitreally injected with saline or different amounts of insulin. Refraction, axial length, and corneal curvature were measured. Effects of insulin on vitreal glucose concentration, on retinal ZENK and glucagon mRNA levels, and on the number of ZENK-immunoreactive glucagon amacrine cells were studied.

results. Insulin injections (0.3 nmol) caused only a small myopic shift in control chicks. When positive lenses were worn, insulin injections (0.3; 0.03 nmol) not only blocked hyperopia but rather induced high amounts of axial myopia. Insulin also enhanced myopia that was induced by negative lenses. Axial elongation was mostly due to an increase in anterior chamber depth and a thickening of the crystalline lens. Insulin temporarily reduced vitreal glucose levels. Insulin increased retinal ZENK mRNA levels, whereas the number of ZENK-immunoreactive glucagon amacrine cells was reduced, a finding that is typically linked to the development of myopia.

conclusions. Given that insulin is used in therapy for human metabolic disorders and has been proposed to treat corneal epithelial disease, its powerful myopiagenic effect, which is mostly due to its effects on the optics of the anterior segment of the eye, merits further investigation.

Approximately 25% of the world population is myopic, and this number is expected to rise. ¹ Although most researchers agree that both the environment and genetics play a role in the development of myopia, the relative contribution of both factors is still a matter for debate. In animal models, myopia can be artificially generated by placing negative lenses in front of the eye, whereas hyperopia can be induced by positive lenses. ² Similar refraction changes were induced by lens-wearing in several other animal models, including tree shrews, ³ marmosets, ⁴ and rhesus monkeys. ^{5 6} Studies in the chick model showed that the retina itself is able to detect the sign of imposed defocus. ^{7 8} Neither accommodation nor image processing in the brain seem necessary, ^{9 10} although the emmetropization set point appears to be shifted to more hyperopic refractions after optic nerve transsection. ¹¹  

Animal studies have shown that visually guided emmetropization involves changes in protein and gene expression throughout the different fundus layers of the eye. Among the biochemical changes induced in the retina of chicks by spectacle lens treatment are alterations in the abundance of the mRNA and protein of the transcription factor ZENK (also called Egr-1, Zif268, or Krox-24 in other species), especially in the glucagon-expressing amacrine cells. ^{12 13} Positive lens wear increases, whereas negative lens wear decreases the number of ZENK-expressing cells after only 40 minutes of exposure to defocus. ¹⁴ A link between Egr-1 (called ZENK in chicks) expression levels and retinal image quality has also been described in mice ¹⁵ and macaques. ¹⁶ Moreover, glucagon mRNA and peptide levels in the retina are altered by treatment with spectacle lenses in a sign-of-defocus-dependent manner. Glucagon, as well as glucagon agonists, inhibited lens-induced myopia development. ^{17 18 19} Glucagon itself may therefore act as a STOP signal for eye growth, at least in chickens. 

Recently, it has been speculated that refractive errors are associated with a metabolic disturbance in the blood sugar levels ²⁰ and it has been shown that a Western diet modulates insulin signaling. ²¹ Since glucagon and insulin have opposite effects on blood glucose levels and on the control of cell proliferation in the retina, ²² we investigated whether they also affect eye growth in an antagonistic manner. 

It has been traditionally thought that, in adult vertebrates, insulin transcription is restricted to β-cells of the pancreatic islets of Langerhans. However, there is growing evidence that insulin and insulin receptors are also expressed in the brain and in the neural retina of vertebrates. ^{23 24 25 26 27} At least in rats and chickens, it has been shown that the retina itself is capable of synthesizing insulin. ^{28 29 30} Since in chicks the levels of retinal insulin and insulin-like growth factor IGF)-I binding by far exceed that observed in the brain, the retina is most probably a major target of both. ²⁷  

In liver, muscle, and fat tissue, insulin plays a role in the regulation of glucose homeostasis. In contrast, neurons metabolize glucose in an insulin-independent manner and have therefore traditionally been thought to be nonresponsive to insulin in vivo. Recent data have challenged this concept ³¹ and suggest that insulin has additional functions. In neural tissues, it seems to regulate growth and development and seems to act as a neuromodulator. ^{26 32} As the primary anabolic peptide hormone, insulin makes a major contribution to normal vision. ³³ Proinsulin-specific staining in embryonic retina was shown in patches of ganglion cells and insulin-specific granular staining appeared in the outer layers of the retina and in the choroid. ³⁴ Insulin receptor immunoreactivity was found in chicken embryos throughout the retina. In 3-day-old posthatching chicks, the receptor was present in the ganglion cell layer and in amacrine cells and their processes. ³⁵ Moreover, some studies have suggested that Müller cells express functional insulin receptors. ^{28 36 37} Insulin not only plays a role in the retina but, together with insulin-like growth factors (IGFs), it is also a putative regulator of cell proliferation and differentiation during lens development. ^{38 39}  

There is more than one reason to study the role of insulin in myopia development: (1) Since insulin is an antagonist of glucagon (which is considered a STOP signal for eye growth in chicks), it is possible that insulin acts in the opposite direction and stimulates myopia development. (2) A recent hypothesis ²⁰ states that nutrition has changed to diets high in refined starches and that this change may lead to increased insulin levels in humans. High insulin suppresses insulin-binding protein-3 which was assumed to disturb the tight coordination of eyeball lengthening and lens growth. (3) Some neural tissues are apparently not dependent on insulin when they regulate their carbohydrate metabolism, and it is important to find out whether this is also true of the retina. (4) Since changes in the number of ZENK-immunoreactive amacrine cells are observed when visually induced changes in axial eye growth are induced and since insulin is a known modulator of ZENK expression in several tissues, it is important to find out whether intravitreal insulin also has an effect on retinal ZENK mRNA and protein expression. (5) Since insulin is regularly applied to diabetic patients (and even directly to the eye in rats to treat diabetic keratopathy ⁴⁰ ), its potential effects on refractive development cannot be neglected. In addition, insulin has been discussed as a possible pharmacologic treatment for early diabetic retinopathy (Singh RS, et al. IOVS 2007;48:ARVO Abstract 5807), although it has been reported to aggravate this condition. ⁴¹ Some of the results of our study have been presented in abstract form (Feldkaemper MP, et al. IOVS 2007;48:ARVO Abstract 5924). 

