Journal: Redox biology

Article Title: Mitochondrial aldehyde dehydrogenase-2 coordinates the hydrogen sulfide - AMPK axis to attenuate high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system.

doi: 10.1016/j.redox.2023.102994

Figure Lengend Snippet: Fig. 3. ALDH2 activation prevents high-glucose-induced G6PD downregulation via CDK5 inhibition (A) INS-1 cells were treated with high glucose (30 mM) with ALDH2 activator ALDA1 (25uM) for 48h. The cell extracts were harvested and tested for ALDH2 activity (*P < 0.005 vs. Control, **P < 0.05 vs. Control; ***P < 0.005 vs. HG). (B) INS-1 cells were treated with high glucose (30 mM) with ALDA1 (25uM) for 48h and protein levels were quantified by immunoblotting (*P < 0.001 vs. Control, **P < 0.001 vs. HG). (C) G6PD activity was measured after above experimental conditions (*P < 0.005 vs. Control, **P < 0.001 vs. Control; ***P < 0.001 vs. HG). (D) Measurement of relative GSH/GSSG ratios in INS-1 cells after ALDA1 (25uM) treatment (*P < 0.001 vs. Control, **P < 0.001 vs. HG). (E–F) cellular ROS and TMRE assay after ALDA1 (25uM) treatment (*P < 0.001 vs. Control, **P < 0.005 vs. HG). (G) Representative Immunoblots for cytochrome c and cleaved caspase-3 after ALDA1 (25uM) treatment (*P < 0.001 vs. Control, **P < 0.001 vs. HG). (H-K) INS-1 cells were treated with 4-HNE (25uM) with ALDH2 activator ALDA1 (25uM) for 24h. The cell extracts were harvested and tested for ALDH2, G6PD activity and immunoblotting (*P < 0.001 vs. Control, **P < 0.005 vs. 4HNE). Data are expressed as means ± SEM of at least three independent experiments.

Article Snippet: Reagents were sourced commercially as follows: L-buthionine sulfoximine(BSO), roscovitine and NaHS from Sigma-Aldrich; BML-275 from Enzo Life Sciences Inc., NY, USA; 4-HNE, ALDA1 and SF7-AM from Cayman, USA; and AGK2 from SelleckChem.

Techniques: Activation Assay, Inhibition, Activity Assay, Control, Western Blot

Journal: Redox biology

Article Title: Mitochondrial aldehyde dehydrogenase-2 coordinates the hydrogen sulfide - AMPK axis to attenuate high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system.

doi: 10.1016/j.redox.2023.102994

Figure Lengend Snippet: Fig. 4. ALDH2 activation rapidly increases endogenous H2S in β-cell (A) INS-1 cells were treated with ALDA1 (25 μM) and NaHS (100 μM) for the indicated time periods, and intracellular H2S levels were measured using SF7-AM dye (*P < 0.001 vs. Control). (B) INS-1 cells were treated with ALDA1 (25 μM) for 3 h and then exposed to high glucose concentrations (30 mM) for 36 h; thereafter, H2S levels were measured using SF7-AM dye (*P < 0.001 vs. Control, **P < 0.001 vs. HG). (C) INS-1 cells were treated with NaHS (100 μM) for the indicated time periods, and intracellular H2S levels were measured using SF7-AM dye (*P < 0.001 vs. Control). (D) INS-1 cells were treated with NaHS (100 μM) for 3 h and then exposed to high glucose concentrations (30 mM) for 48 h; thereafter, ALDH2 activity assays were performed (*P < 0.001 vs. Control, **P < 0.005 vs. HG). (E) INS-1 cells were treated with NaHS (100 μM) for 3 h and then exposed to high glucose concentrations (30 mM) for 48 h. Cell extracts were harvested and tested for protein levels using the indicated antibodies. (F–I) G6PD activity, GSH/GSSG ratios, cellular ROS levels, and mitochondrial membrane potential were measured in cells treated with NaHS (100 μM) and high glucose concentrations (30 mM) (*P < 0.005 vs. Control, **P < 0.001 vs. HG). (J & K) Representative Immunoblots for cytochrome c, cleaved caspase-3, and viability of NaHS-treated cells.

Article Snippet: Reagents were sourced commercially as follows: L-buthionine sulfoximine(BSO), roscovitine and NaHS from Sigma-Aldrich; BML-275 from Enzo Life Sciences Inc., NY, USA; 4-HNE, ALDA1 and SF7-AM from Cayman, USA; and AGK2 from SelleckChem.

Techniques: Activation Assay, Control, Activity Assay, Membrane, Western Blot

Journal: Redox biology

Article Title: Mitochondrial aldehyde dehydrogenase-2 coordinates the hydrogen sulfide - AMPK axis to attenuate high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system.

doi: 10.1016/j.redox.2023.102994

Figure Lengend Snippet: Fig. 5. ALDH2 induced AMPK activation blocks G6PD degradation and death in β-cells (A) INS-1 cells were treated with high glucose concentrations (30 mM) with or without ALDA1 (25 μM) for the indicated time periods. ALDA1-induced AMPKα (Thr172) and ACC (Ser79) phosphorylation was analyzed using western blotting (*P < 0.05 vs. Control, **P < 0.005 vs. HG). (B) INS-1 cells were treated with NaHS (100 μM) for 3h and then exposed to high glucose (30 mM) for 48h. The cell extracts were harvested and tested for AMPKα (Thr172) and ACC (Ser79) phos phorylation. (C) INS-1 cells were treated with ALDA1 (25 μM) or NaHS (100 μM) with high glucose (30 mM) for 48h. Phospho PKC-ε and NAMPT protein were quantified by immunoblotting. (D) NAD+ levels after ALDA1 (25 μM) or NaHS (100 μM) treated cells with high glucose (30 mM) (*P < 0.001 vs. control; **P < 0.005 vs. HG). (E) INS-1 cells were treated with high glucose (30 mM) with ALDA1 (25uM) for 48h with or without the AMPK inhibitor BML-275(10uM). (*P < 0.005 vs. control; **P < 0.001 vs. HG; ***P < 0.005 ALDA1). (F) INS-1 cells were treated with high glucose (30 mM) with NaHS (100 μM) for 48h with or without the AMPK inhibitor BML-275(10uM) (*P < 0.005 vs. control; **P < 0.001 vs. HG; ***P < 0.005 NaHS). (G–H) Phospho PKC-ε and NAMPT protein were quantified by immunoblotting with or without the AMPK inhibitor BML-275(10uM). (I) NAD+ levels after ALDA1 (25 μM) with or without the AMPK inhibitor BML-275(10uM) (*P < 0.005 vs. control; **P < 0.001 vs. HG; ***P < 0.005 ALDA1). Data are expressed as means ± SEM of at least three independent experiments.

Article Snippet: Reagents were sourced commercially as follows: L-buthionine sulfoximine(BSO), roscovitine and NaHS from Sigma-Aldrich; BML-275 from Enzo Life Sciences Inc., NY, USA; 4-HNE, ALDA1 and SF7-AM from Cayman, USA; and AGK2 from SelleckChem.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control

Journal: Redox biology

Article Title: Mitochondrial aldehyde dehydrogenase-2 coordinates the hydrogen sulfide - AMPK axis to attenuate high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system.

doi: 10.1016/j.redox.2023.102994

Figure Lengend Snippet: Fig. 6. SIRT2 Inhibition reverse ALDA1 protective effect in β-cells (A-B) INS-1 cells were treated with high glucose (30 mM) with ALDA1 (25uM) for 48h with or without the SIRT2 inhibitor AGK2 (3uM). Western blots of total G6PD, acetylated G6PD and G6PD activity were measured (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.001 ALDA1). (C) Measurement of relative GSH/GSSG ratios in INS-1 cells after ALDA1 treatment with AGK2 (3uM) in high glucose conditions (*P < 0.005 vs. control; **P < 0.001 vs. HG; ***P < 0.001 ALDA1). (D–F) Cellular ROS production and mitochondrial membrane potential after AGK2 treatment (*P < 0.01 vs control; **P < 0.01 vs HG; ***P < 0.01 vs ALDA1). Cytosolic cytochrome c, cleaved casapase-3 protein levels were quantified by immunoblotting (*P < 0.01 vs control; **P < 0.05 vs HG; ***P < 0.01 vs ALDA1). (G–I) INS-1 cells were treated with high glucose (30 mM) with NaHS (100uM) for 48h with or without the SIRT2 inhibitor AGK2 (3uM) (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.005 NaHS). The results represent the mean ± SEM of three independent experiments. (J–N) INS-1 cells were treated with high glucose (30 mM) with ALDA1 (25uM) for 48h with or without glutathione synthesis inhibitor BSO (100uM). (J) SIRT2, G6PD protein levels were quantified by immunoblotting (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.005 BSO). (K) Measurement of G6PD activity in INS-1 cells after ALDA1 treatment with BSO (100uM) in high glucose conditions after 48h (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.001 BSO). (L) Cellular ROS production and (M) mitochondrial membrane potential after BSO treatment (*P < 0.01 vs control; **P < 0.01 vs HG; ***P < 0.01 vs BSO). (H) Cytosolic cytochrome c, cleaved casapase-3 protein levels were quantified by immunoblotting (*P < 0.01 vs control; **P < 0.05 vs HG; ***P < 0.01 vs BSO). Data are represented as mean ± SEM of three independent experiments.

