The skeletal muscle response to high-intensity training assessed by single-nucleus RNA-sequencing is blunted in individuals with type 2 diabetes

Abstract

Training can improve insulin sensitivity in individuals with type 2 diabetes, but a clear understanding of the mechanisms remains elusive. To further our knowledge in this area, we aimed to examine the effect of type 2 diabetes and of high-intensity interval training (HIIT) on the nuclear transcriptional response in skeletal muscle. We performed single-nucleus RNA-sequencing (snRNA-seq) and immunofluorescence analysis on muscle biopsies from the trained and the untrained legs of participants with and without type 2 diabetes, after 2 weeks of one-legged HIIT on a cycle ergometer. Surprisingly, the type 2 diabetes condition only seemed to have a minor effect on transcriptional activity in myonuclei related to major metabolic pathways when comparing the untrained legs. However, while in particular the type IIA myonuclei in the control group displayed a considerable metabolic response to HIIT, with increases in genes related to glycogen breakdown and glycolysis primarily in the type IIA myonuclei of the trained leg, this response was blunted in the diabetes group, despite a marked increase in glucose clearance in both groups. Additionally, we observed that fibre type distribution assessed by immunofluorescence significantly correlated with the proportion of myonuclei in the snRNA-seq analysis. In conclusion, the type 2 diabetes condition blunts the metabolic transcriptional response to HIIT in the type IIA myonuclei without affecting the improvement in insulin sensitivity. Additionally, our results indicate that snRNA-seq can be used as a surrogate marker for fibre type distribution in sedentary middle-aged adults. KEY POINTS: The study utilized single-nucleus RNA sequencing (snRNA-seq) to analyse 38 skeletal muscle biopsies, revealing distinct transcriptional profiles in myonuclei from individuals with and without type 2 diabetes (T2D) after 2 weeks of HIIT. snRNA-seq identified significant differences in gene expression, with 14 differentially expressed genes (DEGs) in type IIA myonuclei of the control group, specifically related to glycogen breakdown and glycolysis, which were blunted in the T2D group. In the control group, HIIT induced a substantial transcriptional response in type IIA myonuclei, enhancing metabolic pathways associated with insulin sensitivity, while the T2D group showed minimal transcriptional changes despite improved insulin sensitivity. The T2D group exhibited a blunted response in metabolic gene expression, indicating that the training effect on muscle adaptation was significantly impaired compared to healthy controls. Overall, the findings highlight the differential impact of HIIT on muscle metabolism, emphasizing the need for tailored exercise interventions for individuals with T2D.

Keywords: HIIT; glucose metabolism; snRNA‐seq; training; type 2 diabetes.

Figure 1. An overview of the workflow

Created in BioRender. Hansen, M. (2025) https://BioRender.com/q05o255. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Cell type distribution

A, UMAP of the skeletal muscle cell clusters. B, the proportions of the different cell types in each muscle sample. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Stacked violin plots of canonical marker gene expression across the 11 snRNA‐seq clusters used for cell type identification

Figure 4. Myonuclei

A, UMAP of myonuclei subclusters. B, The proportions of the different myonuclei in each muscle sample. Abbreviation: MTJ, myotendinous junction. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Immunofluorescence analysis and snRNA‐seq assessment of myofibre type composition and training response in control and type 2 diabetes muscle

A, a representative image of immunostaining of a muscle biopsy cross‐section, with type I and type II fibres denoted. Scale bar = 50 µm. B, type I myofibre proportion observed by immunofluorescence (IF) analysis in the trained and untrained legs of the control group (CON) and the type 2 diabetes (T2D) group. C, correlation between the proportion of type I fibres from the IF analysis and the proportion of type I myonuclei (% of the sum of type I, IIA and IIX myonuclei) from the snRNA‐seq. D, the expression of MYH2 (type IIA myosin) in type IIX (MYH1+) myonuclei in the trained and untrained legs of CON and T2D. Box plots are shown as median, 25th and 75th percentiles. The number and percentage of cells expressing MYH2 are printed above the respective condition. The red arrow denotes a significant difference between the trained and untrained legs in CON. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Differentially expressed genes (DEGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways

A–C, number of differentially expressed genes (DEGs) in the untrained (UT) legs between the control group (CON) and the diabetes group (T2D) (A), and between the trained (T) and untrained legs of CON (B) and T2D (C). D–F, the top three differentially expressed KEGG pathways in the untrained legs between T2D and CON (D), and between the trained and untrained legs of CON (E) and T2D (F). There were no significantly regulated KEGG pathways between the trained and untrained legs in the type I myonuclei of the control group. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7. Differentially expressed genes in metabolic pathways in type I, IIA, and IIX myonuclei

Differentially expressed genes (DEGs) related to metabolic pathways between CON and T2D in the untrained legs (A) and between the trained and untrained legs within the groups (B). Additionally, DEGs related to insulin‐related KEGG pathways between CON and T2D in the untrained legs (C) and between the trained and untrained legs within the groups (D). E, a Venn diagram of all genes related to the KEGG insulin signalling and KEGG insulin resistance pathways. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 8. Differential expression of genes in type IIA fibres from trained and untrained legs in controls and in patients with Type 2 diabetes, with pathway context

Relative expression levels of the seven genes that were differentially expressed in type IIA fibres between the trained and untrained legs of the control group (see Fig. 7B ), along with an overview of their position in the glycogenolysis and glycolysis pathways. Abbreviations: CON, controls; F6P, fructose 6‐phosphate; F1,6P, fructose‐1,6‐bisphosphate; G1P, glucose 1‐phosphate; G6P, glucose 6‐phosphate; T, trained leg; T2D, type 2 diabetes; UT, untrained leg. [Colour figure can be viewed at wileyonlinelibrary.com]