Lactate-mediated lactylation in human health and diseases: Progress and remaining challenges.

Alterations in the lactate level are a typical characteristic of the occurrence and development of many diseases. Previous studies have shown that lactate is a metabolic waste [23] , [64] . However, recent studies have shown that lactate can regulate the progression of various diseases through lactylation ( Fig. 3 ). Its substrates include both histones and non-histones, and the modification sites and mechanisms are also diverse. Fig. 3 Lactylation contributes to various diseases. Lactylation is involved in the regulation of cancer progression, inflammation and immune related diseases, fibrosis related diseases, Alzheimer’s disease, myocardial infarction, insulin resistance, pulmonary hypertension, nonalcoholic fatty liver disease, cerebral infarction, ocular neovascularization, endometriosis, viral infection and many others. NAFLD: nonalcoholic fatty liver disease. HCC: Hepatocellular carcinoma. ccRCC: clear cell renal cell carcinoma. CRC: colorectal cancer. GC: gastric cancer. NSCLC: non-small cell lung cancer. PCa: prostate cancer. SAKI: sepsis-induced acute kidney injury. Lactylation contributes to various diseases. Lactylation is involved in the regulation of cancer progression, inflammation and immune related diseases, fibrosis related diseases, Alzheimer’s disease, myocardial infarction, insulin resistance, pulmonary hypertension, nonalcoholic fatty liver disease, cerebral infarction, ocular neovascularization, endometriosis, viral infection and many others. NAFLD: nonalcoholic fatty liver disease. HCC: Hepatocellular carcinoma. ccRCC: clear cell renal cell carcinoma. CRC: colorectal cancer. GC: gastric cancer. NSCLC: non-small cell lung cancer. PCa: prostate cancer. SAKI: sepsis-induced acute kidney injury.

From the above, we can see that lactylation plays important regulatory roles in various pathological and physiological processes. Now, through lactylome and proteome analysis, researchers have found that lactylation occurs widely in proteins [140] , [141] . According to the kind of substrate proteins, lactylation can be divided into histone and nonhistone lactylation. The effects of lactylation on different proteins vary. Generally, histone lactylation mostly alters the transcription of target genes, while nonhistone lactylation has diverse effects on proteins. The basic unit of chromatin is the nucleosome, an octamer formed by DNA winding histones. Histones are among the main components of chromatin, and their chemical modification after translation is one of the important epigenetic modifications. Common histone modifications include acetylation, butyrylation, methylation, and phosphorylation [142] . These modifications change the relationship between histones, and between histones and DNA, controlling the opening and compression of chromatin, and allowing gene expression or silencing [143] . Current research has shown that the primary effect of histone lactylation is to activate chromatin and stimulate the transcription of downstream target genes. Histones are mainly classified into five categories based on their amino acid composition and molecular weight: H1, H2A, H2B, H3, and H4. Among them, histones H1, H2B, H3 and H4 can be lactylated ( Table 1 ). Elevated histone H1 lactylation has been correlated with stress-associated neural excitation stimulation and reduced social behavior [41] . However, its specific lactylation sites have yet not been reported. For histone H2B, the only identified site of lactylation is K6. H2BK6la and H4K80la cooperatively regulate the expression of miR-155-5p, playing an important role in dyslipidaemia [144] . For histone H3, many lactylation sites,including K9, K18, K23, and K56, [37] , [77] have been discovered. Among them, H3K18 is the most common and extensively studied site in histone lactylation, and has the most diverse regulatory target genes [145] . At present, the downstream target genes activated by H3K18la transcription include reparative genes Lrg1, Vegf-a, and IL-10 [26] , pluripotency genes Oct4, Sall4, and Mycn [36] ;the transcription factors JunB [45] , FOXP3 [101] ,c-MYC [75] ,HIF-1α [146] ,YBX1 and YY1 [91] ;the proinflammatory factor HMGB1 [49] , [139] ; the m6A-related regulatory proteins METTL3 [95] ,YTHDF2 [66] ,ALKBH3 [147] ,YTHDF1 [110] and FTO [132] ,the fibrosis-related genes FN1, SERPINE1 [111] ,α-SMA、Col1a1 and Timp 1 [112] ;the signaling pathway key proteins Notch1 [133] ;the chemokine CXCL1 and CXCL5 [83] ;the non-coding RNA LINC01127 [80] ;the autophagy enhancer protein RUBCNL [90] ; the PASMC proliferation genes Bmp5, Trpc5, and Kit [123] ,PDGFRβ [72] ,HSPA6 [137] and ZGA genes [38] . In addition, by exerting synergistic effects with H4K5la, H3K18la can also regulate the expression of PD-L1, playing a significant role in immunosuppression in AML [97] . Interestingly, the underlying mechanism by which H3K18la regulates the expression of so many genes remain unclear, and the special characteristics of the H3K18 site are still unknown. H3K9 and H3K56 lactylation have been suggested to promote the hyperplasia in liver cancer cells, but their downstream target genes have not yet been identified [77] . Individually, H3K9la upregulates Neu2 expression to play a role in myogenesis [60] . K8, K12, and K18 are the lactylation sites of histone H4. Among them, H4K8 lactylation regulates the expression of the metabolism-related genes HK-1 and IDH3G in lung cancer cells [68] , H4K12 lactylation activates the transcription of the glycolytic genes HIF-1α [53] , PKM2 [62] and CCNB1 [89] , and H4K18 lactylation reduces the binding efficiency of the repressor YY1 with the LINC00152 promoter [82] . Table 1 Histone lactylation sites and biological effects. Histone Lactylation sites Downstream target genes Biological processes Reference H1 N/A Neural excitation [41] H2BK6, H4K80 miR-155-5p Dyslipidemia [144] H3K18 Oct4/Sall4 Cell pluripotency [36] N/A Neural development [39] JunB Osteoblast differentiation [45] HMGB1 Pyroptosis [49] Foxp3 Inflammation [101] Lrg1, Vegf-a, and IL-10 Myocardial infarction [26] FN1 and SERPINE1 Preeclampsia [111] YTHDF2 Ocular melanoma [66] PDGFRβ ccRCC [72] Mettl3 Colorectal cancer [95] Bmp5, Trpc5, and Kit Hypoxic pulmonary hypertension [123] RUBCNL Chemotherapy drug resistance [90] HSPA6 Viral infection [137] c-Myc Breast cancer [75] LINC01127 Gliomas [80] ALKBH3 Ocular melanoma [147] HMGB1 Endometriosis [139] α-SMA、Col1a1 and Timp 1 Liver fibrosis [112] Ythdf1 Arsenite-related idiopathic pulmonary fibrosis [110] CXCL1 and CXCL5 Colorectal cancer liver metastases [83] HIF-1α Prostate cancer [146] YBX1 and YY1 Cisplatin resistance [91] FTO Diabetic retinopathy [132] ZGA genes Zygotic genome activation [38] Notch1 Myopia [133] H3K18/H4K5 PD-L1 Immunosuppression [97] H3K23/H3K18 N/A Embryonic development [37] H3K9 Neu2 Myogenesis [60] H3K9/H3K56 N/A Liver cancer [77] H4K12 PKM2 Alzheimer’s disease [62] CCNB1 Chemotherapy drug resistance [89] HIF-1α Decidualization [53] H4K18 LINC00152 Colorectal cancer [82] H4K8 HK-1 and IDH3G NSCLC [68] Histone ARG1,PDGFA,THBS1 and VEGFA Lung fibrosis [109] Histone lactylation sites and biological effects. Apart from histone lactylation, researchers have also found that many important functional nonhistone proteins exhibit lactylation. There are various effects of lactylation on nonhistone proteins ( Table 2 ). In summary, lactylation can affect nonhistone proteins in six ways. Table 2 Nonhistone lactylation sites and biological effects. Nonhistone lactylation site