INTRODUCTION

Lipid infiltration into skeletal muscle in conditions such as obesity and type II diabetes is hypothesized to interfere with proteostatic signalling, leading to increased proteolysis and atrophy(1). Evidence from other cell types/tissues suggests lipid infiltration can inhibit autophagy(2), a cellular recycling process essential for the maintenance of skeletal muscle mass and quality(3), however this has not been investigated in human skeletal muscle. Therefore, we aimed to investigate the effect of high-fat exposure on autophagic flux and associated signalling events in immortalised human primary myotubes.

METHODS

Immortalised human primary myotubes (C25/KM155 line) were exposed to palmitate (PAL, 500uM), or control media (CTRL), for 24h. Myotubes were then either immediately collected (BASAL, n=7 per condition) or media changed to EBSS for 5h to deprive cells of nutrients (STARVE, n=5 per condition), to activate autophagy. A subset of cells were incubated with Bafilomycin A1 (100nM) for 2h prior to collection to assess pre-lysosomal autophagic flux. Immunoblotting was then completed for LC3 (autophagic flux) and a range of associated signalling targets. Two-factor independent measures ANOVAs were used to assess differences between conditions, with significance threshold set at p<0.05. Data is presented as Mean ± SD.

RESULTS

Main effects of condition and nutrient deprivation (both p<0.001) were observed for pre-lysosomal autophagic flux whereby STARVE elevated flux in both CTRL and PAL (2.2-fold & 3-fold respectively) but overall was lower in PAL. This was not mirrored by more commonly used LC3II/I ratio, where no differences were observed, suggesting this ‘static’ measure of autophagy does not reflect flux. Further main effects of condition showed AMPK-mediated ULK1^Ser555 phosphorylation and Beclin1 protein content to be lower in PAL irrespective of nutrient availability (p≤0.036), indicating a potential impairment of autophagy induction in PAL. Markers of mTORC1 activity (p-ULK1^Ser757, p-RPS6^{Ser240/244 & Ser235/236}) displayed main effects of nutrient deprivation whereby STARVE was lower than BASAL irrespective of condition (p≤0.026). However, when expressed relative to basal mean values, the reduction in p-RPS6^{Ser240/244 & 235/236} following STARVE was less pronounced with PAL (72±2% vs. 83±6%, p=0.009 & 66±4% vs. 74±3%, p=0.005 respectively).

CONCLUSION

We show that palmitate exposure impairs absolute pre-lysosomal autophagic flux but not responsiveness to nutrient withdrawal. This impairment may be due to reduced autophagy induction displayed by lower Beclin1 content and AMPK-mediated ULK1^Ser555 phosphorylation. These data provide initial evidence of a potential detrimental effect of high-fat/lipid exposure on autophagy in human skeletal muscle which warrants further research in vivo.