Glibenclamide targets MDH2 to relieve aging phenotypes through metabolism-regulated epigenetic modification

Abstract

Mitochondrial metabolism-regulated epigenetic modification is a driving force of aging and a promising target for therapeutic intervention. Mitochondrial malate dehydrogenase (MDH2), an enzyme in the TCA cycle, was identified as an anti-aging target through activity-based protein profiling in present study. The expression level of MDH2 was positively correlated with the cellular senescence in Mdh2 knockdown or overexpression fibroblasts. Glibenclamide (Gli), a classic anti-glycemic drug, was found to inhibit the activity of MDH2 and relieve fibroblast senescence in an MDH2-dependent manner. The anti-aging effects of Gli were also further validated in vivo, as it extended the lifespan and reduced the frailty index of naturally aged mice. Liver specific Mdh2 knockdown eliminated Gli's beneficial effects in naturally aged mice, reducing p16^INK4a expression and hepatic fibrosis. Mechanistically, MDH2 inhibition or knockdown disrupted central carbon metabolism, then enhanced the methionine cycle flux, and subsequently promoted histone methylation. Notably, the tri-methylation of H3K27, identified as a crucial methylation site in reversing cellular senescence, was significantly elevated in hepatic tissues of naturally aged mice with Mdh2 knockdown. Taken together, these findings reveal that MDH2 inhibition or knockdown delays the aging process through metabolic-epigenetic regulation. Our research not only identified MDH2 as a potential therapeutic target and Gli as a lead compound for anti-aging drug development, but also shed light on the intricate interplay of metabolism and epigenetic modifications in aging.

Fig. 1

MDH2 regulates cellular senescence. a Schematic diagram of ABPP. b Relative MDH2 and p16^INK4a level in Blk and Dox-induced senescent MRC-5 cells. c Quantification of b. d Relative MDH2 and p16^INK4a level in replicative senescent MEFs form P7 to P11. e Quantification of d. f SA-β-gal staining of sh-Scr/sh-Mdh2 MEFs (P11) transfected at P4. g Quantification of f. h Relative MDH2 and p16^INK4a level in sh-Scr/sh-Mdh2 MEFs (P7) transfected at P4. i Quantification of h. j SA-β-gal staining of OE-NC/OE-Mdh2 MEFs (P10) transfected at P4. k Quantification of j. l Relative MDH2 and p16^INK4a level in OE-NC/OE-Mdh2 MEFs (P8) transfected at P4. m Quantification of l. Error bars represent the standard deviation (± SEM.). The significance of differences of c, g, i, k, m was analyzed with two-sided Student’s t-test. The significance of difference of e was analyzed with Dunnett’s multiple comparisons tests (*p < 0.05, **p < 0.01, ***p < 0.005, n. s., not significant)

Fig. 2

Gli relieves aging phenotypes in vitro and in vivo. a Structure of Gli and the chemical probe of Gli (Gli-P). b MDH2 immunoblotting of proteins pulldown from in situ labeling of MRC-5 cells using Gli-P. c Binding constant (K_D) of Gli to MDH2 measured by Grating-Coupled Interferometry. d SA-β-gal staining of Blk and Dox-induced senescent MRC-5 cells treated with -/Chl (200 μM)/Gli (100 μM)/Met (100 μM) for 7 days. e Quantification of d. f Relative p16^INK4a level in Blk and Dox-induced senescent MRC-5 cells treated with -/Gli (100 μM)/Met (100 μM) for 7 days. g Quantification of f. h Relative MMP1 level in Blk and Dox-induced senescent MRC-5 cells treated with -/Gli (100 μM)/Met (100 μM) for 7 days. i Quantification of h. j IL-6 level in the medium of Blk and Dox-induced senescent MRC-5 cells treated with -/Gli (100 μM)/Met (100 μM) for 7 days. k IL-1β level in the medium of Blk and Dox-induced senescent MRC-5 cells treated with -/Gli (100 μM)/Met (100 μM) for 7 days. l SA-β-gal staining of MEFs (P6) treated with -/Gli (100 μM)/Met (100 μM) for 15 days. m Quantification of l. n Relative p16^INK4a and γH2AX level in MEFs (P6) treated with -/Gli (100 μM)/Met (100 μM) for 5 days. o Quantification of p16^INK4a in n. p Quantification of γH2AX in n. q Diagram of assays on naturally aged mice treated with -/Gli (10 mg/kg)/NMN (500 mg/kg) daily. r Lifespan curves of mice in different groups (Ctrl, n = 10; Gli, n = 12; NMN, n = 10). s Frailty index of mice in different groups (Ctrl, n = 10; Gli, n = 12; NMN, n = 10 when the test began at 18-month age). For s, every dot in the plot presents the data of 1 mouse. Error bars represent the standard deviation (± SEM.). Log-rank tests were used for analyzing the significant differences of r. Other differences were analyzed with Tukey’s multiple comparations tests (*p < 0.05, **p < 0.01, ***p < 0.005, n. s., not significant)

Fig. 3

Gli alleviates cellular senescence dependent on MDH2. a Diagram of the MDH2 knockdown or overexpression assay in cells. b Relative p16^INK4a level in sh-Scr/sh-Mdh2 MEFs (P7) treated with -/Gli (100 μM)/Met (100 μM) for 5 days. c Quantification of b. d SA-β-gal staining of sh-Scr/sh-Mdh2 MEFs (P7) treated with -/Gli (100 μM)/Met (100 μM) for 15 days. e Quantification of d. f Relative p16^INK4a level in OE-NC/OE-Mdh2 MEFs (P7) treated with -/Gli (100 μM)/Met (100 μM) for 5 days. g Quantification of f. h SA-β-gal staining of OE-NC/OE-Mdh2 MEFs (P7) treated with -/Gli (100 μM)/Met (100 μM) for 15 days. i Quantification of h. Error bars represent the standard deviation (± SEM.). The significance of differences (*p < 0.05, **p < 0.01, ***p < 0.005, n. s., not significant) of all panels were analyzed with Tukey’s multiple comparations tests

