Myristic acid selectively augments β-tubulin levels in C2C12 myotubes via diacylglycerol kinase δ

Abstract

Effective amelioration of type II diabetes requires therapies that increase both glucose uptake activity per cell and skeletal muscle mass. Myristic acid (14:0) increases diacylglycerol kinase (DGK) δ protein levels and enhances glucose uptake in myotubes in a DGKδ-dependent manner. However, it is still unclear whether myristic acid treatment affects skeletal muscle mass. In this study, we found that myristic acid treatment increased the protein level of β-tubulin, which constitutes microtubules and is closely related to muscle mass, in C2C12 myotubes but not in the proliferation stage in C2C12 myoblasts. However, lauric (12:0), palmitic (16:0) and oleic (18:1) acids failed to affect DGKδ and β-tubulin protein levels in C2C12 myotubes. Moreover, knockdown of DGKδ by siRNA significantly inhibited the increased protein level of β-tubulin in the presence of myristic acid, suggesting that the increase in β-tubulin protein by myristic acid depends on DGKδ. These results indicate that myristic acid selectively affects β-tubulin protein levels in C2C12 myotubes via DGKδ, suggesting that this fatty acid improves skeletal muscle mass in addition to increasing glucose uptake activity per cell.

Keywords: diacylglycerol kinase; myotube; myristic acid; type II diabetes; β-tubulin.

Fig. 1

Effects of myristic acid on several protein levels in C2C12 myotubes. (A) C2C12 myotubes at 48 h of culture in differentiation medium were treated for 24 h with 0.1 mm myristic acid (14:0). DGKδ, β‐tubulin, β‐actin, GAPDH, cyclin D1, cyclin D3, myogenin and MyHC in the C2C12 cells treated with 0.1 mm myristic acid were detected by western blot. (B) The protein levels of each protein were normalized to the GAPDH level. The protein levels for the control were set as 100%. Values are presented as the means ± SD (n = 8). **P < 0.01 and ***P < 0.001 versus untreated cells, two‐tailed t test. Representative images (A) and graphs (B) of DGKδ, β‐tubulin, β‐actin and GAPDH are shown in left side for Section 3.1; Representative images (A) and graphs (B) of cyclin D1, cyclin D3, myogenin and MyHC are shown in right side for Section 3.2.

Fig. 2

Effects of various fatty acids on the levels of several proteins in C2C12 myotubes. (A) C2C12 myotubes grown for 48 h in differentiation medium were treated for 24 h with 0.1 mm lauric (12:0), myristic (14:0), palmitic (16:0) or oleic (18:1) acid. DGKδ, β‐tubulin, β‐actin, GAPDH, cyclin D1, cyclin D3, myogenin and MyHC in the C2C12 cells treated with each fatty acid were detected by western blot. (B) The expression levels of each protein were normalized to the GAPDH level. The protein levels for the control were set as 100%. Values are presented as the means ± SD (n = 7–12). *P < 0.05, **P < 0.01 and ***P < 0.005 versus untreated cells, ANOVA followed by Dunnett's test. Representative images (A) and graphs (B) of DGKδ, β‐tubulin, β‐actin and GAPDH are shown in left side for Section 3.1; Representative images (A) and graphs (B) of cyclin D1, cyclin D3, myogenin and MyHC are shown in right side for Section 3.2.

Fig. 3

Effects of myristic acid on several protein levels in C2C12 cells during proliferation and at the early differentiation stage. (A) C2C12 myoblasts grown for 24 h in growth medium were treated for 24 h with 0.1 mm myristic acid. DGKδ, β‐tubulin, β‐actin and GAPDH in the C2C12 cells treated with 0.1 mm myristic acid were detected by western blot. (B) The expression levels of each protein were normalized to the GAPDH level. The protein levels for the control were set as 100%. Values are presented as the means ± SD (n = 4). *P < 0.05 and ***P < 0.005 versus untreated cells, two‐tailed t test. (C) After initiation of differentiation (at 0 h of differentiation), C2C12 cells were treated for 48 h with 0.1 mm myristic acid. DGKδ, β‐tubulin, β‐actin and GAPDH in the C2C12 cells treated with 0.1 mm myristic acid were detected by western blot. (D) The expression levels of each protein were normalized to the GAPDH level. The protein levels for the control were set as 100%. Values are presented as the means ± SD (n = 8). *P < 0.05 and ***P < 0.005 versus untreated cells, two‐tailed t test.

Fig. 4

Effects of DGKδ silencing on β‐tubulin protein levels in C2C12 myotubes in the presence or absence of myristic acid. (A) C2C12 myoblasts transfected with control siRNA or DGKδ‐specific siRNA were grown for 48 h in growth medium; then, the medium was replaced with differentiation medium. C2C12 myotubes at 48 h after differentiation were treated for 24 h with 0.1 mm myristic acid, and DGKδ, β‐tubulin, β‐actin and GAPDH were detected by western blot. (B) The expression levels of each protein were normalized to the β‐actin level. The protein levels for the control (myristic acid, control siRNA) were set as 100%. Values are presented as the means ± SD (n = 4). *P < 0.05, **P < 0.01 and ***P < 0.005, ANOVA followed by Student–Newman–Keuls test.