Article

Transcriptomic Profiling Reveals Lactate-Responsive Signaling Networks Associated with Cellular Adaptation

Bok Sil Hong^1,*, Youngseo Lee², Min Seo Jeong², Myoung-Rye Kim²

Abstract

Background/Objectives: Lactate has traditionally been regarded as a metabolic byproduct generated during anaerobic glycolysis. However, accumulating evidence indicates that lactate functions as a signaling metabolite involved in cellular adaptation, energy metabolism, and skeletal muscle remodeling. Despite increasing recognition of its signaling role, the global transcriptional responses induced by lactate in skeletal muscle cells remain incompletely understood. This study aimed to investigate lactate-responsive transcriptomic changes and associated signaling networks in C2C12 myoblasts.

Methods: C2C12 myoblasts were treated with lactate for 30, 60, and 120 min. RNA sequencing was performed, followed by differential gene expression, hierarchical clustering, functional annotation, and KEGG pathway analyses.

Results: A total of 2,462 differentially expressed genes (DEGs) were identified at |FC| > 1.5, including 439 DEGs at |FC| > 2.0. Lactate altered the expression of genes involved in oxidative stress regulation, apoptosis, cell cycle progression, fatty acid metabolism, and skeletal muscle adaptation. KEGG pathway analysis identified enrichment of PI3K-Akt signaling and cell cycle pathways, while several DEGs were associated with fatty acid metabolism and skeletal muscle adaptation.

Conclusions: Lactate induced extensive transcriptomic remodeling in C2C12 myoblasts and regulated genes associated with cellular adaptation. These findings support the concept that lactate functions as a signaling metabolite and may contribute to skeletal muscle adaptation through coordinated regulation of adaptive signaling networks.

Keywords

Lactate, Transcriptomics, Cellular adaptation, Oxidative stress, PI3K-Akt signaling, Cell cycle, C2C12 myoblasts

Introduction

Lactate has traditionally been regarded as a metabolic byproduct generated during anaerobic glycolysis. For decades, lactate accumulation in skeletal muscle was considered a consequence of oxygen deficiency and was frequently associated with muscle fatigue and impaired performance [1]. However, growing evidence has fundamentally changed this perspective and established lactate as an important metabolic intermediate and signaling molecule involved in cellular adaptation, energy metabolism, and inter-organ communication [2-5].

The lactate shuttle theory proposed by Brooks demonstrated that lactate is continuously produced and utilized by multiple tissues, including skeletal muscle, heart, liver, and brain [2-4]. Beyond serving as an energy substrate, lactate has been reported to regulate gene expression, mitochondrial biogenesis, angiogenesis, inflammation, and cellular stress responses [5]. Recent studies have further suggested that lactate functions as a signaling metabolite capable of influencing intracellular pathways involved in cell survival, proliferation, and metabolic adaptation [6].

Skeletal muscle is a major site of lactate production and utilization. During exercise, elevated lactate concentrations are accompanied by extensive metabolic and transcriptional remodeling that contributes to muscle adaptation and recovery [7, 8]. Several studies have reported that lactate activates signaling pathways associated with cellular growth and adaptation, including PI3K-Akt signaling and redox-regulatory pathways [9]. In addition, exercise-induced lactate accumulation has been linked to changes in reactive oxygen species (ROS) homeostasis and antioxidant responses [10, 11]. Despite increasing recognition of lactate as a signaling molecule, the global transcriptional responses induced by lactate in skeletal muscle cells remain incompletely understood.

Recent advances in RNA sequencing (RNA-seq) technologies have enabled comprehensive characterization of transcriptome-wide responses to metabolic stimuli [12, 13]. Transcriptomic profiling provides a powerful approach for identifying differentially expressed genes (DEGs) and elucidating molecular pathways involved in cellular adaptation.

Therefore, the present study investigated the transcriptomic responses of C2C12 myoblasts following lactate treatment using RNA-seq analysis. Differential gene expression profiles were examined at multiple treatment periods, and functional annotation and pathway analyses were performed to identify biological processes associated with lactate-induced transcriptional remodeling. We hypothesized that lactate regulates genes involved in oxidative stress responses, apoptosis, cell cycle progression, and adaptive signaling pathways that may contribute to skeletal muscle adaptation.

Materials and Methods

Cell culture

Murine C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [14]. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

Lactate treatment

C2C12 myoblasts were treated with sodium lactate at the indicated concentration. Cells were harvested after 30, 60, and 120 min of lactate exposure. Untreated cells served as controls. Three independent biological replicates were prepared for each experimental condition.

RNA extraction and RNA sequencing

Total RNA was isolated using an RNA extraction kit according to the manufacturer’s instructions. RNA quantity and quality were evaluated using spectrophotometric and electrophoretic methods. RNA libraries were prepared using a stranded mRNA library preparation kit and sequenced using an Illumina sequencing platform [15].

Differential gene expression analysis

Raw sequencing reads were subjected to quality control and adaptor trimming. Clean reads were aligned to the mouse reference genome. Gene expression levels were quantified and normalized prior to differential expression analysis [16].

Genes exhibiting an absolute fold change (|FC|) > 1.5 or |FC| > 2.0 relative to control samples were considered differentially expressed. Up-regulated and down-regulated genes were identified for each treatment period.

Hierarchical clustering analysis

Hierarchical clustering analysis was performed to visualize global transcriptional changes induced by lactate treatment. Heatmaps were generated using normalized gene expression values, and clustering was performed using Euclidean distance and complete linkage methods [17].

Functional annotation and pathway analysis

Differentially expressed genes were subjected to functional annotation and pathway enrichment analyses. Genes associated with oxidative stress, apoptosis, cell cycle regulation, and skeletal muscle adaptation were further examined. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to identify signaling pathways affected by lactate treatment [18,19].

Results

Lactate induces time-dependent transcriptomic remodeling in C2C12 myoblasts

To characterize the transcriptional responses induced by lactate exposure, RNA-seq analysis was performed in C2C12 myoblasts treated with lactate for 30, 60, and 120 min (Figure 1A).

At a threshold of |FC| > 1.5, a total of 2,462 DEGs were identified across all treatment groups. Among these genes, 223 commonly up-regulated genes and 255 commonly down-regulated genes were shared among the three treatment periods, accounting for 19.6% of all DEGs (Figure 1B). In contrast, four genes displayed differential temporal expression patterns across the three treatment periods.

Application of a more stringent threshold (|FC| > 2.0) reduced the number of DEGs to 439 genes. Notably, 66 commonly up-regulated genes and 61 commonly down-regulated genes remained consistently altered across all treatment periods, representing 29.2% of the identified DEGs. Under this criterion, Abl was the only gene exhibiting a distinct temporal expression pattern. The increased proportion of shared DEGs at the higher fold-change threshold suggests the existence of a core lactate-responsive transcriptional program that is maintained throughout the treatment period.

Analysis of DEG distributions further demonstrated that lactate induced rapid and substantial transcriptional responses during the early treatment period, followed by progressive stabilization of gene expression patterns (Figure 1C). These findings indicate that lactate acts as an early signaling stimulus capable of triggering widespread transcriptional remodeling in skeletal muscle cells.

Hierarchical clustering reveals coordinated gene expression responses to lactate

To further characterize the transcriptional landscape induced by lactate treatment, hierarchical clustering analysis was performed using all identified DEGs (Figure 2).

The resulting heatmap revealed distinct clusters of genes exhibiting coordinated up-regulation or down-regulation following lactate exposure. Several genes associated with transcriptional regulation, chromatin remodeling, and cellular proliferation were consistently enriched among the up-regulated clusters. Representative genes included Fos, Nfkbia, Ect2, Hist1h2ah, Hist1h2ak, and Hist1h3a, which displayed sustained induction throughout the treatment period.

Among the down-regulated clusters, genes such as Oasl1, Chad, Slc43a3, Hpgds, and Pnoc exhibited marked suppression following lactate treatment. The coordinated expression patterns observed in these clusters suggest that lactate-mediated transcriptional responses are regulated through interconnected biological networks rather than isolated gene-specific mechanisms.

Interestingly, immediate early response genes, including Fos, were among the most strongly induced genes, indicating rapid activation of transcriptional programs associated with cellular adaptation.

Lactate modulates oxidative stress-, apoptosis-, and cell cycle-related genes

To identify biological processes potentially affected by lactate exposure, genes associated with oxidative stress, apoptosis, and cell cycle regulation were examined (Figure 3).

Among oxidative stress-related genes, Gpx3, Gstm2, Nqo1, Sod3, and Xdh were consistently down-regulated across treatment periods. These genes are involved in antioxidant defense and cellular redox homeostasis. In contrast, JunB and Mt1a exhibited increased expression following lactate treatment. JunB is known to participate in cellular stress adaptation and transcriptional regulation, whereas Mt1a plays an important role in metal ion homeostasis and protection against oxidative stress.