Biological effects Biological processes Reference PKM2 K62 The lactylation of PKM2 inhibits its tetramer-to-dimer transition, promotes its pyruvate kinase activity and reduces nuclear distribution Macrophage phenotype transition [103] AK2 K18 Lactylation at K28 inhibits the function of adenylate kinase 2 Liver cancer [70] CCNE2 K348 Lactylated CCNE2 promotes HCC cell growth Liver cancer [71] β-catenin site:N/A β-catenin lactylation enhanced the protein stability and expression of β-catenin Colorectal cancer [78] HIF-1α site:N/A HIF-1α lactylation stabilizes HIF-1α under normoxia Prostate cancer [86] METTL3 K281/K345 Lactylation on zinc-finger domain of METTL3 enhances its capture of m6A-modified RNA Colorectal cancer [95] MOESIN K72 Lactylation of Lys72 in MOESIN improves MOESIN interaction with transforming growth factor b (TGF-b) receptor and downstream SMAD3 signaling Tumorigenesis [96] HMGB1 Site:N/A HMGB1 lactylation promotes its exosomal release in polymicrobial sepsis Sepsis [105] Snail1 Site:N/A Lactylation of Snail1, a TGF-β transcription factor, activates TGF-β/Smad2 signal after hypoxia/MI. Myocardial infarction [117] FASN K673 Lactylation at the K673 site of FASN inhibited FASN activity NAFLD [126] LCP1 Site:N/A Inhibiting the glycolysis decreased the lactylation levels of LCP1 and resulted in the degradation of LCP1 Cerebral infarction [128] YY1 K183 YY1 lactylation in microglia plays an important role in retinal neovascularization by upregulating FGF2 expression Ocular neovascularization [131] α-MHC K1897 α-MHC K1897la enhanced the interaction of α-MHC with Titin Heart failure [118] Fis1 K20 Fis1 K20la promoted excessive mitochondrial fission Sepsis-induced acute kidney injury [106] eEF1A2 K408 eEF1A2K408la boosted translation elongation and enhanced protein synthesis Colorectal cancer [28] Ikzf1 K164 Ikzf1 K164la promoted TH17 differentiation by directly modulating the expression of TH17-related genes TH17 differentiation [102] VEGFR2/VE-cadherin Site:N/A VEGFR2 and VE-cadherin lactylation increased their protein expression Vasculogenic mimicry [87] PFKP K688 Lactylation of PFKP attenuates its enzyme activity Colorectal cancer [150] Mecp2 k271 Mecp2 k271la repressed the expression of epiregulin (Ereg) to inhibit atherosclerosis Atherosclerosis [135] MRE11 K673 MRE11 lactylation promotes its binding to DNA Homologous recombination [55] METTL16 K229 METTL16 lactylation at site K229 promotes FDX1 accumulation via m6A modification on FDX1 mRNA Cuproptosis [57] SHMT2 Site:N/A Hypoxia triggered lactylation of the SHMT2 protein and enhanced its stability Esophageal cancer [79] PDHA1 K336 CPT2 K456/7 PDHA1 K336la and CPT2 K456/7 la inhibit the activity of these two enzymes Oxidative phosphorylation [31] YAP K90 TEAD1 K108 Lactylation of YAP 85 at K90 and TEAD1 at K108 activate downstream target gene expression Gastric cancer [30] p53 K120 /K139 Lactylation of p53 impairs p53 LLPS and DNA binding Tumorigenesis [32] Vps34 K356/k781 Vps34 lactylation enhance its association with Atg14L, Beclin1 and UVRAG, thereby increasing the lipid kinase activity of Vps34 Macroautophagy [27] CENPA K124 The lactylation of CENPA at K124 promotes CENPA activation Hepatocellular carcinoma [156] Nonhistone lactylation sites and biological effects. (1) Affects the stability of proteins. Protein stability is regulated by various factors, and lactylation is one of them [148] . The effect of lactylation on protein stability is not fixed, as it may either increase [79] , [87] or decrease protein stability. For example, hypoxia induced glycolysis was shown to stimulate β-catenin lactylation, and further enhance its stability and expression, thus exacerbating the malignancy of CRC [78] . In addition, under normoxia, HIF-1α could be stabilized through lactylation via the import of lactate into PCa cells by MCT1 [86] . However, research has shown that the inhibition of glycolysis lessened LCP1 lactylation, which increases its degradation, and eventually alleviates CI progression [128] . (2) Affects the catalytic activity of proteases. Most enzymes are proteins. When lactylation occurs precisely at the active sites of an enzyme, its activity may be affected accordingly [149] . Similarly, lactylation may either increase [57] or inhibit the activity of proteases [31] , [150] . For example, PKM2 lactylation at the K62 site facilitates the function of pyruvate kinase [103] , while lactylation at the K28 site inhibits adenylate kinase 2 activity [70] . Additionally, MPC1 knockout could mediate the lactylation of FASN at the K673 site, which suppressed FASN activity, and downregulated liver lipid accumulation [126] , [151] , while Vps34 lactylation (K356 and K781) has been suggested to increase its own integration with Beclin1, Atg14L, and UVRAG, thereby promoting the lipid kinase activity of Vps34 [27] . (3) Affects the interactions between proteins and other molecules. Interactions between molecules are influenced by various factors [152] , and lactylation has been shown to affect interaction between proteins and other biological macromolecules [32] , [55] . For example, the interaction between METTL3 and m6A modified RNA was found to be enhanced by METTL3 lactylation on the zinc-finger domain at the K281 and K345 sites [95] . Additionally, MOESIN lactylation at Lys72 could strengthen its association with TGF-β receptor I and activate downstream SMAD3 signaling, which further modulates Treg cell generation [96] . Moreover, α-MHC K1897la was found to enhance the interaction of α-MHC with Titin, which alleviated heart failure [118] . (4) Affects the distribution of proteins. Proteins are known to be the bearers of life activities, and for proteins to perform their corresponding functions, they must reach their designated locations [153] . Lactylation also regulates the localization and distribution of proteins. For example, researchers found that PKM2 nuclear distribution is decreased due to lactylation at the K62 site. [103] In addition, in polymicrobial sepsis, lactate can promote the exosomal release of macrophage HMGB1 [105] . (5) Affects the structure of proteins. The structure of a protein determines its biological function [154] , and research has shown that lactylation can also alter protein structure. For example, the transformation of PKM2 from a tetramer to a dimer occurs through PKM2 lactylation at the K62 site [103] , which promotes its pyruvate kinase activity and reduces its nuclear distribution. (6) Affects the function of proteins. Transcription factors are a class of protein molecules with special structures that regulate gene expression [155] . Various transcription factors can be lactylated [30] , [102] , [135] , which affects their functions. For example, lactylation of Snail1 via interaction with CBP/p300 could induce the transformation of cardiac endothelial cells into mesenchymal cells after MI [117] . Moreover, increased YY1 lactylation in microglia under hypoxia accelerated angiogenesis [131] . In addition, the lactylation of mitochondrial fission 1 protein (Fis1) lysine 20 (Fis1 K20la) was found to promote excessive mitochondrial fission, thereby enhancing SAKI [106] . In addition to those of transcription factors, the functions of some other proteins are also enhanced after lactylation [28] , [57] . For example, CCNE2 lactylation at the K348 site could promote the malignant proliferation of HCC cells [71] . In addition, Liao et al . [156] found that CENPA can be lactylated at lysine 124 (K124), which promotes CENPA activation, leading to enhanced expression of its target genes.