Methods

Animals and Treatment

Male White Leghorn chickens (n = 172) were raised in groups of six to eight animals under a 12/12 hour light/dark cycle (lights on at 7 AM and off at 7 PM). The treatments were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the university commission for animal welfare (reference AK 6/05). Intravitreal injections were performed while animals were under diethyl ether anesthesia. In the lens-treated groups, Velcro rings were glued to the periorbital feathers. Lenses were binocularly attached to these rings and could be removed easily for measurements and cleaning of the lenses. The details of the various treatment groups in this study are summarized in Table 1 . 

Experiment 1: Effects of Intravitreal Insulin Injections on Refractive Development, Axial Length, and Corneal Curvature

The chicks were intravitreally injected in both eyes with either saline (group 1) or different amounts of insulin (injection volume 12.5 μL, containing 0.05 U, 0.005 U, or 0.0005 U insulin; Insuman Rapid, Aventis, Frankfurt, Germany), corresponding to 0.3, 0.03, and 0.003 nmol (group 2), at the age of 7 days after hatching. The same injection was repeated 2 days later. In addition, from day 7, the chicks were bilaterally treated with +7-D lenses and injected with saline (group 3) or with one of the three different insulin concentrations (group 4). Finally, the chicks were bilaterally treated with negative lenses and injected with saline (group 5) or with 0.3 nmol insulin (group 6). Since the experiment with negative lenses was supposed to show that the myopiagenic effects of insulin persisted also with this type of lenses, only the dose that was most effective in the experiments with positive lenses was tested (0.3 nmol insulin). 

Before the insulin injections and beginning of the lens treatments and 4 days later, refractive states and axial lengths were determined. Moreover, the effects of insulin injections (0.3 nmol) on corneal radius of curvature were measured. 

Experiment 2: Recovery from Intravitreal Insulin Injections and Lens Wear

Some of the chicks (group 3rec, initially saline-injected, and group 4rec, initially injected with 0.03 nmol insulin) wore the positive lenses for another 3 days without further injections. Thereafter, the lenses were removed, and the chicks were allowed to recover from the lens treatment for another 2 days. Refraction and axial length were measured at four time points: (1) at the beginning of the experiment, (2) 4 days after the beginning (after the two insulin injections), (3) at day 7 (after an additional 3 days of lens wearing and 5 days after the last insulin injection) and (4) at day 9 (2 days after lens removal and 7 days after the last insulin injection). 

Experiment 3: Short and Long-Term Effects of Lens Wear and of Insulin Injections on Intravitreal Glucose Level

Short-Term Time Kinetics.

Chickens, aged 8 days, were intravitreally injected with insulin in both eyes (0.3 nmol, group 7). The eyes were removed, and the vitreous was cut with a razor blade into small pieces. The intravitreal glucose concentration was measured 0, 15, 120, and 240 minutes after the insulin injection (Accu Check; Roche, Mannheim, Germany). 

Short-Term Effects of Lens Wear on Intravitreal Glucose Level.

Chicks at the age of 8 days wore a positive or a negative lens over one eye, and the fellow eye served as the control (groups 8b and 9b). Intravitreal glucose concentrations were measured either after 1 hour of negative lens wear (group 8a) or after 1 hour of positive lens wear (group 9a). 

Long-Term Effects of Intravitreal Insulin on Glucose Level.

Intravitreal glucose concentration was measured 2 days after the last injection took place (after two injections of saline or 0.3 nmol insulin; groups 1 and 2). Moreover, the combined effects of lens wear and insulin injection on intravitreal glucose concentration were measured in chicks that were treated with either positive or negative lenses (0.3 nmol, groups 3–6). 

Experiment 4: Effects of Insulin on ZENK mRNA Expression Levels and on the Number of ZENK-Immunoreactive Glucagon Amacrine Cells

Short-term effects of insulin (0.03 or 0.3 nmol) on ZENK mRNA expression were studied (group 11). Saline-injected chicks served as the control (group 10). Chicks were treated binocularly with positive lenses. Before the lens attachment, either saline (group 12) or insulin (0.3 or 0.03 nmol, group 13) was injected. Chicks were killed one hour after saline or insulin injection. One eye of each chick was used for mRNA quantification by real-time RT-PCR, and the other eye was used for immunostaining. 

Measurements of Refractive States and Ocular Dimensions

The refractive state of the eyes was determined by automated infrared photoretinoscopy. ⁴² Axial lengths were measured by A-scan ultrasonography. ⁴³ The term axial length refers to the distance from the surface of the cornea to the vitreoretinal interface and is the sum of anterior chamber depth, lens thickness, and vitreous chamber depth. Infrared photokeratometry was used to measure corneal radius of curvature. ⁴⁴  

Tissue Preparation, RNA Isolation, and cDNA Synthesis

All animals were killed between 1 and 3 PM by an overdose of diethylether. Fundus punches of 8 mm were obtained from the posterior pole. The retinas were carefully separated on ice under a dissecting microscope and immediately snap frozen in liquid nitrogen. Total RNA was isolated from retinal tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. All samples were subjected to treatment with RNase free DNase I (Roche, Mannheim Germany), and the integrity of the purified RNA was verified by agarose gel electrophoresis. One microgram of total RNA was reverse transcribed with M-MLV reverse transcriptase (Promega, Mannheim, Germany), with 0.5 μg oligo(dT)₁₅ primer and 50 ng of a random primer mixture (Invitrogen, Solingen, Germany). 

