|Molecular Vision 2006;
Received 21 July 2005 | Accepted 10 January 2006 | Published 18 January 2006
The effect of insulin and glucose levels on retinal glial cell activation and pigment epithelium-derived fibroblast growth factor-2
Christopher J. Layton, Simone
Becker, Neville N. Osborne
Nuffield Laboratory of Ophthalmology, Oxford, UK
Correspondence to: Christopher J. Layton, Nuffield Laboratory of Ophthalmology, Walton Street, Oxford OX2 6AW, UK; Phone: +44 1865 248 996; FAX: +44 1865 794 508; email: firstname.lastname@example.org
Purpose: The diabetic retina exhibits decreases in endogenous nonangiogenic neurotrophins. This study hypothesized that deficiencies in systemic and retinal pigment epithelium-derived (RPE) neurotrophic factors also influence retinal changes in diabetes.
Methods: Diabetes was established in Listar hooded rats with streptozotocin. Reverse transcriptase coupled polymerase chain reaction (RT-PCR) and immunoblotting were used to determine the expression of fibroblast growth factor-2 (FGF-2) in the retina and RPE, and glial fibrillary acid protein (GFAP) in the retina. In addition, primary human RPE cultures and a transformed Müller cell line were used to determine the effect of insulin, glucose, and insulin-like growth factor (IGF) on the expression of these substances.
Results: FGF-2 and GFAP were increased in retina, but FGF-2 was decreased in the RPE of diabetic animals. Retinal GFAP correlated with RPE FGF-2 expression in these animals. Insulin produced a dose-dependent increase in FGF-2 in RPE cells and decrease in GFAP in Müller cells grown in 15 mM glucose. In 5 mM glucose, insulin had no effect on expression of either protein. Physiological levels of insulin inhibited changes induced by 15 mM glucose. The effect of 9 nM insulin on each culture was mimicked by 1 nM IGF, and blocked with an IGFR-1 inhibitor.
Conclusions: It is suggested that decreased systemic insulin and high glucose levels contribute to decreased FGF-2 production in the RPE and increased glial cell activation in the diabetic retina. Addition of insulin and IGF act to reverse this effect through the IGFR-1. These mechanisms may contribute to the development of diabetic retinopathy.
The traditional view of diabetic retinopathy, based on ophthalmoscopic observations, is of a microangiopathic complication associated with diabetes beginning after a long period of the disease . However, more recent investigations into the effect of diabetes on retinal neural tissue have shown that the disease damages neurons in the inner [2-4] and outer retina , induces glial cell activation [4,6-8], and reduces oscillatory potentials on the electroretinogram  prior to the onset of microvascular disease. Such studies suggest that virtually all cell types in the retina are affected in the diabetic process before the onset of retinopathy . This phenomenon can be described as diabetic retinal neuropathy.
Animal studies of the diabetic retina in the pre-retinopathic stage show that neuronal cell death is accompanied by a reduction in nonangiogenic neurotrophic factors from endocrine (insulin and erythropoietin [EPO])  and paracrine (brain-derived neurotrophic factor-BDNF  and pigment epithelium-derived factor, PEDF) [12,13] sources, with an accompanying increase in locally produced angiogenic neurotrophic factors such as vascular endothelial growth factor (VEGF) [14,15]. Studies involving the supplementation of locally produced nonangiogenic neurotrophic factors to reduce the neuronal and glial signs of diabetes in the retina support this view [2,11,16]. In humans, supplementation of endocrine neurotrophic factors has also had some success, with EPO administration reducing signs of diabetic retinopathy in a small cohort . Moreover, the Diabetes Control and Complications Trial (DCCT) can be interpreted to show that exogenous administration of the neurotrophic factor insulin prevents diabetic retinopathy .
The retinal pigment epithelium (RPE) is a neuroectodermal monolayer of cells situated between the photoreceptors and the choroid. The functions of the RPE are complex, but one of its major roles is to support retinal integrity. It achieves this in part by regulating nutrients , phagocytosing rod outer segments , and by contributing to the production of cytokines in the eye, including neurotrophic factors such as fibroblast growth factor-2 (FGF-2)  and angiogenic factors such as VEGF . While retinal and systemic production of trophic factors have been well studied in diabetes, the growing understanding of the contribution of different retinal cell types to the diabetic process has led to no studies which have investigated whether the RPE contributes to the retinal trophic support in diabetes.
This investigation focuses on the expression of FGF-2 in diabetes. FGF-2 is produced by the RPE  and its levels are actively modulated in the tissue in response to a variety of insults . Originally thought to be important in angiogenesis , the function of FGF-2 is now thought to be not primarily angiogenic in nature  and its main role in the central nervous system is now thought to be neurotrophic and neuroprotective. Constitutive FGF-2 production in the healthy adult is confined to the brain and retina [26,27], where it is expressed widely . Continuous production of the neurotrophin is essential to the health of the retina, and inhibition of FGF-2 signaling in the normal retina induces retinal degeneration in a transgenic model . This is supported by in vitro studies, which have shown that the protein enhances survival and neurite outgrowth in a range of neural preparations [30,31]. In vivo studies in models of CNS injury reflect the neuroprotective nature of the compound, showing FGF-2 is upregulated, and its distribution altered in models of cortical [32-34] and retinal [35-37] injury. FGF-2 also shows unusual properties in the diabetic retina: Unlike most neurotrophic factors with little angiogenic activity, FGF-2 is elevated in diabetic retinopathy [38,39]. Indeed, laser photocoagulation, a treatment known to be beneficial for diabetic retinopathy , causes an increase in FGF-2 production in the retina together with its more accepted role of decreasing oxygen consumption . It therefore seems attractive to postulate that the upregulation of FGF-2 in the diabetic retina is an endogenous mechanism for protecting neural tissue from injury and that if a deficiency in FGF-2 production occurs in the diabetic RPE than this could contribute to diabetic retinal neuropathy.
The investigations performed here used the well characterized model of the streptozotocin-induced diabetic rat. This model is useful because retinal neovascularization does not occur  but signs of neural injury and glial cell activation similar to those reported in human retinas (diabetic retinal neuropathy) become evident as the disease progresses [3,7]. Thus, it can be argued that any changes in the levels of neurotrophic factors in this model are predominant associated with a response analogous to diabetic retinal neuropathy, rather than as markers of incipient angiogenesis.
Therefore, an aim of this investigation was to isolate the RPE from a rat model of diabetes and measure the expression of the neurotrophic factor, FGF-2, in the tissue. In addition, experiments were performed to explore the mechanism of any changes in FGF-2 levels in the RPE during diabetes by investigating possible paracrine and endocrine factors likely to be involved. These included retinal FGF-2 expression, glial cell activation, environmental glucose levels, insulin, and insulin-like growth factor (IGF).
All experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eighteen age-matched adult male Listar hooded rats (200-250 g) were housed in a temperature- and humidity-controlled room with a 12 h light/12 h dark cycle and provided with food and water ad libitum. Random blood glucose concentrations were measured using a Precision PCx Glucometer (Medisense UK Ltd., Abingdon, Oxfordshire, UK). Nine rats were randomly assigned to the diabetic group and given an intraperitoneal injection of 62.5 mg/kg of streptozotocin in 10 mM citrate buffer. A control group of nine rats received an intraperitoneal injection of the citrate buffer alone. Retinas were taken after 15 weeks of diabetes for analysis. One to three RPE sheets from each eye were isolated by washing the eye cup in phosphate-buffered saline (PBS) and then incubating the whole cup in 0.5% trypsin at 37 °C for 5 min. A flame-blunted Pasteur pipette was then used to scrape the RPE sheets from each eye into 1 ml of unsupplemented Hams F-10 containing soybean trypsin inhibitor (type I-S, 5 mg/7.5 ml). Samples were centrifuged at 2000x g at 4 °C for 5 min. The resultant pellet was washed repeatedly in PBS (100 mM, pH 7.4) and centrifuged again to provide samples for analysis.
The levels of cyclophilin, FGF-2, and GFAP mRNA were determined using a semiquantitative reverse transcriptase-polymerase chain reaction technique (RT-PCR) as described elsewhere . Primer sequences are listed in Table 1. Briefly, total RNA was isolated, and first strand cDNA synthesis performed on 2 μg of DNAse-treated RNA. Aliquots of the resultant cDNA species were amplified in PCR buffer with 4 mM MgCl2. Reactions were initiated by incubating at 94 °C for 10 min and PCRs (94 °C, 15 s; 52 °C, 30 s; 72 °C, 30 s) performed for a suitable number of cycles followed by a final extension at 72 °C for 3 min. PCR products were separated on 1.5% agarose gels using ethidium bromide for visualization. The relative abundance of each PCR product was determined by quantitative analysis of digital photographs of the gels viewed under UV light using Labworks software (UVP Products, Upland, CA).
Retinal and RPE proteins were isolated simultaneously with RNA using the standard Tri-Reagent technique (Sigma, Poole, UK). After processing, samples were solubilized in homogenization buffer with protease inhibitors (20 mM Tris HCl, pH 7.4, containing 2 mM EDTA, 0.5 mM EGTA, 1% SDS, 0.1 mM phenylmethylsulphonyl fluoride, 50 μg/ml aprotinin, 50 μg/ml leupeptin, and 50 μg/ml pepstatin A). An equal volume of sample buffer (62.5 mM Tris HCl, pH 7.4, containing 4% SDS, 10% glycerol, 10% mercaptoethanol, and 0.002% bromophenol blue) was then added. Electrophoresis of samples was performed using 10% polyacrylamide gels containing 0.1% SDS and proteins blotted onto nitrocellulose (Sigma). Blots were incubated for 3 h at room temperature with primary antibodies against actin (Chemicon, Chandler's Ford, UK; monoclonal antibody, 1:2000), FGF-2 (Santa Cruz Biotechnology, through Insight Biotechnology, Wemberly, UK; polyclonal rabbit sc-79, 1:200), and GFAP (DAKO, Ely, UK; polyclonal rabbit 1:400). Development was then performed using an avidin-biotin peroxidase complex kit (Vector Labs, Peterborough, UK; 1:100) with appropriate secondary antibodies and subsequently processed according to the manufacturer's directions. The final nitrocellulose blots were developed with a 0.016% w/v solution of 3-amino-9-ethylcarbazole (AEC) in 50 mM sodium acetate (pH 5.0) containing 0.05% (v/v) Tween-20 and 0.03% (v/v) H2O2. The color reaction was stopped with 0.05% sodium azide solution and scanned at 800 dpi by an Epson Perfection 1200u scanner. Quantitative analysis of the files was performed using Labworks software (UVP Products, CA).
RPE cell culture
Postmortem donor human eyes (donors aged 26 and 58 years) were obtained without their cornea (for transplantation purposes) from Bristol Eye Bank (Bristol, England) up to 48 h after enucleation and were processed immediately. Culture procedures were undertaken in a sterile laminar flow hood (ICN Flow, Thames, UK). Cultures of RPE cells were prepared and characterized by labeling for cytokeratin (KG 8.13) as described previously . Culture medium consisted of Hams-F10 supplemented with 5 mM glucose, 10% (v/v) fetal bovine serum, 0.4% glucose, 2 mM glutamine, amphotericin B (25 μg/mL), and gentamicin (100 μg/mL). Primary cultures were grown in 25 cm2 culture flasks and passaged in a ratio of 1:3, and thereafter, in 75 cm2 flasks. While growing, cultures were kept in an incubator at 37 °C, with saturating humidity and an atmosphere of 5% carbon dioxide to 95% air. Experiments were performed between the third and seventh passages.
Culture of transformed Müller cells
The RMC-1-transformed Müller cell line was obtained from Dr. V. Sarthy (Northwestern University, Chicago, IL). These cells were grown in modified Eagle's medium (MEM) supplemented with 5 mM glucose, 10% FBS, 2 mM glutamine, 2.5 mg/ml amphotericin B, and 100 μg/ml gentamicin. Cells were passaged at a ratio of 1:3 every 48-72 h and used for the outlined studies when 80% confluent.
Some cells from each culture were transferred to 13 mm glass coverslips in 24 well plates. After appropriate treatments, cells were fixed in 4% paraformaldehyde in sodium phosphate buffer (100 mM, pH 7.4) for 30 min. Cells were washed in PBS and PBS plus Triton X-100 (0.1%, v/v (PBS-T)), and nonspecific antibody binding was blocked with bovine serum albumin in PBS (0.5%, w/v; PBS-B). Cultures were then incubated in PBS-B with primary antibodies against FGF-2 (Santa Cruz Biotechnology; sc-79 polyclonal rabbit, 1:200), vimentin (Sigma, monoclonal 1:1000) and GFAP (Dako, polyclonal rabbit; 1:200). After cells were washed three times (5 min each) in PBS-T, they were immunolabeled with fluoroscein isothiocyanate (FITC)-linked antirabbit or antimouse antisera (Sigma, Poole, UK; 1:100). Visualization was by using a Zeiss epifluorescence microscope (Göttingen, Germany).
Experimental protocol in cell culture experiments
Experiments designed to investigate the mechanism of in vivo findings in the RPE were performed on cells grown to 80% confluence in both RPE and Müller cell cultures. To remove insulin from the FBS used in the growth media, appropriate cultures were washed three times in PBS (100 mM, pH 7.4) and maintained for two days in a hormonally defined media (HDM) of Ham's F10 (for RPE cells) or MEM (for Müller cells) supplemented with 5 mM glucose, 2 mM glutamine, 2.5 mg/ml amphotericin B, 100 μg/ml gentamicin plus 1 g/l transferrin, 96.6 μg/ml putresceine, 300 nM sodium selenate, 200 nM progesterone, and 10 pM estrogen, together with treatments of glucose, mannitol, insulin, IGF-1, and the IGFR-1 inhibitor, AG1024.
Cultures used for semiquantitative analysis of protein levels were grown in 6 well plates and samples collected for analysis by dislodging cells with a cell scraper, centrifuging the cell suspension at 1000x g for 5 min and then resuspending the pellet in 100 μl of homogenization buffer and processed for immunoblotting as already described.
All analyses were performed with the SPSS statistical package (v. 12; SPSS Inc., Chicago, IL). Rat weights and blood glucose levels were analyzed using Student's unpaired t-tests. Other analyses of difference were performed with Student's unpaired t-tests. Intra- and interclass correlations between the two eyes of each animal were accounted for using repeated measures analysis of variance. Bonferroni's correction to the p value was included where multiple endpoints were measured from the same samples. Correlation coefficients were obtained from linear regressions by the least squares method and 95% prediction intervals calculated. All results are presented as mean±standard error of the mean (SEM). Assay readings and analysis of protein levels in cultures were corrected for osmotic control levels, and the results of varying replications in four to six independent cultures were compared using Student's t-test or repeated measures analysis of variance where appropriate. An α level of 0.05 was chosen.
Streptozotocin induces diabetes in the rat
Diabetic rats displayed polyuria, polydipsia, and had unformed feces throughout the experiment. Cataracts became evident in some rats in the diabetic group after nine weeks of diabetes, and all rats displayed cataracts on gross examination after 12 weeks of the experiment. No cataracts were evident in the control group at any time. Rats in the diabetic group showed reduced weight gain, displaying an average gain of 4.5±1.6 g/week compared with 11.2±0.4 g/week in the control group averaged across the experiment (p<0.001). Final random blood glucose concentrations were 6.7±0.5 mM in the control group and 22.3±3.1 mM in the diabetic group (p<0.001). No rats from either group died during the experiment.
Figure 1 shows analysis of retinal and RPE samples taken from diabetic and control rats. Figure 1A shows that the RPE samples contained no detectable neurofilament light (an inner retinal marker found in ganglion cells) or von Willebrand factor (vWF), a marker of endothelial cells as found in the choroid, revealing the relative purity of the sample. Figure 1B,C show that FGF-2 protein and mRNA levels in retinal samples were significantly increased by 25±6% and 34±4%, respectively, in the diabetic group after 15 weeks of diabetes. GFAP levels were also significantly increased in the diabetic retina, with a 68±37% increase in mRNA and 98±16% increase in protein expression. Figure 1B,C also show that RPE FGF-2 protein and mRNA levels were distinctly reduced in diabetes by 25±6% and 36±7%, respectively.
In order to determine if retinal production of either protein could relate to a decrease in FGF-2 expression in the RPE, the amount of FGF-2, and GFAP protein in individual eyes were correlated. There was no significant correlation between retinal and RPE FGF-2 levels (r2=0.01, p<0.87; Figure 1D). In contrast, there was a significant correlation between RPE FGF-2 expression and retinal glial cell activation as indicated by GFAP protein expression (r2=0.31, p<0.01; Figure 1E).
Studies on RPE and Müller cell cultures in low (5 mM) or high (15 mM) glucose environment were undertaken in order to determine if the relationship between GFAP and RPE FGF-2 production in the diabetic retina in vivo was causal or if a separate dose-dependent mechanism was responsible for these phenomena.
The characteristics of Müller and RPE cell cultures used in these studies are shown in Figure 2. RPE cultures at the passages used in these studies showed immunolabeling for cytokeratin (KG 8.13) an RPE specific protein , and FGF-2 (Figure 2B,D). Müller cell cultures were positively labeled for vimentin, GFAP, and FGF-2 (Figure 2A).
In "type 1 diabetic" conditions (15 mM glucose and no insulin for 1 day), immunolabeling for FGF-2 in RPE cells (Figure 2D) showed less intense staining than in conditions of 5 mM glucose. Addition of 9 nM insulin resulted in more intense FGF-2 labeling in 15 mM of glucose than in 5 mM glucose (Figure 2D). In the Müller cell cultures, GFAP staining in 15 mM glucose and no insulin was more intense than when insulin was present in either 5 mM glucose or 15 mM glucose. (Figure 2C) In contrast, the intensity of FGF-2 staining in the Müller cell culture appeared unaffected by insulin in either a low or high glucose environment (results not shown).
Figure 3 quantifies the relationships described in Figure 2 for 6 h, 24 h, and 48 h treatment periods using immunoblotting. A dose-dependent relationship exists between insulin and FGF-2 production in RPE cells and GFAP suppression in Müller cells, respectively. These relationships are evident only after a 24 h period and only for cultures in the 15 mM when results are normalized with what occurs in 5 mM glucose in the absence of insulin.
After two days of exposure to high glucose in the absence of insulin, RPE cells contained 65±23% less FGF-2 than cells in 5 mM glucose (p<0.05) and 71±26% less FGF-2 than cells in 15 mM glucose and treated with physiological (36-179 pM)  levels of insulin (p<0.05; Figure 3C). When cultures exposed to 15 mM glucose are treated with 9 nM insulin, FGF-2 levels are increased 64±28% (p<0.05) relative to the same treatment in cultures exposed to 5 mM glucose (Figure 3C). Insulin had no significant effect on FGF-2 expression in cultures exposed to an environment of 5 mM glucose (Figure 3A-C). FGF-2 levels in cells exposed to 15 mM of glucose and treated with physiological levels of insulin (90 pM) were also not significantly different to the levels in cultures maintained in 5 mM glucose (Figure 3A-C).
Insulin treatment had the opposite effect on expression of GFAP in Müller cell cultures (Figure 3D-F). After two days of exposure to high glucose in the absence of insulin, Müller cells produced 83±27% more GFAP than cells in 5 mM glucose (p<0.05) and 81±26% less GFAP than cells in 15 mM glucose that were treated with physiological levels of insulin (p<0.05; Figure 3F). When treated with 9 nM insulin, GFAP levels decreased 44±15% relative to the same treatment in cultures exposed to 5 mM glucose. Insulin had no significant effect on GFAP expression in cultures in an environment of 5 mM glucose. Cells exposed to 15 mM glucose and treated with physiological levels of insulin did not display significantly different FGF-2 or GFAP levels than the cells maintained in 5 mM glucose. Müller cell FGF-2 levels were not significantly affected by either glucose or insulin treatment in the ranges tested (Figure 3G-I).
Figure 4 shows the results of investigations designed to explore the possible mechanism by which insulin influences RPE FGF-2 levels and Müller GFAP expression in an environment of 15 mM glucose. Figure 4A shows that in RPE cultures a significant increase (121±24%, p<0.05) in FGF-2 occurs after 24 h of treatment with 9 nM insulin in a high (15 mM) glucose environment. This effect is reduced to levels not significantly different to the insulin-free baseline by addition of 30 μM AG1024 which is known to specifically block IGFR-1 receptor signaling . Higher concentrations of AG1024 (120 μM) also blunts the effect of insulin. This concentration of AG1024 is known to block both IGFR-1 and insulin receptors , and from the data shown in Figure 4A had no clear effect on FGF-2 expression. In addition, treatment with 1 nM IGF led to an increase in FGF-2 expression of 128±28% (p<0.05) which was not significantly different to that induced by treatment with 9 nM insulin (Figure 4A). This IGF-induced increase in FGF-2 production in the RPE was reduced to basal levels by 30 μM AG1024.
Figure 4B demonstrates a similar effect in Müller cell cultures. In the presence of 15 mM glucose, 9 nM insulin reduced GFAP expression by 48±11%, (p<0.05) and this was significantly inhibited by 30 μM AG1024, increasing GFAP expression to baseline levels. Addition of 120 μM AG1024 had no additional effect on GFAP expression. Treatment with 1 nM IGF caused a decrease in GFAP expression of 58±9% (p<0.05), similar to that induced by 9 nM insulin. The IGF-induced reduction of GFAP was inhibited by 30 μM AG1024 to levels observed in the absence of insulin.
The experiments detailed here show that FGF-2 expression is decreased in the RPE after 15 weeks of diabetes in an experimental model. Surprisingly, this decrease is not correlated with retinal FGF-2 production but showed instead a strong correlation with retinal GFAP levels, a marker of glial cell activation. While a causal effect of one phenomenon upon the other is a possibility, it is more likely that the statistical correlation results from a dose-dependent effect of a separate factor responsible for both observations.
