Journal: Nature Communications

Article Title: Reducing Akt2 in retinal pigment epithelial cells causes a compensatory increase in Akt1 and attenuates diabetic retinopathy

doi: 10.1038/s41467-022-33773-0

Figure Lengend Snippet: a Representative immunoblots and quantification of ( b ) phospho- Akt2 , ( c ) total- Akt2 , ( d ) phospho-Akt1 and ( e ) total Akt1 in RPE lysates obtained from diabetic and nondiabetic Akt2 fl/fl and cKO mice. Diabetes increased the level of phospho- Akt2 but did not affect the levels of total Akt2 in Akt2 fl/fl RPE cells compared to the nondiabetic control. RPE-specific Akt2 cKO reduced the levels of both phospho- and total- Akt2 in the RPE; induction of diabetes did not further affect the levels of either phospho- or total Akt2 in Akt2 cKO RPE. Induction of diabetes in Akt2 fl/fl mice decreased the level of phospho-Akt1 but did not affect the levels of total Akt1 in the RPE cells compared to nondiabetic control. Akt2 cKO increased the levels of phospho-Akt1 in diabetic Akt2 cKO mice, but not in nondiabetic cKO mice compared to diabetic or nondiabetic Akt2 fl/fl mice, respectively. The total Akt1 in the RPE was upregulated in both nondiabetic and diabetic Akt2 cKO mice compared to Akt2 fl/fl mice. f Representative immunoblots. g The ratio of phospho-GSK3β/total GSK3β was lower in diabetic Akt2 fl/fl mice, relative to nondiabetic control mice. However, it was increased in diabetic Akt2 cKO mice. h In addition, Akt2 cKO rescued the diabetes-induced increase in the ratio of p-NF-κB p65/total NF-κB p65. i Representative immunoblots. Diabetes induction increased the protein level of ( j ) ICAM-1 and ( k ) iNOS in Akt2 fl/fl mice, whereas in diabetic Akt2 cKO mice this diabetes-induced increase in inflammatory protein levels was significantly rescued. In ( b – e , g , h , j , k ), n = 6 mice per group, the data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 denotes changes versus Akt2 fl/fl nondiabetic (N) control. † p < 0.05, †† p < 0.01, ††† p < 0.001, and †††† p < 0.0001 denotes changes with respect to Akt2 fl/fl diabetic (D) mice. ### p < 0.001 and #### p < 0.0001 shows changes versus nondiabetic Akt2 cKO group. Statistical test used in this study is One-way ANOVA followed by a Tukey’s post hoc test. Exact p values are: b p = 0.025 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p < 0.0001 ( Akt2 cKO N vs. Akt2 fl/fl N , Akt2 cKO D vs. Akt2 fl/fl D). c p < 0.0001 ( Akt2 cKO N vs. Akt2 fl/fl N, Akt2 cKO D vs. Akt2 fl/fl D). d p = 0.0109 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0009 ( Akt2 cKO D vs. Akt2 fl/fl N), p < 0.0001 ( Akt2 cKO D vs. Akt2 fl/fl D), p = 0.0007 ( Akt2 cKO D vs. Akt2 cKO N). e p = 0.0005 ( Akt2 cKO N vs. Akt2 fl/fl N), p < 0.0001 ( Akt2 cKO D vs. Akt2 fl/fl N). p < 0.0001 ( Akt2 cKO D vs. Akt2 fl/fl D). g p = 0.0326 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0001 ( Akt2 cKO D vs. Akt2 cKO N), p < 0.0001 for the rest. h p = 0.0003 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0283 ( Akt2 cKO D vs. Akt2 fl/fl D). j p = 0.0004 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.011 ( Akt2 cKO D vs. Akt2 fl/fl D). k p < 0.0001 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0076 ( Akt2 cKO D vs. Akt2 fl/fl D). Source Data is provided in the file.

Article Snippet: An Akt1 inhibitor (Selleck Chemicals, S2670) was dissolved in sterile H 2 O at a stock concentration of 10 mM, and Akt2 cKO diabetic RPE flatmounts were treated with the Akt1 inhibitor at a final concentration of 40 nM for 48 h. The flatmount culture medium was then collected, and the same Multi-Analyte ELISArray Kit mentioned above was used to measure the inflammatory cytokines, while RPE flatmount tissue was lysed for western blot analysis.

