Endogenous insulin signaling in the RPE contributes to the maintenance of rod photoreceptor function in diabetes

Abstract

In diabetes, there are two major physiological aberrations: (i) Loss of insulin signaling due to absence of insulin (type 1 diabetes) or insulin resistance (type 2 diabetes) and (ii) increased blood glucose levels. The retina has a high proclivity to damage following diabetes, and much of the pathology seen in diabetic retinopathy has been ascribed to hyperglycemia and downstream cascades activated by increased blood glucose. However, less attention has been focused on the direct role of insulin on retinal physiology, likely due to the fact that uptake of glucose in retinal cells is not insulin-dependent. The retinal pigment epithelium (RPE) is instrumental in maintaining the structural and functional integrity of the retina. Recent studies have suggested that RPE dysfunction is a precursor of, and contributes to, the development of diabetic retinopathy. To evaluate the role of insulin on RPE cell function directly, we generated a RPE specific insulin receptor (IR) knockout (RPEIRKO) mouse using the Cre-loxP system. Using this mouse, we sought to determine the impact of insulin-mediated signaling in the RPE on retinal function under physiological control conditions as well as in streptozotocin (STZ)-induced diabetes. We demonstrate that loss of RPE-specific IR expression resulted in lower a- and b-wave electroretinogram amplitudes in diabetic mice as compared to diabetic mice that expressed IR on the RPE. Interestingly, RPEIRKO mice did not exhibit significant differences in the amplitude of the RPE-dependent electroretinogram c-wave as compared to diabetic controls. However, loss of IR-mediated signaling in the RPE reduced levels of reactive oxygen species and the expression of pro-inflammatory cytokines in the retina of diabetic mice. These results imply that IR-mediated signaling in the RPE regulates photoreceptor function and may play a role in the generation of oxidative stress and inflammation in the retina in diabetes.

Keywords: Diabetic retinopathy; Insulin; Oxidative stress; Photoreceptor; Retinal pigment epithelium.

Figure 1.. Loss of IR expression in the RPE of RPEIRKO mice.

(A) RPE/choroid flatmounts were prepared from non-diabetic IR flox and RPEIRKO mice at 6 weeks of age. Tissue was stained with mouse anti-Cre antibody (green), rabbit anti-IRβ antibody (red) and counterstained with DAPI. Scale bar = 500μm. (B) RPE lysates were prepared from IR flox, Best1-Cre and RPEIRKO mice. Western blotting was performed using anti-IRβ, anti-actin (top) and anti-cre recombinase (bottom) antibodies.

Figure 2.. Diabetic RPEIRKO mice exhibit reduced plasma insulin and elevated blood and retinal glucose concentrations.

(A) Systemic non-fasting blood glucose concentration. (B) Body weight of each cohort of mice measured at the time of each electroretinogram session (2 weeks and 4 weeks). (C) Plasma insulin levels measured by ELISA after 4 weeks of diabetes. (D) Retinal glucose levels measured by hexokinase assay at 4 weeks of diabetes. (E) Retinal insulin levels relative to protein concentration at 2 weeks of diabetes. Data points indicate the mean ± SEM. At each time point, n≥3 for each group. *p≤0.05, **p≤0.001, ***p≤0.0001 by one-way ANOVA with Tukey’s post hoc analysis.

Figure 3.. IR flox, Best1-cre and RPEIRKO mice display normal retinal histology under normal physiological conditions and following 4 weeks of diabetes.

(A) Representative light micrographs from semi-thin sections through the optic nerve of control and diabetic IR flox, Best1-cre and RPEIRKO mice 4 weeks following onset of diabetes, stained with toluidine blue O. OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer. All images were taken at 40x magnification. Scale bar=20 μm. Thickness of the OS (B), IS (C) and ONL (D) was measured from three locations in each image and at least three images per mouse (n≥3 for each group). Data was compiled and analyzed by one-way ANOVA with Tukey post-hoc test.

Figure 4.. Diabetes does not affect diameter or area of RPE cells in IR flox and RPEIRKO mice.

(A) Representative flat mount images from IR flox and RPEIRKO mice at 2 weeks of diabetes stained with phalloidin. At least 20 cells per flatmount were analyzed by Image J software for RPE cell area (B) and diameter (C). (D) High magnification images from semi-thin sections of each mouse genotype/treatment at 4weeks following onset of diabetes. Scale bar = 10μm. Images were analyzed for RPE thickness with Image J software (E). Statistical significance was determined by one-way ANOVA with Tukey post hoc test. n≥3 for each group.

Figure 5.. Diabetes induces reductions in the a- and b-wave of RPEIRKO but not IR flox or Best1-cre mice.

