mTORC1-Independent Reduction of Retinal Protein Synthesis in Type 1 Diabetes

Diabetes reduces retinal protein synthesis and degradation independent of retinal weight, total protein, total RNA, and ribosomal RNA content. Retinas from 3-month diabetic STZ rats treated or not with systemic insulin and age-matched controls were harvested for assessment of the impact of diabetes on retinal protein synthesis (A), degradation rate (B), and composition (C–F). Diabetes reduced retinal protein synthesis (A) and retinal degradation (B) as measured by the pulse-chase method without affecting retinal wet weight (C), protein content (D), total RNA content (E), and ribosomal RNA content (F) (n ≥ 20/group). *Significantly different from control.

Diabetes reduces retinal protein synthesis through an impairment in both peptide chain initiation and elongation while affecting specific mRNA translation rates. The impact of diabetes on polysome profiles and RNA pools of the subpolysomal and polysomal fractions was also analyzed. Representative polysome profiles are presented and show that translation efficiency is due to impairment in both peptide chain initiation and elongation in the retina during diabetes (A). RNA isolated from both subpolysomal and polysomal fractions was analyzed by quantitative RT-PCR and demonstrated the decreased translation of α-A-crystallin (cryaa) and β-B3-crystallin (crybb3) mRNA and increased translation of synaptic genes Snap25 and synapsin 1 (syn1) during diabetes (B). *Significantly different from control.

Blood glucose normalization reverses both retinal protein synthesis and retinal protein degradation induced by diabetes, whereas local insulin only reverses the reduction in protein synthesis. Rats with 3 months of diabetes were treated by administration of phloridzin twice daily or insulin subconjunctivally once daily for the last 4 days. Protein synthesis rate was then measured using the flooding dose of phenylalanine method, and results show that both treatments partially reverse the protein synthesis decrease induced by diabetes (A). Similarly, retinas from diabetic and age-matched control rats treated with either phloridzin twice daily or insulin subconjunctivally once daily for the last 4 days were harvested and incubated ex vivo with radiolabeled methionine to measure protein synthesis rate (B) followed by pulse-chase method to measure protein degradation rate (C). Phloridzin treatment for 4 days reversed both protein synthesis and protein degradation rates, whereas ocular insulin only reversed the protein synthesis defects induced by diabetes (n ≥ 8/group). *Significantly different from control (P < 0.05). #Significantly different from diabetic (P < 0.05). LOC, local.

Diabetes reduces total retinal mTOR activity and mTORC2 associated mTOR phosphorylation. Rats with 3 months of diabetes were treated with either phloridzin twice daily or insulin subconjunctivally once daily for the last 4 days. Retinal mTOR activity was then measured, which demonstrated that mTOR activity was significantly reduced in the retina during diabetes and was reversed by the combined treatment (A). Retinal mTOR activity was also measured in 3-month diabetic Ins2^Akita mice and demonstrated a similar reduction (B) (n ≥ 8/group). *Significantly different from control (P < 0.05). #Significantly different from diabetic (P < 0.05). Specific activity of the mTORC1 and mTORC2 complexes was assessed by immunoprecipitation of rictor and raptor followed by assessment of the bound mTOR activity using an antibody against pS2481 (C). Diabetes did not reduce raptor-bound mTOR phosphorylation, but it significantly reduced rictor-bound mTOR phosphorylation. This reduction was reversed by both phloridzin and local insulin treatment. C, control; D, diabetic; D+I, diabetic + insulin; D+P, diabetic + phloridzin; D+P+L, diabetic + phloridzin + local insulin; IP, immunoprecipitation. ***Significantly different from control (P < 0.001); #Significantly different from diabetic (P < 0.05).

Diabetes does not affect the expression and phosphorylation of downstream targets of mTORC1. Rats with 3 months of diabetes were treated with either phloridzin twice daily or insulin subconjunctivally once daily for the last 4 days. Analysis of the levels of expression of 4E-BP1 and its phosphorylation on threonines 37 and 46 (A) and S6 ribosomal protein and its phosphorylation on serines 240/244 and serines 235/236 (B), downstream targets of TORC1, showed that neither was reduced by diabetes (n ≥ 5/group). C, control; D, diabetic; D+I, diabetic + insulin; D+P, diabetic + phloridzin; D+P+L, diabetic + phloridzin + local insulin.