Semiquantitative Real-Time RT-PCR

The primers used for measurements of β-actin, ZENK, and glucagon expression levels have been described. ^{13 17} All PCRs were performed on a thermal cycler (iCycler iQTM Multi-Color Real Time PCR Detection System; Bio-Rad, Munich, Germany). The volume of a single reaction added up to 15 μL containing 1 ng template from a PCR kit (SYBR Green; Bio-Rad) and a final primer concentration of 0.6 μM each. After an initial heat activation (15 minutes at 95°C), 40 cycles of 15 seconds at 94°C, 30 seconds at 59°C, and 45 seconds at 72°C were run with cyclic fluorescence measurements at the end of the annealing phase. Melting curve analysis was applied to control for contamination by unspecific byproducts. For all PCR run evaluations, cycle thresholds (C_T) were calculated for the different products, as described elsewhere. ¹⁷ Real-time PCR efficiencies were 1.98 for glucagon, 1.95 for β-actin, and 1.97 for ZENK. All PCR reactions for a given sample were analyzed in three replicates, and the averages of the replicates were used for statistical data processing. Mean normalized expressions (MNEs) of the target genes were calculated using β-actin as the housekeeping gene. ^{13 17}  

Immunohistochemistry

Eyes were enucleated and cut with a razor blade perpendicular to the anterior-posterior axis. The anterior segment of the eye was discarded, and the vitreous was removed. Eyes were fixed for 20 minutes in 4% paraformaldehyde, and 12-μm-thick vertical sections were double-stained for ZENK and glucagon, as previously described. ¹⁴ Antibodies and their working dilutions included anti-Egr-1 rabbit polyclonal antibody at 1:12,000 (Egr-1 (588)x, sc-110x; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-glucagon antibody (mouse monoclonal at 1:400; CURE Digestive Diseases Research Center, University of California at Los Angeles, Los Angeles, CA). Cy3-conjugated goat anti-rabbit IgG (1:1000 GE Healthcare, Freiburg, Germany) and Oregon Green conjugated goat- anti-mouse IgG (Invitrogen-Molecular Probes, Leiden, The Netherlands) were used as secondary antibodies. ZENK-immunoreactive glucagon amacrine cells were counted in at least four successive transverse sections taken from the center of the retina. The percentage of glucagon cells that were ZENK positive was determined by dividing the number of glucagon cells that were also immunoreactive for ZENK by the total number of glucagon cells per section and multiplying by 100. 

Statistics

All data are expressed as the mean of results obtained from several animals ± SEM. In our statistical tests, both eyes of a chick were taken into account, because interindividual variability of changes in ocular parameters was similar to the variability between both eyes of the same animals. The data were adjusted for normal distributions with a Shapiro-Wilk test. Different types of analyses of variance (ANOVA) were performed. The text indicates whether a single-factor ANOVA or a two-way ANOVA was used. A significant ANOVA was followed by a paired Student’s t-test for post hoc analysis. Statistical tests were performed with commercial software (JMP 4; SAS Institute, Cary, NC). 

Results

Experiment 1: Effects of Insulin on Normal Eye Development and Lens-Induced Hyperopia or Myopia

Effects of Insulin on Refractive Development.

Refractive development in chickens that were injected with insulin and had either a normal visual experience or wore spectacle lenses is shown in Figure 1 . Lenses (either +7 or −7 D) were attached bilaterally for 4 days, and intravitreal saline or insulin injections were performed twice, at the beginning of the experiment and 2 days later. The difference in refraction between the beginning and the end of the experiment is shown for each group. A two-way ANOVA revealed that the mean changes in refraction were significantly affected by insulin concentration as well as lens type and that the interaction of both was also significant (for all: P < 0.0001). The highest dose of insulin (0.3 nmol) induced a small but significant myopic shift in chickens with normal visual experience, compared with the saline-injected control chicks (one-way ANOVA, paired Student’s t-test post hoc, P < 0.05), whereas lower amounts of insulin had no effect. As in previous studies, covering saline-injected eyes with positive lenses (+7 D) induced hyperopia, whereas treatment with negative lenses (−7 D) induced myopia. The lowest dose of insulin used (0.003 nmol) prevented positive lens compensation completely, whereas higher doses (0.3 nmol; 0.03 nmol) not only prevented hyperopia but even induced high amounts of myopia (positive lens + saline versus positive lens + 0.3 nmol insulin; refraction at day 4: +6.90 ± 0.42 D vs. −5.78 ± 0.86 D; Table 1 ). Both insulin doses, 0.3 and 0.03 nmol, were similarly effective. In negative lens-treated eyes, insulin (0.3 nmol) had an additional myopiagenic effect (negative lens + saline versus negative lens + insulin; refraction at day 4: −2.03 ± 0.66 vs. −6.48 ± 1.02 D, Table 1 ). Insulin (0.3 nmol) seemed to override signals derived from the sign of the lenses completely, since there was no difference in the refractions of chicks after treatment with positive or with negative lenses; all became similarly myopic (one-way ANOVA followed by Student’s t post hoc test, P > 0.05). Only the insulin dose that was most effective in the experiment with positive lenses was also tested with negative lenses. 

Effects of Insulin on Axial Eye Growth.

A two-way ANOVA revealed that the mean changes in all ocular dimensions, except for vitreous chamber depth, were significantly affected by insulin concentration as well as by lens type, with significant interactions between both variables (for all P < 0.0001). The myopic shift induced by insulin, shown in Figure 1 , correlated with an increase in axial length. In saline-injected chickens, positive lenses decreased and negative lenses increased axial length, compared with chicks without lenses. Chicks with normal visual experience developed longer eyes than saline-injected controls with the highest insulin dose (Fig. 2 , one-way ANOVA, Student’s t post hoc test, P < 0.01). In lens-wearing chicks, insulin caused a substantial increase in axial length, no matter what type of lens was worn, and there was no difference in the axial lengths between chicks that were treated with positive or negative lenses (one-way ANOVA, Student’s t post hoc test, P > 0.05). 

Effects of Insulin on Vitreous Chamber Depth.

Figure 3shows that the increase in axial length in insulin-injected chicks without lenses was not due to an increase in vitreous chamber depth. Instead, at lower insulin concentrations (0.03 and 0.003 nmol) vitreous chamber depth was actually reduced (one-way ANOVA, Student’s t post hoc test, P < 0.01). On the other hand, as found in previous studies, treatment with positive lenses in saline-injected chicks reduced vitreous chamber depth. This compensatory reduction in vitreous chamber depth was completely suppressed by insulin at all used doses. Treatment with negative lenses induced the expected increase in vitreous chamber depth in saline-injected chicks, but insulin injections had no additional effect. Unlike axial length, there was a statistically significant difference in vitreous chamber depth between chicks with combined insulin (0.3 nmol) and plus-lens treatment, versus combined treatment with insulin (0.3 nmol) and minus lenses (one-way ANOVA, Student’s t post hoc test, P < 0.001). There was some variability in vitreous chamber depth changes in the saline-injected control chicks, which most likely can be attributed to interbatch variability. For each dose of insulin, a different batch had to be used because the number of animals per batch was limited. 

Effects of Insulin on Anterior Chamber Depth Growth.

Injections of 0.3 nmol insulin stimulated an increase in anterior chamber depth (Fig. 4) , compared with the saline-injected control (one-way ANOVA, Student’s t post hoc test, P < 0.01). Lower insulin doses were ineffective. Treatment with positive lenses alone had no effect on anterior chamber depth. However, treatment with negative lenses produced deeper anterior chambers. Insulin injections (0.3 and 0.03 nmol) increased anterior chamber depth in combination with both positive and negative lenses. At the lowest dose of insulin (0.003 nmol), no effect was observed on anterior chamber depth in any of the different treatment conditions. 

Effects of Insulin on the Growth of the Crystalline Lens.

Figure 5shows the effects of insulin on crystalline lens thickness. Treatment with positive lenses did not change lens thickness, whereas treatment with negative lenses decreased it. High insulin doses (0.3 nmol) increased the lens thickness in animals without lenses and in those with positive- or negative lenses (one-way ANOVA, Student’s t post hoc test, P < 0.01). Lower amounts of insulin (0.03 nmol) had no effect in chicks with normal vision but increased lens thickness when positive lenses were worn in addition (positive lenses and saline injections versus positive lens and insulin injections, one-way ANOVA, Student’s t post hoc test, P < 0.001). The lowest insulin concentration used in this study (0.003 nmol) had no effect on crystalline lens thickness, with or without lenses. 

Effects of Insulin on Corneal Radius of Curvature.

At a dose of 0.3 nmol, insulin reduced the corneal radius of curvature, both in negative- and positive lens-treated eyes (saline, negative lenses versus insulin, negative lenses: P < 0.01; saline, positive lenses versus insulin, positive lenses: P < 0.05; Table 2 ). However, insulin had no effect on corneal curvature in chickens with normal visual experience. 

Experiment 2: Recovery from Intravitreal Insulin Injections and Lens Wear

To find out whether the effects of insulin on eye growth were only transient or permanent, we allowed some chicks to recover (Fig. 6) . Saline injections as well as insulin injections were terminated after the second injection. Five days after the last insulin injection, at the age of 14 days, the chicks were still very myopic, even though they wore positive lenses. At this time point, the lenses were taken off. The chicks recovered completely from the myopia within 2 days and became slightly hyperopic, similar to the refractions in untreated chicks. Moreover, lens thickness returned to normal. The anterior chambers recovered to normal, becoming more shallow and actually shrinking in depth, after the insulin treatment was terminated (result not shown). It should be kept in mind that recovery in this experiment occurred after two different treatments: the removal of the plus lenses and the termination of the action of insulin. Insulin must have lost its efficacy, at least partly, 7 days after the last injection. Otherwise, the chicks would have stayed myopic, as insulin resulted in an additional myopic shift in negative lens-treated eyes (Fig. 1) . 

Experiment 3: Short and Long-Term Effects of Lens Wear and Insulin Injections on Intravitreal Glucose Levels

Short-Term Effects of Insulin Injections.

Insulin acts on cells throughout the body to stimulate uptake, utilization, and storage of glucose. To learn whether insulin also controls glucose levels in the vitreous of the eye, we measured glucose content 15 minutes, 2 hours, and 4 hours after an insulin injection (0.3 nmol). Intravitreal glucose levels dropped significantly after the injections, reaching a minimum after approximately 2 hours (−12.5%, one-way ANOVA, Student’s t post hoc test, P < 0.05). Subsequently, it returned to baseline levels within the next 2 hours. 

Short-Term Effects of Lens Wear on Intravitreal Glucose Level.

Intravitreal glucose levels were measured in eyes without insulin injections, after just 1 hour of positive or negative lens wear (Table 3) . Surprisingly, glucose levels were found to be increased in response to both types of lenses. 

Long-Term Effects of Intravitreal Insulin Injections and Lens Wear on Glucose Level.

Glucose levels were also measured after 4 days of lens treatment. During this time, insulin or saline was injected every other day as in experiments described earlier. No long-term effects of insulin or saline injections were found on glucose levels in chicks without lenses (group 1 vs. group 2). Of interest, insulin always reduced glucose levels when lenses (positive or negative lenses) were worn in addition to the injections (Table 3) . 

Experiment 4: Effects of Insulin on ZENK mRNA Expression Levels and on the Number of ZENK-Immunoreactive Glucagon Amacrine Cells

Effects of Insulin on Retinal ZENK and Glucagon mRNA Expression Levels.

Since both ZENK and glucagon have been shown to represent a potential STOP signal for myopia development in chicks, ^{14 18} the short-term effects of insulin (0.03 and 0.3 nmol) were studied on retinal ZENK mRNA levels, in animals with or without positive lenses. Insulin injections (0.3 nmol) substantially enhanced ZENK mRNA expression in the retina. This effect was even higher in animals with normal vision (by a factor of 7.6; Fig. 7A ) than in animals that wore positive lenses (5.6-fold). Even in chicks that were injected with the lower insulin concentration (0.03 nmol), ZENK mRNA levels were increased, although to a lesser extent. In contrast to ZENK, no significant effects of insulin was detected on glucagon mRNA expression in birds with no lenses (Fig. 7B) . In accordance with former studies, 1 hour of treatment with positive lenses increased glucagon mRNA levels. This effect could be prevented by an insulin injection (Fig. 7B) . 

Effects of Insulin on the Number of ZENK-Immunoreactive Glucagon Amacrine Cells.

The short-term effect of insulin on the expression of ZENK in glucagon amacrine cells was investigated in double-labeled transverse cryostat sections of the retina. In saline-injected chicks, approximately 46% of the glucagon amacrine cells were labeled for ZENK (Fig. 8) , which is comparable to observations in published experiments (∼50% ¹⁴ ). One hour of treatment with positive lenses (which normally increases the number of double-labeled cells), had no significant effect. Instead, insulin injections in positive lens-treated eyes reduced the number of ZENK-positive glucagon amacrine cells, an effect that is normally seen with negative lenses (one-way ANOVA, P < 0.05). 

