Abstract
Glutamine is an amino acid previously linked with improved skeletal muscle metabolism and insulin signaling, however, past observations often use cell culture models with only supraphysiological concentrations. Additionally, past reports have yet to simultaneously investigate both metabolic outcomes and insulin signaling. The present report utilized cell culture experiments and measured the effects of both physiological and supraphysiological levels of glutamine on myotube metabolism and insulin signaling/resistance. It was hypothesized the addition of glutamine at any level would increase cell metabolism and related gene expression, as well as improve insulin signaling versus respective control cells. C2C12 myotubes were treated with glutamine ranging from 0.25 mM-4 mM (or media control) for 24 h to capture a range of physiological and supraphysiological concentrations. qRT-PCR was used to measure metabolic gene expression. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Insulin sensitivity (indicated by pAkt:Akt) and metabolism following glucose/insulin infusion were also assessed. Glutamine treatment consistently increased mitochondrial and glycolytic metabolism versus true controls (cells treated with media void of glutamine), however, supraphysiological glutamine did not enhance metabolism beyond that of cells with physiological levels of glutamine. Neither physiological nor supraphysiological levels of glutamine altered insulin signaling regardless of insulin stimulation or insulin resistance when compared with respective controls. These data demonstrate excess glutamine does not appear to alter myotube metabolism or glucose disposal when base levels of glutamine are present. Moreover, glutamine does not appear to alter insulin sensitivity regardless of level of insulin resistance or presence of insulin stimulation.
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Abbreviations
- Akt:
-
Protein kinase B
- CS:
-
Citrate synthase
- ECAR:
-
Extracellular acidification rate
- FCCP:
-
Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone
- GLUT4:
-
Glucose transporter 4
- HK:
-
Hexokinase
- LDHa:
-
Lactate dehydrogenase A
- LDHb:
-
Lactate dehydrogenase B
- NRF1/2:
-
Nuclear respiratory factor 1/2
- OCR:
-
Oxygen consumption rate
- PDH:
-
Pyruvate dehydrogenase
- PFK:
-
Phosphofructokinase
- PI3K:
-
Phosphatidylinositol-3-kinase
- PGC-1α:
-
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha
- PK:
-
Pyruvate kinase
- PPARα:
-
Peroxisome proliferator-activated receptor alpha
- TBP:
-
TATA Binding Protein
- TFAM:
-
Mitochondrial transcription factor A
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Acknowledgements
Support for this work was provided by the Department of Exercise Science within the Congdon School of Health Sciences. We would like also to thank the Department of Physical Therapy (Congdon School of Health Sciences) for the use of shared lab space and equipment. RMW and SEL were supported by the HPU Natural Science Fellows Program. Instrumentation support was provided by the Shimadzu Partnership for Academics, Research and Quality of Life (SPARQ) Program. All authors read and approved the final manuscript. Authors and contributors declare no conflict of interest.
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Support for this work was provided by the Department of Exercise Science within the Congdon School of Health Sciences.
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MJK, CNR, MER, RMW, SEL, AJW, and RAV conducted experiments and assisted with manuscript preparation. RAV conceived the study, conducted and oversaw experiments, performed statistical analyses, and oversaw manuscript preparation.
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The authors declare that all data were generated in-house and that no paper mill was used. All authors read and approved the final manuscript. Authors and contributors declare no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. All authors have read and approved the final manuscript. Additional declarations for articles in life science journals that report the results of studies involving humans and/or animals: Not applicable. Authors and contributors declare no conflict of interest.
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726_2022_3131_MOESM1_ESM.tif
Supplementary file1 (TIF 133 KB) Figure s1 Effect of glutamine treatment continuum on nuclei content from Seahorse assays. Effect of glutamine continuum 0mM, 0.25mM, 0.5mM, 1mM, 2mM, and 4mM for 24 hours on myotube relative nuclei staining (indicated by DAPI staining). Notes: DAPI staining was measured in quadruplicate and the average used (less the background). Values were normalized to the control mean (relative) to reduce inter-assay variability. Relative nuclei content was analyzed using one-way ANOVA with Bonferroni's correction for multiple comparisons using n=15-16 for each of 2 independent experiments for a total of n=29-32 per group for the final analyses (one value was excluded from the 4mM group as an outlier)
726_2022_3131_MOESM2_ESM.tif
Supplementary file2 (TIF 787 KB) Figure s2 Effect of glutamine treatment continuum on mitochondrial and glycolytic metabolism. (a) Correlation between glutamine continuum treatment concentration (0mM, 0.25mM, 0.5mM, 1mM, 2mM, and 4mM for 24 hours) and basal (left) and peak (right) mitochondrial metabolism. (b) Basal (left) and peak (right) mitochondrial metabolism of cells treated as described in “a” after normalizing mitochondrial metabolism to relative nuclei content. (c) Correlation between glutamine continuum treatment concentration and basal (left) and peak (right) glycolytic metabolism. (d) Basal (left) and peak (right) glycolytic metabolism of cells treated as described in “a” after normalizing mitochondrial metabolism to relative nuclei content. Notes: Basal and peak mitochondrial and glycolytic metabolism were analyzed using one-way ANOVA with Bonferroni's correction for multiple comparisons using n=15-16 for each of two independent experiments for a total of n=29-32 per group for the final analyses (one value was excluded from the 4mM group as an outlier due to nuclei staining). Dashed lines within each correlation represent 95% CI with solid line representing linear regression line of best fit
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Krone, M.J., Rivera, C.N., Rivera, M.E. et al. Excess glutamine does not alter myotube metabolism or insulin sensitivity. Amino Acids 54, 455–468 (2022). https://doi.org/10.1007/s00726-022-03131-x
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DOI: https://doi.org/10.1007/s00726-022-03131-x