Exercise-induced protein lactylation: From molecular mechanisms to precision health applications

Summary

Exercise-induced lysine lactylation of proteins is an important metabolic regulatory mechanism. This review systematically assesses how different exercise modes impact lactate post-translational modifications in a tissue-specific manner that in turn helps orchestrate energetic metabolic homeostasis, boost neuroprotection, and optimize cardiovascular adaptations. This article aims to put forward novel strategies for exercise prescriptions, tailored to individuals’ metabolic profiles, with an eye on consolidated molecular interventions aimed at modulating and modifying these metabolic pathways. Such proposals may well offer precision in sports medicine.

Graphical abstract

Biochemistry; Human metabolism

Introduction

Lactylation is an emerging post-translational modification (PTM) that represents a significant advancement in our understanding protein modification and the regulation of cellular metabolism. In 2019, Zhang et al. first reported this modification in Nature, demonstrating that lactate functions not only as a metabolite but also covalently binds to lysine(K) residues of histones, thereby forming a protein lysine lactylation modification (Kla).¹ This discovery enhances our comprehension of lactate’s functionality and underscores its critical role in cell signaling and the regulation of gene expression.

As early as the 1920s, German biochemist Otto Warburg observed that tumor cells preferentially convert glucose to lactate via the glycolytic pathway rather than generating energy through the more efficient oxidative phosphorylation pathway, even under aerobic conditions.² This phenomenon is known as aerobic glycolysis. Since then, the understanding of the physiological role of lactic acid has evolved, prompting the exploration of its various physiological functions. Research conducted over the past century has revealed that the Warburg effect is not only evident in tumor cells but also plays a significant role in other pathological conditions such as inflammation, infection, and hypoxia.³

In contrast, most studies on protein lactylation in recent years have focused on pathological environments such as tumor microenvironments and diseased tissues. However, relatively few investigations have been conducted regarding the exercise-induced high-lactic acid environment and its role in triggering protein lactylation. Exercise serves as a crucial means of maintaining and enhancing health, with benefits that are extensive and far-reaching. Numerous studies have demonstrated that exercise significantly reduces the risk of cardiovascular disease,⁴ metabolic disorders,⁵ and cancer,⁶ while also improving cognitive function,⁷ cardiorespiratory endurance, and muscle strength.⁴ Recent findings indicate that exercise not only enhances health through traditional metabolic pathways but also regulates metabolic pathways and energy balance by increasing lactate production and inducing lactylation in various tissues and cells. Research in this domain not only deepens our understanding of the physiological mechanisms underlying exercise but also offers potential targets for the development of novel therapeutic strategies.

Exercise-induced lactylation

Exercise and lactate

During exercise, muscle demand for ATP exceeds the supply capacity of oxidative phosphorylation, leading to a significant increase in glycolysis rate as glucose is rapidly broken down into pyruvate. When oxygen supply is insufficient (or mitochondrial entry is restricted), pyruvate cannot enter the tricarboxylic acid cycle. Instead, it is reduced to lactate by lactate dehydrogenase (LDH), accompanied by the reoxidation of NADH → NAD⁺, thereby sustaining the ongoing glycolytic process. After production, lactate accumulates briefly within muscle fibers before being transported into the interstitial fluid via monocarboxylate transporters (MCT1/MCT4) and subsequently entering the bloodstream. MCT4 primarily facilitates the export of lactic acid from high-output muscle fibers (fast-twitch muscles) into the blood; MCT1, conversely, reabsorbs lactic acid from the blood into energy-demanding tissues such as cardiac muscle, slow-twitch muscles, and the liver.⁸ Research indicates that elevated lactate concentration is a necessary prerequisite for the occurrence of lactylation.⁹ This review will focus on exercise-induced lactylation.

Overview of lactylation

Lactylation refers to the formation of ε-N-lactyl lysine through an acyl transfer reaction involving lactic acid, wherein the carboxyl group of lactic acid forms an amide bond (-CO-NH-) with the ε-amino group of lysine, resulting in an increase in protein mass of 72.021 Da.¹ Currently, all reported protein lactylation modifications specifically occur on the ε-amino group of lysine residues, with no direct evidence of lactylation occurring on other amino acids. This specificity is likely due to the reliance of the reaction on the free amino group of the lysine side chain, as other amino acids do not possess a similar nucleophilicity or spatial conformation conducive to stable amide bond formation. Lysine, owing to the chemical reactivity of its ε-amino group, which is significantly more reactive than that of other amino acid side chains is the sole amino acid in proteins capable of modification by multiple acyl groups (acetyl, propionyl, butyryl, succinyl, etc.).^10,11 The pathways for lactylation modification can be categorized into enzymatic and non-enzymatic processes. In the enzymatic pathway, the primary “writer” in eukaryotes is histone acetyltransferase p300/CBP, which recognizes several of acyl-coenzyme A molecules and participates in lactylation (Figure 1).¹² The substrate source, lactyl-CoA is catalyzed by enzymes such as YdiF, which can provide lactoyl groups.¹³ Histone deacetylases (HDAC1-3) and SIRT3 function as the “eraser” of lactylation.^14,15 In the non-enzymatic pathway, lactoylglutathione (LGSH) modifies glycolytic enzymes through spontaneous acyl transfer, with regulation dependent on glyoxalase 2 (GLO2) (Figure 2).¹³

Figure 1.

The process of histone lactylation and non-histone lactylation(enzymatic pathway)

This figure illustrates the primary processes involved in the lactylation of histones and non-histone proteins. The histone types are represented as H2A, H2B, H3, and H4, while K denotes lysine residues and la signifies lactyl groups. Within the cellular environment, lactate cannot directly serve as a donor of lactylation; it must first be converted to lactyl-CoA. This compound is then transferred to histones and non-histone proteins by the action of “writers,” which facilitate the initiation of the lactylation process as lactylation donors. Conversely, “erasers” are responsible for removing the lactyl group from lactylated proteins.¹⁸

Figure 2.

Non-enzymatic lysine lactoylation of glycolytic enzymes

This diagram illustrates the non-enzymatic pathway of lactylation modification.¹³ K denotes lysine residues, and la represents the lactyl group. Methylglyoxal (MGO), an intermediate in glycolysis, accumulates intracellularly. It subsequently reacts with glutathione (GSH) via glyoxalase 1 (GLO1) to form the highly reactive intermediate lactoylglutathione (LGSH). LGSH undergoes a non-enzymatic rearrangement, releasing a free lactyl group. This free lactyl group spontaneously attacks lysine residues (via nucleophilic attack), completing the lactylation modification. LGSH is then recycled back to GSH and converted into D-lactic acid by glyoxalase 2 (GLO2).

Sites of histone lactylation

Histones are a class of basic proteins located in the nucleus of eukaryotic cells, primarily responsible for packaging and regulating DNA, which in turn influences protein transcription and translation. There are five main types of histones: H1, H2A, H2B, H3, and H4, with two copies of each of the latter four forming the histone octamer.¹⁶ Histone octamers package DNA by forming nucleosomes, the fundamental unit of chromatin. Among these, H3 and H4 serve as core particles that constitute nucleosomes, while the N-terminal tails of H3 and H4 extend from the nucleosome’s exterior and are targets for various PTMs, such as methylation, acetylation, ubiquitination, and phosphorylation.¹⁷ The histone lactylation induced by exercise interventions in the current study specifically focused on histone H3 (Figure 1). Han et al. found that moderate-intensity aerobic exercise (AE) enhanced H3s lactylation, particularly H3 K18 lactylation, in the microglia of the mouse hippocampus and cerebral cortex.¹⁹ Wu et al. reported that high-intensity interval training (HIIT) upregulated histone H3 K18 lactylation in bone marrow mesenchymal stem cells (BMSCs).²⁰ Additionally, Dai et al. observed that following lactic acid injection in mice, there was an upregulation of myoblast differentiation processes in histone lactylation, particularly with respect to H3 K9 lactylation levels, suggesting a new perspective on the process of motor adaptation in skeletal muscle.²¹

Sites of non-histone lactylation

In addition to histone lactylation, an increasing number of studies in recent years have reported lactylation modifications of non-histone proteins. Wu et al. identified a characteristic fragment ion, cycIm, which appeared in mass spectrometry (MS/MS) spectra of Klac-containing peptides.²² They subsequently validated the sensitivity and specificity of the cycIm ion for the detection of Klac. This diagnostic method was used extensively to mine potential histone and non-histone lactylation sites, which are conserved across different cell lines and tissue samples. Non-histone lactylation is widely found in key enzymes of various physiological pathways, including glycolysis, lipid metabolism, autophagy, immune response, and functional pathways of the nervous system.²³ For instance, the negative feedback regulation of intracellular lactate concentration is mediated by lactate-induced K147 lactylation in fructose bisphosphate aldolase A (ALDOA), which plays a crucial role in the regulation of the glycolytic pathway.²⁴ Wang et al. found that K62 lactylation of pyruvate kinase M2 (PKM2), a key enzyme in the glycolytic pathway, enhances its enzymatic activity and facilitates the conversion of pro-inflammatory macrophages into a reparative phenotype thereby, modulating immune processes.²⁵ Chen et al. reported that fatty acid synthase (FASN), a key target protein for HIIT-induced lactylation, contains lactylation sites at four lysine residues, which were affected by in vitro lactic acid treatment.²⁶ Furthermore, Wang et al. revealed through multi-omics analysis that moderate-intensity exercise induces hepatic hypoxia and lactic acid accumulation, resulting in significant alterations in the transcriptional levels, protein expression, and lactylation modifications of key metabolic genes ALDOA, Acsl1, and HADHB.²⁷ Notably, lactylation levels at K42 of the key metabolic enzyme ALDOA and at K273 of HADHB were significantly downregulated, thereby enhancing glycolytic and fatty acid oxidation (FAO) capacities. This process further established a metabolic-epigenetic regulatory network mediated by transcription factors such as peroxisome proliferator activated receptor γ (PPARγ).