Analysis of apoptosis-related genes demonstrated significant induction of Birc3 and Bcl2, both of which are recognized anti-apoptotic regulators. Increased expression of these genes suggests activation of survival-promoting mechanisms in response to lactate exposure.

Furthermore, several cell cycle-associated genes exhibited marked up-regulation. Notably, Ccnb1, Cdc20, Cdc6, Cdk1, and Chek1 were consistently induced across treatment periods. These genes are involved in DNA replication, checkpoint regulation, mitotic entry, and cell cycle progression. Conversely, Cdkn1a displayed a relative reduction compared with proliferative regulators, suggesting a shift toward cell cycle activation.

Collectively, these findings indicate that lactate regulates genes involved in oxidative stress responses, cell survival, and cell cycle progression.

Lactate alters genes associated with fatty acid metabolism and skeletal muscle adaptation

To further investigate the biological processes influenced by lactate, genes associated with fatty acid metabolism and skeletal muscle function were examined. Several genes involved in lipid metabolism exhibited significant transcriptional changes following lactate treatment. Notably, Acsl4, Acsl6, Scd1, Acadsb, and Prkaa1 were differentially expressed across treatment periods, suggesting that lactate may influence pathways involved in fatty acid utilization and energy homeostasis.

Acsl4 and Acsl6 are involved in long-chain fatty acid activation, whereas Acadsb participates in mitochondrial fatty acid β-oxidation. In addition, Prkaa1, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK), was altered following lactate exposure. These findings suggest that lactate may contribute to metabolic reprogramming by regulating genes associated with lipid metabolism and cellular energy sensing.

Genes associated with skeletal muscle contraction and calcium signaling were also affected by lactate treatment. Among these, Tpm4, Atf3, Igfbp4, Igfbp5, Ramp1, and Ryr3 exhibited altered expression patterns. Tpm4 is involved in actin filament regulation and muscle contraction, whereas Ryr3 encodes a ryanodine receptor that mediates intracellular calcium release. The observed transcriptional changes suggest that lactate may influence molecular pathways associated with muscle remodeling and contractile adaptation.

IB-DK143 is associated with a decreasing trend in ERK1/2 signaling

Because computational analysis prioritized ERK2/MAPK1 and MEK1/MAP2K1, ERK1/2 expression and/or phosphorylation was evaluated as an early signaling readout. IB-DK143-treated cells showed a decreasing trend in ERK1/2 signaling. This observation is consistent with the proposed model that IB-DK143 may suppress ERK-mediated survival signaling. However, quantitative phospho-ERK1/2 and total ERK1/2 Western blotting is required to determine whether ERK suppression is statistically significant and whether it occurs before caspase activation.

Lactate-responsive genes are enriched in PI3K-Akt signaling and cell cycle pathways

To identify signaling pathways associated with lactate-induced transcriptional changes, KEGG pathway analysis was performed (Figures 4 and 5).

Multiple DEGs were mapped to the PI3K-Akt signaling pathway, including genes associated with cell proliferation, apoptosis regulation, stress adaptation, and metabolic regulation. The widespread distribution of DEGs throughout this pathway suggests that PI3K-Akt signaling may serve as a central regulatory hub mediating lactate-induced cellular responses.

In addition, pathway analysis revealed extensive regulation of genes involved in cell cycle progression. Differentially expressed genes were distributed throughout multiple stages of the cell cycle, including DNA replication, checkpoint control, G1/S transition, and G2/M progression. Up-regulation of Ccnb1, Cdc20, Cdk1, and Chek1 supports the hypothesis that lactate promotes transcriptional programs associated with cellular proliferation and adaptation.

Taken together, these results demonstrate that lactate induces broad transcriptional remodeling and influences multiple biological pathways associated with stress adaptation, cell survival, and cell cycle regulation.

Discussion

The present study investigated transcriptome-wide responses to lactate treatment in C2C12 myoblasts and demonstrated that lactate induces extensive transcriptional remodeling involving oxidative stress regulation, apoptosis-associated genes, cell cycle progression, and PI3K-Akt signaling pathways.

Traditionally, lactate has been regarded as a metabolic byproduct generated during anaerobic glycolysis. However, increasing evidence supports the concept that lactate functions as a signaling molecule capable of regulating cellular adaptation [4,5]. The present findings further support this concept by demonstrating that lactate exposure rapidly altered the expression of hundreds of genes, many of which were consistently regulated across multiple treatment periods.

One notable observation was the existence of a conserved lactate-responsive transcriptional signature. Although thousands of DEGs were identified at the |FC| > 1.5 threshold, the proportion of shared genes increased substantially under the more stringent |FC| > 2.0 criterion. This finding suggests that a subset of genes may represent robust and reproducible molecular targets of lactate signaling.

The regulation of oxidative stress-related genes represents another important finding. Several antioxidant-associated genes, including Gpx3, Gstm2, Nqo1, and Sod3, were down-regulated following lactate treatment. Although this observation may initially appear paradoxical, previous studies have suggested that moderate increases in ROS can function as signaling molecules that promote adaptive cellular responses [10,11,20]. Therefore, the observed transcriptional changes may reflect lactate-induced redox signaling rather than oxidative damage.

Lactate treatment also increased the expression of anti-apoptotic genes such as Birc3 and Bcl2. These findings suggest that lactate promotes cell survival pathways and enhances resistance to cellular stress. Consistent with this interpretation, pathway analysis identified significant involvement of PI3K-Akt signaling, a pathway widely recognized for its role in promoting survival, growth, and adaptation [21,22].

Perhaps the most striking observation was the induction of multiple cell cycle regulators, including Ccnb1, Cdc20, Cdc6, Cdk1, and Chek1. These genes are essential components of DNA replication and cell cycle progression [23,24]. Their coordinated up-regulation suggests that lactate may facilitate proliferative responses and cellular remodeling processes. The enrichment of the cell cycle pathway further supports this interpretation.

In addition to oxidative stress regulation and cell cycle-associated pathways, lactate treatment altered the expression of genes involved in fatty acid metabolism. Differential expression of Acsl4, Acsl6, Acadsb, Scd1, and Prkaa1 suggests that lactate may participate in metabolic reprogramming by modulating pathways associated with lipid utilization and energy homeostasis. Previous studies have proposed that lactate functions not only as a metabolic intermediate but also as a signaling molecule capable of coordinating substrate utilization in response to changing energetic demands [4,5,25]. The transcriptional changes observed in the present study support this concept and suggest a potential role for lactate in fine-tuning metabolic adaptation in skeletal muscle cells.

Furthermore, several genes associated with muscle contraction and calcium regulation, including Tpm4 and Ryr3, were differentially expressed following lactate treatment. Skeletal muscle adaptation to exercise involves coordinated regulation of calcium signaling, contractile proteins, and metabolic pathways [7-9]. Together with the induction of Fos, JunB, and multiple cell cycle regulators, these findings suggest that lactate may contribute to transcriptional programs associated with skeletal muscle remodeling and exercise-induced adaptation. Although functional validation is required, the present results provide transcriptomic evidence supporting the emerging view that lactate acts as an exercise-responsive signaling metabolite capable of regulating both metabolic and structural adaptation in skeletal muscle cells.

Interestingly, immediate early response genes such as Fos and JunB were among the most responsive genes identified in this study. Both genes have been implicated in exercise-induced transcriptional adaptation and skeletal muscle remodeling [7,8,26]. Together with the observed activation of PI3K-Akt signaling and cell cycle-associated genes, these findings suggest that lactate may contribute to molecular programs associated with exercise adaptation rather than functioning solely as an energy substrate.

Several limitations should be acknowledged. First, the present study was performed using cultured C2C12 myoblasts and may not fully reflect physiological responses in vivo. Second, transcriptomic alterations do not necessarily correspond to protein-level changes. Finally, functional validation experiments were not performed. Future studies should investigate the protein expression and physiological consequences of the identified lactate-responsive genes.

In conclusion, lactate induced extensive transcriptional remodeling in C2C12 myoblasts and regulated genes involved in oxidative stress responses, apoptosis, PI3K-Akt signaling, and cell cycle progression. These findings further extend the lactate shuttle concept proposed by Brooks by suggesting that lactate functions not only as a metabolic intermediate but also as a transcriptomic regulator capable of coordinating adaptive gene expression programs. Collectively, the present data support an expanded view of lactate as a multifunctional signaling metabolite involved in the regulation of cellular adaptation, metabolic remodeling, and exercise-associated responses.

Conclusion

Lactate induced extensive transcriptomic remodeling in C2C12 myoblasts and regulated genes associated with oxidative stress responses, cell survival, fatty acid metabolism, and cell cycle progression. KEGG pathway analysis identified PI3K-Akt signaling and cell cycle pathways as major lactate-responsive networks. In addition, lactate altered the expression of genes related to metabolic adaptation and skeletal muscle function. These findings support the concept that lactate acts as a signaling metabolite rather than merely a glycolytic byproduct and may contribute to cellular adaptation through coordinated regulation of multiple signaling pathways.

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