Glucose is a primary source of energy for the body. Depending on whether oxygen is present, glucose metabolism can be divided into anaerobic glycolysis and oxidative phosphorylation [19] . Typically, lactate is a metabolic product of anaerobic glycolysis under hypoxic conditions. Glucose metabolism involves multiple steps and requires the participation of various enzymes [20] . Specifically, after cells take up glucose through glucose transporters (GLUTs), the conversion of intracellular glucose is catalyzed by hexokinase (HK) to glucose-6-phosphate. Furthernore, through the catalysis of several enzymes such as pyruvate kinase (PKM), glucose-6-phosphate is converted to pyruvate. Under normal aerobic conditions, the generated pyruvate enters the mitochondria and is converted to acetyl-CoA by pyruvate dehydrogenase (PDH). Acetyl-CoA then enters the TCA cycle and is completely oxidized to carbon dioxide and water, releasing a large amount of ATP to provide energy for the organism（ Fig. 1 ）, that’s the so called “oxidative phosphorylation”. However, under hypoxic conditions, the generated pyruvate does not enter mitochondria for oxidation but is instead converted to lactate by lactate dehydrogenase (LDH), leading to lactate accumulation within the cell, that’s the so called “anaerobic glycolysis”. Importantly, under certain special pathophysiological conditions, such as exercise, tumors, sepsis, trauma, and heart failure [1] , even when oxygen is sufficient within the cell, glucose still primarily undergoes glycolysis and produces a large amount of lactate, which is known as “aerobic glycolysis” [21] . Apart from being produced by cellular metabolism, intracellular lactate can also originate from the extracellular microenvironment. Extracellular lactate cannot directly enter the cell but requires the involvement of the lactate transporter MCT1 and the coordination of CD147 [22] . At the same time, intracellular lactate can also be transported to the extracellular space by another lactate transporter MCT4 to perform regulatory functions such as signaling molecules [22] , [23] . Fig. 1 Lactate metabolism and lactylation. Lactate can be divided into exogenous and endogenous lactate. Exogenous lactate is transported into cells by MCTs and then catalyzed to lactyl-CoA, which provides lactoyl groups for histone and nonhistone lactylation. Endogenous lactate is produced from glycolysis. Under aerobic conditions, the glucose metabolite pyruvate enters mitochondria and is broken down by the TCA cycle. MCTs: monocarboxylate transporters. GLUTs: glucose cotransporters. LDH: lactate dehydrogenase.HK : hexokinase. PDH: pyruvate dehydrogenase. PDK: pyruvate dehydrogenase kinase. HDACs: histone deacetylases. SIRTs: sirtuins. TCA: tricarboxylic acid. AARS 1/2: Alanyl-tRNA synthetase 1/2. Lactate metabolism and lactylation. Lactate can be divided into exogenous and endogenous lactate. Exogenous lactate is transported into cells by MCTs and then catalyzed to lactyl-CoA, which provides lactoyl groups for histone and nonhistone lactylation. Endogenous lactate is produced from glycolysis. Under aerobic conditions, the glucose metabolite pyruvate enters mitochondria and is broken down by the TCA cycle. MCTs: monocarboxylate transporters. GLUTs: glucose cotransporters. LDH: lactate dehydrogenase.HK : hexokinase. PDH: pyruvate dehydrogenase. PDK: pyruvate dehydrogenase kinase. HDACs: histone deacetylases. SIRTs: sirtuins. TCA: tricarboxylic acid. AARS 1/2: Alanyl-tRNA synthetase 1/2. In addition to serving as an energy molecule and signaling molecule [2] , recent studies have revealed that lactate can participate in epigenetic regulation through a novel protein modification method: lactylation [24] . Lactylation is the process of covalent modification of proteins with lactoyl groups [25] . During this process, lactate is typically first catalyzed into lactyl-CoA, which then transfers the lactoyl groups to substrate proteins under the action of “writer” proteins. The known lactylation “writer” proteins are mainly histone acetyltransferases, including P300 [16] , GCN5 [26] ,TIP60 [27] and KAT8 [28] （ Fig. 1 ）. Lactylation is a reversible biological process, and the known “erasers” for removing lactylation modifications are primarily histone deacetylases (HDACs or SIRTs) [29] . Moreover, scientists recently discovered a new class of lactylation “writer” proteins, alanyl-tRNA synthetase, which includes the members AARS1 [30] and AARS2 [31] . Due to the similar chemical structures of lactate and alanine, AARS1 can directly recognize lactate and, with the participation of ATP, convert it to lactyl-AMP and covalently modify it onto substrate proteins [32] .

High lactate is a marked feature of the tumor microenvironment [65] . There are two main reasons for its occurrence. First, compared to normal tissue, tumor tissue possesses abnormal blood vessels, which leads to insufficient blood supply and low oxygen content. Second, tumor cells still decompose glucose mainly by glycolysis even under aerobic conditions, which is called the “Warburg effect”. Lactate is an inducing factor for lactylation. Currently, many studies have proposed that [32] , [66] , [67] , [68] the abundance of lactylation in tumor tissue is significantly greater than that in adjacent tissue, and is closely related to the poor prognosis of patients, indicating the potential of lactylation level as a tumor diagnostic marker. In addition, lactate mediated lactylation accelerates tumor development through various underlying mechanisms ( Fig. 4 ). Fig. 4 Lactylation drives oncogenesis via multiple mechanisms. On the one hand, lactylation promotes cancer cell proliferation or metastasis, cancer stem cell proliferation, tumor angiogenesis and drug resistance. On the other hand, lactylation contributes to the formation of the tumor immune microenvironment by regulating tumor-infiltrating myeloid cells, Treg cells and the expression of PD-L1 in cancer cells. Lactylation drives oncogenesis via multiple mechanisms. On the one hand, lactylation promotes cancer cell proliferation or metastasis, cancer stem cell proliferation, tumor angiogenesis and drug resistance. On the other hand, lactylation contributes to the formation of the tumor immune microenvironment by regulating tumor-infiltrating myeloid cells, Treg cells and the expression of PD-L1 in cancer cells. Tumor cells exhibit prominent malignant proliferation properties [69] , which are highly correlated with lactylation. Through in-depth lysine lactylome and proteome analysis, Yang et al . [70] successfully identified 9275 Kla sites and 9140 proteins from a total of 52 tumor and adjacent liver tissue samples collected from hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients. In particular, Kla was found mainly in enzymes involved in metabolic pathways, such as the TCA cycle. The suppression of adenylate kinase 2 function by K28 lactylation has been verified as the probable mechanism for promoting the proliferation and metastasis of HCC cells. Moreover, the lactated modified “eraser” protein SIRT3 has been reported to be expressed at low levels in liver cancer tissue, and is thus unable to effectively exert its inhibitory effect on tumor cell growth in liver cancer cells by removing cyclin E2 (CCNE2) K348 lactylation [71] . Another study revealed that [67] in ocular melanoma, the level of tumor tissue lactylation was significantly increased. H3K18la upregulated the expression of the m6A reader YTHDF2 in tumor cells, while YTHDF2 recognized and facilitated the degradation of the PER1 and TP53 mRNAs, accelerating the occurrence of ocular melanoma. In addition, in ccRCC, the lactylation of inactive the VHL-triggered histone H3K18 could promote the progression of ccRCC by activating PDGFRβ mRNA transcription [72] . In addition, in lung cancer, Wang et al . [73] reported that BZW2 promoted the malignant progression of LUAD by promoting glycolysis-mediated lactate production and the lactylation of IDH3G. In bladder cancer, Xie et al . [74] reported that histone H3K18 la promotes cancer cell proliferation and migration by upregulating the expression of LCN2, while CircXRN2 functions as a tumor suppressor gene by inhibiting this process through activating the Hippo pathway. In colorectal cancer, Xie et al . [28] reported that protein lactylation is significantly increased in tumor tissues and is closely related to poor patient prognosis. Through lactylation proteomics analysis, the authors revealed that the translation elongation factor eEF1A2 undergoes lactylation at the K408 site. eEF1A2 K408la enhances protein translation, thereby promoting tumor cell proliferation, and KAT8 is the “writer” molecule that catalyzes eEF1A2 K408la. In breast cancer, Pandkar et al . [75] reported that lactate can induce histone H3K18la and upregulate the expression of c-Myc, while the increased c-Myc further promotes breast cancer cell proliferation through the c-Myc-SRSF10 axis. In gastric cancer, Ju et al . [30] reported that alanyl-tRNA synthetase AARS1, in addition to its classic function as an alanyl-tRNA synthetase, can also serve as a lactyltransferase. It directly utilizes lactate as a lactoyl donor to catalyze the lactylation of key components of the Hippo signaling pathway, YAP and TEAD, activating downstream gene transcription and expression, and promoting malignant proliferation of gastric cancer cells. Collectively, these studies suggest that lactylation is a positive regulatory factor for tumor cell proliferation. Tumor stem cells are known as the “seed” cells of tumor occurrence and development, and are the “root” of continuous proliferation, metastasis, and chemotherapy resistance of tumor cells [76] . Recently, a study revealed that [77] lactate mediated lactylation could promote the proliferation and self-renewal of tumor stem cells, while demethylzeylasteral (DML) could inhibit the generation of lactate and the lactylation of histones H3K9 and H3K56 in liver tumor stem cells, thereby repressing the genesis of liver cancer. In addition, hypoxia or lactate stimulation has been shown to increase the level of the β-catenin protein in colorectal cancer (CRC) cells and increase its constancy, resulting in enhanced tumor cell stemness and proliferation ability [78] . In addition, Qiao et al . [79] reported in their research on esophageal cancer (EC) that the hypoxic tumor microenvironment can induce lactylation of the serine hydroxymethyl transferase 2 (SHMT2) protein, enhancing its stability. SHMT2 promotes the stemness of esophageal cancer cells by interacting with and increasing the expression of MTHFD1L. Furthermore, in glioblastoma, Li et al . [80] reported that histone H3K18la upregulates the expression of LINC01127, which in turn regulates MAP4K4 in a cis -acting manner to activate the JNK signaling pathway, thereby promoting the self-renewal of glioblastoma stem cells. Overall, these researches indicate that lactylation is a positive regulatory factor for the self-renewal of cancer stem cells. Tumor metastasis is the main cause of tumor recurrence, and is also a major factor in the failure of tumor treatment [81] . Lactylation is closely related to tumor metastasis. In the colorectum, LPS derived from gut microbes has been found to induce lactylation of tumor cell histone H4K8, inhibit the binding of the transcription factor YY1 to the LINC00152 promoter and upregulate its expression, thus assisting in tumor cell invasion and migration [82] . In liver cancer, lactylation at K28 restricts the enzyme activity of AK2, and facilitates tumor cell proliferation and metastasis [70] . In addition, in a colorectal cancer liver metastasis model, Zhou et al . [83] reported that orphan G protein-coupled receptor 37(GPR37) could activate the Hippo pathway, thereby promoting LDHA expression and glycolysis. This leads to increased lactylation of H3K18la, resulting in upregulation of CXCL1 and CXCL5, which promotes colorectal cancer liver metastasis. Collectively, these studies indicate that lactylation is a positive regulatory factor for tumor cell metastasis. Intratumoral angiogenesis not only provides nutrients for cancer cell proliferation, but also provieds conditions for the development and metastasis of tumors [84] . Therefore, inhibiting angiogenesis can obviously prevent the development, diffusion, and metastasis of tumor tissue [85] . In prostate cancer [86] , Luo et al . reported that lactate introduced into PCa cells through MCT1 stabilized HIF-1α under normal oxygen conditions via HIF-1α lactylation. Moreover, HIF-1α lactylation increased the transcription of KIAA1199, a binding protein of hyaluronic acid (HA), promoting tumor angiogenesis by increasing the secretion of VEGFA. In addition, in glioblastoma (GBM), Zhang et al . [87] reported that the functional peptide P4-135aa encoded by the pseudogene MAPK6P4 promotes the phosphorylation of the transcription factor KLF15 at the S238 site and stabilizes its protein expression. Subsequently, the transcription factor KLF15 enters the nucleus and upregulates the transcription level of LDHA, leading to an increase in lactate levels and promoting the lactylation of the angiogenic markers VEGFR2 and VE-cadherin. This, in turn, increases their protein expression and promotes tumor angiogenesis. Overall, these researches suggest that lactylation is a positive regulatory factor for tumor angiogenesis. Drug resistance is a crucial factor in cancer recurrence and metastasis [88] . By studying the mechanisms related to pemetrexed resistance in lung cancer brain metastatic cells, Duan et al . [89] reported that aldo–keto reductase family 1 B10 (AKR1B10) is highly expressed in lung cancer brain metastatic cells. Knocking down AKR1B10 significantly increased the sensitivity of lung cancer brain metastasis cells to the chemotherapy drug pemetrexed. Mechanistically, the authors discovered that AKR1B10 promotes glycolysis and lactate production in lung cancer brain metastasis cells by upregulating LDHA expression, which further induces H4K12la, leading to the upregulation of the cell cycle regulatory gene CCNB1 and ultimately causing drug resistance in cells. Furthermore, in studying the mechanisms of bevacizumab resistance in colorectal cancer, Li et al. [90] found that tumor tissues from bevacizumab-resistant patients with colorectal cancer had increased levels of lactylation. The inhibition of lactylation hindered the development and progression of colorectal cancer. Mechanistically, researchers have shown that lactate in the tumor microenvironment induces histone H3K18 lactylation, upregulates the expression of the autophagy enhancer protein RUBCNL, promotes cell autophagy, and contributes to the tumorigenesis and progression of colorectal cancer. Recently, by single-cell sequencing, Li et al . [91] discovered that cisplatin resistance in bladder cancer (BCa) is closely related to histone lactylation. They found that cisplatin-resistant subpopulations of bladder cancer cells exhibit significant glycolytic metabolic characteristics. These cells induce H3K18 lactylation, upregulate the expression of the transcription factors YBX1 and YY1, and subsequently promote cisplatin resistance in BCa. In summary, these studies indicate that lactylation contributes to drug resistance for cancer treatment. In addition to directly participating in regulating the malignant phenotypes of tumor cells such as proliferation and metastasis, lactylation is also closely related to the formation of an immunosuppressive tumor microenvironment [92] . The tumor microenvironment (TME) is a complex combination of tumor cells, immune cells, and various interstitial cells [93] . Among them, CD3 + T cells, CIK cells, and NK cells are the main tumor killing cells that inhibit tumor progression. In contrast, immunosuppressive cells such as tumor-infiltrating myeloid cells (TIMs) and regulatory T (Treg) cells can inhibit the killing effect of CD3 + T cells on tumor cells, playing an important role in maintaining the immunosuppressive tumor microenvironment [94] . In CRC, H3K18la upregulated the expression of METTL3 in TIMs, which further boosted the immunosuppressive functions of myeloid cells via the m6A/JAK1/STAT3 axis, stimulating the development of CRC [95] . Lactate stimulation can also increase the maintance and function of Treg cells, whereas lactate degradation decreases Treg cell induction, increased antitumor immunity, and reduces tumor growth in mice [96] . Mechanistically, lactate promoted MOESIN K72 lactylation, which improved the interaction between MOESIN and TGF-β receptor I, and facilitated SMAD3 signal transduction, thus regulating the generation of Treg cells. In addition to regulating immune cells, in AML, Huang et al . [97] reported that the transcription factor STAT5 promotes glycolysis in AML by upregulating the expression of glycolysis-related genes, leading to lactate accumulation. In turn, elevated lactate upregulates PD-L1 expression by inducing the lactylation of varies histones, thereby promoting tumor immune evasion. Collectively, these studies indicate that lactylation contributes to the formation of an immunosuppressive tumor microenvironment and promotes the development of cancer by controlling the function of immunosuppressive cells or upregulating PD-L1 expression in cancer cells. Inflammation is a protective response of body tissue to harmful stimuli such as pathogenic microorganisms and dead cells, and is a complex biological process mediated by immune cells [98] . Typically, the function of inflammation is to eliminate cell damage factors and initiate tissue repair. However, excessive or prolonged inflammatory reactions are detrimental to the body and require anti-inflammatory responses to maintain homeostasis. Recent research has revealed that one of the notable features of the inflammatory microenvironment is elevated lactate, which could induce immune cell anti-inflammatory capacity by regulating their reprogramming through lactylation [99] , [100] . In an inflammatory microenvironment, lactate was found to induce proinflammatory Th17 cells to metabolically reprogram into regulatory T cells expressing FOXP-3 by catalyzing H3K18la [101] . In addition, the accumulation of lactate are involved in the dysregulation of CD4 + T cell differentiation. In experimental autoimmune uveitis, through in-depth lysine lactylome analysis, Fan et al . [102] found that transcription factor Ikzf1 k164la promoted TH17 differentiation by directly modulating the expression of TH17-related genes, including RUNX1, TLR4, interleukin-2(IL-2), and IL-4. Moreover, lactate stimulation can also promote the proinflammatory to reparative transformation of macrophages through PKM2 lactylation [103] . Inflammation regression is a necessary step in the process of heart repair after myocardial infarction (MI). The anti-inflammatory and angiogenic functions of monocyte macrophages are regulated by histone lactylation via the promotion of repair gene transcription, which is beneficial for improving the repair environment and cardiac function after MI [26] . In addition, in sepsis, an inflammation-related disease, a high circulating level of lactate is a typical feature. By comparing the collected samples from healthy and diseased populations, researchers have shown that lactylation existed in both healthy and sick people, but to different degrees. The level of H3K18la might be positively related to the severity of sepsis and infection [104] . Similarly, the significant increase in HMGB1 lactylation might be positively correlated with the severity and mortality of sepsis. HMGB1 lactylation is mediated by macrophages ingesting extracellular lactate via MCTs through a p300/CBP-dependent mechanism. Lactylated HMGB1 is then released from macrophages via exosome secretion which increases endothelial permeability, and exacerbates sepsis [105] . Recently, researchers have shown that lactate modification is also closely related to sepsis-induced acute kidney injury (SAKI) [106] . Through a multi-modifiedomics strategy (acetylation and lactylation modification), researchers discovered that the acetylation of the α subunit of pyruvate dehydrogenase E1 (PDHA1) promotes an increase in lactate levels, which in turn promotes the lactylation of the nonhistone protein Fis1 K20 and regulates mitochondrial fission to regulate the occurrence of SAKI. Taken together, these studies suggest that lactylation regulates the occurrence of inflammatory diseases and their complications through multiple mechanisms. Fibrosis is a pathological process characterized by necrotic parenchymal cells, and abnormally increased and excessively deposited extracellular matrix in tissue [107] . Progressive fibrotic processes can lead to structural damage and functional decline of organs, ultimately resulting in organ failure, which is the main cause of disability and mortality in many diseases [108] . Lactylation has also been implicated in fibrosis. In pulmonary fibrosis, myofibroblasts metabolize abnormally, producing and secreting a large amount of lactate, which further leads to the presence of histone Kla in the promoter regions of macrophage fibrogenic genes. This finding is in accordance with the upregulation of epigenetic modifications in fibrotic lung myofibroblasts [109] . In arsenite-related idiopathic pulmonary fibrosis, Wang et al . [110] discovered a novel mechanism by which lactylation promotes lung fibrosis. They found that lactate in the IPF microenvironment is transported into alveolar epithelial cells (AECs) through MCT1, leading to H3K18la and upregulation of YTHDF1. YTHDF1 further upregulates NREP expression through RNA m6A modification, and NREP in alveolar epithelial cells facilitates the fibromyxoid transformation (FMT) of pulmonary lymphoid follicles (PLFs) by regulating TGF-β1. In addition, compared with normal pregnant placentas, placentas from women with preeclampsia had greater lactate levels. This elevated lactate could regulate the expression of fibrosis-related genes such as FN1 and SERPINE1 through histone lactylation, thereby resulting in placental fibrosis [111] . Furthermore, recently, Rho et al . [112] reported that lactylation is also involved in the development of liver fibrosis. Specifically, they found that hexokinase 2 (HK2) upregulates lactate levels and induces histone H3K18la, which in turn upregulates the expression of genes such as α-SMA, COL1A1, and TIMP1, thereby governing the activation of hepatic stellate cells (HSCs) and resulting in liver fibrosis. In summary, these studies suggest that lactylation contributes to the development of fibrosis related diseases through multiple mechanisms. Alzheimer's disease (AD) is characterized by the proinflammatory activation of microglia, which involves the transition from oxidative phosphorylation to glycolysis [113] . Significant increases in lactate and lactylation levels have been found in AD model mice and patient brain tissue [62] , [114] , [115] . Mechanistically, histone H4K12 lactylation activated glycolytic gene transcription in AD microglia, leading to its proinflammatory activation. This finding correlated with the generation of AD, and intervention in related signaling pathways could markedly improve the symptoms of AD mice. Overall, these studies show that lactylation contributes to the development of AD, indicating that inhibiting related signaling pathways can improve AD symptoms. High lactate levels are positively associated with prognosis and mortality in patients with heart attack [116] . Through the MCT-dependent signaling pathway, lactate can be transported into cells and prompt Snail1 lactylation, which upregulates the endothelial-to-mesenchymal transition of the heart after MI, thereby accelerating cardiac fibrosis and aggravating cardiac dysfunction [117] . Apart from