Techniques: Western Blot, Control

Journal: Nature Communications

Article Title: Reducing Akt2 in retinal pigment epithelial cells causes a compensatory increase in Akt1 and attenuates diabetic retinopathy

doi: 10.1038/s41467-022-33773-0

Figure Lengend Snippet: a Diabetes-induced increase of retinal VEGF was inhibited in Akt2 cKO mice. b Diabetes increased the RPE/retinal expression of inflammatory cytokines, including IL-1β, IL-6, IL-17A, IFN-γ and TNF-α, in Akt2 fl/fl mice compared to Akt2 fl/fl nondiabetic animals. Akt2 cKO inhibits diabetes-induced elevation of these inflammatory cytokines. c RPE explants were obtained from Akt2 fl/fl and Akt2 cKO nondiabetic and diabetic mice and cultured in 5 mM and 25 mM glucose medium, respectively, for 48 h. The spent medium was collected and ELISA assays performed. As expected, Akt2 cKO inhibited the release of inflammatory cytokines caused by diabetes, including IL-1β, IL-6, IL-12, IL-17A, IFN-γ and TNF-α. Such inhibitory effects were abolished if an Akt1 inhibitor was added to the RPE explants (isolated from Akt2 cKO diabetic mice) culture. d RPE explant culture was collected for western blot analysis. e – j Akt2 cKO increased the level of phospho-Akt1 in the RPE from diabetic mice and inhibited the diabetes-induced reduction in the ratio of p-GSK3β/total GSK3β, as well as the diabetes-induced elevation in the ratio of p-NF-κB p65/total NF-κB p65. The presence of the Akt1 inhibitor in the Akt2 cKO diabetic mouse RPE explants significantly reversed this protective effect for the levels of p-Akt1, p-GSK3β/total GSK3β and p-NF-κB p65/total NF-κB p65. In ( a – c , e – j ), data are shown as Mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and *** p < 0.0001 versus the Akt2 fl/fl nondiabetic mice. † p < 0.05, †† p < 0.01, ††† p < 0.001, and †††† p < 0.0001 versus Akt2 fl/fl diabetic mice. # p < 0.05, ## p < 0.01, ### p < 0.001, and ### p < 0.0001 versus Akt2 cKO diabetic group. Statistical tests used in ( a , e – j ) is One-way ANOVA followed by a Tukey’s post hoc test, n = 6 mice for each group. Statistical test used in ( b , c ) is two tailed, unpaired t -test, n = 6 animals; 3 samples in each group; each sample was composed of 2 animals. Exact p values are: a p = 0.0001 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0049 ( Akt2 cKO D vs. Akt2 fl/fl D), b IL-1β: p = 0.0342, 95% CI 0.4993 to 2.146, R 2 = 0.8326 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0128, 95% CI −2.095 to −0.4477, R 2. = 0.8212 ( Akt2 cKO D vs. Akt2 fl/fl D); IL-6: p = 0.0352, 95% CI 0.026 to 0.4347, R 2 = 0.7101 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0483, 95% CI −0.4111 to −0.002517, R 2 = 0.6639 ( Akt2 cKO D vs. Akt2 fl/fl D); IL-17A: p = 0.0388, 95% CI 0.01557 to 0.3558, R 2 = 0.6966 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0374, 95% CI −0.3671 to −0.01823, R 2 = 0.706 ( Akt2 cKO D vs. Akt2 fl/fl D); IFN-γ: p = 0.0028, 95% CI 0.1819 to 0.4488, R 2 = 0.915 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0026, 95% CI −0.4227 to −0.1746, R 2 = 0.9178 ( Akt2 cKO D vs. Akt2 fl/fl D); TNF-α: p = 0.0291, 95% CI 0.04679 to 0.5145, R 2 = 0.7351 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0418, 95% CI −4694 to −0.01459, R 2 = 0.6858 ( Akt2 cKO D vs. Akt2 fl/fl D). c IL-1β: p = 0.0154, 95% CI 0.06971 to 0.373, R 2 = 0.8042 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.048, 95% CI −0.3297 to −0.002349, R 2 = 0.6684 ( Akt2 cKO D vs. Akt2 fl/fl D), p = 0.0274, 95% CI 0.2393 to 0.2387, R 2 = 0.7424 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 cKO D), p = 0.0042, 95% CI 0.09867 to 0.2747, R 2 = 0.8966 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N); IL-6: p = 0.0154, 95% CI 0.06357 to 0.3391, R 2 = 0.8045 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.044, 95% CI −0.2824 to −0.006241, R 2 = 0.678 ( Akt2 cKO D vs. Akt2 fl/fl D), p = 0.041, 95% CI 0.05295 to 0.1464, R 2 = 0.8977 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 cKO D), p = 0.007, 95% CI 0.1109 to 0.2024, R 2 = 0.9576 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N); IL-12: p = 0.0122, 95% CI 0.02614 to 0.1185, R 2 = 0.8254 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N), p = 0.0012, 95% CI 0.05197 to 0.1054, R 2 = 0.9436 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 cKO D); IL-17A: p = 0.0097, 95% CI 0.03451 to 0.1368, R 2 = 0.8439 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0358, 95% CI −0.1173 to −0.006687, R 2 = 0.7077 ( Akt2 cKO D vs. Akt2 fl/fl D); IFN-γ: p = 0.0114, 95% CI 0.0195 to 0.08512, R 2 = 0.8308 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0227, 95% CI −0.08379 to −0.01087, R 2 = 0.7646 ( Akt2 cKO D vs. Akt2 fl/fl D), p = 0.0055, 95% CI 0.03739 to 0.1153, R 2 = 0.881 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 cKO D), p = 0.0031, 95% CI 0.0458 to 0.1169, R 2 = 0.9099 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N); TNF-α: p = 0.0404, 95% CI 0.00717 to 0.1955, R 2 = 0.6909 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0392, 95% CI −0.1894 to −0.007913, R 2 = 0.6949 ( Akt2 cKO D vs. Akt2 fl/fl D), p = 0.0038, 95% CI 0.03767 to 0.1017, R 2 = 0.9014 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 cKO D), p = 0.0078, 95% CI 0.03166 to 0.113, R 2 = 0.8591 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N). e p = 0.0003 ( Akt2 cKO N vs. Akt2 fl/fl N), p < 0.0001 for the rest. f p = 0.0018 ( Akt2 cKO N vs. Akt2 fl/fl N), p = 0.0016 ( Akt2 cKO D vs. Akt2 fl/fl D), p = 0.0009 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl D). g p = 0.0124 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0019 ( Akt2 cKO D vs. Akt2 fl/fl N), p = 0.0043 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N), p = 0.018 ( Akt2 cKO N vs. Akt2 fl/fl D), p < 0.0001 for the rest. h p = 0.0007 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N), p < 0.0001 for the rest. i p = 0.003 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p = 0.0004 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N, Akt2 cKO D vs. Akt2 fl/fl D), p = 0.0002 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 cKO D). j p = 0.0002 ( Akt2 fl/fl D vs. Akt2 fl/fl N), p < 0.0001 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 fl/fl N), p = 0.0012 ( Akt2 cKO D vs. Akt2 fl/fl D), p = 0.0006 ( Akt2 cKO D + Akt1 inhibitor vs. Akt2 cKO D). Source Data is provided in the file.

