Skeletal muscle hypertrophy rewires glucose metabolism: An experimental investigation and systematic review

Abstract

Background: Proliferating cancer cells shift their metabolism towards glycolysis, even in the presence of oxygen, to especially generate glycolytic intermediates as substrates for anabolic reactions. We hypothesize that a similar metabolic remodelling occurs during skeletal muscle hypertrophy.

Methods: We used mass spectrometry in hypertrophying C2C12 myotubes in vitro and plantaris mouse muscle in vivo and assessed metabolomic changes and the incorporation of the [U-¹³C₆]glucose tracer. We performed enzyme inhibition of the key serine synthesis pathway enzyme phosphoglycerate dehydrogenase (Phgdh) for further mechanistic analysis and conducted a systematic review to align any changes in metabolomics during muscle growth with published findings. Finally, the UK Biobank was used to link the findings to population level.

Results: The metabolomics analysis in myotubes revealed insulin-like growth factor-1 (IGF-1)-induced altered metabolite concentrations in anabolic pathways such as pentose phosphate (ribose-5-phosphate/ribulose-5-phosphate: +40%; P = 0.01) and serine synthesis pathway (serine: -36.8%; P = 0.009). Like the hypertrophy stimulation with IGF-1 in myotubes in vitro, the concentration of the dipeptide l-carnosine was decreased by 26.6% (P = 0.001) during skeletal muscle growth in vivo. However, phosphorylated sugar (glucose-6-phosphate, fructose-6-phosphate or glucose-1-phosphate) decreased by 32.2% (P = 0.004) in the overloaded muscle in vivo while increasing in the IGF-1-stimulated myotubes in vitro. The systematic review revealed that 10 metabolites linked to muscle hypertrophy were directly associated with glycolysis and its interconnected anabolic pathways. We demonstrated that labelled carbon from [U-¹³C₆]glucose is increasingly incorporated by ~13% (P = 0.001) into the non-essential amino acids in hypertrophying myotubes, which is accompanied by an increased depletion of media serine (P = 0.006). The inhibition of Phgdh suppressed muscle protein synthesis in growing myotubes by 58.1% (P < 0.001), highlighting the importance of the serine synthesis pathway for maintaining muscle size. Utilizing data from the UK Biobank (n = 450 243), we then discerned genetic variations linked to the serine synthesis pathway (PHGDH and PSPH) and to its downstream enzyme (SHMT1), revealing their association with appendicular lean mass in humans (P < 5.0e-8).

Conclusions: Understanding the mechanisms that regulate skeletal muscle mass will help in developing effective treatments for muscle weakness. Our results provide evidence for the metabolic rewiring of glycolytic intermediates into anabolic pathways during muscle growth, such as in serine synthesis.

Keywords: Warburg effect; lactate; metabolomics; resistance exercise; serine synthesis pathway.

Figure 1

(A) Volcano plots displaying the false discovery rate (FDR) values (−log10) versus log10 fold changes of all features (i.e., both unannotated and annotated metabolites) of LC‐MS/MS measurements (negative ion mode) between insulin‐like growth factor‐1 (IGF‐1) and vehicle control (left) and between vehicle control and rapamycin (right) treatment in differentiated C2C12 muscle cells; features with FDR < 0.2 are above the purple box. (B) Principal component analysis (PCA) of metabolite intensities of LC‐MS/MS measurements (negative ion mode). (C) Heatmap of significant amino acid level changes after vehicle control, IGF‐1 or rapamycin treatment in vitro. Crosses indicate that the metabolite is below the detection limit. *Significant differences between treatments (IGF‐1 or rapamycin) compared with vehicle control. ^#Significant differences between IGF‐1 compared with rapamycin, unpaired two‐tailed Student's t‐test (FDR < 0.1). (D) Enrichment analysis with MetaboAnalyst 5.0 between IGF‐1 and rapamycin treatments. CON, control; RAP, rapamycin.

Figure 2

Metabolomic changes in differentiated C2C12 myotubes treated with vehicle control, insulin‐like growth factor‐1 (IGF‐1; 100 ng/mL) or rapamycin (10 ng/mL) for 48 h (n = 3). Metabolites highlighted in bold were detected by untargeted metabolomics. Frames show significant metabolite level changes between conditions (FDR < 0.2). Metabolite intensity (MI) is represented on a log₂ scale. CON, control; ND, not detected; P, phosphate; RAP, rapamycin; TCA, tricarboxylic acid cycle.

Figure 3

(A) Gene expressions of enzymes of glycolysis and the pentose phosphate pathway in C2C12 myotubes, including hexokinase 2 (Hk2), glucose‐6‐phosphate 1‐dehydrogenase X (G6pdx) and phosphoribosyl pyrophosphate synthetase 2 (Prps2). (B) Gene expressions of key enzymes of the serine synthesis pathway in C2C12 myotubes: phosphoglycerate dehydrogenase (Phgdh) and serine hydroxymethyltransferase‐2 (Shmt2). (C) Volcano plots displaying the false discovery rate (FDR) values (−log10) versus log10 fold changes of all features between overloaded and control mice plantaris muscles (negative ion mode); features with FDR < 0.2 are above the purple box. CON, control; IGF‐1, insulin‐like growth factor‐1; Phosph. sugar, phosphorylated sugar; RAP, rapamycin. *Significant differences between indicated conditions, one‐way ANOVA with Tukey's HSD post hoc test (P < 0.05). Data are expressed as the mean ± standard error of the mean (SEM).

Figure 4

(A) Metabolic changes in response to skeletal muscle hypertrophy stimulation (log2 fold change vs. rest); black cross: in vivo studies; red cross: in vitro studies. (B) Venn diagram showing overlap and unique metabolites associated with muscle growth between studies performed in mice in vivo (animals) or in vitro (cell culture). (C) Enrichment analysis with MetaboAnalyst 5.0. DHA, docosahexaenoic acid; GlcNAc‐6P, N‐acetylglucosamine‐6‐phosphate; NADP+, oxidized nicotinamide adenine dinucleotide phosphate; P, phosphate; UDP, uridine diphosphate.

Figure 5

(A) ¹³C‐enrichment of glucose, lactate, alanine and glycine between insulin‐like growth factor‐1 (IGF‐1) and vehicle control in conditioned media after 24 h. (B) Isotopologues of proline in media. (C) Serine concentration in conditioned media. (D) Fractional synthesis rate (FSR) based upon ¹³C‐alanine incorporation. CON, control; RAP, rapamycin. *Significant differences between groups, unpaired two‐tailed Student's t‐test (P < 0.05). Data are expressed as the mean ± standard error of the mean (SEM).

Figure 6

(A) Protein synthesis determined using puromycin; (B) total protein concentration of C2C12 myotubes; and (C) lactate dehydrogenase (LDH) activity in conditioned media measured, including NCT‐503 inhibitor or vehicle control (CON) after 48 h. *Significant differences between groups, unpaired two‐tailed Student's t‐test (P < 0.05). Data are expressed as the mean ± standard error of the mean (SEM).