Types of exercise interventions that induce lactylation

Lactate availability is the decisive factor limiting lysine lactylation induction, and different exercise modalities exhibit significant differences in regulating lactate production, tissue distribution, and subsequent lactylation dynamics. This study systematically compared three primary exercise interventions: HIIT, AE, and resistance training (RT). Despite differing in intensity, energy metabolism patterns, and physiological adaptations, all three modulate lactylation by up- or down-regulating systemic or local lactate concentrations. Among these, core intensity metrics for humans are typically expressed as percent maximal oxygen consumption (VO₂ max), percent maximum heart rate (MHR max), or blood lactate concentration. In animal studies, intensity is generally described using treadmill speed (m/min). There are no strict standards for exercise intervention methods and intensity settings, leading to variations across different studies.

HIIT

HIIT is a training method that alternates short bursts of high-intensity exercise with low-intensity recovery periods. This approach is highly effective in enhancing cardiorespiratory fitness, increasing muscular endurance, and promoting fat oxidation. Due to its high-intensity nature, HIIT results in a rapid production of lactate that exceeds its removal, leading to a significant accumulation of lactate in both the blood and muscle tissues.²⁸ Research indicates that accumulating longer training sessions at intensities close to VO₂ max (typically ≥90% of VO₂ max) is most effective for maximizing aerobic adaptation capacity.²⁹ A population study indicates that male college students with a resting blood lactate concentration of 1.91 ± 0.20 mmol/L can reach a peak blood lactate concentration of 14.16 ± 1.79 mmol/L following HIIT.³⁰ During HIIT in mice, the use of dichloroacetate (DCA) to inhibit lactate production resulted in a significant decrease in the lactylation levels of subcutaneous inguinal white adipose tissue (iWAT), confirming that lactate produced during HIIT is a key factor inducing its lactylation.²⁶ Huang et al. found that after a single session of HIIT, the blood lactate concentration in mice could exceed 5 mmol/L (while the control group remained at 2 mmol/L), and was significantly higher than that of the control group within 5 min.³¹ This significance disappeared after 15 min, and lactylation was induced in various tissues of the mice, including the iWAT, brown adipose tissue (BAT), soleus muscle, and liver, which are metabolically active in lactate uptake. The study indicated that the energy expenditure in mice remained elevated for 24 h post-HIIT, coinciding closely with the peak lactylation time, suggesting that lactylation may be one of the molecular mechanisms driving excess oxygen consumption after exercise. Lactylation levels peaked within 24 h after HIIT and returned to baseline by 72 h, highlighting the dynamic nature of lactylation as a regulatory mechanism responding to metabolic demands during and after exercise. Lei et al. examined long-term HIIT in aged mice with in vitro lactate intervention and found that the hippocampal region sampled 48 h after the last intervention was unaffected by lactylation.³² This further suggests the time and tissue specificity of lactylation when HIIT is used as an exercise intervention.

AE

AE is a prevalent type of exercise intervention characterized by prolonged durations during a single session, typically manifested in forms such as platform running, swimming, or cycling. The maximal lactate steady state (MLSS) is defined as the highest exercise intensity at which blood lactate concentration remains stable, generally corresponding to 70%–80% VO₂ max.³³ When exercise intensity is below the MLSS, the production and clearance of lactate achieve a dynamic equilibrium, thus maintaining a constant blood lactate concentration. However, as exercise intensity escalates, a greater number of skeletal muscles engage in contraction, resulting in a relatively hypoxic state within the body. This condition increases the reliance on glycolysis, leading to a rapid elevation of lactate concentration in the internal environment.³⁴ Consequently, researchers often employ moderate to high-intensity AE, such as MLSS exercise intensity, fixed-time treadmill running at 16–18 m/min (for mice), and exhaustive force tests to effectively elevate circulating blood lactate levels for the investigation of exercise-induced lactylation. In the population, long-term AE training can improve VO₂ max and endurance performance. Untrained individuals experience an increase in muscle lactate and blood lactate concentrations at approximately 60%–65% VO₂ max, while endurance athletes need to reach 80%–90% VO₂ max.³⁵ However, some studies have also found that low-intensity AE (10 m/min treadmill exercise) can significantly increase serum, liver, and skeletal muscle lactate levels, reversing the significant decline in lactate levels caused by chronic restraint stress (CRS), and promoting the lactylation process of synaptic-related protein 91 (SNAP91) in the medial prefrontal cortex (mPFC).³⁶ Furthermore, Xu et al. discovered that a continuous swimming training regimen lasting 4 weeks reduced lactate levels in myocardial tissues of mice, concomitantly downregulating lactylation levels.³⁷ Prolonged swimming training was also found to enhance cardiac function and improve the contractility and pumping efficiency of cardiomyocytes. This improved functionality contributes to more efficient blood transportation and metabolic waste removal, including lactate clearance. These findings suggest that exercise-induced lactylation is closely related to the specific mode and intensity of exercise.

RT

RT builds muscle strength and volume primarily through the application of external resistance, such as dumbbells, barbells, or resistance bands. The intensity and duration of RT can be tailored to meet specific training goals; however, it typically involves prolonged muscle contractions and higher muscle loads. In current research, RT is typically studied using human subjects, with a notable lack of animal models. In rodent studies, bilateral synergistic muscle resection is often employed to induce gastrocnemius hypertrophy, thereby simulating the effects of RT. In investigations exploring the role of RT in promoting muscle hypertrophy, the accumulation of lactate produced during RT is referred to as “metabolic stress.”³⁸ Some researchers suggest that the mechanical tension response pathways and metabolic stress during RT may have a synergistic effect.³⁹ Although some articles indicate that training intervals in RT can minimize lactate production,⁴⁰ RT can still significantly elevate lactate levels, particularly during high-intensity, multiple-set training sessions.⁴¹ Mattingly et al. conducted a single session of RT followed by a continuous 6-week RT regimen, performing quadriceps muscle biopsies.^42,43 In a separate experiment, mice underwent surgical removal of half of the gastrocnemius muscle and the entire soleus muscle to induce plantar muscle hypertrophy, and were sacrificed after 10 days. The results indicated that while a single session of RT significantly increased blood lactate levels (12.1 ± 0.8 mM vs. 1.7 ± 0.8 mM, p < 0.001), there was no significant lactylation of nuclear or cytoplasmic proteins. Moreover, the 6-week RT did not lead to significant lactylation of cytoplasmic proteins (nuclear protein data were not available). The 10-day simulated RT also failed to induce lactylation of cytoplasmic proteins and instead reduced the level of lactylation in nuclear proteins. Furthermore, no other studies on lactylation in the context of RT were found. Considering the tissue-specific nature of lactylation, the lactate uptake metabolism in human quadriceps and mouse soleus muscles may not be active enough to produce significant lactylation. Additionally, overload-induced skeletal muscle hypertrophy is associated with an increase in acetylation levels,⁴⁴ which may competitively reduce lactylation sites on lysine residues. Therefore, there is still a considerable amount of related work that remains to be perfected, and RT continues to be a valuable intervention for studying lactylation in skeletal muscle.

Physiological functions of exercise-induced lactylation

Exercise-induced lactylation mediates exercise adaptation by regulating three core dimensions: metabolic regulation, cell signaling, and gene expression. This section explores its unique, interconnected roles in exercise adaptation based on existing research. Metabolic regulation represents the most direct level of action, as lactylation originates from lactate—the end product of glycolysis—and bidirectionally modulates key enzymes in glycolysis, oxidative phosphorylation, and lipid metabolism,^25,45,46 establishing timely feedback to finely tune energy homeostasis. Cell signaling constitutes an intermediate layer, where lactylation acts as a molecular switch in receptor-mediated signaling pathways and intracellular signaling cascades. This layer integrates mechanical and metabolic stimuli, including calcium flux and pathways such as mitogen-activated protein kinase (MAPK) and NF-κB,^19,47,48 to coordinate rapid adaptive responses. Gene expression regulation constitutes the epigenetic regulatory layer, which reprograms chromatin accessibility through histone lactylation and establishes long-term transcriptional memory.⁴⁹ These three dimensions from immediate metabolic feedback to sustained transcriptional remodeling collaboratively orchestrate the systemic benefits of exercise, ensuring that acute metabolic challenges translate into enduring functional improvements in muscle, brain, and cardiovascular tissues.