Although the number of ZENK-immunoreactive glucagon cells was not significantly influenced by insulin injections in control animals (Fig. 9) , ZENK expression was stimulated in many other cells of the inner nuclear layer (INL; Fig. 9 ). Their location within the INL ⁴⁵ indicates that these cells are probably Müller and/or bipolar cells. The increase in the amount of ZENK-expressing cells in the INL is in line with the observations from real-time PCR which also showed a heavy increase in retinal ZENK mRNA levels after insulin injections (Fig. 7) . 

Discussion

The effects of intravitreal insulin injections on myopia development and eye growth were investigated in the chicken model. Insulin was studied, because it was known to act antagonistically to glucagon, and glucagon was previously proposed as a STOP signal for axial eye growth. The following results were obtained:

1.  
   Intravitreal insulin stimulated the development of myopia. High doses of insulin (0.3 nmol) were necessary to induce small myopic shifts in chicks with normal visual exposure.
2.  
   The myopia-promoting effect was much enhanced when spectacle lenses were worn in addition. In combination with positive lenses, insulin not only inhibited the expected hyperopia but rather reversed the refractive error and induced high amounts of myopia. In the case of negative lenses, the development of myopia was further enhanced.
3.  
   Myopic refractive errors induced by insulin correlated with an increase in axial length in all treatment groups. However, the changes in eye length were mostly due to an increase in anterior chamber depth, as well as a thickening of the crystalline lens. The corneal radius of curvature did not change in animals without lenses but decreased in insulin-injected, lens-treated eyes. The induced form of myopia is therefore different from the type of myopia typically seen in humans, which is mainly due to an increase in vitreous chamber depth. It is most likely that insulin caused myopia not only through a retinal mechanism but also through a direct effect on the anterior segment and the crystalline lens.
4.  
   Insulin had a powerful stimulatory effect on ZENK mRNA and ZENK protein expression in control animals as well as positive lens-treated chicks. In contrast, the number of a specific cell population, the ZENK-immunoreactive glucagon amacrine cells, was reduced by the insulin injections, at least in positive lens-treated animals.
5.  
   Since the end-point refractions reached with positive or negative lenses were indistinguishable after 4 days when high doses of insulin (0.3 nmol) were injected, we hypothesize that insulin renders the retina insensitive to the sign of imposed defocus. Perhaps, image degradation activates signaling pathways that are shared by the insulin signaling cascade.
6.  
   The dose-response curve for insulin was steep: at the two highest insulin concentrations (0.3 and 0.03 nmol), similar amounts of myopia were induced but, at the lowest dosage (0.003 nmol), insulin inhibited only the development of hyperopia.

Although there is no direct experimental evidence, the antagonistic effects of glucagon and insulin on refraction and eye growth in lens-treated chicks suggest an interaction between glucagon- and insulin-dependent mechanisms. This assumption is supported by a recent study (Zhu X, et al. IOVS 2007;48:ARVO Abstract 5925) in which glucagon and insulin were applied together. As a result, glucagon partially blocked the effects of insulin on ocular length, and insulin partially blocked the effects of glucagon on choroidal thickness. 

Compared to most other substances that affect refractive development in animal models by modulating vitreous chamber depth, insulin was different because it affected primarily the anterior segment of the eye. Minor effects on the anterior segment have also been found to be induced by melatonin ⁴⁶ and the nitric oxide synthase inhibitor l-NAME. ⁴⁷  

It is already known in other tissues that insulin may induce cell swelling, ⁴⁸ and it could be that the thickening of the crystalline lens in our experimental paradigm was due to cell swelling as well. In humans, lens swelling has been reported during the initial therapy in diabetes. ⁴⁹ Moreover, insulin is known to have a mitogenic effect on the lenticular epithelium and may induce stimulated lens growth. ⁵⁰ In the present study, the strong myopiagenic effect of insulin is due to its effects on anterior chamber depth, corneal curvature, and lens thickness. Insulin also had some effect on vitreous chamber depth (VCD), but this was variable among the different treatment groups: in the case of positive lenses, insulin inhibited the decrease in VCD that normally occurs during plus lens wear. This effect was most likely due to inhibition of the choroidal response to the defocus. Lower amounts of insulin inhibited vitreous chamber growth in animals without lenses, whereas, in the case of negative lenses, no effect on vitreous chamber depth was observed. In summary, although the relative contribution of the changes in the different ocular dimensions was variable, these changes always added up to produce a dramatic increase in myopia. The effects of insulin were reversible. Chicks recovered completely from their myopia and became slightly hyperopic, with refractions similar to those of untreated chicks, as soon as the lens treatment and the insulin injections were terminated. Also lens thickness and anterior chamber depth returned to normal. 

A recent study provided data for comparison (Zhu X, et al. IOVS 2007:48;ARVO Abstract 5925). The authors found that insulin increased the rate of ocular elongation in eyes wearing positive lenses, and they also measured an increase in lens thickness and anterior chamber depth. Different from our study, the highest dose of insulin caused marked axial elongation also when no lenses were worn. These differences may be due to slight differences in the treatment protocols: insulin was injected every day rather than every other day, the injection volumes were larger, and the insulin concentrations were twice as high as those in our study. 

Vitreal and Retinal Concentration of Insulin and Possible Binding Sites

Insulin content is low in ocular tissues, with only approximately 0.58 ng/mg of protein in the embryonic chick retina at day 14. ³⁰ Insulin mRNA was detectable only by reverse transcription coupled to polymerase chain reaction, but not by in situ hybridization in the retinas of embryonic chicks. ⁵¹ Assuming a total volume of the vitreous of approximately 300 μL, ⁵² the three doses used in the present study produced intravitreal concentrations of 10⁻⁶, 10⁻⁷, and 10⁻⁸ M. Even the lowest concentration (10⁻⁸ M) was still above the half-maximum binding concentration in chicken scleral tissue (0.4 × 10⁻⁹ M). ⁵³ The half-life of insulin in the retina is not known, but it has been found that insulin is removed from the circulation approximately 70 minutes after its initial release. ⁵⁴ In the retina, insulin-degrading enzymes are not highly expressed. ⁵⁵ Because of the high binding capacity for insulin and insulin-like growth factor I (IGF-I), the retina can be considered a major target of both. ²⁷ A potential target cell population for insulin in the chick retina are the progenitor cells at the retinal margin. ⁵⁶ Proliferation of these cells was increased when myopia was induced. ⁵⁷ Insulin increased the proliferation of these progenitor cells, whereas glucagon suppressed their proliferation. ²² The proliferating cells were first detected only in the CMZ (circumferential marginal zone) but later also in the outer nuclear layer (ONL) and INL, up to 800 μm away from the CMZ. Most of these cells were glutamine-synthetase-positive Müller glia cells. Moreover, insulin stimulates the transdifferentiation of Müller glia in combination with fibroblast growth factor-2. ⁵⁶  

