Accumulation of branched-chain amino acids deteriorates the neuroinflammatory response of Müller cells in diabetic retinopathy via leucine/Sestrin2-mediated sensing of mTOR signaling.

Aims: This study aimed to investigate branched-chain amino acid (BCAA) catabolism in diabetic retinopathy (DR).Wild-type and <italic>db/db</italic> mice were fed BCAAs (5 or 10 mg/kg/day) for 12 weeks, and hyperglycemia-exposed Müller cells were treated with BCAAs (2 or 5 mmol/L) for 24 and 48 h. BCAA levels were measured using MS/MS. Western blotting was performed to detect proteins. Flow cytometry, oxygen consumption rate, and Cell Counting Kit-8 assays were used to evaluate Müller cell viability. Each experiment was conducted at least thrice.BCAAs and branched-chain α-keto acids (BCKAs) were increased in the retina and systemic tissues of diabetic mice, and these changes were further enhanced to approximately 2-fold by extra BCAAs compared to wild-type group. In vitro, BCAAs and BCKAs were induced in hyperglycemic Müller cells, and augmented by BCAA supplementation. The aberrant BCAA catabolism was accompanied by mTORC1 activation and subsequently induced TNF-ɑ, VEGFA, GS, and GFAP in retinas and Müller cells under diabetic conditions. The cell apoptosis rate increased by approximately 50%, and mitochondrial respiration was inhibited by hyperglycemia and BCAA in Müller cells. Additionally, mTORC1 signaling was activated by leucine in Müller cells. Knockdown of Sestrin2 or LeuRS significantly abolished the leucine-induced mTORC1 phosphorylation and protected Müller cell viability under diabetic conditions.We found that BCAA catabolism is hindered in DR through mTORC1 activation. Leucine plays a key role in inducing mTORC1 by sensing Sestrin2 in Müller cells. Targeting Sestrin2 may ameliorate the toxic effects of BCAA accumulation on Müller cells in DR.Methods: This study aimed to investigate branched-chain amino acid (BCAA) catabolism in diabetic retinopathy (DR).Wild-type and <italic>db/db</italic> mice were fed BCAAs (5 or 10 mg/kg/day) for 12 weeks, and hyperglycemia-exposed Müller cells were treated with BCAAs (2 or 5 mmol/L) for 24 and 48 h. BCAA levels were measured using MS/MS. Western blotting was performed to detect proteins. Flow cytometry, oxygen consumption rate, and Cell Counting Kit-8 assays were used to evaluate Müller cell viability. Each experiment was conducted at least thrice.BCAAs and branched-chain α-keto acids (BCKAs) were increased in the retina and systemic tissues of diabetic mice, and these changes were further enhanced to approximately 2-fold by extra BCAAs compared to wild-type group. In vitro, BCAAs and BCKAs were induced in hyperglycemic Müller cells, and augmented by BCAA supplementation. The aberrant BCAA catabolism was accompanied by mTORC1 activation and subsequently induced TNF-ɑ, VEGFA, GS, and GFAP in retinas and Müller cells under diabetic conditions. The cell apoptosis rate increased by approximately 50%, and mitochondrial respiration was inhibited by hyperglycemia and BCAA in Müller cells. Additionally, mTORC1 signaling was activated by leucine in Müller cells. Knockdown of Sestrin2 or LeuRS significantly abolished the leucine-induced mTORC1 phosphorylation and protected Müller cell viability under diabetic conditions.We found that BCAA catabolism is hindered in DR through mTORC1 activation. Leucine plays a key role in inducing mTORC1 by sensing Sestrin2 in Müller cells. Targeting Sestrin2 may ameliorate the toxic effects of BCAA accumulation on Müller cells in DR.Results: This study aimed to investigate branched-chain amino acid (BCAA) catabolism in diabetic retinopathy (DR).Wild-type and <italic>db/db</italic> mice were fed BCAAs (5 or 10 mg/kg/day) for 12 weeks, and hyperglycemia-exposed Müller cells were treated with BCAAs (2 or 5 mmol/L) for 24 and 48 h. BCAA levels were measured using MS/MS. Western blotting was performed to detect proteins. Flow cytometry, oxygen consumption rate, and Cell Counting Kit-8 assays were used to evaluate Müller cell viability. Each experiment was conducted at least thrice.BCAAs and branched-chain α-keto acids (BCKAs) were increased in the retina and systemic tissues of diabetic mice, and these changes were further enhanced to approximately 2-fold by extra BCAAs compared to wild-type group. In vitro, BCAAs and BCKAs were induced in hyperglycemic Müller cells, and augmented by BCAA supplementation. The aberrant BCAA catabolism was accompanied by mTORC1 activation and subsequently induced TNF-ɑ, VEGFA, GS, and GFAP in retinas and Müller cells under diabetic conditions. The cell apoptosis rate increased by approximately 50%, and mitochondrial respiration was inhibited by hyperglycemia and BCAA in Müller cells. Additionally, mTORC1 signaling was activated by leucine in Müller cells. Knockdown of Sestrin2 or LeuRS significantly abolished the leucine-induced mTORC1 phosphorylation and protected Müller cell viability under diabetic conditions.We found that BCAA catabolism is hindered in DR through mTORC1 activation. Leucine plays a key role in inducing mTORC1 by sensing Sestrin2 in Müller cells. Targeting Sestrin2 may ameliorate the toxic effects of BCAA accumulation on Müller cells in DR.Conclusions: This study aimed to investigate branched-chain amino acid (BCAA) catabolism in diabetic retinopathy (DR).Wild-type and <italic>db/db</italic> mice were fed BCAAs (5 or 10 mg/kg/day) for 12 weeks, and hyperglycemia-exposed Müller cells were treated with BCAAs (2 or 5 mmol/L) for 24 and 48 h. BCAA levels were measured using MS/MS. Western blotting was performed to detect proteins. Flow cytometry, oxygen consumption rate, and Cell Counting Kit-8 assays were used to evaluate Müller cell viability. Each experiment was conducted at least thrice.BCAAs and branched-chain α-keto acids (BCKAs) were increased in the retina and systemic tissues of diabetic mice, and these changes were further enhanced to approximately 2-fold by extra BCAAs compared to wild-type group. In vitro, BCAAs and BCKAs were induced in hyperglycemic Müller cells, and augmented by BCAA supplementation. The aberrant BCAA catabolism was accompanied by mTORC1 activation and subsequently induced TNF-ɑ, VEGFA, GS, and GFAP in retinas and Müller cells under diabetic conditions. The cell apoptosis rate increased by approximately 50%, and mitochondrial respiration was inhibited by hyperglycemia and BCAA in Müller cells. Additionally, mTORC1 signaling was activated by leucine in Müller cells. Knockdown of Sestrin2 or LeuRS significantly abolished the leucine-induced mTORC1 phosphorylation and protected Müller cell viability under diabetic conditions.We found that BCAA catabolism is hindered in DR through mTORC1 activation. Leucine plays a key role in inducing mTORC1 by sensing Sestrin2 in Müller cells. Targeting Sestrin2 may ameliorate the toxic effects of BCAA accumulation on Müller cells in DR. [ABSTRACT FROM AUTHOR]