Metabolic regulation

Regulation of glycolysis

During exercise, particularly in HIIT and AE, muscle cells generate significant amounts of lactic acid through glycolysis. The production and accumulation of lactate not only reflect the activity of glycolysis but also induce the protein lactylation through various mechanisms, which subsequently feedback to regulate the glycolytic process. Research has demonstrated that lactylation of PKM2 enhances the stability of its tetrameric form and inhibits its transition from tetramer to dimer. In contrast, the tetrameric form of PKM2 exhibits high enzymatic activity and efficiently converts phosphoenolpyruvate (PEP) to pyruvate, thereby diminishing the accumulation of glycolytic intermediates.²⁵ Furthermore, studies indicate that increased PKM2 activity promotes the de novo synthesis of oxaloacetate (OAA), which inhibits the Warburg effect in the glycolytic pathway and reduces lactate production.⁵⁰ Consequently, during exercise, the accumulation of lactic acid may enhance the activity of glycolytic enzymes via lactylation, thereby accelerating the glycolytic process and providing a rapid energy supply to the cells.

OXPHOS

During moderate to high-intensity exercise, the supply of oxygen to muscle cells increases, yet localized hypoxia may still occur. On one hand, under exercise-induced hypoxic conditions (i.e., high-intensity exercise exceeding oxygen supply capacity), skeletal muscle cells inhibit regulation of oxidative phosphorylation (OXPHOS) due to insufficient oxygen as the terminal electron acceptor in the mitochondrial electron transport chain. At this time, cells rapidly upregulate glycolytic metabolism to meet energy demands, as this anaerobic pathway can produce ATP more efficiently without relying on oxygen. This metabolic shift from oxidative phosphorylation to glycolysis is a key adaptive response of the body to hypoxic stress, ensuring an immediate energy supply for muscle contraction.⁵¹ On the other hand, under exercise-induced hypoxic conditions, lactate is transmitted between cells via the “lactate shuttle” mechanism. Once inside the mitochondria, lactate is oxidized to pyruvate, which then enters the tricarboxylic acid cycle and the electron transport chain, supporting oxidative phosphorylation to enhance energy supply.⁵² The pyruvate dehydrogenase complex (PDHc), with PDHA1 as its catalytic subunit, is crucial for converting pyruvate into acetyl-CoA, which serves as a substrate for OXPHOS.⁵³ Carnitine palmitoyltransferase 2 (CPT2) facilitates the transport of long-chain fatty acids into the mitochondrial matrix for β-oxidation and is a key enzyme in FAO, an essential energy source for skeletal muscle.⁵⁴ Mao et al. demonstrated that hypoxia induced by high-intensity endurance running leads to the accumulation of mitochondrial alanine tRNA synthetase (AARS2).⁴⁵ This accumulation results in PDHA1 K336 lactylation and CPT2 K457/8 lactylation, inactivating both enzymes and thereby inhibiting OXPHOS by limiting the influx of acetyl-CoA from pyruvate and fatty acid oxidation. Additionally, the exercise-induced elevation of lactate, mediated through AARS2 lactate, restricts OXPHOS and contributes to the prevention of cellular hypoxia and oxidative stress injuries resulting from excessive oxygen consumption. Furthermore, the study revealed that the expression of the “eraser” sirtuin-3 (SIRT3) increased during training, suggesting its role in training adaptation.⁴⁵

Regulation of lipid metabolism

Lipids serve as a crucial fuel source during exercise, and exercise also exerts a modulatory effect on lipid metabolism. A substantial body of literature illustrates the bidirectional relationship between exercise and lipid metabolism.⁵⁵ Studies on lactylation provide new insights into the regulation of lipid metabolism by exercise. Huang et al. reported that lactylation levels in mouse iWAT and BAT were significantly elevated 24 h post HIIT.³¹ Quantitative proteomics analysis revealed that lactylated proteins in iWAT were predominantly involved in the tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation pathways. This suggests that lactylation enhances the efficiency of lipid metabolism by remodeling energy metabolism networks. Furthermore, HIIT-induced lactate significantly increased the lactylation level of FASN in iWAT, inhibited the catalytic activity of FASN by altering its spatial conformation, and reduced the synthesis of palmitic acid (C16:0) and triglycerides. This process concurrently downregulated the activities of lipogenesis-related enzymes, such as ATP-citrate lyase (ACLY), resulting in a multi-level inhibition of lipid synthesis.²⁶ Jia et al. demonstrated that lactate mediates the lipokinase VPS34 K356/781 lactylation through the acyl transferase KAT5/TIP60.⁴⁶ This modification enhances the interaction between Vps34 lipokinase and beclin1 as well as autophagy-related gene Atg14L, subsequently improving the synthesis of phosphatidylinositol 3-phosphate (PtdIns(3)P). This enhancement promotes the formation of autophagic vesicles and facilitates lysosomal degradation. This mechanism is crucial in the mouse forced swimming model for maintaining skeletal muscle and mitochondrial homeostasis. Furthermore, lipophagy, a key pathway in autophagy, is responsible for the breakdown of intracellular lipid droplets. Through the lysosomal degradation of these droplets, lipophagy releases free fatty acids (FFAs), which serve as substrates for energy metabolism.⁵⁶ This process is particularly vital during periods of increased metabolic demands associated with moderate to high-intensity exercise.

Cell signaling

Receptor-mediated signaling

The exercise-induced lactylation regulates physiological functions by binding to specific receptors and activating downstream signaling pathways. Lactate, an exercise-induced metabolite, signals through the activation of G protein-coupled receptors (GPCRs). Released by skeletal muscle contraction during exercise, lactate acts on GPR81 (HCAR1) located on the surface of adipocytes and neurons via blood circulation. This action inhibits lipolysis and reduces inflammatory responses while promoting muscle hypertrophy.⁵⁷ The lactylation also influences cell signaling by modulating epidermal growth factor receptor (EGFR) activity. Wang et al. demonstrated that in an atherosclerosis model, exercise-induced endothelial methylation reader protein (MeCP2) K271 lactylation decreased the release of vascular inflammatory factors (e.g., VCAM-1, IL-6) by inhibiting epiregulin (EREG) expression and reducing EGFR phosphorylation levels, which subsequently inhibited the MAPK signaling pathway.⁴⁸ Upon entering the central nervous system, lactate activates HCAR1 in the hippocampus, upregulating the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF), thereby promoting neurogenesis and synaptic plasticity.¹⁹ Furthermore, lactate’s regulation of glycolipid metabolism is linked to metabolic sensing receptors (e.g., PPARγ) in the liver and muscle. High-intensity exercise or HIIT enhances mitochondrial function in both muscle and liver through the lactate-PPARγ-PGC-1α axis.⁴⁵

Regulation of intracellular signaling pathways

The ability of lactylation to influence the dynamic homeostasis of metabolism, inflammation, as well as mitochondrial function is evident. It achieves this through direct modulation of intracellular signaling molecule activity and transcription factor function, with a particular emphasis on intracellular enzymatic responses, transcriptional regulation, and epigenetic modifications. Exercise-induced lactate inhibits the NF-κB signaling pathway downstream of the toll-like receptor (TLR), reducing the release of pro-inflammatory factors IL-1β and TNF-α, while simultaneously promoting the expression of the anti-inflammatory factor Arg1. This process alleviates neuroinflammation and enhances cognitive function via the catalytic action of histone H3 lactylation in mouse microglia, a model for Alzheimer’s disease.¹⁹ Lei et al. conducted a long-term HIIT study with lactate intervention in aged mice, discovering that mice hippocampal protein lactylation enhances the interaction between peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and sirtuin 1 (SIRT1), activates mitochondrial biosynthesis-related genes (NRF1 and TFAM), and upregulates the expression of brain-derived neurotrophic factor (BDNF).³² This cascade improves metabolic homeostasis and neural plasticity through the intracellular NAD+/SIRT1 axis. Additionally, Chen et al. demonstrated that AE induces methyl-CpG binding protein 2 (MeCP2) lactylation in mouse macrophages, which protects atherosclerotic plaque stability by enhancing H3K36 trimethylation binding, increasing chromatin openness, inhibiting runt-related transcription factor 1 (RUNX1) transcriptional activity, and subsequently promoting the polarization of M2-type anti-inflammatory phenotypes.⁵⁸

Regulation of intercellular communication

Lactate is crucial for the regulation of systemic homeostasis and the maintenance of synergistic multi-organ functions via intercellular metabolite delivery. Transporting lactate between tissues allows for communication between different types of cells. Muscles produce lactate, which is picked up into circulation via monocarboxylate transporters (MCTs), subsequently taken up by the heart and the liver where it undergoes oxidation to produce energy. This is referred to as the “lactate shuttle” phenomenon.⁴⁷ Moreover, lactate acts as a myokine, mediating inter-organ communication through the circulation. Lactate released during skeletal muscle exercise inhibits lipolysis and reduces the secretion of pro-inflammatory factors by activating HCAR1 on the surface of adipocytes, while concurrently promoting hepatic gluconeogenesis. However, lactylation during exercise may lead to adverse health effects. Liu et al. found that excessive exercise-induced hepatic fibrosis may be associated with SH3 domain-containing 3 (SORBS3) lactylation in skeletal muscle cells.⁵⁹ The excessive accumulation of lactic acid in skeletal muscle triggers SORBS3 lactylation and liquid-liquid phase separation (LLPS), which, in turn, promotes the sorting of F box protein 2 (FBXO2) into lactosomes. This process can activate hepatic stellate cells through the MCL1–BAX/BAK signaling pathway, ultimately leading to liver fibrosis.

Regulation of gene expression

Exercise induces protein lactylation modifications in both histones and non-histone proteins by increasing lactate production, facilitating a multilevel regulation of gene expression. Specifically, exercise-induced lactate enhances lactylation modifications at histone H3K9, H3K14, and H3K18 sites through the mediation of lactyl-CoA, which is notably enriched in the promoter regions of genes (±3 kb from the transcription start site). This process directly activates genes involved in DNA repair and protein homeostasis, thereby counteracting skeletal muscle aging.⁴⁹ Furthermore, Chang et al. demonstrated that 6 weeks of moderate-intensity running training significantly reduced the lactylation of mitochondrial ATPase subunits (Mtatp8, Atp5mg, and Atp5po), which was accompanied by an increase in protein expression within mouse muscle.⁶⁰ Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the reduction in lactylation activated the OXPHOS and thermogenesis pathways, and multi-omics correlation analyses indicated that exercise significantly modified the 2-oxocarboxylate metabolism and sphingolipid signaling pathways, collectively enhancing the efficiency of energy metabolism.

The lactate-GPR81 axis synergizes with lactylation to regulate gene expression. Lactate activates GPR81 on the membranes of adipocytes and immune cells, inhibiting the cyclic AMP (cAMP)/PKA pathway, reducing lipolysis and FFA release, and improving insulin sensitivity.^61,62 Lactate enhances the transcription of anti-inflammatory genes (e.g., Arg1 and VEGF) through the acetyltransferase p300/CBP-mediated enrichment of histone H3 K18 lactylation in their promoter regions. Additionally, it inhibits the nuclear translocation of NF-κB, promotes M2 polarization of microglial cells, and attenuates neuroinflammation.^19,63 Lactylation has an important role in the metabolic-epigenetic-immune dialogue: exercise-induced elevation of lactylation directly remodels chromatin accessibility and activates repair genes (BRCA1 and Arg1) through histone lactylation (H3 K18 lactylation), while also optimizing metabolic pathways via non-histone lactylation (ATPase PKM2) and synergizing with GPR81 signaling to suppress inflammation. This multilevel regulation ensures the synchronous adaptation of gene expression and immune metabolism, jointly maintaining tissue homeostasis.

Exercise-induced lactylation and other PTMs

Existing research indicates that exercise-induced lactylation is connected to other PTMs, and these interactions are not random. Instead, they form a multi-layered and dynamic metabolic signaling network centered around lactylation. This section will analyze this network based on existing evidence at three progressive levels.

Metabolic substrate competition and synergy between lactylation and acylation modifications

Lysine acylation is under competitive pressure from shared precursor and modification site availability. Exercise enhances lactate production through glycolysis while concurrently depleting acetyl-CoA levels. An elevated concentration of lactate promotes the production of lactyl-CoA, which in turn competitively inhibits acetyl coenzyme A-dependent modifications. The p300/CBP acts as a shared “writer,” catalyzing both lysine acetylation (Kac) and lactylation.^64,65 Pyruvate is the end product of glycolysis, situated at the crossroads of cellular energy metabolism. Its fate determines the flow of the carbon skeleton, directly influencing the substrate supply for lactylation and acetylation.⁶⁶ Pyruvate itself does not directly interfere with lactylation, but the metabolic fork it occupies (LDH vs. pyruvate dehydrogenase complex [PDH]) serves as a critical regulatory node determining the relative abundance of intracellular lactate (lactylation precursor) versus acetyl-CoA (acetylation precursor). Exercise intensity is the primary factor determining this metabolic diversion. During moderate-to-low intensity AE, pyruvate predominantly enters the tricarboxylic acid (TCA) cycle; whereas during high-intensity anaerobic exercise, pyruvate is largely converted into lactate.⁶⁷ During exercise, intracellular concentrations of acetyl-CoA reach the micromolar (μM) range,⁶⁸ whereas intracellular concentrations of lactyl-CoA remain extremely low. One study detected lactyl-CoA levels in multiple tissues that were 20–350 times lower than acetyl-CoA levels in the same tissues.⁶⁹ This substantial concentration gradient implies that, from the perspective of substrate availability, the probability of p300’s active site being occupied by acetyl-CoA is significantly higher than that of lactyl-CoA. In macrophages, lactate accumulation induced by swimming results in an increased proportion of lactylation at the histone H3K18 site, supplanting the original acetylation modification and facilitating M2-type polarization.⁵⁸ Both succinyl-CoA and lactyl-CoA originate from intermediates of the TCA cycle.⁷⁰ The exercise-induced acceleration of TCA cycling may promote both modifications; however, the sites that are common to both may be mutually exclusive due to spatial site resistance or changes in charge. The enzymes responsible for demodification are similarly cross-regulated. HDAC1-3 and SIRT1-3 are capable of removing both acetylation and lactylation modifications.^14,71 SIRT3 activates PDHA1 and CPT2 through de-lactylation, thereby restoring OXPHOS function in muscle cell mitochondria following exercise.⁴⁵ Additionally, HDAC3 regulates MeCP2 lactylation levels, enhancing chromatin accessibility in a model of exercise-improved atherosclerosis.⁵⁸ During prolonged endurance exercise or ketosis, the liver produces large amounts of ketone bodies, among which β-hydroxybutyrate (BHB) can be converted into β-hydroxybutyryl-CoA. This drives a novel modification—lysine β-hydroxybutyrylation (Kbhb)—which bears a strong resemblance to lactylation. p300 utilizes the activated form of BHB, β-hydroxybutyryl-CoA, to modify lysine residues, with its “erasers” including HDAC1/2.⁷² Although direct experimental evidence for BHB acting as a competitive inhibitor of lactylation is currently lacking, the emergence of high BHB levels may exhibit greater “context-dependence” compared to the ubiquitous presence of acetyl-CoA. Lactylation and acylation modifications compete with and synergize with metabolic substrates, potentially exhibiting significant differences across various types of exercise.

Epigenetic cascades of lactylation and methylation

Research indicates that exercise-induced lactylation significantly reshapes the epigenetic landscape by modulating the activity of methylated reading proteins. Exercise enhances the lactylation on the K271 site of the MeCP2 protein, which facilitates its selective recognition and binding to the H3 K36 trimethylation-tagged promoter region of RUNX1. This interaction results in a decrease in H3 K36 trimethylation methylation levels and inhibits the transcription of proatherosclerotic genes. In the ApoE−/− mouse model, this cascade effectively suppresses the macrophage inflammatory response and diminishes arterial plaque formation.⁵⁸ Furthermore, Xiong et al. identified two lactylation modification sites, K281 and K345, within the zinc finger structural domain of methyltransferase-like 3 (METTL3).⁷³ Their findings suggest that lactylation is crucial for capturing target RNAs and for the N6-methyladenosine (m6A) modification of Janus kinase 1 (JAK1) mRNA. Additionally, histone H3 K18 lactylation binds to the promoter region of the RNA methylation reader YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2), thereby promoting YTHDF2 expression, which has negative health implications. Conversely, sustained exercise training has been shown to reduce local levels of lactate and lactylation, thereby indirectly influencing methylation levels.^37,74

Integration of lactylation and phosphorylation/ubiquitination signaling pathways

High-intensity exercise significantly increases phosphorylation levels in skeletal muscle.⁷⁵ During exercise-induced hypoxia, PDHA1 is inhibited by both phosphorylation and lactylation: PDK1 catalyzes the phosphorylation of serine residues (S232, S293, and S300) on PDHA1, thereby blocking the conversion of pyruvate to acetyl-CoA. Additionally, AARS2 catalyzes the lactylation of K336 on PDHA1, further reinforcing the inhibition of OXPHOS and prolonging muscular endurance.⁴⁵ This dual modification acts as a “metabolic brake,” preventing exercise-induced acidosis and the overproduction of reactive oxygen species (ROS). Furthermore, exercise induces lactylation of MeCP2 at K271, enhancing its binding affinity for H3 K36 trimethylation and silencing pro-inflammatory genes. In contrast, kinases such as STAT1 phosphorylate MeCP2, antagonizing the lactylation effect and dynamically regulating the inflammatory response.⁵⁸ Numerous studies indicate that high-intensity exercise alters proteasome activity and the skeletal muscle ubiquitinome, leading to a reduction in global ubiquitination.⁷⁶ Exercise-induced hypoxia activates Hypoxia inducible factor 1α (HIF-1α), whose stability is modulated by phosphorylation, ubiquitination, and lactylation, potentially interacting with other PTMs to enhance HIF-1α transcriptional activity and promote glycolytic gene expression.⁶⁵ Lactylation of the autophagy transcription factor TFEB at K91 inhibits its interaction with the E3 ubiquitin ligase WWP2, subsequently inhibiting ubiquitination and proteasomal degradation of TFEB, resulting in increased autophagic flux.⁷⁷ Moreover, in the brain tissue of Alzheimer’s disease (AD) patients, the accumulation of aberrant lactylation of hyperphosphorylated tauopathy (tau) protein, due to the Warburg effect, increases phosphorylation and cleavage while inhibiting ubiquitination, which may contribute to the progression of AD.⁷⁸