In addition to a possible retinal function of insulin, other sites of action of insulin may be the lens, the cornea and the sclera: on the one hand, because insulin receptors are present in these tissues as well, and on the other hand, because insulin had a strong effect on lens thickness and anterior chamber depth. Furthermore, it was recently shown that the choroid responds to insulin (Zhu X, et al. IOVS 2007;48:ARVO Abstract 5925). Zhu et al. found that intravitreal insulin injections had no influence on choroidal thickness in chicks with normal vision but caused choroidal thinning in chicks treated with positive lenses. Insulin can also exert its effects by binding to another receptor, the IGF-I receptor, where it has approximately 1% of the potency of IGF-I. ^{27 58} Studies on the pharmacologic effects of insulin and IGF-I in the retina suggest even more than 10% cross-reactivity. ⁵⁹ Some of the effects of insulin on eye growth may therefore also be mediated by stimulation of the IGF-I receptor. 

Possible Mechanisms of Insulin

Binding of insulin to its receptor leads to autophosphorylation of the β-subunits and the tyrosine phosphorylation of insulin receptor substrates (IRS). The metabolic effects of insulin are mediated through activation of PI3K (phosphoinositol-3 kinase) and activation of mitogen-activated protein (MAP) kinase pathways. Insulin is a hormone that stimulates glucose uptake and storage as glycogen, especially in liver and muscle cells. Of interest, we found that lens treatment alone increased vitreal glucose content. The underlying mechanism is unknown. One hypothesis is that the retina is less active in the absence of high contrast and high spatial frequencies as occurs during defocus and that it may therefore consume less glucose. 

Insulin injections had only a small effect on vitreal glucose content (maximum reduction, ∼12.5%). Therefore, insulin in the eye may have additional functions other than the regulation of glucose uptake. Another interesting factor is that birds are known to be hyperglycemic. Infusion of insulin causes only minor decreases in blood glucose levels. ⁶⁰ Apparently, birds display a high degree of resistance to insulin, ⁶⁰ and it is likely that the major role of insulin is not the modulation of glucose metabolism but rather a function as a neuromodulator. It is known that neurons metabolize glucose in an insulin-independent manner. It has been shown that, in the rat retina, retinal insulin receptor kinase activity does not fluctuate with the feeding-fasting cycle, which is different with systemic insulin. Thus, the basal insulin receptor activity in the retina is different from that in the liver, muscle, and fat. Moreover, recent studies have demonstrated a tissue-specific signaling pathway for insulin in retinal tissue. ^{33 61}  

It is already known that insulin and/or IGF-I play a critical role during retinal development and are crucial for the survival and/or differentiation of different neuronal cells. ^{62 63 64 65 66} There is also evidence that, in the mammalian brain, insulin plays an important role in learning, memory, and cognition. ⁶⁷ The present study adds to these findings: Insulin was found to change ocular growth, probably by changing retinal image processing. This conclusion can be made because the effects of insulin on eye growth were much enhanced when lenses were worn. 

The effects of glucagon, the physiological antagonist of insulin, are mediated by the glucagon receptor. Glucagon binding leads to increased cellular levels of cAMP and activation of PKA (protein kinase A). Insulin injections had no effect on glucagon mRNA expression in chicks with normal vision but, in positive lens-treated chicks, the typical increase in glucagon mRNA levels was suppressed by insulin. Therefore, insulin appears to interact with glucagon mRNA only when the retinal image is defocused. 

Potential Target Genes for Insulin Action in the Retina

Glucose and insulin are important transcriptional regulators of many genes, ⁶⁸ including several transcription factors. In various tissues it has been found that insulin increases the amount of Egr-1 protein (also called ZENK, Krox-24, and NGFI-A), which itself has numerous target genes. ⁶⁹ Also in the present study, ZENK mRNA content and the number of ZENK-immunoreactive cells were heavily increased by insulin. Their location within the INL indicates that ZENK expression was most probably induced in bipolar and/or Müller cells. Changes in ZENK protein expression are particularly clearly detectable when the analysis is confined to glucagon amacrine cells. ^{12 14} In this case, the changes are also dependent on the sign of imposed defocus (upregulation with positive lenses and downregulation with negative lenses). Using immunohistochemistry, we found that insulin injections in positive lens-treated eyes reduced the amount of ZENK in glucagon amacrine cells. A reduction was previously found when myopia was induced by negative lenses or diffusers, so that myopia seems to be linked to reduced ZENK levels in the case of insulin injections, as well. The large increase in ZENK mRNA expression in the whole retina probably obscured the small decrease in ZENK in glucagon amacrine cells in positive lens-treated eyes. In summary, insulin has differential effects on the ZENK expression in different retinal cells. We did not measure gene expression changes in the lens, anterior chamber, and cornea. Further studies will show whether the marked effects of insulin on these compartments were mostly due to swelling, or whether gene expression changes may have occurred in the anterior segment as well. 

Metabolic Conditions, Diabetes, and Myopia

Since some of the risk factors for myopia tend to occur in combination, it is important to study their effects separately. ⁷⁰ Recently, Cordain et al. ^{20 71} have hypothesized that diet-related chronic hyperinsulinemia may play a key role in the pathogenesis of juvenile-onset myopia. According to them, high insulin levels could lead to an increase in circulating IGF-I and a subsequent decline in the levels of insulin-binding protein-3. It was further proposed that this would disturb the coordinated growth of the eyeball and the crystalline lens. At present, this hypothesis it is not sufficiently supported by data and is very speculative. Nevertheless, since we showed that insulin can induce high myopia it would be interesting to test whether retinal insulin levels are influenced by nutrition and/or by metabolic disorders such as diabetes. Then again, the hypothesis is challenged by our results, since higher doses of insulin induced mostly an “anterior segment” form of myopia that is not typically seen in humans. 

