Fig. 7. OXPHOS drives lactylation to feedback limit OXPHOS and endurance running ability.

a OXPHOS induces intracellular hypoxia. HIF1α and AARS2 levels of C2C12 cells in DMEM base and glucose- (25 mM), palmitate- (200 μM) and Me-lac- (2 mM) supplemented DMEM media were tested in the presence or absence of 1 μM rotenone which inhibits OXPHOS. b Lactate injection induces mouse muscle hypoxia. HIF1α and AARS2 levels of mouse hind leg thighs were detected before and after 2 g/kg lactate was injected into gastrocnemius muscle (n = 3). c Lactate production is essential for inducing hypoxia and lactylation in mouse leg muscles. The HIF1α, AARS2, and lactylation levels of mouse hind leg thighs of untreated mice and mice pre-treated with FX-11 were assessed when they started to run. d Running leads to the accumulation of lactate in mouse leg skeletal muscles. Relative lactate levels in type I and type II mouse skeletal muscles were determined before and after 30 min of running (n = 6). e Running induces more pronounced lactylation in type I muscle. HIF1α and AARS2, as well as Lac-K336 and Lac-K457/8 levels in type I and type II mice skeletal muscles, were determined when started to run. f Drop of ATP in type I skeletal muscle is correlated with an increase in Lac-K336 and Lac-K457/8. ATP levels in type I and II mouse leg skeletal muscles were monitored after running for indicated times (n = 6). Changes in Lac-K336 and Lac-K457/8 are shown (Fig. 5j). g Basal AARS2 levels in mouse leg skeletal muscle are inversely correlated with their running exhaustion time. High-intensity running exhaustion time and resting leg muscle AARS2 levels were correlated (n = 30). h Lac-K336 and Lac-K457/8 inhibit the influx of mouse skeletal muscle Ac-CoA. The ¹³C-Ac-CoA levels in wild-type, AARS2 overexpressing and Sirt3^−/− mouse leg skeletal muscle were detected 1 h after each mouse received a ¹³C-glucose injection via the tail vein (n = 6). i, j Lactylation reduces mouse skeletal muscle OCRs and ATP production. OCR (i) and ATP levels (j) in mitochondria in the leg skeletal muscles of resting, AARS2 overexpressing, and Sirt3^−/− mice, were analyzed (n = 6). k Lac-K336 and Lac-K457/8 inhibit mouse running exhaustion time. Mouse high-intensity running exhaustion time was measured in wild-type, muscle AARS2-overexpressing, and Sirt3^−/− mice (n = 9). l Lactate and β-alanine inversely regulate mouse running exhaustion time. High-intensity running exhaustion times of untreated mice or treated with lactate or β-alanine via gastrocnemius muscle injection were measured, respectively (n = 9). m β-alanine prolongs mice exhaustion time in an AARS2-dependent manner. High-intensity running exhaustion times of wild-type, AARS2 overexpressing, and Aars2^−/− mice with or without gastrocnemius muscle β-alanine injection (n = 10) were measured. n, o Loss of lactylation results in higher ROS damage. ROS levels (n) and induced malondialdehyde levels (o) of wild-type and Aars2^−/− mice at the time of running exhaustion (n = 6) were compared. p Diagram showing that exercise promotes lactate oxidation through OXPHOS, which promotes intracellular hypoxia that induces AARS2 and Lac-K336 and Lac-K457/8 to feedback inhibit Ac-CoA influxes from lactate/glycolysis and FAO and OXPHOS. The mechanisms revealed in this study are marked by red arrows. All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test and two-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance.