Because the protective effect of oleate against palmitate-induced insulin resistance may be lessened in skeletal muscle once cell metabolism is overloaded by fatty acids (FAs), we examined the impact of varying amounts of oleate on palmitate metabolic channeling and insulin signaling in C2C12 myotubes. Cells were exposed to 0.5 mM of palmitate and to increasing doses of oleate (0.05, 0.25 and 0.5 mM). Impacts of FA treatments on radio-labelled FA fluxes, on cellular content in diacylglycerols (DAG), triacylglycerols (TAG), ceramides, acylcarnitines, on PKCθ, MAPKs (ERK1/2, p38) and NF-ΚB activation, and on insulin-dependent Akt phosphorylation were examined.

Low dose of oleate (0.05 mM) was sufficient to improve palmitate complete oxidation to CO₂ (+ 29%, P < 0.05) and to alter the cellular acylcarnitine profile. Insulin-induced Akt phosphorylation was 48% higher in that condition vs. palmitate alone (p < 0.01). Although DAG and ceramide contents were significantly decreased with 0.05 mM of oleate vs. palmitate alone (− 47 and − 28%, respectively, p < 0.01), 0.25 mM of oleate was required to decrease p38 MAPK and PKCθ phosphorylation, thus further improving the insulin signaling (+ 32%, p < 0.05). By contrast, increasing oleate concentration from 0.25 to 0.5 mM, thus increasing total amount of FA from 0.75 to 1 mM, deteriorated the insulin signaling pathway (− 30%, p < 0.01). This was observed despite low contents in DAG and ceramides, and enhanced palmitate incorporation into TAG (+ 27%, p < 0.05). This was associated with increased incomplete FA β-oxidation and impairment of acylcarnitine profile. In conclusion, these combined data place mitochondrial β-oxidation at the center of the regulation of muscle insulin sensitivity, besides p38 MAPK and PKCθ.