Article Snippet: Reagents were sourced commercially as follows: L-buthionine sulfoximine(BSO), roscovitine and NaHS from Sigma-Aldrich; BML-275 from Enzo Life Sciences Inc., NY, USA; 4-HNE, ALDA1 and SF7-AM from Cayman, USA; and AGK2 from SelleckChem.

Techniques: Inhibition, Western Blot, Activity Assay, Control, Membrane

Journal: Redox biology

Article Title: Mitochondrial aldehyde dehydrogenase-2 coordinates the hydrogen sulfide - AMPK axis to attenuate high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system.

doi: 10.1016/j.redox.2023.102994

Figure Lengend Snippet: Fig. 7. ALDH2 expression is reduced in T2D, while its activation improves survival under high glucose (A-C) Representative images of immunostaining for ALDH2 in brown (A) or triple fluorescent staining for ALDH2 in red, insulin in green and DAPI in blue of pancreatic sections from C57Bl/6J mice fed a ND or HFD for 17 weeks (A, B) and (C) from organ donors from autopsy of non-diabetic controls (n = 4) and patients with T2D (n = 4; scale bar depicts 10 mm). (D) Human pancreatic 1.1B4 cells were incubated with ALDA1 (25 μM) for 48 h under high-glucose conditions (30 mM) (*P < 0.005 vs. Control, **P < 0.001 vs. HG). (E) Human pancreatic 1.1B4 cells were incubated with ALDA1 (25 μM) for 48 h under high-glucose conditions (30 mM). CDK5R1, P-CDK5, P-AMPK, P-ACC, SIRT2, and G6PD were analyzed using western blotting (*P < 0.001 vs. Control, **P < 0.005 vs. HG). (F & G) G6PD activity and relative GSH/GSSG ratios (*P < 0.005 vs. Control, **P < 0.001 vs. HG). (H & I) Cellular ROS production and mitochondrial membrane potential after ALDA1 treatment (*P < 0.001 vs. Control, **P < 0.001 vs. HG). (J) Cytosolic cytochrome c and cleaved caspase-3 were quantified by immunoblotting (*P < 0.001 vs. Control, **P < 0.005 vs. HG). The data represent means ± SEM of three independent experiments. (K) Effect of the ALDH2 activation and CDK5 inhibition on insulin secretion (*P < 0.001 vs. 2.8 mM glucose; **P < 0.005 vs. 16.6 mM glucose; ***P < 0.005 vs. 16.6 mM glucose). (L) Effect of SIRT2 inhibition on the ALDH2 agonist ALDA1-amplified insulin secretion (*P < 0.001 vs. 2.8 mM glucose; **P < 0.005 vs. 16.6 mM glucose; ***P < 0.001 vs.AGK2).

Article Snippet: Reagents were sourced commercially as follows: L-buthionine sulfoximine(BSO), roscovitine and NaHS from Sigma-Aldrich; BML-275 from Enzo Life Sciences Inc., NY, USA; 4-HNE, ALDA1 and SF7-AM from Cayman, USA; and AGK2 from SelleckChem.

Techniques: Expressing, Activation Assay, Immunostaining, Staining, Incubation, Control, Western Blot, Activity Assay, Membrane, Inhibition, Amplification