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Lactate Is a Metabolic–Epigenetic Signal That Links HIIT to miRNA-Centered Remodeling of the Skeletal-Muscle Methylome and Transcriptome

ABSTRACT: Lactate, a major exercise-derived metabolite, has been proposed to couple cellular metabolism to gene regulation, yet its direct impact on skeletal muscle remains unclear. Male mice underwent six-week interventions (Control, lactate, HIIT, MCT1/2 inhibition, or HIIT+inhibition). We profiled gastrocnemius DNA methylation, mRNA-seq and miRNA-seq, and assessed signaling proteins, metabolites, and running performance. Lactate and HIIT each induced broad, site-specific remodeling of the methylome and transcriptome with substantial overlap at promoter CpGs and among DEGs. Promoter methylation changes showed weak coupling to steady-state mRNA, whereas integrative analyses revealed robust anti-directional miRNA–mRNA networks and enriched for chromatin/epigenetic regulators, identifying a lactate-driven miRNA axis as a proximate regulator of transcriptional output. At the protein level, lactate selectively increased TET2 and DNMT3A and activated PAX7, VEGF, and AKT–S6 signaling, consistent with integrative miRNA–mRNA findings. HIIT increased TET1/2 and DNMT3A with DNMT3B reduction and uniquely enhanced mitochondrial/antioxidant signaling. Blocking MCT1/2 abrogated HIIT-induced methylome and miRNA remodeling and blunted transcriptomic and protein adaptation, demonstrating that intact lactate flux is required for exercise-evoked epigenetic and transcriptomic reprogramming. Despite molecular convergence, chronic lactate did not improve running performance, indicating that lactate is necessary but not sufficient for the full physiological adaptation of training. These findings position a lactate–miRNA axis linking metabolic perturbation to epigenetic and transcriptional remodeling in skeletal muscle.

ORGANISM(S): Mus musculus

PROVIDER: GSE308673 | GEO | 2025/12/17

REPOSITORIES: GEO

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Project description:The methylome and transcriptome signatures following exercise that are physiologically and metabolically relevant to sporting contexts such as team sports or health prescription scenarios (e.g. high intensity interval training/HIIT) has not been investigated. To explore this, we performed two different sport/exercise relevant high-intensity running protocols in 5 male sport team members using a repeated measures design of: 1) Change of direction (COD) versus; 2) straight line (ST) exercise with a wash-out period of at least 2 weeks between trials. Skeletal muscle biopsies collected from the vastus lateralis 30 minutes and 24 hours post exercise, were assayed using 850K methylation arrays and a comparative analysis with recent (subject-unmatched) sprint and acute aerobic exercise meta-analysis transcriptomes was performed. Despite COD and ST exercise being matched for classically defined intensity measures (speed x distance and number of accelerations/decelerations), COD exercise elicited greater movement (GPS-Playerload), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) measures compared with ST exercise, suggesting COD exercise evoked a higher exercise intensity. The exercise response alone across both conditions evoked extensive alterations in the methylome 30 mins and 24 hrs post exercise, particularly in MAPK, AMPK and axon guidance pathways. COD evoked a considerably greater hypomethylated signature across the genome compared with ST exercise, particularly at 30 minutes post exercise, enriched in: Protein binding, MAPK, AMPK, insulin, and axon guidance pathways. Comparative methylome analysis with sprint running transcriptomes identified considerable overlap, with 49% of genes that were altered at the expression level also differentially methylated after COD exercise. After differential methylated region analysis, we observed that VEGFA and its downstream nuclear transcription factor, NR4A1 had enriched hypomethylation within their promoter regions. VEGFA and NR4A1 were also significantly upregulated in the sprint transcriptome and meta-analysis of exercise transcriptomes. We confirmed increased gene expression of VEGFA, and considerably larger increases in the expression of canonical metabolic genes, PPARGC1A (that encodes PGC1-α) and NR4A3 in COD vs. ST exercise. Overall, we demonstrate that increased physiological/metabolic load via change of direction exercise in human skeletal muscle evokes considerable epigenetic modifications that are associated with changes in expression of genes responsible for adaptation to exercise.

Project description:Many cancers rely on glycolytic metabolism to fuel rapid proliferation. This has spurred interest in designing drugs that target tumor glycolysis such as AZD3965, a small molecule inhibitor of Monocarboxylate Transporter 1 (MCT1) currently undergoing Phase I evaluation for cancer treatment. Since MCT1 mediates proton-linked transport of monocarboxylates such as lactate and pyruvate across the plasma membrane (Halestrap and Meredith, 2004), AZD3965 is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show that MCT1 inhibition impairs proliferation of glycolytic breast cancer cells that express MCT4 via disruption of pyruvate rather than lactate export. We found that MCT1 expression is elevated in glycolytic breast tumors and cell lines as well as in malignant breast and lung tissues. High MCT1 expression predicts poor prognosis in breast and lung cancer patients. Stable knockdown and AZD3965-mediated inhibition of MCT1 promote oxidative metabolism. Acute inhibition of MCT1 reduces pyruvate export rate but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that also express MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest that MCT1 expression is elevated in glycolytic cancers to promote pyruvate export, which when inhibited enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors that further supports their use as anti-cancer therapeutics. Since MCT1 levels are elevated in glycolytic and malignant breast tumors, we hypothesized that MCT1 may contribute to the Warburg effect metabolic phenotype. To test this hypothesis, we generated whole genome microarray data from breast cancer cell lines either a) expressing a short hairpin (sh)RNA-mediated stable knockdown of MCT1; or b) treated for 24 hours with an MCT1 inhibitor (AZD3965). Scramble shRNA or DMSO were used as controls, and all conditions were analzed in triplicate. The cell lines used â HS578T, SUM149PT, and SUM159PT â are among the most glycolytic in a panel of 31 breast cancer cell lines.

Project description:Background: Exercise mimetics is a proposed class of therapeutics that specifically mimics or enhances the therapeutic effects of exercise. Muscle glycogen and lactate extrusion are critical for physical performance. The mechanism by which glycogen and lactate metabolism are manipulated during exercise remains unclear. This study aimed to assess the effect of miR-92b on the upregulation of exercise training-induced physical performance. Methods: Adeno-associated virus (AAV)-mediated skeletal muscle miR-92b overexpression in C57BLKS/J mice, and global knockout of miR-92b mice were used to explore the function of miR-92b in glycogen and lactate metabolism in skeletal muscle. AAV-mediated UGP2 or MCT4 knockdown in WT or miR-92 knockout mice was used to confirm whether miR-92b regulates glycogen and lactate metabolism in skeletal muscle through UGP2 and MCT4. Body weight, muscle weight, grip strength, running time and distance to exhaustion, and muscle histology were assessed. The expression levels of muscle mass-related and functionrelated proteins were analysed by immunoblotting or immunostaining. Results: Global knockout of miR-92b resulted in normal body weight and insulin sensitivity, but higher glycogen content before exercise exhaustion (0.8538 ± 0.0417 vs 1.043 ± 0.040, **P=0.0087), lower lactate levels after exercise exhaustion (4.133 ± 0.2589 vs 3.207 ± 0.2511, *P=0.0279), and better exercise capacity (running distance to exhaustion, 3616 ± 86.71 vs 4231 ± 90.29, ***P=0.0006; running time to exhaustion, 186.8 ± 8.027 vs 220.8 ± 3.156, **P=0.0028), as compared to those observed in the control mice. Mice skeletal muscle overexpressing miR-92b (both miR-92b-3p and miR-92b-5p) displayed lower glycogen content before exercise exhaustion (0.6318 ± 0.0231 vs 0.535 ± 0.0194, **P=0.0094), and higher lactate accumulation after exercise exhaustion (4.5 ± 0.2394 vs 5.467 ± 0.1892, *P=0.01), and poorer exercise capacity (running distance to exhaustion, 4005 ± 81.65 vs 3228 ± 149.8, ***P＜0.0001; running time to exhaustion, 225.5 ± 7.689 vs 163 ± 6.476, **P=0.001). Mechanistic analysis revealed that miR-92b-3p targets UDP-glucose pyrophosphorylase 2 (UGP2) expression to inhibit glycogen synthesis, while miR-92b-5p represses lactate extrusion by directly target monocarboxylate transporter 4 (MCT4). Knockdown of UGP2 and MCT4 reversed the effects observed in the absence of miR-92b in vivo. Conclusions: This study revealed regulatory pathways, including miR-92b-3p/UGP2/glycogen synthesis and miR-92b-5p/MCT4/lactate extrusion, which could be targeted to control exercise capacity.

Project description:Ten healthy male participants were recruited and successfully completed all preliminary testing and the two exercise bouts (Figure 1A). This randomized crossover design permitted signaling responses to workload- and duration-matched HIIT and MICT exercise to be mapped and interrogated within the same participant. Baseline whole-body anthropometric and blood glucose and lipid measurements confirmed these participants (age 25.4 ± 3.2 y; BMI 23.5 ± 1.6 kg/m2) were metabolically healthy, and maximal exercise capacity testing confirmed they were recreationally active but relatively untrained (relative V̇O2 peak 37.9 ± 5.2) in line with our recruitment strategy to maximize detection of skeletal muscle signaling responses to exercise (Figure 1B). Peak power output (Wpeak; 207.5 ± 40.3 W) was used to prescribe the relative work-matched intensities for HIIT and MICT. Lean mass from the DXA scan and resting metabolic rate (Figure 1B) were used to prescribe a standardized meal for each participant to consume prior to each exercise trial day. Following consumption of a standardized dinner meal the night before the HIIT or MICT exercise trial, blood and skeletal muscle biopsy samples were collected in the fasted state at baseline and at 5 min and 10 min of each respective exercise intensity. Participants completing the acute bout of HIIT, which consisted of 10 min total of 1-min ‘on’ intervals at 85 ± 0.1% of individual Wpeak (176 ± 34 W) and 1-min ‘off’ intervals at 50 W, displayed increased plasma lactate concentrations at 5 and 10 min of exercise relative to pre-exercise baseline (Figure 1C; P < 0.0001 and P < 0.001, respectively; main effect of time P < 0.0001) and no changes in plasma glucose (Figure 1D). In response to total work- and duration-matched acute bout of MICT at 55 ± 2% individual Wpeak (113 ± 17 W), participants displayed increased plasma lactate concentrations at 5 and 10 min of exercise versus baseline (Figure 1C; P < 0.001; main effect of time P < 0.0001) and no changes in plasma glucose (Figure 1D), with a main effect of higher plasma lactate levels in response to HIIT (Figure 1C; P < 0.05). To map the signaling networks regulated by HIIT and MICT, proteins from each skeletal muscle biopsy sample were extracted, digested to peptides with trypsin, isobarically labelled prior to phosphopeptide enrichment, fractionation and LC-MS/MS phosphoproteomic analysis (Figure 2A).

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Lactate Is a Metabolic–Epigenetic Signal That Links HIIT to miRNA-Centered Remodeling of the Skeletal-Muscle Methylome and Transcriptome

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