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Metabolic effects of physiological levels of caffeine in myotubes

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Abstract

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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Abbreviations

ACAD:

Fatty acyl-coA dehydrogenase

ACC:

Acetyl-coA carboxylase

AMPK:

AMP-activated protein kinase

CaMKI/II:

Ca2+/calmodulin-dependent protein kinases I/II

ChREBP:

Carbohydrate-responsive element-binding protein

CREB:

cAMP response element-binding protein

CS:

Citrate synthase

ERRα:

Estrogen-related receptor alpha

FAS:

Fatty acid synthase

Foxo1:

Forkhead box protein O1

GLUT4:

Glucose transporter 4

LDHa:

Lactate dehydrogenase a

LDHb:

Lactate dehydrogenase b

MEF2:

Myocyte enhancer factor 2

NRF1:

Nuclear respiratory factor 1

PGC-1α:

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PPARα:

Peroxisome proliferator-activated receptor alpha

PPARβ/δ:

Peroxisome proliferator-activated receptor beta/delta

PPARγ:

Peroxisome proliferator-activated receptor gamma

SCD1:

Stearoyl-CoA desaturase

SREBP-1c:

Sterol receptor element-binding protein-1c

SIRT3:

NAD+-dependent deacetylase sirtuin 3

TFAM:

Mitochondrial transcription factor A

UCP:

Uncoupling protein

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Acknowledgments

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.

Funding

Support for this work was provided by the Department of Exercise Science within the Congdon School of Health Sciences.

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Authors and Affiliations

  1. Department of Exercise Science, High Point University, One University Parkway, High Point, NC, 27268, USA

    Jamie K. Schnuck, Lacey M. Gould, Hailey A. Parry, Michele A. Johnson, Kyle L. Sunderland & Roger A. Vaughan

  2. School of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Jamie K. Schnuck & Nicholas P. Gannon

  3. School of Kinesiology, Auburn University, Auburn, AL, 36849, USA

    Hailey A. Parry

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  1. Jamie K. Schnuck
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Contributions

All authors read and approved the final manuscript. JKS, LMG, HAP, MAJ, and NPG assisted with experiments and manuscript preparation. KLS assisted with experimental design and manuscript preparation. RAV performed and oversaw all experiments, statistical analyses, manuscript preparation, and designed/conceived the experiments.

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Correspondence to Roger A. Vaughan.

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The authors declare that they have no conflict of interest.

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Figure S1

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

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