myocardial infarction, lactate and lactylation have also been implicated in regulating heart failure. Recently, by lactylation omics analysis, researchers discovered a significant decrease in lactate concentration in myocardial cells during heart failure, which further led to a decrease in the lactylation level of K1897 on the α-myosin heavy chain (α-MHC) and a significant reduction in the interaction between α-MHC and titin, ultimately resulting in heart failure [118] . Collectively, these findings indicate that lactylation is closely related to the development of cardiac-related diseases and may serve as a target for intervention. The phenomenon of reduced sensitivity of surrounding tissue to insulin, elevated blood sugar, and islet function compensation for increased insulin secretion is called insulin resistance. Previous findings have shown that circulating lactate levels were often increased in obese and insulin resistant individuals [119] , [120] . A recent study indicated that [121] lactate-mediated lactylation in skeletal muscle is connected with insulin resistance in humans. The research indicates that lactate may regulate insulin resistance through lactylation, but the underlying mechanisms remain unclear. Pulmonary hypertension (PH) is a progressive disease characterized by elevated mROS and enhanced glycolysis [122] . Hypoxia is considered to induce an increase in mROS, which further restrains HIF-1α hydroxylation, and subsequently activates the HIF-1α/PDK1&2/p-PDH-E1α axis. This triggered hypoxic glycolysis in PASMCs, leading to lactate accumulation and increased histone lactylation, which upregulated downstream target genes of HIF-1α, thus promoting the proliferation of PASMCs. Furthermore, in hypoxic PH rats, drug intervention with an LDH inhibitor reduced the histone lactylation, and enhanced PASMC proliferation and vascular remodeling [123] . In summary, these studies indicate that lactylation promotes pulmonary hypertension, suggesting that targeting lactylation could be a potential therapeutic strategy for related diseases. The mitochondrial pyruvate carrier (MPC) is a protein complex in the inner mitochondrial membrane that transports pyruvate from outside the mitochondria into the mitochondrial matrix to produce acetyl-CoA [124] . The underlying relationships between MPC1 and fibrosis, inflammation or insulin sensitivity in obese or NASH mice have been reported in a few studies [125] . However, whether MPC1 can control the levels of lactate and lactylation in NAFLD remains unclear. Recently, MPC1 was found to decrease the level of lactate in hepatocytes, and was negatively associated with lactylation of several proteins, especially fatty acid synthase (FASN). Mechanistically, high MPC1 expression decreased FASN K673 lactylation, activated FASN thereafter, and promoted liver lipid accumulation in NAFLD [126] . Overall, the research found that MPC1 promotes NAFLD through regulating FASN lactylation, indicating that targeting MPC1 may be an effective treatment for non-alcoholic fatty liver disease (NAFLD). Cerebral infarction (CI) is associated with high morbidity and mortality. Impaired cerebral blood circulation and local tissue ischemia and hypoxia damage are commonly observed after CI. It often injures neurons, and eventually leads to paralysis, aphasia and other neurological deficit symptoms [127] . Recently, lactylation has been suggested to regulate CI progression. Lymphocyte cytosolic protein 1 (LCP1) lactylation is reduced by glycolysis inhibition, which facilitates LCP1 degradation, and ultimately relieves CI progression [128] . The research demonstrates that lactylation promotes cerebral infarction, and inhibiting lactylation can improve the condition. The eyes are the windows of the soul, and lactylation has also been found to be related to the occurrence of many eye diseases. For example, ocular neovascularization is one of the major characteristics of proliferative retinopathy [129] . Retinal microglia are tightly associated with hypoxia-induced angiogenesis and vasculopathy [130] , but the potential underling mechanisms remain to be explored. Wang et al . [131] developed a lactylation map of microglia under normal and hypoxic conditions, via the application of 4D proteomics and lactylation omics technology. They revealed that the elevation of YY1 (K183 site) lactylation in microglia under hypoxia and the subsequent increase in FGF2 transcription and expression are ways to regulate angiogenesis in endothelial cells. These results provide a novel theoretical basis for the study of the pathogenesis and treatment of proliferative retinopathy and other critical diseases that cause blindness. In addition, lactylation is closely related to diabetic retinopathy. Recently, Chen et al . [132] reported that retinal lactate homeostasis is disrupted under diabetic retinopathy (DR) conditions, and that lactate-mediated H3K18la can affect the stability of CDK2 mRNA by regulating FTO expression, ultimately impacting endothelial cell function and retinal homeostasis, revealing the importance of histone lactylation in the pathogenesis of DR. Moreover, scleral hypoxia is a significant factor contributing to myopia, but how hypoxia induces myopia is poorly understood. Recently, Lin et al . [133] reported that a hypoxia induced glycolysis/lactate/histone lactylation cascade drives fibroblast to-myofibroblast transdifferentiation (FMT), resulting in myopia. They demonstrated that the suppression of glycolysis, lactate production, or Notch1 expression can mitigate FMT and myopia development. Taken together, these researches suggest that lactylation plays important roles in the development and progression of eye diseases. Atherosclerosis is a serious cardiovascular disease, and current research has shown that exercise is highly beneficial for improving cardiovascular health, but the underlying mechanisms are not entirely clear [134] . Recently, Wang et al . [135] reported that lactate produced during exercise can inhibit the occurrence of atherosclerosis by inducing the lactylation of methylated CpG-binding protein 2 (Mecp2) at the K271 site in vascular endothelial cells. Furthermore, through RNA-sequencing and Chip-qPCR, they found that Mecp2 k271la inhibited the expression of epiregulin (Ereg), which altered the mitogen-activated protein kinase (MAPK) signaling pathway by regulating the phosphorylation of epidermal growth factor receptors, thereby affecting the expression of Vcam-1, Icam-1, Mcp-1, IL-1β, IL-6, and Enos in ECs, which in turn promoted the regression of atherosclerosis. The research indicates that lactate-mediated lactylation is beneficial for improving atherosclerosis. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that has devastated the swine industry worldwide for more than 30 years [136] . Previous studies have found that cells infected with this virus exhibit high lactic acid levels, but the specific role and mechanism are unclear. Recently, Pang et al . [137] reported that infection with PRRSV can induce the lactylation of histone H3K18, which upregulates the expression of HSPA6. In turn, HSPA6 reduces IFN-β induction by hindering the interaction between TRAF3 and IKKɛ, ultimately leading to increased viral replication. The research suggests that lactylation promotes viral infection. The molecular mechanisms of endometriosis have long been unclear [138] . Recently, Chen et al . [139] reported that compared with normal endometrial tissue and stromal cells, endometriotic tissue and stromal cells exhibit increased LDHA expression and lactate levels. Furthermore, they discovered that lactate promotes the proliferation, invasion, and migration of endometrial stromal cells by inducing histone H3K18 lactylation, which upregulates the expression of HMGB1, ultimately promoting the progression of endometriosis. This research indicates that lactylation contributes to the development and progression of endometriosis.

Overall, we can see that lactylation plays an important regulatory role in various pathophysiological processes, with diverse target proteins and biological effects. Targeting lactylation might be a novel therapeutic strategy for related diseases. Studies ( Table 3 ) have indicated that lactylation is regulated by numerous metabolic modulators ( Fig. 5 ). According to different mechanisms, lactylation targeting can be divided into two main categories: Table 3 Metabolic modulators regulating lactate-mediated lactylation. Target Metabolic modulators Drug dosage Effects on lactylation Biological processes Reference MCTS CHC N/A Down Myocardial infarction [117] AZD3965 0.5 µM Myocardial infarction [26] CD147 MEM-M6/1 1.25–10  μg/ml Down Cancer progression [158] LDH Oxamate 20 mM Down Insulin resistance [121] 8 mM ccRCC [72] 50 μM Hypoxic pulmonary hypertension [123] 20 m M Sepsis [105] GSK2837808A 100 pM Embryonic development [37] FX-11 N/A Myocardial Infarction [26] LDHi 20 mg/kg Cancer progress [96] PDH Rotenone 5 nM Up Myocardial Infarction [26] 500 nM Colorectal cancer [95] 1.5 mg/kg Ocular neovascularization [131] PDK DCA 5 mM Down Inflammation [101] 5 mM Myocardial Infarction [26] 20 mM Ocular neovascularization [131] PKM2 FGS 10–50 μM Down NSCLC [157] HK 2-DG 5 mM 10 mM Down Insulin resistance [121] 20 mM Cerebral Infarction [128] 1 mM Inflammation [101] 4 mM ccRCC [72] P300 C646 100 µ M Down Colorectal cancer [95] 5 µM sepsis [105] A485 20 μM Ocular neovascularization [131] TIP60 MG149 20 μM Down Autophagy [27] HDACs TSA 1 µM Up Colorectal cancer [95] MS-275 1 µM Neural development [39] SIRT3 Honokiol 10 mg/kg Down hepatocellular carcinoma [71] AARS1 β-alanine 1.2 %β-alanine Down Tumorigenesis [32] Fig. 5 Metabolic modulators and their targets in the regulation of lactylation. Lactylation can be targeted in two ways: ① by targeting the production or transport of lactate and ② by targeting “writers” or “erasers” of lactylation. The production or transport of lactate relies on many enzymes or functional proteins, which can be targeted by drugs. Lactylation is catalyzed by “writers” and removed by “erasers”. The “writers” or “erasers” of lactylation can also be targeted by drugs. Metabolic modulators regulating lactate-mediated lactylation. Metabolic modulators and their targets in the regulation of lactylation. Lactylation can be targeted in two ways: ① by targeting the production or transport of lactate and ② by targeting “writers” or “erasers” of lactylation. The production or transport of lactate relies on many enzymes or functional proteins, which can be targeted by drugs. Lactylation is catalyzed by “writers” and removed by “erasers”. The “writers” or “erasers” of lactylation can also be targeted by drugs. Typically, lactate is a donor of lactyl-CoA and a promoting factor of lactylation. Therefore, targeted regulation of intracellular lactate production or transportation can control the process of lactylation. The production of intracellular lactate is a complex physiological process catalyzed by multiple enzymes and involves multiple steps. Stimulation of cells with the HK inhibitor 2-DG or the PKM inhibitor FGS [157] commonly leads to reduced glycolysis, lactate levels, and lactylation, since HK and PKM are considered the critical enzymes that promote the conversion of glucose to pyruvate, the upstream substance of lactate [72] , [101] , [128] . Under anaerobic conditions, LDH participates in the translation of pyruvate to lactate, and the LDH inhibitors oxamate [72] , LDHi [96] , FX-11 [26] and GSK2837808A [37] were found to significantly reduce intracellular lactate and lactylation levels. In contrast, under aerobic conditions, pyruvate is usually converted to acetyl-CoA via PDH, while the use of rotenone, an inhibitor of PDH, blocks this process, causing pyruvate metabolism in the direction of lactate, thus promoting lactylation [26] , [95] . Pyruvate dehydrogenase kinases (PDKs) can also restrict the activity of PDH. The PDK inhibitor DCA was therefore effective in facilitating acetyl-CoA production from pyruvate, leading to a decrease in the levels of lactate and lactylation [26] , [101] , [131] . In addition, exogenous lactate plays a vital role in inducing lactylation. These findings indicate that MCT1 is necessary for the entry of exogenous lactate into cells. CD147 was also involved in the regulation of lactate transport, as it coexists with MCTs on the cell membrane and controls their expression. Accordingly, the application of the MCT inhibitors CHC [117] and AZD3965 [26] , as well as the CD147 antibody MEM-M6/1 [158] could reduce intracellular lactate and lactylation levels. In addition to requiring the participation of lactate, lactylation itself is also a reversible enzymatic reaction. Therefore, modulating enzyme activity can also target lactylation. The enzymes that catalyze protein lactylation are known as “writers”, and the acetyltransferases P300, GCN5, TIP60 and KAT8 [28] were recently discovered. Both the P300 inhibitors C646 [95] , [105] and A485 [131] , and the TIP60 inhibitor MG149 [27] can suppress lactylation. Excitingly, the alanyl-t RNA synthetase,AARS1 was recently found to act as a lactyl-transferase to promote lactylation [30] , [32] , while β-alanine could disrupt lactate binding to AARS1 and inhibit global lactylation. In contrast, “erasers” refer to enzymes that erase protein lactylation. Histone deacetylases (HDACs and SIRTs) are “erasers” identified at present, and the use of the HDAC inhibitors TSA [95] and MS-275 [39] stimulates lactylation, while the SIRT3 activator honokiol [71] reinforces delactylation. Before applying drugs to clinical disease treatments, the primary considerations must be their efficacy and safety. Among the aforementioned drugs targeting lactylation, MCT inhibitor AZD3965 is an anti-tumor candidate drug that has entered phase I clinical trials. Current clinical results indicate that it is safe and well-tolerated [159] , while in vitro and animal experiments have demonstrated its effectiveness in treating tumors [160] , indicating that AZD3965 is a highly promising anti-tumor drug. Additionally, HDAC inhibitor MS-275 (entinostat) is a candidate anti-cancer drug that has entered phase III clinical trials. Previous phase I and II clinical trials have shown that MS-275, either alone or in combination with other drugs, is safe and effective for treating solid tumors and lymphomas [161] , [162] , [163] , [164] . Excitingly, recent phase III results showed that entinostat combined with exemestane significantly enhanced progression-free survival (PFS) compared to exemestane monotherapy in patients with HR + /HER2 – advanced breast cancer (ABC) who progressed following endocrine therapy [165] . This combination could represent a novel treatment option for Chinese patients with ABC. In contrast to AZD3965 and entinostat, honokiol, an activator of SIRT-3 [166] , is a natural compound derived from the Chinese and Japanese traditional medicine Magnolia officinalis, which has been used for thousands of years. In recent years, various food safety authorities have evaluated honokiol and deemed it safe [167] . Honokiol possesses multiple pharmacological effects, including anti-infective, anxiolytic, and antioxidant properties [168] . It has also been found to have anti-tumor activity in recent years, making it a potential anti-cancer drug [169] . Besides these, other lactylation regulatory molecules also exhibited certain pharmaceutical activities in in vitro and animal trials [167] , [168] , [169] . However, their safety, efficacy, and indications still require further research.

Due to the existence of lactylation, lactate, a primary product of glycolysis in mammals, has gracefully transformed from an “ugly duckling” into a “beautiful swan.” Currently, lactate-mediated lactylation participates in the regulation of numerous pathophysiological processes, including cell pluripotency, embryonic development, neural excitation, cancer, inflammation and immune related diseases, and fibrosis related diseases. Lactylation substrate proteins and sites are diverse, and their biological effects are varied. Generally, histone lactylation mostly alters the transcription of target genes, while nonhistone lactylation affects protein functions. Considering the important role of lactylation in the development of diseases, targeting lactylation may be a promising therapeutic strategy for treating diseases. In general, the physiological and pathological processes regulated by lactylation are accompanied by abnormal lactate levels. However, is this a necessary condition for lactylation to participate in the regulation of biological processes? We believe the answer is no. For example, in the process of cuproptosis, the upstream condition that triggers METTL16 lactylation is Cu 2+ , but not lactate [57] . However, due to the network of signal regulatory pathways in organisms [170] , we cannot rule out the existence of unknown signaling pathways linking copper ion and lactate metabolism. This also means that some physiological or pathological processes may still be regulated by lactylation even if there is no change in the lactate level. As a novel epigenetic regulation way, lactylation is becoming a hotspot and leading edge of research, and its functions and mechanisms under other physiological and pathological conditions are being revealed gradually. However, the remaining questions related to lactylation are still numerous ( Fig. 6 ): Fig. 6 The remaining questions related to lactylation. Despite the progress in our understanding of lactylation in recent years, many questions remain regarding basic aspects, such as whether there is an enzyme that specifically catalyzes lactylation and whether lactylation can regulate gene expression beyond histone lactylation. Solid arrows: reported biological processes; Dashed arrows: physiological processes that may exist but have not yet been reported. The remaining questions related to lactylation. Despite the progress in our understanding of lactylation in recent years, many questions