Korean J Physiol Pharmacol.  2014 Jun;18(3):255-261. 10.4196/kjpp.2014.18.3.255.

The Stimulatory Effect of Essential Fatty Acids on Glucose Uptake Involves Both Akt and AMPK Activation in C2C12 Skeletal Muscle Cells

• Park SY
• Kim MH
• Ahn JH
• Lee SJ
• Lee JH
• Eum WS
• Choi SY
• Kwon HY

• KMID: 2285518
• DOI: http://doi.org/10.4196/kjpp.2014.18.3.255

Abstract

Essential fatty acid (EFA) is known to be required for the body to function normally and healthily. However, the effect of EFA on glucose uptake in skeletal muscle has not yet been fully investigated. In this study, we examined the effect of two EFAs, linoleic acid (LA) and alpha-linolenic acid (ALA), on glucose uptake of C2C12 skeletal muscle cells and investigated the mechanism underlying the stimulatory effect of polyunsaturated EFAs in comparison with monounsaturated oleic acid (OA). In palmitic acid (PA)-induced insulin resistant cells, the co-treatment of EFAs and OA with PA almost restored the PA-induced decrease in the basal and insulin-stimulated 2-NBDG (fluorescent D-glucose analogue) uptake, respectively. Two EFAs and OA significantly protected PA-induced suppression of insulin signaling, respectively, which was confirmed by the increased levels of Akt phosphorylation and serine/threonine kinases (PKCtheta and JNK) dephosphorylation in the western blot analysis. In PA-untreated, control cells, the treatment of 500 microM EFA significantly stimulated 2-NBDG uptake, whereas OA did not. Phosphorylation of AMP-activated protein kinase (AMPK) and one of its downstream molecules, acetyl-CoA carboxylase (ACC) was markedly induced by EFA, but not OA. In addition, EFA-stimulated 2-NBDG uptake was significantly inhibited by the pre-treatment of a specific AMPK inhibitor, adenine 9-beta-D-arabinofuranoside (araA). These data suggest that the restoration of suppressed insulin signaling at PA-induced insulin resistant condition and AMPK activation are involved at least in the stimulatory effect of EFA on glucose uptake in C2C12 skeletal muscle cells.

Keyword

AMPK; C2C12 cells; Essential fatty acid; Glucose uptake; Insulin signaling

MeSH Terms

Acetyl-CoA Carboxylase
Adenine
alpha-Linolenic Acid
AMP-Activated Protein Kinases*
Blotting, Western
Fatty Acids, Essential*
Glucose*
Insulin
Linoleic Acid
Muscle, Skeletal*
Oleic Acid
Palmitic Acid
Phosphorylation
Phosphotransferases
AMP-Activated Protein Kinases
Acetyl-CoA Carboxylase
Adenine
Fatty Acids, Essential
Glucose
Insulin
Linoleic Acid
Oleic Acid
Palmitic Acid
Phosphotransferases
alpha-Linolenic Acid

Figure

• 

  Fig. 1 Concentration-dependent effect of palmitic acid (PA) on 2-NBDG uptake of C2C12 cells in the presence or absence of 100 nM insulin (A). Effects of oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) on PA-induced impairment of 2-NBDG uptake in C2C12 cells (B). Cells were incubated with different kinds of 500 µM fatty acids either alone or in combination for 18 h. And then, cells were incubated with or without 100 nM insulin. Glucose uptake activity was measured using a fluorescent 2-NBDG as described in Methods. The results are presented as means±SEM of 6 independent experiments. **p<0.01, ***p<0.001 compared to untreated control group and #p<0.05, ##p<0.01, ###p<0.001 compared to insulin-untreated group.

• 

  Fig. 2 Effects of palmitoleic acid (POA, C16:1), oleic acid (OA, C18:1), linoleic acid (LA, C18:2), and α-linolenic acid (ALA, C18:3) on the levels of phosphorylation of Akt in palmitic acid (PA)-treated C2C12 cells. Cells were incubated with different kinds and concentrations of fatty acids either alone or in combination for 18 h. And then, cells were incubated with or without 100 nM insulin. The level of p-Akt was detected by Western blotting. The results are presented as means±SEM of 4 independent experiments. **p<0.01, ***p<0.001 compared to untreated control group, #p<0.05, ##p<0.01 compared to insulin-treated group, and §p<0.05, §§p<0.01 compared to PA-treated group in the presence of 100 nM insulin.

• 

  Fig. 3 Effects of palmitoleic acid (POA, C16:1), oleic acid (OA, C18:1), linoleic acid (LA, C18:2), and α-linolenic acid (ALA, C18:3) on the levels of phosphorylation of PKCθ (A) and JNK (B) in palmitic acid (PA)-treated C2C12 cells. Cells were incubated with different kinds and concentrations of fatty acids either alone or in combination for 18 h. The levels of p-PKCθ and p-JNK were detected by Western blotting. The results are presented as means±SEM of 4 independent experiments. **p<0.01, ***p<0.001 compared to untreated control group, and §p<0.05, §§p<0.01, §§§p<0.001, compared to PA-treated group.

• 

  Fig. 4 Effects of oleic acid (OA), linoleic acid (LA), α-linolenic acid (ALA), and metformin (Met) on 2-NBDG uptake of C2C12 cells in the presence or absence of an AMPK inhibitor, araA (A) and the levels of phosphorylation of AMPK and ACC (B~D). Cells were incubated with different kinds of 500 µM fatty acids and 1 mM Met for 18 h. A specific AMPK inhibitor, araA (2 mM) was added to medium at 20 min before incubation of fatty acid. Glucose uptake activity was measured using a fluorescent 2-NBDG as described in Methods. The levels of p-AMPK and ACC were detected by Western blotting. The results are presented as means±SEM of 6 independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to untreated control group, #p<0.05, ##p<0.01 compared to araA-untreated corresponding group.

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