Glucose-Sensing ChREBP Protein in the Pathogenesis of Diabetic Retinopathy

Figures

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  Figure 1.. Immunohistochemical analysis of human control and diabetic retina. A: Retinas were subjected to treatment with anti-ChREBP antibody. The ChREBP immunoreactivity in human control (a &c) and diabetic (b & d) retinas. In the diabetic retina, robust staining of ChREBP (green) was detected in the photoreceptors, the cells of the INL (e), endothelial cells (f & h), and retinal pigment epithelial cells (g). Co-localization of ChREBP in the nuclei (red) of retinal cells is indicated in yellow. B: The MondoA immunoreactivity in human control (a) and diabetic (b) retinas is shown in green. In the diabetic retina, robust staining was detected in cones (c) and the cells of the INL (d). Localization of MondoA in the nuclei is shown in yellow (d).

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  Figure 2.. Expression of Mondo family proteins in hyperglycemic retinas. A: The RNAscope technique revealed enhanced ChREBP mRNA expression (green) in 12-week-old db/db retinas. The retinal ganglion cells (RGCs), Müller cells (highlighted in right inserts), and photoreceptors are responsive to hyperglycemia, showing increased ChREBP expression. B: Retinal explants were cultured in a medium supplemented with either high glucose or an equimolar concentration of mannitol (control) for 24 hours. High glucose, but not mannitol, culture conditions resulted in significant increases in both Chrebpα and Chrebpβ mRNAs, indicating that high glucose stimulates ChREBP expression ex vivo. C: To confirm ex vivo findings, diabetic retinas were isolated for qRT-PCR analysis to evaluate ChREBP and MondoA gene expression. The qRT-PCR results show that retinas from 12-week-old db/db and Akita mice exhibit increased expression of both Chrebp and MondoA mRNAs. Statistical significance is indicated as p*<0.05 and p**<0.01 (n=4 per group).

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  Figure 3.. Transgenic expression of constitutively active ChREBP in rods leads to vision loss. A: i-Cre-mediated recombination in the rod photoreceptors of caChREBPRP transgenic mice resulted in deletion of eGFP and expression of FLAG-tagged caChREBP, detected by anti-FLAG antibody, shown in red. The nuclei are shown in blue. B: Expression of caChREBP in rod photoreceptors leads to reduction in the scotopic a- and b-wave amplitudes in caChREBPRP versus control (eGFPflox/wt-caChREBP or CghREBPf/wt) mice at postnatal day 35. Representative waveforms are shown on the right. p*<0.05, n=6 per group.

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  Figure 4.. Increased ChREBP activity in rods alters retinal proteomics and KEGG signaling in caChREBPRP mice. A: Heatmap showing the major altered proteins in caChREBPRP compared with eGFPflox/wt-caChREBP control (Control) retinas (n=3-4 per group), high-lighting significant changes in retinal protein expression. B: Expression of constitutively active ChREBP (caChREBP) in rods leads to an increase in TXNIP protein levels at postnatal day 35 (P35). Statistical significance is indicated by p*<0.05, with n=3-4 per group. C: The major downregulated KEGG signaling pathways in caChREBP-expressing retinas are identified, showing significant pathway alterations due to sustained ChREBP activity in rods.

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  Figure 5.. Overexpression of ChREBP in ARPE-19 Cells. A: Images of ARPE-19 cells overexpressing human ChREBP and control cells treated with empty virus. Direct fluorescence emitted by GFP indicates successful infection and ChREBP expression. B: The heatmap displays the major altered proteins in ARPE-19 cells with sustained ChREBP expression (N=3-4 per group), highlighting significant protein expression changes due to ChREBP overexpression. C: Results from the proteomic analysis were analyzed using the Shiny GO program to generate diagrams of altered KEGG pathways. Both decreased and increased pathways are shown, reflecting the impact of sustained ChREBP expression on cellular signaling networks in ARPE-19 cells.

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  Figure 6.. ChREBP Overexpression Alters Molecular Function and Affects Cellular Components of ARPE-19 Cells. A: The molecular functions that are decreased or increased due to ChREBP overexpression in ARPE-19 cells are shown, highlighting the functional alterations induced by sustained ChREBP expression. B: The cellular components affected by ChREBP expression in ARPE-19 cells are presented, demonstrating how ChREBP overexpression leads to changes in the cellular architecture and composition (n=3-4 per group).