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. 2017;13(8):98.
doi: 10.1007/s11306-017-1236-5. Epub 2017 Jul 7.

Effect of Everolimus on the Glucose Metabolic Pathway in Mouse Skeletal Muscle Cells (C2C12)

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Free PMC article

Effect of Everolimus on the Glucose Metabolic Pathway in Mouse Skeletal Muscle Cells (C2C12)

Kayoko Yoshida et al. Metabolomics. .
Free PMC article

Abstract

Introduction: Everolimus selectively inhibits mammalian target of rapamycin complex 1 (mTORC1) and exerts an antineoplastic effect. Metabolic disturbance has emerged as a common and unique side effect of everolimus.

Objectives: We used targeted metabolomic analysis to investigate the effects of everolimus on the intracellular glycometabolic pathway.

Methods: Mouse skeletal muscle cells (C2C12) were exposed to everolimus for 48 h, and changes in intracellular metabolites were determined by capillary electrophoresis time-of-flight mass spectrometry. mRNA abundance, protein expression and activity were measured for enzymes involved in glycometabolism and related pathways.

Results: Both extracellular and intracellular glucose levels increased with exposure to everolimus. Most intracellular glycometabolites were decreased by everolimus, including those involved in glycolysis and the pentose phosphate pathway, whereas no changes were observed in the tricarboxylic acid cycle. Everolimus suppressed mRNA expression of enzymes related to glycolysis, downstream of mTOR signaling enzymes and adenosine 5'-monophosphate protein kinases. The activity of key enzymes involved in glycolysis and the pentose phosphate pathway were decreased by everolimus. These results show that everolimus impairs glucose utilization in intracellular metabolism.

Conclusions: The present metabolomic analysis indicates that everolimus impairs glucose metabolism in muscle cells by lowering the activities of glycolysis and the pentose phosphate pathway.

Keywords: CE-TOFMS; Everolimus; Glycometabolism; Hyperglycemia; Metabolomics; mTOR inhibitor.

Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

This article does not contain any studies with human participants performed by any of the authors.

Research involving human participants and/or animals

This article does not contain any study using human or animals.

Figures

Fig. 1
Fig. 1
Changes in extracellular and intracellular glucose concentrations. At 0 h, 500 ng/mL of everolimus was added to the cultured media (EVE). a The cultured media were collected for measurement of extracellular glucose at 48 h. The original (0 h) concentration of cultured media was 4.5 mg/mL. The open bar represents the glucose concentration of the 48 h control sample and the filled bar denotes a sample exposed to everolimus for 48 h. Data represent the mean ± S.D. (n = 3). Statistically significant difference between EVE and control: **p < 0.01 (Student’s t-test). b Intracellular glucose concentration at 48 h. Open bars represent 48 h control cells and filled bars denote 48 h everolimus-exposed cells. Data represent the mean ± S.D. (n = 3). Statistically significant difference between EVE and control: *p < 0.05 (Student’s t-test)
Fig. 2
Fig. 2
Metabolic profiling of intracellular glycometabolism. Changes in metabolite levels after 48 h exposure to everolimus (closed bars, EVE) were compared with those without everolimus at 48 h (Control). The data represent the mean ± S.D. (n = 3). Statistically significant differences between the EVE and control: *p < 0.05; **p < 0.01 (Student’s t-test). Abbreviations: G6P glucose 6-phosphate, F6P fructose 6-phosphate, F1,6P fructose 1,6-bisphosphate, GAP glyceraldehyde phosphate, DHAP dihydroxyacetone phosphate, 1,3-Bis-PG 1,3-Bis phosphoglycerate, 3PG 3-phosphoglycerate, 2PG 2-phosphoglycerate, PEP phosphoenolpyruvate, Ac-CoA acetyl CoA, Ru5P ribulose 5-phosphate, R5P ribose 5-phosphate, Xu5P xylulose 5-phosphate, mTOR mammalian target of rapamycin, HIF1a hypoxia-inducible factor-1α, GLUT glucose transporter, HK1 hexokinase-1, GPI glucose-6-phosphate isomerase, PFKM 6-phosphofructokinase, ALDOA aldolase, TPI1P2 triosephosphate isomerase, GAPDH glyceraldehyde 3-phosphate dehydrogenase, PGK1 phosphoglycerate kinase-1, ENO1 enolase-1, PKM2 pyruvate kinase-2, LDHA_lactate dehydrogenase A, PDHA1 pyruvate dehydrogenase α1, G6PD 6-phosphate dehydrogenase, PGD 6-phosphogluconate dehydrogenase, RPIA ribose-5-phosphate isomerase A, TKT transketolase, TALDO1 transaldolase-1, CS citrate synthase, ACO1 aconitase-1, IDH1 isocitrate dehydrogenase-1, OGDH α-ketoglutarate dehydrogenase, DLST dihydrolipoamide succinyltransferase, SUCLG2 succinyl-CoA ligase, SDHA succinate dehydrogenase A, FH fumarate hydratase, MDH1 malate dehydrogenase-1
Fig. 3
Fig. 3
Metabolic profiling of intracellular amino acid and TCA cycle. Changes in metabolite levels after 48 h exposure to everolimus (closed bars, EVE) were compared to those without everolimus at 48 h (Control). The data represent mean ± S.D. (n = 3). Statistically significant difference between EVE and control: *p < 0.05; **p < 0.01 (Student’s t-test). Abbreviations: Ala alanine, Arg arginine, Asn asparagine, Asp aspartic acid, Cys cysteine, Glu glutamic acid, Gln glutamine, Gly glycine, His histidine, Ile isoleucine, Leu leucine, Lys lysine, Met methionine, Phe phenylalanine, Pro proline, Ser serine, Thr threonine, Trp tryptophan, Tyr tyrosine, Val valine
Fig. 4
Fig. 4
mRNA expression of enzymes and factors involved in glycometabolism. Changes in expression levels after 48 h exposure to everolimus (closed bars, EVE) were compared with those without everolimus at 48 h (Control). The data represent the mean ± S.D. (n = 3). Statistically significant difference between EVE and control: *p < 0.05; **p < 0.01 (Student’s t-test). Abbreviations are the same as Fig. 2
Fig. 5
Fig. 5
Activity for key enzymes involved in glycolysis and pentose phosphate pathway. Changes in enzyme activity after 48 h exposure to everolimus (closed bars, EVE) were compared with those without everolimus at 48 h (Control). The data represent the mean ± S.D. (n = 3). Statistically significant difference between EVE and control: **p < 0.01 (Student’s t-test). Abbreviations: LDH lactate dehydrogenase, PFK phosphofructokinase, HK hexokinase, PKM pyruvate kinase, PGD 6-phosphogluconate dehydrogenase. The other abbreviations are the same as Fig. 2

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