We have investigated transcription of methyltransferases and methylation capacity (Met/Hcy ratio) on data from a 12 w training intervention described previously 26. After acute and long-term exercise, more than 200 genes with known or probable functions in methyltransferase reactions were transcribed in SkM. Differential gene expression analyses of several methyltransferases in response to acute and long-term exercise in SkM demonstrated that these were primarily involved in the biosynthetic processes, histone- and lysine-methylation (just after exercise), RNA processing (2 h after exercise) as well as cellular communication and developmental processes (long-term exercise). The Met/Hcy ratio was reduced in response to acute exercise but increased slightly following long-term exercise. Several methyltransferases were associated with improved in insulin sensitivity measured by clamp (GIR) and VO2max after the long-term intervention.
The most upregulated methyltransferase just after and 2 h after acute exercise was NNMT, which encodes nicotinamide N-methyltransferase, an enzyme catalyzing the N-methylation of the vitamin B3-derivative nicotinamide to methylnicotinamide. This finding is partly in line with a previous study demonstrating upregulation of NNMT in response to exercise and suggested that methylnicotinamide may be a signal for WAT lipolysis 17. In addition, NNMT mRNA levels have been shown to be higher in WAT of patients with type 2 diabetes compared to healthy controls 27. We did not observe altered NNMT trancription in response to long-term exercise in SkM or WAT. Therefore, change in transcription of NNMT was not associated with improved GIR, VO2max, muscle hypertrophy or body fat. Thus, our results support that NNMT may be involved in the acute response to exercise but not necessarily phenotypical improvements.
Few other genes remained differentially expressed throughout the recovery period suggesting a potential shift in biological processes requiring methyltransferase activities from just after to 2 h after exercise. Just after exercise, regulation of several biosynthetic processes as well as peptidyl and histone-lysine methylation were prominent which are important in epigenetic regulation of gene expression which has received considerable attention for its role in SkM adaptations to exercise together with DNA methylation 11–16, 28,29. In particular, DOT1L, PHF1, and SETD2 were present in most of the enriched GOs, all of which are involved in histone methylation and epigenetic regulation according to OMIM. In particular these methyltransferases are involved in histone H3 methylation, which indeed seems to play a role in the epigenetic regulation of gene expression in response to exercise 28. E.g. WDR82 is part of at least four methyltransferase complexes 30 that trimethylates histone H3 in the lysine 4 position. This particular histone modification occurred in exercising mice 31 and was linked to upregulated expression of PGC-1α, a crucial regulator of exercise-induced changes in mitochondrial biogenesis and energy metabolism 13,32. The downregulated GOs just after exercise also included protein methylation, including the three protein arginine methyltransferases, PRMT2, PRMT6, and PRMT7. In particular, PRMT7 is highly transcribed in SkM 19 but has not previously been shown to be affected by acute exercise. Notably, PRMTs are also involved in arginine methylation of histones 19, and the GO enrichment analyses thus collectively suggest differential methylation of specific histone amino acid residues just after exercise. These results may aid future research in identifying more targeted approaches in studying the role of epigenetic modifications in SkM during exercise 29,33,34, and we note that differential expression largely involved histone methyltransferases and not DNA methyltransferases.
After 2 h rest, GO biological processes including histone methylation and epigenetic regulation were no longer enriched. Instead, analyses suggested that SkM methyltransferases involved in RNA and non-coding RNA processing and methylation, as well as ribonucleoprotein complex biogenesis were upregulated. These processes are important for translational regulation in response to exercise. One study showed that high-intensity interval training increased ribosome abundance and subsequent protein translation, and suggested that this is one of the adaptive mechanisms in SkM to long-term high-intensity interval training 35. Another study showed that trained individuals had higher expression of ribosomal proteins than untrained controls 36. However, an important distinction was that these relationships were assessed after long-term exercise or in a cross-sectional manner, and not after acute exercise bouts. To our knowledge, the qualitative contribution of methyltransferases to these mechanisms is not known in the immediate recovery phase.
After the 12 w exercise intervention, only 11 out of 210 transcripts with methyltransferase activities were increased. The most upregulated methyltransferase was PRDM1, which is involved in B cell transcriptional repression through histone modifications 37. The change in PRDM1 was positively correlated to the improvements in VO2max and tended to be correlated to improvements in GIR and increased m. vastus lateralis cross-sectional area. This is in line with a previous GWAS suggesting that a single nucleotide polymorphism in the PRDM1-encoding region was a significant predictor of improved VO2max 38. However, PRDM1 was not present in the enriched GOs including cell communication and signaling. In these GOs, 8 of the 11 upregulated methyltransferases were present and included genes mostly involved in signaling pathways, histone modifications, and epigenetic regulation such as DNMT1, MECOM, PRMT2, and PRMT6. The change in PRMT2 was strongly and positively associated with improved insulin sensitivity and VO2max in the present study, which is in contrast to observations in Prmt2−/− models exhibiting lower fasting blood glucose and an overall beneficial metabolic profile 39.
The majority of differentially expressed methyltransferases after long-term exercise were downregulated and associated with broad, unspecific GO terms. The most downregulated transcript was METTL21C, an SkM-specific non-histone protein-lysine methyltransferase involved in regulation of myogenesis, muscle function, and protein catabolism 40,41. Furthermore, METTL21C has been described as important for regulation of ATPase activity and depletion impairs voluntary running in mice 41. The physiological and clinical relevance of METTL21C in humans remain to be established, but we note that it was present in several of the enriched downregulated GOs after long-term exercise. Moreover, there was a trend for an inverse association between METTL21C and improvements in GIR, suggesting that subjects with a more negative change in METTL21C mRNA levels had more improved GIR. Notably, METTL21C was the second most upregulated gene 2 h after exercise, whereas it was significantly downregulated after the 12 w exercise intervention. These findings indicate that METTL21C is not only important in the acute and long-term SkM response to exercise, but may also contribute to beneficial phenotypical responses after a long-term exercise intervention.
Because most methyltransferase reactions depend on methionine we evaluated the effects of exercise on the plasma Met/Hcy ratio, which is increasingly used as a plasma indicator of intracellular methylation capacity 7,8. We observed a ~ 40 % reduction in plasma Met/Hcy ratio in response to the acute exercise indicating that tissue consumption of methionine for methylation reactions are induced by exercise. Importantly, the decrease in Met/Hcy was driven by the reduction in methionine. Animal studies have shown that SAM consumption increases in liver and the endothelium after exercise promoting increased plasma concentrations of total homocysteine concentrations 24,25, but no studies that we are aware of have characterized methionine metabolism in SkM during exercise. The reduced plasma Met/Hcy ratio and increased expression of LAT1 and MAT2A in our study indicate that SkM uptake and metabolism of methionine could be increased after exercise and involved in methyltransferase reactions. One previous study demonstrated an increase in SkM methionine after exercise in both type I and type II SkM fibers 42 supporting that increased methionine uptake may occur post-exercise in addition to protein degradation. However, some caution should be shown in interpreting these findings considering that the LAT1 membrane transporter is not specific to methionine. In addition, methyltransferases are generally expressed to a large extent in the liver, and thus the Met/Hcy might not be a good marker of methionine metabolism in SkM.
The strengths of this study include the controlled design, supervised acute and long-term exercise regimen, and deep phenotypical data including improvements in oxygen uptake and systemic insulin sensitivity. This allows for detailed descriptions of the SkM and WAT transcriptomic response just after exercise, in the early recovery phase and after long-term exercise training.
There are also some limitations to the study, including that we only have data on the transcriptomic level, making it impossible to infer on changes in protein activity and the regulation of targeted mechanisms. However, the aims of this study were strictly explorative and its purpose to inform future research seeking to unravel the relationship between methyltransferase reactions and exercise further. With regards to plasma markers, future studies focusing on methylation and methylation reactions should monitor essential intermediates and co-factors for these reactions including SAM and S-adenosylhomocysteine, vitamin B12, folate, choline and betaine.
In conclusion, acute exercise and the immediate recovery phase led to differential expression of methyltransferases related to highly variable biological processes including epigenetic modification and RNA processing, whereas long-term changes in expression were related to developmental processes. In addition, the plasma Met/Hcy ratio decreased in response to acute exercise but increased after long-term exercise training. These results suggest that SkM transcriptomic response to exercise may extend beyond DNA methylation and epigenetic regulation to the processing of other macromolecules and phenotypical improvements.