From human studies, it is known that the duration of diabetes has a marked effect on the thickness of the crystalline lens, which becomes larger in diabetes. ⁵⁰ Patients with insulin-dependent (type I) diabetes undergo considerable variations in systemic insulin and glucose levels. Depending on their treatment regimens, they may have high insulin levels immediately after injections, followed by a decrease in glucose levels. Given that insulin appears to interact with eye growth control, there is some chance that emmetropization is affected, in particular in young subjects. Studies of the short-term effects of insulin treatment on refraction have often revealed a transient shift in the hyperopic direction in patients until hyperglycemia is under control. A reduction of the overall refractive index in intraocular tissues, especially in lens, has been proposed to be responsible for this transient hyperopic shift. ⁷² To our knowledge, human vitreous insulin levels have been measured in only one study. ⁷³ The investigators found reduced vitreal insulin levels in type I diabetes, whereas patients with hyperinsulinemia type II had normal or even elevated vitreal insulin concentrations. Recently, a new study ⁷⁴ suggested glucose to be the primary mediator of the myopic shift. Altogether, more studies are necessary to resolve the question of whether elevated glucose and/or insulin levels can induce myopia in humans. 

Outlook

The findings of this study suggest that insulin is a potent stimulator of myopia, especially if the retinal image is defocused in addition. Insulin changed the growth pattern of the eye at different places that have to be addressed separately. Another question to be resolved is whether insulin acts predominantly by binding to the insulin receptor or the insulin-like growth factor receptor. It is important to elucidate further its role in the eye, since insulin is currently viewed as a possible pharmacologic treatment, especially corneal epithelial healing in diabetes mellitus. 

 Supported in part by European Union Marie Curie Research training Network MYEUROPIA Grant MRTN-CT-2006-034021) and German Research Council Grant Scha 518/13-1).

 Submitted for publication January 8, 2008; revised March 7, April 24, May 26, and June 18, 2008; accepted October 30, 2008.

 Disclosure: M.P. Feldkaemper, None; I. Neacsu, None; F. Schaeffel, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Summary of Treatment Groups

Experiment Group	Lens	Drug (Dose)	Experiment Duration
Experiment 1: Effects of Intravitreal Insulin Injections on Refractive Development, Axial Length, and Corneal Curvature
1	No lens	Saline, every second day	4 days
2	No lens	Insulin (0.3; 0.03; 0.003 nmol), every second day	4 days
3	+7 D	Saline, every second day	4 days
4	+7 D	Insulin (0.3; 0.03; 0.003 nmol), every second day	4 days
5	−7 D	Saline, every second day	4 days
6	−7 D	Insulin (0.3 nmol), every second day	4 days
Experiment 2: Recovery from Intravitreal Insulin Injections and Lens Wear
3rec	+7 D	Saline, every second day	4 days
	+7 D	No injection	3 days
	No lens	No injection	2 days
4rec	+7 D	Insulin (0.3 nmol), every second day	4 days
	+7 D	No injection	3 days
	No lens	No injection	2 days
Experiment 3: Short and Long-Term Effects of Lens Wear and of Insulin Injections on Intravitreal Glucose Level
7	No lens	Insulin (0.3 nmol)	0, 15, 120 and 240 minutes
8a	−7 D	No injection	1 hour
8b	No lens	No injection	1 hour
9a	+7 D	No injection	1 hour
9b	No lens	No injection	1 hour
Experiment 4: Effects of Insulin on ZENK mRNA Expression Level and on the Number of ZENK Immunoreactive Glucagon Amacrine Cells
10	No lens	Saline	1 hour
11	No lens	Insulin (0.03; 0.3 nmol)	1 hour
12	+7 D	Saline	1 hour
13	+7 D	Insulin (0.03; 0.3 nmol)	1 hour

 In all treatment groups, except 8 and 9, both eyes of the chicks received the same treatment. Chicks in groups 8 and 9 were treated unilaterally with lenses, with the contralateral eye serving as the control.

Figure 1.

 

Effects of insulin and saline injections on refractive development in chickens with normal visual experience (control) and in chickens wearing +7- or −7-D lenses. Data are the differences in refractive state between the beginning (start-up level) and end (4 days later) of the experiment (mean ± SEM). Insulin (0.3 nmol) induced a relative myopic shift in chicks with normal visual experience but induced severe myopia in all lens-wearing chicks, no matter whether positive or negative lenses were worn.

Figure 2.

 

Effects of insulin and saline injections on axial eye growth in chickens without lenses (control) or with +7- or −7-D lenses. Data are the differences in axial length between the beginning (start-up level) and end (4 days later) of the experiment (mean ± SEM). Intravitreal insulin (0.3 nmol) enhanced axial eye growth in chicks with normal visual experience, but had a much more pronounced growth-stimulating effect in chicks that were treated with spectacle lenses (tested doses with negative lenses: 0.3 nmol; with positive lenses: 0.3 nmol, 0.03 nmol, and 0.003 nmol).

Figure 3.

 

Effects of intravitreal insulin and saline injections on vitreous chamber growth in chickens without lenses (control) or with +7- or −7-D lenses. Differences in VCD between the beginning of the lens treatment (start-up level) and the end, 4 days later, are shown for saline and insulin-injected eyes (mean ± SEM). The highest dose of insulin (0.3 nmol) had no effect on vitreous chamber depth in eyes with normal visual experience and in negative lens-treated eyes. With positive lens treatment, the normally occurring inhibition of vitreous chamber growth was suppressed by insulin. In line with previous observations, positive lens treatment reduced vitreous chamber depth in saline-injected eyes.

Figure 4.

 

Effects of insulin and saline injections on ACD growth in chickens without lenses (control) or with +7- or −7-D lenses. Data are differences in ACD between the beginning of the lens treatment (start-up level) and the end, 4 days later (mean ± SEM). Insulin injections (0.3 nmol) caused the ACD to increase, compared with the respective saline-injected groups.

Figure 5.

