Skeletal muscle interstitial fluid metabolomics at rest and associated with an exercise bout: application in rats and humans

Abstract

Blood or biopsies are often used to characterize metabolites that are modulated by exercising muscle. However, blood has inputs derived from multiple tissues, biopsies cannot discriminate between secreted and intracellular metabolites, and their invasive nature is challenging for frequent collections in sensitive populations (e.g., children and pregnant women). Thus, minimally invasive approaches to interstitial fluid (IF) metabolomics would be valuable. A catheter was designed to collect IF from the gastrocnemius muscle of acutely anesthetized adult male rats at rest or immediately following 20 min of exercise (~60% of maximal O₂ uptake). Nontargeted, gas chromatography-time-of-flight mass spectrometry analysis was used to detect 299 metabolites, including nonannotated metabolites, sugars, fatty acids, amino acids, and purine metabolites and derivatives. Just 43% of all detected metabolites were common to IF and blood plasma, and only 20% of exercise-modified metabolites were shared in both pools, highlighting that the blood does not fully reflect the metabolic outcomes in muscle. Notable exercise patterns included increased IF amino acids (except leucine and isoleucine), increased α-ketoglutarate and citrate (which may reflect tricarboxylic acid cataplerosis or shifts in nonmitochondrial pathways), and higher concentration of the signaling lipid oleamide. A preliminary study of human muscle IF was conducted using a 20-kDa microdialysis catheter placed in the vastus lateralis of five healthy adults at rest and during exercise (65% of estimated maximal heart rate). Approximately 70% of commonly detected metabolites discriminating rest vs. exercise in rats were also changed in exercising humans. Interstitium metabolomics may aid in the identification of molecules that signal muscle work (e.g., exertion and fatigue) and muscle health.

Keywords: beta-oxidation; interstitium; metabolome.

Fig. 1.

Intramuscular catheter used to collect interstitial fluid from the gastrocnemius muscle of adult male rats. A: schematic of catheter outer dimensions, inner dimensions, length, and through holes. B: magnified photograph of catheter tip. C: photograph of entire catheter-syringe apparatus. D: photograph of catheter inserted into gastrocnemius muscle of an anesthetized rat.

Fig. 2.

Plots of partial least squares (PLS)-discriminant analysis scores highlight that variances in concentrations of select metabolites in muscle interstitial fluid (A) and blood plasma (B) can readily discriminate the rested state from the immediate postexercise state in adult male rats. Each score from individual rats is shown. Confidence regions of group clusters are presented as 95% confidence ellipses based on Hotelling’s T² statistic. Exercise samples were collected in anesthetized animals ~10 min after cessation of the acute exercise bout.

Fig. 3.

Heat maps depicting log fold concentration changes in muscle interstitial fluid metabolites in adult male rats following an acute exercise bout relative to the rested state. Only metabolites that were contributing factors in the interstitial fluid partial least squares-discriminant analysis model (see Fig. 2A) and were annotated are shown. Mean concentration values from each condition were used to generate heat map results (from 9 rats). Metabolites were classified according to the main classes of the metabolite databases at the National Institutes of Health Metabolomics Data Repository (http://www.metabolomicsworkbench.org).

Fig. 4.

Heat maps depicting log fold concentration changes in blood plasma metabolites in adult male rats following an acute exercise bout relative to the rested state. Only metabolites that were contributing factors in the interstitial fluid partial least squares-discriminant analysis model (see Fig. 2B) and that were annotated are shown. Mean concentration values from each condition were used to generate heat map results (from 10 rats). Metabolites were classified according to the main classes of the metabolite databases at the National Institutes of Health Metabolomics Data Repository (http://www.metabolomicsworkbench.org). *Not classified in the database.

Fig. 5.

Concurrence between adult male rat muscle interstitial fluid and rat blood plasma metabolites that were detected using the gas chromatography-time-of-flight analytical platform (A), rat metabolites included in partial least squares-discriminant analysis models that discriminated resting from exercised states (B), and detected human interstitial fluid metabolites (C). Numbers within each section of the Venn diagram represent the number of metabolites, and percentages refer to the fraction of all detected metabolites within that section. Comparator lists may be found in Supplemental Tables S4 (rat comparisons) and S6 (rat vs. human comparison).