Fig. 4

Gli targets the TCA cycle and regulates central carbon metabolism. a mtROS level in MEFs treated with -/Gli (100 μM)/LW6 (10 μM) for 2/4/8/12/24 h. b Relative fluorescence ratio indicating subcellular lactate level in H1299 cells treated with -/LW6 (10 μM)/Gli (100 μM)/oxamate (10 mM) for 2 h. c KEGG pathway analysis of differential metabolites in MRC-5 cells treated with -/Gli (100 μM) for 2 h. d Relative level of glycolysis intermediates in MRC-5 cells treated with -/Gli (100 μM) for 2 h. e Relative level of TCA cycle intermediates in MRC-5 cells treated with -/Gli (100 μM) for 2 h. f Relative level of amino acids in MRC-5 cells treated with -/Gli (100 μM) for 2 h. g KEGG pathway analysis of differential metabolites in MRC-5 cells treated with -/Gli (100 μM) for 24 h. h Relative level of glycolysis intermediates in MRC-5 cells treated with -/Gli (100 μM) for 24 h. i Relative level of TCA cycle intermediates in MRC-5 cells treated with -/Gli (100 μM) for 24 h. j Relative level of amino acids in MRC-5 cells treated with -/Gli (100 μM) for 24 h. For metabolome assays, 6 replicates were tested. Error bars represent the standard deviation (± SEM.). The significance of differences of a and b were analyzed with Tukey’s multiple comparisons tests, and other significance of differences in metabolome assays were analyzed with Sidak’s multiple comparisons tests (*p < 0.05, **p < 0.01, ***p < 0.005)

Fig. 5

Gli regulates central carbon metabolism and methionine cycle flux through MDH2, and induces the methylation potential. a KEGG pathway analysis of differential metabolites in sh-Scr and sh-Scr+Gli (100 μM) groups. b KEGG pathway analysis of differential metabolites in sh-Mdh2 and sh-Mdh2+Gli (100 μM) groups. c Relative level of malic acid in different groups (n = 6). d Venn graph of differential metabolites in different groups (SC: sh-Scr; SG: sh-Scr+Gli (100 μM); RC: sh-Mdh2; RG: sh-Mdh2+Gli (100 μM)). Gli-regulated metabolites dependent on MDH2 were marked in orange font. e Relative p16^INK4a level in MEFs treated with metabolites (100 μM) for 5 days. f Relative SAM, SAH, and SAM/SAH level in sh-Scr/sh-Mdh2 MRC-5 cells under different treatments. g Diagram of methionine cycle. h Relative H3K4me3, H3K9me3, and H3K27me3 level in MEFs treated with -/Gli (100 μM)/SAH (100 μM) for 1 day. i Quantification of h. j Relative H3K4me3, H3K9me3, and H3K27me3 level in MEFs treated with -/Gli (100 μM)/SAH (100 μM) for 5 days. k Quantification of j. l CUT&TAG-qPCR assays testing H3K4me3 and H3K27me3 level along the p16^INK4a locus in MEFs treated with -/Gli (100 μM) for 5 days. Error bars represent the standard deviation (± SEM.). The significance of differences (*p < 0.05, **p < 0.01, ***p < 0.005) of all panels were analyzed with Tukey’s multiple comparisons tests

Fig. 6

MDH2 inhibition delays hepatic aging and induces H3K27me3 level in vivo. a Diagram of MDH2 knockdown assay in naturally aged mice (19-month). b AST level of mice in different groups. Mice at 2-month age were used in Young groups. c ALT level of mice in different groups. d HbA1c ratio in serum of mice in different groups. e Serum insulin level of mice in different groups. f Behavior index of hanging endurance in mice of different groups. g Total number of arm entries in Y-maze of mice in different groups. h Alteration rate in Y-maze of mice in different groups. i Relative MDH2 and p16^INK4a level in mouse livers. j Quantification of i. k Sirius red staining of mouse livers. l Relative area of fibrosis in mouse livers, quantification of k. m Relative H3K4me3, H3K9me3 and H3K27me3 level in mouse livers of different groups (n = 5 for all groups). n Quantification of m. Young represents mice at 2-month age, and Old represents mice at 20.5-month age. Sample size for b-h: Young, n = 7; sh-Scr, n = 8; sh-Scr+Gli (10 mg/kg), n = 7; sh-Mdh2, n = 7; sh-Mdh2+Gli (10 mg/kg), n = 8. Sample size for i: n = 5 for all groups. Sample size for k: Young, n = 5; sh-Scr, n = 5; sh-Scr+Gli (10 mg/kg), n = 4; sh-Mdh2, n = 5; sh-Mdh2+Gli (10 mg/kg), n = 3. Error bars represent the standard deviation (± SEM.). Every dot in the plots presents the data of 1 mouse. The significance of differences (*p < 0.05, **p < 0.01, ***p < 0.005) of all panels were analyzed using Tukey’s multiple comparisons tests

Fig. 7

Diagram of the anti-aging effects of metabolic-epigenetic regulation under MDH2 inhibition