remain regarding basic aspects, such as whether there is an enzyme that specifically catalyzes lactylation and whether lactylation can regulate gene expression beyond histone lactylation. Solid arrows: reported biological processes; Dashed arrows: physiological processes that may exist but have not yet been reported. (1) Do dedicated “writers” or “erasers” that catalyze or erase lactylation exist? At present, the discovered “writers” of lactylation include histone acetyltransferases and alanyl-tRNA synthetase, while the “writers” of lactylation are mainly histone deacetylases. The enzymes that specifically catalyze or clear protein lactylation are still unknown. (2) HATs and HDACs act as “writers” and “erasers” of lactylation respectively. What are the factors that regulate their role switching? In other words, when a HAT (histone acetyltransferase) or HDAC (histone deacetylase) encounters a protein, what factors determine whether that protein will undergo acetylation or lactylation? The existence of competition between acetylation and lactoylation and its underlying mechanism are still unclear. (3) The key enzyme that catalyzes the conversion of lactate to lactyl-CoA has been found in prokaryotes [171] , [172] , but has not been detected in animals or humans. Excitingly, AARS1, an alanyl-t RNA synthetase, was recently found to moonlights as a lactate sensor and lactyltransferase to promote P53 lactylation and tumorigenesis. In this process, AARS1 directly recognizes lactate and catalyzes the lactylation of P53, without the involvement of lactyl-CoA. Instead, it utilizes lactyl-AMP [32] . However, the proportions of protein lactylation mediated by histone acetyltransferases and AARS1/2 respectively are still unclear. (4) Histone lactylation may modulate the transcription of downstream genes. However, could lactylation control gene expression at nontranscriptional level, such as protein translation? Gene expression regulation is complex and multilayered and occurs at both the RNA and the protein levels [173] . Typically, histone lactylation regulates gene transcription. However, the regulatory role and mechanisms of lactylation in protein translation remain poorly understood. Excitingly, recent research has revealed evidence that lactylation regulats protein translation. Researchers have shown that the translation elongation factor eEF1A2 undergoes lactylation at the K408 site, which enhances protein translation and thereby promotes tumor cell proliferation [28] . Apart from eEF1A2, protein translation also involves the participation of various other regulators [174] , such as the translation initiation complex composed of EIF4G, EIF4A, and EIF4E. It is still unclear whether these proteins can undergo lactylation and affect protein translation. (5) The regulation of intracellular gene expression is a network structure, and research has discovered the associations of lactylation with RNA m6A modification [95] and other acylations [175] . Among these, the link between acetylation and lactylation is the most common. In polymicrobial sepsis, Yang et al. [105] found that macrophages can uptake extracellular lactate to promote both HMGB1 lactylation and acetylation, which promotes the release of HMGB1 via exosome secretion and increases endothelium permeability. Moreover, Li L et al. [36] showed that transcription factor Gli-like transcription factor 1 (Glis1) can enhance both the levels of acetyl-CoA and lactate and synergistically drove histone acetylation and lactylation to induce pluripotency. Additionally, under low-temperature conditions, Lu et al. [176] found that mitochondrial damage in macrophages leads to metabolic reprogramming, which induces histone acetylation and promotes M1 proinflammatory differentiation. At the same time, metabolic reprogramming results in increased histone lactylation mediated by lactate in M1 macrophages, which initiates repair gene expression. In addition to acetylation, other post-translational acylations, such as crotonylation, have also been reported to crosstalk with lactylation. During neural development, Dai et al. [39] found that histone lysine crotonylation (Kcr) and lysine lactylation (Kla) are widely distributed in the brain, which promote cell-fate transitions in the developing telencephalon. In addition, lactylation may also be linked with succinylation. In gastric cancer, Zhang et al. [177] found that lysine acetyltransferases 2 A (KAT2A) promotes the succinylation of PKM2 at K475 site and accelerate glycolysis, which may induces lysine lactylation in cancer cells. However, what is the “cross talk” between lactylation and other types of gene expression regulation modes? Given the extensive and intricate spectrum of target genes and biological processes regulated by lactylation, it is reasonable to speculate that lactylation engages in “cross-talk” with other types of regulatory modes. However, the precise underlying mechanisms remain to be elucidated. (6) Lactylation plays a prominent regulatory role in various pathological processes, and studies have shown that inhibiting lactate mediated lactylation has antitumor effects [77] , [157] . Nevertheless, no specific therapeutic drugs have been developed for diseases specifically targeting lactylation. At present, we can target lactylation via two approaches: by inhibiting lactate production or its uptake into cells, and by suppressing the “writers” that catalyze lactylation. However, the specificity and efficacy of these two approaches pose a challenge, as these targets are involved not only in lactylation but also in other vital physiological processes [178] . The key enzymes that catalyze the conversion of lactate to lactyl-CoA and AARS1/2 may be good targets for targeting lactylation in the treatment of related diseases. Lactylation has opened up new avenues for epigenetic research, and has also revealed new biological functions and molecular mechanisms related to the role of lactate. Here, we reviewed the roles and mechanisms of lactate-mediated lactylation in human health and diseases, as well as the effects of lactylation on proteins and the strategies for targeting lactylation in the treatment of diseases. Finally, we raised the current issues and challenges surrounding the study of lactylation. With further research, it is believed that in the near future, unsolved questions related to lactylation will be unraveled by researchers, and these studies will provide new ideas and approaches for the diagnosis and treatment of related diseases.

Lactate, a metabolic product of cells undergoing glycolysis under anaerobic conditions, has long been considered a useless metabolic waste product. However, in the 1920 s, Otto Warburg, a German oncologist, discovered that compared to normal cells, tumor cells can absorb glucose more efficiently and still prefer to undergo glycolysis even in the presence of oxygen, producing a large amount of lactate and providing energy. This is known as the “Warburg effect” [1] . In recent years, researchers have found that lactate can not only be transported into cells as an energy source for metabolism, but is also an important ignalling molecule [2] involved in regulating important physiological and pathological processes such as angiogenesis and immune response, the mechanism of which still needs further study. In addition to lactate, there are thousands of metabolites found in biological systems [3] . Recent studies have shown that in addition to their primary role in metabolism, these small molecules produced during cellular metabolic processes can also be covalently modified onto proteins as substrates to participate in epigenetic regulation [4] , [5] . Epigenetics refers to the condition in which the DNA sequence remains unchanged, while gene expression undergoes heritable changes. There are numerous patterns of epigenetic regulation, mainly including DNA methylation, RNA modification, noncoding RNA modification, and a variety of protein modifications [6] . Acetylation, ubiquitination, methylation, and phosphorylation are common types of protein modifications [7] . As the main carrier of life activities, protein modification often has a significant impact on life processes. For example, acetylation of histones typically activates downstream target gene transcription, ubiquitination can affect protein degradation, and phosphorylation can affect protease activity [8] . With the development of science and technology [9] , many novel types of protein modifications have been identified, including crotonylation [10] , methacrylylation [11] , O-glycosylation [12] , succinylation [13] , β-hydroxybutylation [14] , palmitoylation [15] and lactylation [16] . In 2019, Professor Zhao Yingming’s team from the University of Chicago first reported in Nature that [16] lactate could serve as a substrate modified to histone lysine residues, and regulate downstream gene expression. Since then, a new epigenetic regulation pattern – lactylation – has entered the perspective of researchers. Recently, studies have shown that lactylation is an important epigenetic regulatory mode that participates in the regulation of numerous physiological and pathological processes [17] , [18] , such as embryonic development, tumorigenesis, inflammation and many others. These studies suggest that lactate can regulate biological processes through a novel mechanism known as lactylation. Currently, research on lactate-mediated lactylation has become a hot topic. However, some unresolved problems still exist. In this review, we summarize recent progress in lactate-mediated protein lactylation, including the regulatory roles and mechanisms of lactylation in physiological and pathological processes, the types of lactylation and their effects on proteins, and the molecules that regulate lactylation and its targets. Finally, we summarize the current questions that still exist in lactylation research. This review provides direction and a theoretical basis for the research and clinical translation of protein lactylation.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.