(A) Representative strobe flash ERG responses recorded in response to a 1.4 log cd.s/m² white flash stimulus at 2 and 4 weeks of diabetes for each group. (B) Amplitude of the a-wave in response to the 1.4 log cd.s/m² stimulus. (C) Amplitude of the b-wave in response to the 1.4 cd.s/m² stimulus. (D) Relative changes of the b-wave plotted as a function of the relative normalized amplitude of the a-wave. The diagonal line indicates an equivalent reduction in the a- and b-wave. n≥6 mice per group. Data represent mean ± SEM. Statistical analysis was performed using a one-way ANOVA for each time point followed by post hoc Tukey’s test. * p≤ 0.05, ** p≤ 0.001.

Figure 6.. Diabetes elicits changes to the oscillatory potentials of diabetic mice.

(A) Representative filtered OPs from ERG waveforms recorded in response to a 1.4 log cd.s/m² stimulus at 2 (top) and 4 (bottom) weeks of diabetes. (B) Average amplitude for OP1–3 at each time point. n≥3 for each group. Data represent mean ± SEM. *p<0.05.

Figure 7.. Diabetes does not affect the light adapted responses of control or RPEIRKO mice.

(A) Representative ERG waveforms recorded in response to a 1.4 log cd.s/m² stimulus superimposed on a steady light adapting field after 2 and 4 weeks of diabetes. (B) Average amplitude of the light adapted response for each group. n≥6 for each group. Data represent mean ± SEM.

Figure 8.. Diabetes induces reductions in the RPE-dependent c-wave regardless of IR-mediated signaling in the RPE.

(A) Representative c-wave recordings at 2 and 4 weeks of diabetes for each group. Control (CNTL) mice are represented by black traces and STZ-injected diabetic mice by gray traces. (B) Amplitude of the c-wave at each time point. (C) The relative amplitude of the c-wave plotted as a function of the relative a-wave amplitude. n≥5 for each group at each time point.

Figure 9.. RPEIRKO mice do not exhibit diabetes-induced increases in oxidative stress.

(A) Freshly dissected eye cups from CNTL and STZ IR Flox, Best1-Cre, and RPEIRKO mice after 4 weeks of diabetes were frozen and mounted in OCT medium prior to cryosectioning. Cryosections were stained with Dihydroethidium-594 and counterstained with DAPI. Scale bar = 50μm. Relative Fluorescence was calculated by corrected total cell fluorescence using Image J software. At 4 weeks of diabetes, retinas from CNTL and STZ IR flox, Best1-cre and RPEIRKO mice were collected and used for mRNA extraction. mRNA levels Cox2 (B) and Nos2 (C) were assessed by real time quantitative PCR using actin or 18S rRNA as the internal control. n≥3 for each group. Data represent mean ± SEM. *p≤0.05, **p≤0.001, ***p≤0.0001 by one-way ANOVA followed by post hoc Tukey’s test.

Figure 10.. RPEIRKO mice do not exhibit diabetes-induced increases in pro-inflammatory and proangiogenic molecules.

At 4 weeks of diabetes, retinas from diabetic (STZ) IR flox, Best1-cre and RPEIRKO mice, as well as from non-diabetic control (CNTL) animals of each cohort were collected and used for mRNA extraction. mRNA levels of IL-1β (A), TNFα (B), HIF1α (C), and VEGF (D) were assessed by real time quantitative PCR using actin or 18S rRNA as the internal control. n≥3 mice per group. Data represent mean ± SEM. *p≤0.05, **p≤0.001, ***p≤0.0001 by one-way ANOVA followed by post hoc Tukey’s test.

Figure 11.. Proposed signal transduction cascades for IR-mediated signaling in RPE cells.

Under physiological (low, 10nM) insulin concentrations, insulin binding to its cognate receptor induces autophosphorylation of the beta subunit of the receptor, binding of scaffolding proteins such as IRS-1 or c-cbl, and downstream mitogenic and metabolic signaling via MAPK, PI3K and Glut4-mediated glucose transport. Simultaneously, low levels of insulin induce an acute spike in hydrogen peroxide which leads to inhibition of tyrosine phosphatase activity and feeds forward to further potentiate cellular survival and proliferation. However, the hydrogen peroxide can also contribute to the exacerbation of already elevated levels of oxidative stress in a hyperglycemic environment. In our RPEIRKO mice, we found that the loss of IR-mediated signaling both leads to reduced photoreceptor function, and a decrease in oxidative stress. We propose that these processes can occur due to the release of tyrosine phosphatase inhibition, causing a reduction in paracrine signaling to photoreceptors that ensures their proper function. Alternately, the reduction in generation of hydrogen peroxide reduces oxidative stress in the hyperglycemic environment.