Lactylation as an integrative hub for metabolic, epigenetic, and stress signals

In summary, the core functions of lactylation within the network of PTMs can be encapsulated as: a metabolic signal sensor, an epigenetic effect amplifier, and a coordinator of stress responses. Firstly, lactylation directly responds to the most significant metabolic changes during exercise—the fluctuations in lactate levels—by competing with shared metabolic precursors (such as acetyl-CoA and succinyl-CoA) for substrates, as well as through enzymatic competition/synergy with shared “writer” enzymes (p300/CBP) and “eraser” enzymes (HDACs/SIRTs), forming a rapidly responsive “metabolic-modification” switch that provides immediate feedback on energy metabolic pathways (dual inhibition of PDHA1). Secondly, lactylation acts as a trigger for epigenetic reprogramming by directly modifying histones or regulating MeCP2 proteins, initiating downstream methylation and m6A RNA modification cascades that achieve long-term, stable regulation of gene expression programs. Finally, under cellular stress conditions (such as hypoxia and inflammation), lactylation deeply integrates with classical signal modifications like phosphorylation and ubiquitination, finely tuning the activity, stability, and function of key transcription factors HIF−1α,TFEB, and coordinating adaptive processes such as autophagy and inflammatory responses.

Exercise health benefits based on lactylation

The physiological functions of exercise-induced lactylation can be directly translated into systemic health benefits, as this modification regulates the activity of key proteins and signaling pathways essential for maintaining tissue homeostasis and functional integrity (Figure 3). From skeletal muscle performance to cardiovascular protection, lactylation serves as a molecular mediator that links the mechanical and metabolic stimuli of exercise to long-term health adaptations.

Figure 3.

Beneficial health effects of exercise-induced lactylation in the current study

This figure illustrates the beneficial health effects of exercise-induced changes in lactylation levels, which include but are not limited to, the following: improved cardiovascular function, inhibition of atherosclerosis, and heart failure; enhanced muscle contractile function and endurance performance; improved self-repair mechanisms and metabolic regulation; protection of the nervous system, enhanced cognitive function and stress resistance; as well as inhibition of liposynthesis, improved lipid metabolism, and participation in liver metabolic remodeling.

Exercise-induced lactylation improves muscle function

Exercise training has been shown to enhance muscle function by increasing muscle strength and endurance, promoting muscle growth and repair, and optimizing metabolic adaptations in muscle tissue.⁷⁹ In previously mentioned experiments, the levels of lactylation modifications in key muscle contractile proteins, such as mouse skeletal muscle protomyosin (TPM1) and myosin heavy chain (MYH1), were significantly upregulated after 6 weeks of sustained moderate-intensity running exercise. These lactylation modifications not only enhanced the protein expression of these contractile proteins but also significantly improved muscle contractile function and endurance performance.⁶⁰ In vitro experiments conducted by Dai et al. demonstrated that lactate upregulates the expression of neuraminidase 2 (Neu2) through histone H3K9 lactylation, thereby regulating metabolic and signaling pathways in muscle and activating muscle satellite cells, which are crucial for repair and regeneration following muscle injury.²¹ During strenuous exercise, muscle cells utilize ULK1 to promote lactate production through the activation of lactate dehydrogenase A (LDHA), which modifies Vps34 via lactylation. This modification enhances the lipokinase activity of VPS34, thereby promoting autophagy and endosomal-lysosomal transport.⁴⁶ In a recent study, Li et al. found that after a single bout of acute endurance exercise, lactate acts as a direct signaling molecule that lactylates mTOR at K921 in skeletal muscle, inhibiting the activity of the mTORC1 complex and relieving its phosphorylation suppression of the key autophagy initiation kinase ULK1 at the Ser757 site.⁸⁰ Meanwhile, lactylated mTOR also promotes the recruitment of the circadian protein PER2 and the negative regulatory complex TSC1/TSC2 to mTORC1, further consolidating its inhibitory state. These changes collectively “unlock” ULK1’s activity, thereby efficiently initiating the process of autophagy approximately 30 min post-exercise, clearing damaged cellular components such as mitochondria and endoplasmic reticulum, and recycling nutrients. This process aids muscle cells in self-repair and metabolic regulation following exercise, ultimately optimizing the metabolic adaptations of the muscle.

Exercise-induced lactylation improves cognitive functions

Exercise has been shown to enhance cognitive function through mechanisms such as neuroprotection, synaptic plasticity, and immune modulation.⁸¹ Specifically, exercise facilitates the reprogramming of microglia and elevates lactate levels in the mouse brain following moderate-intensity exercise. This increase in lactate induces lactylation of histone H3K18, which contributes to the transformation of microglia into an anti-inflammatory M2 phenotype, thereby reducing neuroinflammation.¹⁹ Furthermore, exercise inhibits the microglia NF-κB pathway via lactylation, leading to a decrease in the expression of interleukin—1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as the suppression of pro-inflammatory factors. These actions collectively mitigate neuroinflammation, protect neurons from inflammation-mediated injury, significantly reduce neuronal apoptosis, and increase the number of surviving neurons, ultimately improving neurological function.⁶³ Yan et al. discovered that the inhibition of synaptic protein SNAP91 lactylation function in the mPFC of mice leads to a synchronous decrease in the affinity of SNAP91 for the clathrin-AP180 complex (CALM), resulting in disrupted synaptic vesicle structure and reduced neuronal activity intensity in the mPFC.³⁶ However, a sustained moderate-intensity exercise regimen (10 m/min) for 2 weeks significantly elevates lactate levels in the mPFC, mediating the lactylation of SNAP91 at K885, thereby improving synaptic structure and stress resistance. Notably, the mechanism of cognitive enhancement mediated by lactylation exhibits spatiotemporal specificity across different brain regions: a study investigating the molecular dynamics of the hippocampus in mice within 24 h following acute high-intensity exercise revealed that, although lactate levels rapidly increased post-exercise, the overall lactylation levels of hippocampal proteins showed a widespread downregulation immediately after exercise. Concurrently, the transcriptional profiles of histone acetyltransferases/deacetylases underwent complex, time-dependent reprogramming.⁸² This suggests that the lactate surge induced by exercise may initially trigger a “reset” of the protein modification environment in the brain, laying the groundwork for precise, functional lactylation reprogramming of key cognition-related proteins (SNAP91) during specific time windows, such as the recovery phase. Therefore, exercise may enhance cognitive function through a lactate-mediated dynamic lactylation network, simultaneously strengthening synaptic structures in the prefrontal cortex and optimizing metabolic and epigenetic states in regions such as the hippocampus, thereby improving cognitive function from multiple dimensions.

Exercise-induced lactylation protects the cardiovascular system

Exercise plays a crucial role in the primary and secondary prevention of cardiovascular disease, safeguarding the cardiovascular system through various mechanisms, including the enhancement of vascular function, regulation of lipid metabolism, and improvement of cardiac adaptability.⁸³ Exercise-induced lactylation enhances vascular function by modulating signaling pathways in vascular endothelial cells. Wang et al. demonstrated that 8 weeks of moderate-to-high-intensity AE increased the level of lactylation modification of MeCP2 in the endothelial cells of mice with high-fat-induced atherosclerosis. This increase inhibited the expression of EREG and the activity of the EGFR/MAPK signaling pathway, leading to a reduction in inflammatory factor expression, improved endothelial function, and a slowdown in atherosclerosis progression.⁴⁸ Furthermore, HIIT-induced lactylation modification influences metabolic processes in adipose tissue, thereby affecting lipid metabolism.³¹ Exercise-generated lactic acid inhibits lipolysis and induces the browning of WAT by activating the GPR81 receptor in adipocytes, which increases thermogenesis and energy expenditure, ultimately improving lipid metabolism and reducing the risk of cardiovascular disease.^84,85 Additionally, lactylation enhances cardiac tolerance to ischemia-reperfusion injury. Four weeks of swimming training improved myocardial lactylation levels, optimized myocardial energy metabolism, increased myocardial tolerance to ischemia-reperfusion injury, and reduced myocardial injury in mice.³⁷ Moreover, Zhang et al. indicated that in patients with heart failure and in mice, lactylation of lactic acid via α-myosin heavy chain (α-MHC K1897) enhances its binding to titin, stabilizing myocardial structure and improving heart failure outcomes.⁸⁶

Exercise-induced lactylation improves metabolic health

Exercise enhances metabolic health by improving insulin sensitivity, promoting lipid metabolism, modulating metabolic enzyme activity, and optimizing metabolic pathways.⁸⁷ Exercise-induced lactylation presents novel insights for the prevention and treatment of metabolic diseases. The acute post-exercise lactate spike (8–12 mM) activates the AMPK/PGC-1α/GLUT4 axis, promoting glucose transport and thereby enhancing insulin sensitivity.⁸⁸ Lactate activates the GPR81 receptor in adipocytes, inhibiting the phosphorylation of hormone-sensitive lipase (HSL) and consequently reducing lipolysis. Furthermore, lactic acid induces the browning of WAT, which increases thermogenesis and energy expenditure, thereby improving lipid metabolism and obesity.⁸⁹ Chen et al. demonstrated that lactic acid following HIIT-induced lactylation of FASN in mouse adipose tissue, inhibited its enzymatic activity, and reduced triglyceride accumulation, thereby suppressing lipid synthesis.²⁶ Six weeks of moderate-intensity exercise downregulates K42 lactylation of mouse hepatic ALDOA (a key enzyme in glycolysis) and enhances glycolysis; it also decreases K273 lactylation of the fatty acid β-oxidase complex subunit (HADHB), promoting fatty acid β-oxidation, which is involved in metabolic remodeling in the liver.²⁷ Additionally, it upregulates the level of the metabolite sphingosine, which positively correlates with the lactylation modification of relevant metabolic enzymes, thereby inhibiting sphingolipid accumulation, maintaining muscle fiber homeostasis and delaying muscle degeneration.⁶⁰

To provide a more intuitive overview of the current state of research on the health effects of exercise-induced lactate accumulation, all relevant studies have been compiled into a single table, as shown in Table 1.

Table 1.

Lactylation in exercise adaptations

Study	Population	Physical activity	Tissue	Testing time point	Lactylation targets	Health impact
Han et al.19	AlCl3/D-gal-induced AD-like mice	treadmill running, 5 days/week for 8 weeks. Speed: 16 m/min for 40 min (after warm-up), increased by 1 m/min weekly	hippocampus, prefrontal cortex	after 8 weeks of exercise	histone H3↑	improved cognitive function, reduced neuron loss, increased reparative microglia
Han et al.19	aging mice (4-, 15-, 20-months old)	treadmill running, 5 days/week for 8 weeks. Speed: 70% of the weekly tested maximum running speed	hippocampus, prefrontal cortex	after 8 weeks of exercise	histone H3↑	improved learning and memory, attenuated age-related neuron loss
After 8 weeks of exercise Wu et al.20	ovariectomized (OVX) mice (postmenopausal osteoporosis model)	high-intensity interval treadmill running, 5 days/week for 8 weeks. Each session: 8 min at 5 m/min, then 10 intervals of high-intensity running (12–16 m/min for 5 min) with 2 min rest. Incline: 10°.	distal femur, blood serum	after 8 weeks of exercise	histone H3↑	increased bone mineral density (BMD) and bone volume/total volume (BV/TV), attenuated osteoporosis
Chen et al.26	C57BL/6 male mice (control, HIIT, DCA+HIIT groups)	HIIT, 5 days/week for 8 weeks. Protocol: after warm-up, 9 intervals of running at 85% of maximal speed for 1.5 min, interspersed with 2 min of active rest at 45% of maximal speed	iWAT, also liver, heart, etc.	after 8 weeks of exercise intervention	FASN↑	HIIT reduced body fat mass; lactate-induced FASN lactylation inhibited FASN activity and de novo lipogenesis
Wang et al.27	healthy adult C57BL/6 J male mice (8 weeks old)	moderate-intensity treadmill running, 5 days/week, 60 min/day, 12 m/min, 5° incline for 6 weeks	liver	within 24 h after the last exercise session	ALDOA_K42 ↓, HADHB_K273 ↓, Acsl1_K676 (↓, No significant change)	promotes fatty acid metabolism and glycolysis; modulates hepatic immune microenvironment
Huang et al.31	male C57/BL6 mice	single bout of HIIT (9 sets of high-intensity intervals)	iWAT, BAT, eWAT, soleus muscle, gastrocnemius muscle, heart, liver, brain	0, 2, 24, 48, and 72 h post-exercise	iWAT pan-Kla ↑ (24 h), BAT pan-Kla ↑ (0, 2, 24 h), soleus pan-Kla ↑ (24 h), liver pan-Kla ↑ (24 h)	may enhance post-exercise energy expenditure, lipid metabolism, and metabolic remodeling in adipose tissue
Lei et al.32	aged male C57BL/6 J mice (25–27 months)	HIIT for 6 weeks (10 cycles of 3 min at 85% max speed, 2 min at 45% max speed)	hippocampus, hemisphere	48 h after the last intervention	pan lactic acid-lysine: no significant change	improved performance in cognitive and behavioral tests; enhanced hippocampal angiogenesis, mitochondrial biomarkers (SDHA), and metabolic pathways.
Yan et al.36	adolescent C57BL/6 J mice (4–5 weeks old)	14 days of treadmill exercise, 1 h daily at 10 m/min	medial prefrontal cortex (mPFC)	after the 14-day intervention period	SNAP91 K885 ↑	confers stress resilience and prevents anxiety-like behaviors by improving synaptic structure and function
Xu et al.37	male C57BL/6 J mice	4-week swimming training (up to 90 min/day)	heart tissue	1, 2, 3, 4 weeks of training	H3K18 ↓; YTHDF2 ↓	induced physiological cardiac hypertrophy; improved cardiac function; lowered heart rate
Mattingly et al.42,43	10 trained college-aged men	acute bout: 4 sets of back squats +4 sets of leg extensions to failure at 30% 1RM	vastus lateralis muscle	pre, 3 h post, 6 h post exercise	cytoplasmic and nuclear protein lactylation (no significant change)	acute resistance exercise significantly increased blood lactate but did not alter muscle protein lactylation
Mattingly et al.42,43	14 trained college-aged men	6-week unilateral leg training (3×/wk): Single-leg leg press and extension, volume progression from 5 to 10 sets of 10 reps at 60% 1RM	vastus lateralis muscle	pre and post (6 weeks)	cytoplasmic protein lactylation (no significant change)	6 weeks of RT induced muscle hypertrophy but did not alter cytoplasmic protein lactylation
Mattingly et al.42,43	16 C57BL/6 mice (5-month old)	10-day plantaris mechanical overload via synergist ablation surgery	plantaris muscle	after 10 days of overload	cytoplasmic protein lactylation (no significant change); nuclear protein lactylation ↓	mechanical overload induced significant muscle hypertrophy but did not increase cytoplasmic protein lactylation; nuclear protein lactylation was significantly decreased.
Mao et al.50,45	C57BL/6 mice (wild-type, Aars2−/−, Sirt3−/−, muscle-specific AARS2 OE)	high-intensity running to exhaustion on treadmill (speed up to 20 m/min, then incline increased 5° every 5 min)	leg skeletal muscle (whole, type 1, type 2 fibers)	pre-run (0 min), during run (5, 10, 15, 20, 25, 30 min), exhaustion	PDHA1 K336 ↑, CPT2 K457/8 ↑	regulates endurance exercise capacity; protects against ROS damage; feedback inhibits OXPHOS
Jia et al.46	C57BL/6 mice	acute swimming exercise for 3 h in 35°C water	skeletal muscle	immediately after 3 h of swimming	VPS34 K356/K781 ↑	promotes cell autophagy to maintain muscle homeostasis
Wang et al.48	C57BL/6 J mice (wild-type)	treadmill running, 60 min/day, 5 days/week, 18 m/min, for 8 weeks	aortic tissue, plasma	after 8 weeks of exercise training	MeCP2k271↑	suppressed atherosclerosis development
Chen et al.58	ApoE−/− mice (atherosclerosis model)	treadmill running, 60 min/day, 5 days/week, 18 m/min, for 8 weeks	aortic root plaque, isolated plaque macrophages, serum	after 8 weeks of exercise training	MeCP2 K271 ↑	reduced plaque area, increased plaque stability, promoted M2 macrophage polarization
Chen et al.58	patients with stable coronary artery disease	moderate-intensity AE, 30–60 min/day, 3–5 days/week, for 12 weeks	peripheral blood mononuclear cells (PBMCs)	at week 12 of exercise program	MeCP2 K271la ↑	associated with atheroprotective epigenetic changes
Liu et al.59	C57BL/6 J mice	treadmill running, 12 weeks. Overtraining: progressive increase to 25 m/min for 60 min, then increased daily frequency in final 4 weeks	serum, liver, muscle, heart, kidney	after 12-week training	not mentioned initially	induced liver fibrosis, collagen deposition, hepatocyte apoptosis, inflammation
Liu et al.59	C57BL/6 J mice	same 12-week overtraining protocol	muscle, serum, liver	after training	SORBS3 lactylation (↑ in muscle)	muscle lactate-induced SEVs (“lactate bodies”) promote liver fibrosis via FBXO2/MCL1 pathway.
Meng et al.49	10-month-old middle-aged C57BL/6 mice	treadmill running at 9 m/min for 30 min per session, for 2 months (with 2–3 days rest after each session).	gastrocnemius muscle	after 2 month training	H3K9 ↑, H3K14 ↑, H3K18 ↑	reduced SA-β-gal+ cells; enhanced DNA repair and proteostasis pathways; improved muscle function.
Chang et al.60	adult male C57BL/6 J mice	moderate-intensity treadmill running; 60 min/day, 5 days/week for 6 weeks	hind leg muscle tissue	24 h after the last training session	Mtatp8 ↓, Atp5mg ↓, Atp5po ↓	may enhance energy metabolism by regulating the oxidative phosphorylation pathway.
Li et al.80	male C57BL/6 J mice; mL DHA KO mice; mTOR K921R mice	acute endurance exercise on treadmill (10° incline); speed gradually increased until exhaustion.	gastrocnemius muscle	after acute exercise	mTOR K921 ↑	promotes autophagy, maintains muscle metabolic homeostasis, and contributes to exercise adaptation
Qian et al.82	male C57BL/6 J mice (15–16 weeks old)	a single bout of high-intensity treadmill running for 60 min (5° incline); speed increased from 6 m/min to 16 m/min	hippocampus	0, 0.5, 1, 4, 7, 24 h post-exercise	total protein lactylation ↓ (at 0 h)	may influence hippocampal functional adaptation and physiological homeostasis

Although current research predominantly describes lactylation characteristics at the tissue level, most studies rely on homogenate analysis, yielding average signals across multiple cell types. The high cellular heterogeneity within tissues implies that different cell types may exhibit distinct lactylation responses to exercise-induced lactate accumulation. For instance, in skeletal muscle, type 2 fibers—characterized by rapid glycolysis—may exhibit significantly higher lactylation levels than oxidative type 1 fibers due to increased lactate production.³¹ Similarly, infiltrating macrophages or activated satellite cells may possess distinct lactylation profiles, regulating inflammatory responses and regenerative processes, respectively.⁵⁸ In adipose tissue, beige/brown adipocytes, preadipocytes, and adipose tissue macrophages (ATMs) exhibit distinct metabolic responses to exercise,^90,91 with their lactylation modifications potentially linked to specific functions such as thermogenesis, differentiation, and immune polarization. However, no studies have yet directly mapped cell type-specific patterns of lactylation within tissues post-exercise using single-cell proteomics (e.g., scCUT&Tag, single-cell mass spectrometry) or spatial proteomics technologies (e.g., matrix-assisted laser desorption/ionization mass spectrometry imaging [MALDI-MSI], 2-Hydroxypent-4-ynoic acid [YnLac] probe imaging). This knowledge gap limits our understanding of the precise functional role of exercise-induced lactylation within the cellular microenvironment.

Applications of lactylation modifications in the health field

In recent years, the potential of lactylation modifications in exercise physiology, as well as in disease prevention and treatment has been extensively explored. These modifications not only serve as a foundation for exercise prescription but also present new targets for pharmacological interventions.

Exercise prescription based on lactylation modification

Personalized exercise prescription involves the development of a tailored exercise regimen based on an individual’s genetic background, metabolic status, and health goals. In the current study, the induction of lactylation modifications through exercise demonstrated a significant dependence on intensity, with various exercise patterns modulating specific molecular targets via differential lactate dynamics. Although human evidence directly linking specific exercise regimens to distinct lactylation modification profiles is still accumulating, the solid mechanistic insights provided by preclinical models lay the foundational principles for its translation to human applications. The core principle that varying lactate dynamics can trigger diverse lactylation modification characteristics has been supported by cross-tissue analyses in rodents. To translate this principle into human applications, it is necessary to map the exercise intensities proven effective in models onto established human physiological benchmarks.

Under HIIT conditions (≥85% VO₂ max), such as treadmill exercise (4 × 4 min at 90% VO₂ max with 3 min of active recovery) or cycling (8 × 1 min all-out sprints with 1 min rest intervals) peak circulating blood lactate levels can reach 8–12 mM. This leads to modifications of target molecules such as CNDP2, FASN, and PDHA1, consequently improving obesity/metabolic syndrome (BHB-Phe-mediated appetite suppression).^26,45,60,92 When applied to human populations, exercising at or above the individually determined lactate threshold or critical power/speed can reproduce this high lactate state and may be preferentially used to target similar metabolic pathways.

In contrast, moderate intensity continuous training (MICT) (60%–75% VO₂ max) exemplified by sustained treadmill exercise (40 min at 60% VO₂ max) or uniform swimming (30 min/repeat), maintains steady-state lactate levels of 3–5 mM and modifies target molecules such as H3K18, SNAP91, and α-MHC. This approach plays an important role in enhancing neurodegenerative disease outcomes and providing cardiovascular protection.^19,36,63,86 When applied to human populations, this corresponds to exercise intensity maintained between the aerobic threshold and the lactate threshold—the classic intensity range for traditional AE.

An individual’s lactate threshold is influenced by factors such as gender, age, exercise capacity, and health status.^93,94 In middle-aged adults, studies have shown that after exhaustive exercise, among individuals matched by age and MHR, the plasma lactate concentration at the third minute of recovery ([la-]p, 3 min) in females is approximately 2.5 mmol/L lower than that in males. This indicates a significant gender difference in the dynamics of lactate accumulation following high-intensity exercise.⁹⁵ The lactate threshold (LT) test—favoring submaximal intensity compared to the 6 min walk test (6 MWT)—is an effective tool for assessing VO2 max in elderly males; its application in physical activity programs for this population can enhance the quality of aerobic training. Notably, research indicates that the corresponding blood lactate concentration for LT in elderly males aged 61–77 is less than 1.5 mmol/L, which is lower than the resting blood lactate concentration in younger males. In maximal cardiopulmonary testing, the average blood lactate concentration is 6.6 ± 1.7 mmol/L, which is only half that of untrained younger males (113.6 ± 0.9 mmol/L).^96,97 Therefore, it may be advisable to reduce MICT to below 60% VO₂ max or to engage in low-intensity AE, which can induce therapeutic lactate accumulation effects while avoiding excessive metabolic burden.

Patients with chronic diseases, particularly those with type 2 diabetes and obesity, commonly exhibit metabolic disorders, especially abnormalities in glucose and lipid metabolism and insulin resistance, which directly impact the balance between lactate production and clearance.⁹⁸ Multiple studies consistently demonstrate that compared to healthy individuals of normal weight, skeletal muscle in obese and/or insulin-resistant individuals exhibits significantly higher levels of protein lactylation and blood lactate levels at rest.⁹⁹ For populations with chronic metabolic disorders, exercise prescription goals should shift toward normalizing lactate metabolism and lactylation dynamics. Metabolic adaptation is a long-term process, requiring exercise prescriptions to emphasize regularity and consistency. Exercise guidelines established by authoritative bodies such as the American Diabetes Association, American College of Sports Medicine, and European Association for Prevention of Cardiovascular Disease recommend maintaining at least 150 min per week of moderate to vigorous intensity AE.¹⁰⁰ For exercise, beginners or patients with poor physical condition, MICT is a safer and more feasible starting option. Training near LT is a classic method for improving aerobic capacity and lactate clearance efficiency.¹⁰¹ It is recommended to perform 30–60 min of MICT per session, maintaining intensity at 60%–75% VO₂ max. Gradually elevating LT through training enables patients to maintain steady-state lactate production at higher exercise intensities, which is crucial for controlling overall lactate load. When selecting HIIT as a strategy to improve lactate metabolism and mitochondrial function in individuals with metabolic disorders, greater attention must be paid to recovery time. While ensuring peak intensity (≥85% VO₂ max) to guarantee exercise efficacy, appropriately extend rest intervals between sets and recovery time between training days. Additionally, low-volume HIIT (LOW-HIIT) is recognized for its significant benefits in improving metabolic syndrome. Patients can achieve this through 5 × 1 min cycling sessions at 95%–105% LT intensity, thereby avoiding the metabolic stress associated with high-intensity exercise.¹⁰² Given substantial individual variability, exercise prescriptions require high personalization and dynamic adjustment based on feedback.

Potential for targeted drug applications based on lactylation modifications

The target ability of lactylation as a core mediator of exercise benefits presents novel therapeutic opportunities for metabolic and degenerative diseases. Potential drug development strategies can be categorized based on their mechanisms of action into three main approaches: modulators of lactate metabolism, direct modulators of lactylation enzymes, and combination therapy strategies. Lactate metabolism modulators indirectly influence lactylation levels by regulating lactate production or clearance. For instance, GPR81 receptor agonists, such as 3,5-dihydroxybenzoic acid (3,5-DHBA), can mimic the lactate-GPR81 signaling axis following exercise, activating the AMPK/PGC-1α pathway, which enhances insulin sensitivity, reduces inflammation in adipose tissues, and improves insulin resistance.¹⁰³ Additionally, pyruvate dehydrogenase kinase inhibitors, such as DCA, can decrease lactate accumulation and reverse the inhibition of lactylation of mitochondrial ATP synthase in heart failure by activating the PDHc.^86,104 Recent studies have identified natural compounds such as chlorogenic acid (CGA) and gallic acid (GA) as regulators of lactic acid metabolism, which can ameliorate high-fat-induced mitochondrial dysfunction and insulin resistance.^105,106 Direct modulators of lactylases can specifically target the “writers” or “erasers” of lactylation modifications. For instance, p300/CBP agonists may mitigate neuroinflammation and enhance cognitive function by promoting H3K9 lactylation in microglia.^19,107 Although p300/CBP plays multiple roles in histone acylation, its agonists do not necessarily activate every type of acylation activity simultaneously. Research has demonstrated that CTPB (N-[4-chloro-3-trifluoromethyl-phenyl]-2-ethoxy-benzamide) and short-chain fatty acids (e.g., propionic acid, butyric acid) promote histone acetylation by altering the p300/CBP protein structure or catalyzing its acetylation, thereby activating its activity.¹⁰⁸ However, agonists that specifically target and enhance p300/CBP lactylation activity remain to be identified. In contrast, histone deacetylases, such as HDACs and SIRTs, can enhance lactylation activity and reverse the pro-carcinogenic effects mediated by lactylation under pathological conditions, indicating their potential in glioma therapy.^14,109 Combination treatment strategies may involve lactylation modulators paired with existing drugs or synergistic interventions combining exercise and medication. For example, a SIRT3 agonist (which activates OXPHOS) combined with metformin synergistically improves mitochondrial function in Type 2 Diabetes Mellitus.^45,110 Additionally, lactylation inhibitors in conjunction with immune checkpoint inhibitors (e.g., PD-1 antibodies) can synergistically enhance antitumor effects.^111,112 Furthermore, preoperative exercise paired with sodium lactate injections can synergize anti-tumor effects by enhancing lactylation in small glial cells, potentially accelerating the recovery of motor function after spinal cord injury.⁶³ Currently, research on exercise-induced lactylation is underdeveloped. Therefore, it is essential to create targeted carriers, such as nanoparticles to prevent systemic administration from disrupting normal physiological lactylation dynamics. Furthermore, future studies should utilize single-cell lactylomics to accurately analyze the spatial-temporal effects of pharmacological interventions.

Clinical translation challenges and prospects for lactylation detection

Currently, research on lactylation, particularly the discovery of new sites and global analysis, relies almost entirely on mass spectrometry-based proteomics techniques, especially liquid chromatography-tandem mass spectrometry (LC-MS/MS).¹¹³ High-resolution mass spectrometry platforms such as Orbitrap and time-of-flight mass spectrometry (TOF-MS) enable precise identification of acylated proteins and their modified lysine residues, providing unparalleled throughput and specificity for exploratory research.^114,115 Recent technological breakthroughs, including direct ion acquisition (DIA) and ion mobility spectrometry (IMS) technologies, have significantly enhanced the analytical depth and reproducibility of low-abundance modified peptides.^116,117 However, the inherent requirements of mass spectrometry-based workflows: specialized instruments, multi-step sample preparation, and extended analysis times—remain major obstacles for routine clinical or point of care testing applications.¹¹⁸ Therefore, while mass spectrometry remains irreplaceable in the discovery and validation phases, its clinical role may still be confined to reference methods. This underscores the necessity of developing complementary technologies tailored to clinical workflows.

Beyond traditional mass spectrometry, spatial analysis techniques offer unique opportunities to address challenges in analyzing large tissue samples. MALDI-MSI enables simultaneous imaging of thousands of molecules—including metabolites, lipids, and peptides—directly on tissue sections without labeling.¹¹⁹ This technique holds a traditional advantage in visualizing small molecules like lactate, directly revealing regional metabolic heterogeneity.¹²⁰ In exercise research, MALDI-MSI can reveal the spatial gradient distribution of lactate accumulation within muscle tissue, such as whether it concentrates near glycolytic muscle fibers or exhibits differential distribution based on capillary density.

However, applying MALDI-MSI technology to detect protein lactylation presents challenges. Lactylation must be detected on peptide fragments following proteolytic digestion, a process that disrupts spatial integrity. Emerging strategies are overcoming this limitation: chemical probes such as YnLac enable metabolic labeling, followed by visualization through click chemistry techniques.¹²¹ Combining immunohistochemical localization with laser capture microdissection, supplemented by LC-MS, integrates spatial information with molecular specificity. Multiple ion beam imaging (MIBI) using validated anti-pan-lactyl antibodies also holds promise for achieving subcellular resolution in lactylation localization.¹²²

These spatial technologies offer unique advantages in addressing critical questions: does exercise establish spatial gradients of lactate within the tissue microenvironment? Is lactate signaling enriched at specific cellular interfaces such as neuromuscular junctions, satellite cell microenvironments, or macrophage-adipocyte contacts? In pathological states like obesity, do spatial patterns of lactate remodeling occur, and can exercise reverse these alterations? Do different muscle fiber types exhibit distinct lactate signaling profiles after exercise?

For clinical translation, extending lactylation detection to peripheral blood is crucial. However, current studies have yet to report lactylated proteins or peptides in peripheral blood as established biomarkers. The protein composition of peripheral blood is extremely complex, with high-abundance proteins (such as albumin and immunoglobulins) accounting for over 99% of total protein mass, masking the presence of low-abundance modified peptides.¹²³ The lactylation process exhibits dynamic reversibility; sample processing may alter modification levels, thereby affecting the reproducibility of detection.¹²⁴

Therefore, attention can be shifted to other potential alternative or indirect biomarkers. For instance, measuring blood lactate levels—the direct precursor of lactate is the most readily achievable substitute indicator. In fact, blood lactate has already become a crucial marker for assessing tissue perfusion and prognosis in critical conditions such as sepsis and shock.¹²⁵ However, elevated blood lactate levels can occur in various physiological and pathological states, limiting its specificity. Therefore, combining it with other metabolites (such as the lactate/pyruvate ratio) is necessary to construct a more specific metabolic signature panel. Exosomes carry protein, nucleic acid, and metabolite information from their source cells, making them a hotspot in liquid biopsy research. Existing studies have focused on acetylation modifications of proteins within exosomes.¹²⁶ Theoretically, exosomes released by tumor cells or immune cells may contain characteristic lactylated proteins. Capturing exosomes expressing specific surface markers via immunological methods and subsequently detecting their internal lactylated proteins holds promise for enhancing specificity and sensitivity.

Looking forward, developing high-affinity recognition elements—diagnostic-grade monoclonal antibodies, nanobodies, or aptamers targeting lactylated lysine—will be essential for clinical translation. Coupled with advanced signal amplification (e.g., digital ELISA, surface-enhanced Raman scattering) and integrated into microfluidic platforms, these tools could enable point of care lactylation assessment.^127,128 Such advances would transform our ability to monitor lactylation dynamics in real time, opening new avenues for precision exercise prescription and therapeutic monitoring.

Discussion

Exercise induces systemic metabolic changes, through complex and diverse mechanisms. Studies on exercise-induced lactylation reveal novel epigenetic and metabolic regulatory dimensions that mediate the benefits of exercise through metabolites.

Exercise-induced lactylation serves as a crucial epigenetic and metabolic bridge linking exercise stimuli to systemic health adaptations. Previous studies have systematically elucidated how different exercise modalities (HIIT, AE, and RT) drive tissue-specific lactylation modifications by shaping differential lactate dynamics, thereby precisely regulating energy metabolic homeostasis, enhancing neuroprotective functions, optimizing cardiovascular adaptations, and improving overall metabolic health. The core evidence suggests that lactylation is not only a dynamic modification responding to the metabolic demands of exercise but also a key molecular switch mediating the long-term benefits of exercise. It directly modifies histones to regulate gene expression while also targeting key non-histone proteins in pathways such as glycolysis, lipid metabolism, and autophagy, playing a significant role in the metabolic-epigenetic-immune cross-talk.

Current research has yet to fully explore the multidimensional interaction networks induced by exercise. This field faces multiple challenges: at the fundamental level, there is a lack of spatiotemporally resolved single-cell dynamics of lactylation, with its upstream regulatory mechanisms and precise temporal dynamics remaining unclear At the translational level, research on lactate response patterns in specific populations with metabolic disorders remains limited. Current exercise prescriptions heavily rely on empirical intensity classifications (e.g., HIIT/MICT), necessitating the development of an actionable standardized protocol based on individual lactate metabolism characteristics and lactate biomarker feedback. Technologically, detection methods have yet to achieve clinical translation, and the functional causality of most modification sites remains to be validated. Future breakthroughs depend on applying cutting-edge single-cell and spatial omics technologies to decipher cell-type-specific lactylated landscapes, thereby establishing quantitative relationship models linking exercise intensity, lactate thresholds, and lactylation modifications. This will lay the theoretical foundation for precision prescriptions that transcend empirical approaches. Ultimately, developing real-time feedback systems for lactate metabolism monitoring and intervention strategies targeting lactylation pathways could transform exercise into a programmable health tool grounded in explicit molecular logic. This would mark a paradigm shift from “black-box” interventions to “lactylation-signal-guided precision exercise medicine.”

Acknowledgments

This work was supported by the National Natural Science Foundation of China (32171136 and 82572947), the Natural Science Foundation of Shanghai (19ZR1452900), and the Key Laboratory of Exercise and Health Sciences (Shanghai University of Sport), Ministry of Education. The authors thank the editors and anonymous reviewers for their valuable comments on the manuscript.

Author contributions

Conceptualization, writing – original draft, W.C.; writing – review and editing, W.C., Y.M., and Q.Z.; funding acquisition, resources, and supervision, R.S.

Declaration of interests

The authors declare no competing interests.