To explore these possibilities, primary cultures of RPE cells and Müller cells (the primary source of GFAP in the diabetic retina) [7,8] were carried out. Addition of a hormonally defined medium to achieve insulin-free culture conditions and high glucose (15 mM) led to significantly reduced levels of FGF-2 in RPE cells and increased GFAP levels in Müller cells, reflecting the findings in vivo. Insulin reversed both effects in cultures in an environment of 15 mM glucose in a dose-dependent manner. The effect of insulin was mimicked by IGF, and both influences were blocked by a specific inhibitor of IGFR-1.
Streptozotocin-induced diabetes in the rat
The animals used in these experiments were pigmented Listar hooded rats, which display pigmented RPE. Although streptozotocin-induced diabetes and diabetic retinopathy is better characterized in other strains, the use of Listar hooded rats was found to be necessary to assist visualizaton and handling of the RPE during the delicate manipulations necessary to separate the RPE sheets while avoiding choroidal contamination. Steptozotocin appeared to induce experimental diabetes in the rats, with increased blood glucose concentrations, polyuria, polydipsia, cataract formation, and a failure to gain weight noted in the diabetic group. In addition, FGF-2 and GFAP, two proteins known to be upregulated in the diabetic retina [4,6-8,38,39], were increased in these rats, giving an indication that diabetic retinal neuropathy was present in the samples after 15 weeks of diabetes.
The isolation of the RPE from rats with minimal contamination from the retina or choroid are shown by a lack of retinal and choroidal markers in the sample. FGF-2 mRNA and protein were detected in the RPE samples, and were downregulated in the diabetic rats. Initially it could be considered that this was the logical result of increased FGF-2 expression in the retina causing a paracrine negative feedback loop with the RPE, but no correlation was found between FGF-2 protein levels in each retina and FGF-2 expression in the adjacent RPE. However, GFAP, a marker known to be induced in Müller cells in diabetes, was found to correlate with RPE FGF-2 protein expression. Cell cultures were chosen to investigate this relationship since possible confounding factors central to the disease, such as ischemia, can be controlled. In these studies, the culture medium was manipulated to reflect the core metabolic insults known to occur in type 1 diabetes: low insulin (<36 pM) and high glucose (>5 mM).
Insulin-free conditions were required for treatment of Müller cell cultures with "diabetic" levels of glucose (15 mM) to mirror the changes in GFAP noted in the streptozotocin-induced diabetic rat retina. The effect was reversed in a dose-dependent manner by the addition of insulin, with GFAP expression returning to levels equivalent to those of cultures grown in 5 mM glucose when treated with physiological concentrations of insulin (36-179 pM) . Cultures displayed less GFAP expression than cultures grown in 5 mM glucose even when treated with supraphysiological levels of insulin. Care should be taken in correlating these results with the conditions of hyperinsulinaemia or type 2 diabetes, where insulin resistance and receptor malfunction is the primary defect .
"Diabetic" glucose levels in insulin-free conditions also decreased FGF-2 production in cultured human RPE cells, mirroring the results presented in the animal model and perhaps indicating that the phenomenon may extend to human disease. Once again, this effect was reduced with the addition of physiological levels of insulin, which restored FGF-2 expression to levels equivalent to those of cultures grown in 5 mM glucose. The results also indicate that the trend continues with supra-physiological insulin treatments, but care should again be taken in extending this analysis to the type 2 diabetic situation for the reasons already given.
The results also demonstrate that no concentration of glucose or insulin tested produced a significant change in Müller cell FGF-2 production. While acknowledging the inherent errors in using monolayers of isolated, transformed, actively dividing or passaged cells in investigations, these results together support the hypothesis that high glucose levels in the absence of insulin contribute to both the increased GFAP expression in the retina (which is mostly of Müller cell origin) , and decreased FGF-2 production in the RPE. Therefore, the combined effects of low insulin and high glucose could account for the correlation between the retinal GFAP and RPE FGF-2 noted in the streptozotocin-induced diabetic rat. Therefore, this may be an important contributor to similar observations in the human condition.
The insulin receptor (IR) is known to be present in the CNS  and in the retina , where it appears to be localized in photoreceptors, other neuronal elements , and in the RPE . However, the retina is unusual in that IGF binding is 10-20 times greater than insulin binding . Like insulin and IGF-1, the IR and IGFR-1 display extensive structural homologies [52,53], with both insulin and IGF acting as ligands for each, although the extent of the modest cross-affinity of each substance to the other receptor is controversial. Binding studies suggest each ligand has a 100 fold increased affinity for its own receptor [54,55]; however, studies of binding and pharmacological effects of the ligands in the retina estimate up to a 30 fold greater cross reactivity than these results predict [51,56,57]. This may be due to nonclassical insulin binding patterns that have been reported in the retina, where a proportion of binding sites have a similar affinity for both IGF and insulin . Given the presence of both the IR and IGFR1 in the RPE and retina, the unusually high cross-reactivity between the two and the fact that the literature supports the strong possibility of a differential regulation of FGF-2 and IGF-1 [58-60], it was hypothesized that the effect of insulin on FGF-2 and GFAP could be modulated through the IGFR-1 receptor.
This hypothesis was supported by the investigations presented here, in which treatment with AG1024 at a concentration known to specifically inhibit IGFR-1  blocked the effect of insulin on FGF-2 expression in cultured RPE cells and its effect on GFAP expression in cultured Müller cells. In addition, treatment with IGF at a concentration ten times lower than that tested for insulin resulted in an effect of the same magnitude on both cell types. This effect was also blocked by the IGFR-1 receptor blocker AG1024. Addition of AG1024 at the much higher concentration necessary to also block the IR  had no further influence on either RPE FGF-2 expression or Müller cell GFAP expression. This evidence may be interpreted to support the tentative conclusion that the dose-dependent effect of insulin on RPE FGF-2 production and Müller cell GFAP production is mediated through the IGFR-1 rather than, as originally expected, the IR.
These results could explain the findings that treatment of cultures with diabetic levels of glucose is a prerequisite for the withdrawal of insulin to induce phenotypical changes in RPE and Müller cells. Previous studies have reported that IGF-induced pancreatic b cell proliferation is glucose dependent, displaying maximal synergy at 15 mM of glucose . Similarly, IGF and glucose have been observed to act synergistically in stimulating fibrosis in renal fibroblasts , contributing to diabetic nephropathy. It appears consistent to argue that, in the culture experiments, insulin acts in synergy with "diabetic" glucose levels through the IGFR-1 receptor to alter RPE FGF-2 expression and Müller cell GFAP expression. This mechanism could induce similar changes consistent with the observations presented here in the streptozotocin-induced rodent model of diabetic retinal neuropathy and with the known manifestations of diabetes in retinal glial cells in human diabetes .
It should be noted that the IGF system is complex in the intact retina and consists of at least insulin, IGF-1, IGF-2, and the primarily inhibitory IGF binding proteins 2 and 3 . In addition, it is known that IGF itself has an important role in diabetes. IGF is downregulated in the retina of diabetic rats . In contrast, IGF is upregulated in the diabetic vitreous after the onset of microvascular disease  and appears to be disproportionally active at this stage of the disease . Additionally, IGF is important in upregulating VEGF expression in the diabetic retina , and IGFR-1 receptors are required for retinal neovascularization in a neonatal hypoxia model . Recent data from a normoglycemic transgenic model overexpressing IGF reported that the model displays most of the signs of human diabetic retinopathy . Together these reported results imply that IGF is decreased in the streptozotocin-induced diabetic retinal neuropathy in the rat and possibly the human, but an increase may mark the onset of neovascularization in both the rat and human . Therefore, changes in IGF appear to be complex and pathogenically important in the diabetic retina. Indeed these experiments do not rule out the possibility that autocrine production of IGF in the culture systems may have contributed to some of the effects noted here; however, it was not the purpose of these experiments to fully investigate the IGF system in the diabetic retina and RPE. It is worth noting that the previously described decrease in retinal IGF levels reported in diabetes would be expected to exacerbate the effects of low insulin on Müller cell GFAP expression and RPE FGF-2 levels in a diabetic environment through this mechanism.
It is concluded that low insulin, high glucose environments in culture mirror phenotypical changes in an animal model of experimental diabetes. It is likely that decreased activation of the IGFR-1 in diabetes contributes, perhaps significantly, to the retinal effects of the disease.
This study was supported by the Rhodes Trust. We would like to thank Dr. Sarthy for providing his Müller cell line, Dr. Glyn Chidlow for helping with primer design, and Dr. John P. M. Wood for his advice on culture methods.
1. Foster DW. Diabetes Mellitus. In: Fauci AS, Braunwald E, Isselbacher KJ, Wilson JD, Martin JB, editors. Harrison's principles of internal medicine. 14th ed. New York: McGraw-Hill, 1998. p. 2060-81.
2. Hammes HP, Federoff HJ, Brownlee M. Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes. Mol Med 1995; 1:527-34.
3. Barber AJ, Lieth E, Khin SA, Antonetti DA, Buchanan AG, Gardner TW. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin. J Clin Invest 1998; 102:783-91.
4. Asnaghi V, Gerhardinger C, Hoehn T, Adeboje A, Lorenzi M. A role for the polyol pathway in the early neuroretinal apoptosis and glial changes induced by diabetes in the rat. Diabetes 2003; 52:506-11.
5. Park SH, Park JW, Park SJ, Kim KY, Chung JW, Chun MH, Oh SJ. Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina. Diabetologia 2003; 46:1260-8.
6. Lieth E, Barber AJ, Xu B, Dice C, Ratz MJ, Tanase D, Strother JM. Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy. Penn State Retina Research Group. Diabetes 1998; 47:815-20. Erratum in: Diabetes 1998; 47:1170.
7. Barber AJ, Antonetti DA, Gardner TW. Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes. The Penn State Retina Research Group. Invest Ophthalmol Vis Sci 2000; 41:3561-8.
8. Mizutani M, Gerhardinger C, Lorenzi M. Muller cell changes in human diabetic retinopathy. Diabetes 1998; 47:445-9.
9. Asi H, Perlman I. Relationships between the electroretinogram a-wave, b-wave and oscillatory potentials and their application to clinical diagnosis. Doc Ophthalmol 1992; 79:125-39.
10. Gardner TW, Antonetti DA, Barber AJ, LaNoue KF, Levison SW. Diabetic retinopathy: more than meets the eye. Surv Ophthalmol 2002; 47 Suppl 2:S253-62.
11. Seki M, Tanaka T, Nawa H, Usui T, Fukuchi T, Ikeda K, Abe H, Takei N. Involvement of brain-derived neurotrophic factor in early retinal neuropathy of streptozotocin-induced diabetes in rats: therapeutic potential of brain-derived neurotrophic factor for dopaminergic amacrine cells. Diabetes 2004; 53:2412-9.
12. Spranger J, Osterhoff M, Reimann M, Mohlig M, Ristow M, Francis MK, Cristofalo V, Hammes HP, Smith G, Boulton M, Pfeiffer AF. Loss of the antiangiogenic pigment epithelium-derived factor in patients with angiogenic eye disease. Diabetes 2001; 50:2641-5.
13. Ogata N, Tombran-Tink J, Nishikawa M, Nishimura T, Mitsuma Y, Sakamoto T, Matsumura M. Pigment epithelium-derived factor in the vitreous is low in diabetic retinopathy and high in rhegmatogenous retinal detachment. Am J Ophthalmol 2001; 132:378-82.
14. Vinores SA, Youssri AI, Luna JD, Chen YS, Bhargave S, Vinores MA, Schoenfeld CL, Peng B, Chan CC, LaRochelle W, Green WR, Campochiaro PA. Upregulation of vascular endothelial growth factor in ischemic and non-ischemic human and experimental retinal disease. Histol Histopathol 1997; 12:99-109.
15. Tanaka Y, Katoh S, Hori S, Miura M, Yamashita H. Vascular endothelial growth factor in diabetic retinopathy. Lancet 1997; 349:1520.
16. Bronson SK, Reiter CE, Gardner TW. An eye on insulin. J Clin Invest 2003; 111:1817-9.
17. Berman DH, Friedman EA. Partial absorption of hard exudates in patients with diabetic end-stage renal disease and severe anemia after treatment with erythropoietin. Retina 1994; 14:1-5.
18. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 1993; 329:977-86.
19. Philp NJ, Ochrietor JD, Rudoy C, Muramatsu T, Linser PJ. Loss of MCT1, MCT3, and MCT4 expression in the retinal pigment epithelium and neural retina of the 5A11/basigin-null mouse. Invest Ophthalmol Vis Sci 2003; 44:1305-11.
20. Young RW, Bok D. Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Biol 1969; 42:392-403.
21. Sternfeld MD, Robertson JE, Shipley GD, Tsai J, Rosenbaum JT. Cultured human retinal pigment epithelial cells express basic fibroblast growth factor and its receptor. Curr Eye Res 1989; 8:1029-37.
22. Adamis AP, Shima DT, Yeo KT, Yeo TK, Brown LF, Berse B, D'Amore PA, Folkman J. Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells. Biochem Biophys Res Commun 1993; 193:631-8.
23. Hackett SF, Schoenfeld CL, Freund J, Gottsch JD, Bhargave S, Campochiaro PA. Neurotrophic factors, cytokines and stress increase expression of basic fibroblast growth factor in retinal pigmented epithelial cells. Exp Eye Res 1997; 64:865-73.
24. Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz D. Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature 1987; 325:257-9.
25. Ozaki H, Okamoto N, Ortega S, Chang M, Ozaki K, Sadda S, Vinores MA, Derevjanik N, Zack DJ, Basilico C, Campochiaro PA. Basic fibroblast growth factor is neither necessary nor sufficient for the development of retinal neovascularization. Am J Pathol 1998; 153:757-65.
26. Shimasaki S, Emoto N, Koba A, Mercado M, Shibata F, Cooksey K, Baird A, Ling N. Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA. Biochem Biophys Res Commun 1988; 157:256-63.
27. Emoto N, Gonzalez AM, Walicke PA, Wada E, Simmons DM, Shimasaki S, Baird A. Basic fibroblast growth factor (FGF) in the central nervous system: identification of specific loci of basic FGF expression in the rat brain. Growth Factors 1989; 2:21-9.
28. Matsuyama A, Iwata H, Okumura N, Yoshida S, Imaizumi K, Lee Y, Shiraishi S, Shiosaka S. Localization of basic fibroblast growth factor-like immunoreactivity in the rat brain. Brain Res 1992; 587:49-65.
29. Campochiaro PA, Chang M, Ohsato M, Vinores SA, Nie Z, Hjelmeland L, Mansukhani A, Basilico C, Zack DJ. Retinal degeneration in transgenic mice with photoreceptor-specific expression of a dominant-negative fibroblast growth factor receptor. J Neurosci 1996; 16:1679-88.
30. Walicke P, Cowan WM, Ueno N, Baird A, Guillemin R. Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc Natl Acad Sci U S A 1986; 83:3012-6.
31. Morrison RS, Sharma A, de Vellis J, Bradshaw RA. Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture. Proc Natl Acad Sci U S A 1986; 83:7537-41.
32. Finklestein SP, Apostolides PJ, Caday CG, Prosser J, Philips MF, Klagsbrun M. Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds. Brain Res 1988; 460:253-9.
33. Gomez-Pinilla F, Cotman CW. Transient lesion-induced increase of basic fibroblast growth factor and its receptor in layer VIb (subplate cells) of the adult rat cerebral cortex. Neuroscience 1992; 49:771-80.
34. Logan A, Frautschy SA, Gonzalez AM, Baird A. A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors (flg) following a localized cortical brain injury. J Neurosci 1992; 12:3828-37.
35. Gao H, Hollyfield JG. Basic fibroblast growth factor: increased gene expression in inherited and light-induced photoreceptor degeneration. Exp Eye Res 1996; 62:181-9.
36. Niu YJ, Zhao YS, Gao YX, Zhou ZY, Wang HY, Yuan CY. Therapeutic effect of bFGF on retina ischemia-reperfusion injury. Chin Med J (Engl) 2004; 117:252-7.
37. Bush RA, Williams TP. The effect of unilateral optic nerve section on retinal light damage in rats. Exp Eye Res 1991; 52:139-53.
38. Frank RN, Amin RH, Eliott D, Puklin JE, Abrams GW. Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthalmol 1996; 122:393-403.
39. Sivalingam A, Kenney J, Brown GC, Benson WE, Donoso L. Basic fibroblast growth factor levels in the vitreous of patients with proliferative diabetic retinopathy. Arch Ophthalmol 1990; 108:869-72.
40. Photocoagulation for diabetic maculopathy. A randomized controlled clinical trial using the xenon arc. British Multicentre Study Group. Diabetes 1983; 32:1010-6.
41. Xiao M, Sastry SM, Li ZY, Possin DE, Chang JH, Klock IB, Milam AH. Effects of retinal laser photocoagulation on photoreceptor basic fibroblast growth factor and survival. Invest Ophthalmol Vis Sci 1998; 39:618-30.
42. Nash MS, Osborne NN. Assessment of Thy-1 mRNA levels as an index of retinal ganglion cell damage. Invest Ophthalmol Vis Sci 1999; 40:1293-8.
43. Wood JP, Osborne NN. Induction of apoptosis in cultured human retinal pigmented epithelial cells: the effect of protein kinase C activation and inhibition. Neurochem Int 1997; 31:261-73.
44. McKechnie NM, Boulton M, Robey HL, Savage FJ, Grierson I. The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo. J Cell Sci 1988; 91 (Pt 2):303-12.
45. Beers MH, Berkow R, editors. The Merck manual of diagnosis and therapy. 17th ed. Rahway (NJ): Merck; 1999. p. 2536.
46. Parrizas M, Gazit A, Levitzki A, Wertheimer E, LeRoith D. Specific inhibition of insulin-like growth factor-1 and insulin receptor tyrosine kinase activity and biological function by tyrphostins. Endocrinology 1997; 138:1427-33.
47. Baskin DG, Porte D Jr, Guest K, Dorsa DM. Regional concentrations of insulin in the rat brain. Endocrinology 1983; 112:898-903.
48. de la Rosa EJ, Bondy CA, Hernandez-Sanchez C, Wu X, Zhou J, Lopez-Carranza A, Scavo LM, de Pablo F. Insulin and insulin-like growth factor system components gene expression in the chicken retina from early neurogenesis until late development and their effect on neuroepithelial cells. Eur J Neurosci 1994; 6:1801-10.
49. Rodrigues M, Waldbillig RJ, Rajagopalan S, Hackett J, LeRoith D, Chader GJ. Retinal insulin receptors: localization using a polyclonal anti-insulin receptor antibody. Brain Res 1988; 443:389-94.
50. Waldbillig RJ, Arnold DR, Fletcher RT, Chader GJ. Insulin and IGF-I binding in developing chick neural retina and pigment epithelium: a characterization of binding and structural differences. Exp Eye Res 1991; 53:13-22.
51. Waldbillig RJ, Chader GJ. Anomalous insulin-binding activity in the bovine neural retina: a possible mechanism for regulation of receptor binding specificity. Biochem Biophys Res Commun 1988; 151:1105-12.
52. Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, Le Bon T, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 1986; 5:2503-12.
53. Yarden Y, Ullrich A. Molecular analysis of signal transduction by growth factors. Biochemistry 1988; 27:3113-9.
54. Schumacher R, Mosthaf L, Schlessinger J, Brandenburg D, Ullrich A. Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors. J Biol Chem 1991; 266:19288-95.
55. Czech MP. Signal transmission by the insulin-like growth factors. Cell 1989; 59:235-8.
56. Barber AJ, Nakamura M, Wolpert EB, Reiter CE, Seigel GM, Antonetti DA, Gardner TW. Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3. J Biol Chem 2001; 276:32814-21.
57. Masuda S, Chikuma M, Sasaki R. Insulin-like growth factors and insulin stimulate erythropoietin production in primary cultured astrocytes. Brain Res 1997; 746:63-70.
58. Li F, Cao W, Steinberg RH, LaVail MM. Basic FGF-induced down-regulation of IGF-I mRNA in cultured rat Muller cells. Exp Eye Res 1999; 68:19-27.
59. Lowe WL Jr, Yorek MA, Karpen CW, Teasdale RM, Hovis JG, Albrecht B, Prokopiou C. Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels. Mol Endocrinol 1992; 6:741-52.
60. Russo VC, Andaloro E, Fornaro SA, Najdovska S, Newgreen DF, Bach LA, Werther GA. Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells. J Cell Physiol 2004; 199:371-80.
61. Hugl SR, White MF, Rhodes CJ. Insulin-like growth factor I (IGF-I)-stimulated pancreatic beta-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells. J Biol Chem 1998; 273:17771-9.
62. Lam S, van der Geest RN, Verhagen NA, van Nieuwenhoven FA, Blom IE, Aten J, Goldschmeding R, Daha MR, van Kooten C. Connective tissue growth factor and IGF-I are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose. Diabetes 2003; 52:2975-83.
63. King JL, Guidry C. Insulin-like growth factor binding proteins modulate Muller cell responses to insulin-like growth factors. Invest Ophthalmol Vis Sci 2004; 45:2848-55.
64. Lowe WL Jr, Florkiewicz RZ, Yorek MA, Spanheimer RG, Albrecht BN. Regulation of growth factor mRNA levels in the eyes of diabetic rats. Metabolism 1995; 44:1038.
65. Grant M, Russell B, Fitzgerald C, Merimee TJ. Insulin-like growth factors in vitreous. Studies in control and diabetic subjects with neovascularization. Diabetes 1986; 35:416-20.
66. Guidry C, Feist R, Morris R, Hardwick CW. Changes in IGF activities in human diabetic vitreous. Diabetes 2004; 53:2428-35.
67. Punglia RS, Lu M, Hsu J, Kuroki M, Tolentino MJ, Keough K, Levy AP, Levy NS, Goldberg MA, D'Amato RJ, Adamis AP. Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes 1997; 46:1619-26.
68. Kondo T, Vicent D, Suzuma K, Yanagisawa M, King GL, Holzenberger M, Kahn CR. Knockout of insulin and IGF-1 receptors on vascular endothelial cells protects against retinal neovascularization. J Clin Invest 2003; 111:1835-42.
69. Ruberte J, Ayuso E, Navarro M, Carretero A, Nacher V, Haurigot V, George M, Llombart C, Casellas A, Costa C, Bosch A, Bosch F. Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease. J Clin Invest 2004; 113:1149-57.