Article Snippet: An Akt1 inhibitor (Selleck Chemicals, S2670) was dissolved in sterile H 2 O at a stock concentration of 10 mM, and Akt2 cKO diabetic RPE flatmounts were treated with the Akt1 inhibitor at a final concentration of 40 nM for 48 h. The flatmount culture medium was then collected, and the same Multi-Analyte ELISArray Kit mentioned above was used to measure the inflammatory cytokines, while RPE flatmount tissue was lysed for western blot analysis.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Mouse Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Reducing Akt2 in retinal pigment epithelial cells causes a compensatory increase in Akt1 and attenuates diabetic retinopathy

doi: 10.1038/s41467-022-33773-0

Figure Lengend Snippet: RPE-specific Akt2 cKO reduced the diabetes-mediated induction of the inflammatory protein level, infiltration of leukocytes and production of ROS in the retina after a 2 month duration of diabetes. Akt2 cKO also showed reduction in retinal capillary degeneration and vascular leakage even after an 8 month duration of diabetes. Such beneficial effects were indicative of the compensatory upregulation of Akt1 in the diabetic Akt2 cKO RPE cells, which downregulated GSK3β/NF-κB mediated inflammatory molecules like ICAM-1, iNOS, VEGF, TNF-α, IFN-γ and IL-1β in cKO RPE cells even after diabetes induction.

Article Snippet: An Akt1 inhibitor (Selleck Chemicals, S2670) was dissolved in sterile H 2 O at a stock concentration of 10 mM, and Akt2 cKO diabetic RPE flatmounts were treated with the Akt1 inhibitor at a final concentration of 40 nM for 48 h. The flatmount culture medium was then collected, and the same Multi-Analyte ELISArray Kit mentioned above was used to measure the inflammatory cytokines, while RPE flatmount tissue was lysed for western blot analysis.

Techniques: