Caffeine has been shown to stimulate multiple major regulators of cell energetics including AMP-activated protein kinase (AMPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Additionally, caffeine induces peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial biogenesis. While caffeine enhances oxidative metabolism, experimental concentrations often exceed physiologically attainable concentrations through diet. This work measured the effects of low-level caffeine on cellular metabolism and gene expression in myotubes, as well as the dependence of caffeine’s effects on the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARβ/δ). C2C12 myotubes were treated with various doses of caffeine for up to 24 h. Gene and protein expression were measured via qRT-PCR and Western blot, respectively. Cellular metabolism was determined via oxygen consumption and extracellular acidification rate. Caffeine significantly induced regulators of mitochondrial biogenesis and oxidative metabolism. Mitochondrial staining was suppressed in PPARβ/δ-inhibited cells which was rescued by concurrent caffeine treatment. Caffeine-treated cells also displayed elevated peak oxidative metabolism which was partially abolished following PPARβ/δ inhibition. Similar to past observations, glucose uptake and GLUT4 content were elevated in caffeine-treated cells, however, glycolytic metabolism was unaltered following caffeine treatment. Physiological levels of caffeine appear to enhance cell metabolism through mechanisms partially dependent on PPARβ/δ.
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Fatty acyl-coA dehydrogenase
AMP-activated protein kinase
Ca2+/calmodulin-dependent protein kinases I/II
Carbohydrate-responsive element-binding protein
cAMP response element-binding protein
Estrogen-related receptor alpha
Fatty acid synthase
Forkhead box protein O1
Glucose transporter 4
Lactate dehydrogenase a
Lactate dehydrogenase b
Myocyte enhancer factor 2
Nuclear respiratory factor 1
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha
Peroxisome proliferator-activated receptor alpha
Peroxisome proliferator-activated receptor beta/delta
Peroxisome proliferator-activated receptor gamma
Sterol receptor element-binding protein-1c
NAD+-dependent deacetylase sirtuin 3
Mitochondrial transcription factor A
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We would like to thank the Department of Physical Therapy (Congdon School of Health Sciences) and the School of Arts and Sciences for the use of shared lab space and equipment.
Support for this work was provided by the Department of Exercise Science within the Congdon School of Health Sciences.
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Cell viability. WST-1 assay time trial of cells treated with or without caffeine at 100 μM for 24 hours (left) and corresponding area-under-the-curve (right). Notes: Viability was performed using 10 replicates per treatment and analyzed using two-way ANOVA with Bonferroni’s correction for multiple comparisons. (JPEG 40 kb)
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Schnuck, J.K., Gould, L.M., Parry, H.A. et al. Metabolic effects of physiological levels of caffeine in myotubes. J Physiol Biochem 74, 35–45 (2018). https://doi.org/10.1007/s13105-017-0601-1
- Skeletal muscle
- Mitochondrial biogenesis