 

Effects of insulin and saline injections on lens thickness in chickens without lenses (control) or with +7- or −7-D lenses. Data are the differences between the beginning of the lens treatment (start-up level) and the end, 4 days later (mean ± SEM). Insulin increased lens thickness at a dose of 0.3 nmol in animals with normal vision. The effects of insulin (0.3 and 0.03 nmol) on lens thickness were enhanced when positive lenses were worn in addition. At the lowest dose of insulin, lens thickness remained unchanged.

Table 2.

 

Effects of Intravitreal Insulin Injections and Treatment with Spectacle Lenses on Refractive Development and Ocular Biometry

Treatment	Saline	Insulin
				0.3 nmol	0.03 nmol	0.003 nmol
No lenses
Refraction (D)	+3.32 ± 0.22	+2.09 ± 0.46	+1.79 ± 1.33	+3.04 ± 0.47
AL (mm)	8.37 ± 0.04	8.63 ± 0.05	8.34 ± 0.07	8.28 ± 0.05
VCD (mm)	5.33 ± 0.03	5.31 ± 0.04	5.23 ± 0.04	5.27 ± 0.05
ACD (mm)	1.00 ± 0.02	1.13 ± 0.02	1.02 ± 0.03	0.93 ± 0.02
Lens (mm)	2.04 ± 0.01	2.19 ± 0.03	2.09 ± 0.02	2.11 ± 0.02
CRC (mm)	3.26 ± 0.03	3.21 ± 0.02
+7-D lenses
Refraction (D)	+6.90 ± 0.42	−5.78 ± 0.86	−6.75 ± 0.92	+1.71 ± 0.97
AL (mm)	8.13 ± 0.05	9.03 ± 0.06	8.96 ± 0.09	8.38 ± 0.06
VCD (mm)	5.08 ± 0.03	5.44 ± 0.04	5.51 ± 0.07	5.30 ± 0.05
ACD (mm)	1.03 ± 0.02	1.28 ± 0.03	1.18 ± 0.01	0.94 ± 0.03
Lens (mm)	2.02 ± 0.03	2.30 ± 0.03	2.23 ± 0.06	2.13 ± 0.03
CRC (mm)	3.28 ± 0.03	3.20 ± 0.02
−7-D lenses
Refraction (D)	−2.03 ± 0.66	−6.48 ± 1.02
AL (mm)	8.34 ± 0.05	8.71 ± 0.07
VCD (mm)	5.27 ± 0.04	5.25 ± 0.06
ACD (mm)	1.09 ± 0.03	1.30 ± 0.03
Lens (mm)	1.96 ± 0.03	2.17 ± 0.03
CRC (mm)	3.21 ± 0.01	3.13 ± 0.02

 The refractive state and ocular dimensions shown were measured after the 4-day lens treatment period (mean ± SEM). Saline-injected chicks were always measured at the same time as the chicks that were injected with one of the three insulin concentrations. n = 6–8/group. AL, axial length; VCD, vitreous chamber depth; Lens: lens thickness; ACD, anterior chamber depth; CRC, corneal radius of curvature.

Figure 6.

 

Recovery of refractive state and lens thickness after termination of the insulin injections (0.3 nmol insulin) and lens wear (mean ± SEM, n = 6). Note that the effects of insulin on refractive state and ocular dimensions were fully reversible.

Table 3.

 

Effects of Insulin Injections on Intravitreal Glucose Level in Lens-Treated Animals

Treatment	Intravitreal Glucose Level (mg/dL)	Treatment	Intravitreal Glucose Level (mg/dL)	P
Contralateral eye (group 8b)	194.2 ± 6.1	1 Hour of minus lens (group 8a)	222.2 ± 6.1	P < 0.01
Contralateral eye (group 9b)	205.5 ± 5.3	1 Hour of plus lens (group 9a)	220.2 ± 3.8	P < 0.05
4 Days, saline (group 1)	178.0 ± 6.9	4 Days, 0.3 nmol insulin (group 2)	169.4 ± 2.9	NS
4 Days +7 D, saline (group 3)	215.4 ± 4.0	4 Days +7 D, 0.3 nmol insulin (group 4)	190.5 ± 6.1	P < 0.01
4 Days −7 D, saline (group 5)	202.8 ± 3.2	4 Days −7 D, 0.3 nmol insulin (group 6)	189.6 ± 2.7	P < 0.01

 Intravitreal glucose levels (mean ± SEM; n = 4–8) were measured after 1 hour of treatment with positive or negative lenses and 4 days after saline or insulin injections (which occurred on days 0 and 2), with or without additional lens treatment. Paired t-tests were used to compare the contralateral with the lens-treated eyes in groups 8 and 9. Unpaired t-tests were used to compare with the other groups.

Figure 7.

 

Effects of insulin on retinal ZENK mRNA and glucagon mRNA levels (mean ± SEM; n = 5–8). MNE of the target genes were calculated using β-actin as the housekeeping gene. Insulin injections (0.3 or 0.03 nmol) caused a substantial increase in ZENK mRNA levels in the retina, in chicks both without and with positive lenses. Glucagon mRNA levels were increased only by treatment with positive lenses. This effect was suppressed by an insulin injection. ***P < 0.001; **P < 0.01; *P < 0.05, one-way ANOVA, paired Student’s t-test post hoc). The significant effects between the four treatment groups at each dose of insulin are shown. Differences between the two dosages (0.3 and 0.03 nmol insulin) were calculated but are not shown.

Figure 8.

 

Effects of insulin on the ratio of glucagon amacrine cells, which also express ZENK (mean ± SEM). A single insulin injection (0.03 nmol) decreased the number of ZENK-immunoreactive glucagon amacrine cells in positive lens-treated eyes (*P < 0.05).

Figure 9.

 

Glucagon and ZENK protein expression in the retina after saline (A) or insulin (B) injection. The number of glucagon amacrine cells (right arrow) was similar in saline- and insulin-injected retinas. Moreover, the number of ZENK-immunoreactive amacrine cells, located directly on the border of the IPL (weaker labeled cells, left arrow) did not change. Insulin (0.3 nmol) prominently increased the number of ZENK-immunoreactive cells (weaker labeled cells, gray arrow, B) in the middle of the INL. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer. Scale bar, 100 μm.