Key points Regulation of autophagy in human muscle in many aspects
Key points Regulation of autophagy in human muscle in many aspects differs from the majority of previous reports based on studies in cell systems and rodent muscle. and frozen in liquid nitrogen and stored at C80C for further analysis. Incubation of intact mouse MK-2866 inhibitor database skeletal muscle (acute exercise study & mouse experiments) or 17,500?(acute exercise and insulin study & training study)]. The supernatant (lysate) was harvested and total protein content was decided using the bicinchoninic acid method (BCA no. 23225, Pierce, Rockford, IL, USA). Next, lysates were diluted in MilliQ ultrapure water and 6 Laemlii buffer (340?mm Tris base, pH 6.8, 11% SDS, 20% glycerol, 0.05% bromophenol blue and 225?mm freshly added dithiothrietol) at the same protein concentration in all samples. Total protein and phosphorylation were measured by standard immunoblotting techniques as described previously (Frosig and and and main effect (and and and em F /em : em n /em ?=?2 in pre ex leg, em n /em ?=?5 in pre rest leg, em n /em ?=?4 in post ex leg, em n /em ?=?4 in post rest leg, em n /em ?=?4 in 4?h post ex leg, em n /em ?=?6 in 4?h post rest leg due to lack of sample. *,**,***/,,/,,Significantly different ( em P /em 0.05, em P /em 0.01, em P /em 0.001) from pre/post/insulin 10; #,##significantly different ( em P /em 0.05, em P /em 0.01) from untrained leg within intervention; $significant different ( em P /em 0.05) from pre independent of leg. AU, arbitrary models; Ex, exercised leg; Ins10, 10?min into the euglycaemicChyperinsulinaemic clamp after MK-2866 inhibitor database the training intervention; Ins90, 90?min into the euglycaemicChyperinsulinaemic clamp after the training intervention; Pre, pre\training intervention/ pre\exercise; Post, post\training intervention/exercise; Rest, rested leg; T, trained leg; UT, untrained leg; 4?h post, 4 hours post\exercise. Discussion This study indicates that regulation of autophagy in human muscle in many aspects differs from the majority of cell and rodent reports. Notably, in human muscle, acute exercise, exercise training and MK-2866 inhibitor database insulin stimulation leads to a reduction in the LC3\II/LC3\I ratio, a commonly used marker of autophagy. Our data further suggest that exercise\induced activation of AMPK leads to regulation of ULK1, but AMPK activation alone is not sufficient to regulate autophagy. In contrast, mTOR signalling via ULK1 is usually a probable signalling axis regulating autophagy in response to insulin stimulation. Autophagy is critical for muscle function and metabolic homeostasis. Consequently, understanding how exercise and insulin stimulation regulates autophagy in human muscle is currently of major interest. During autophagy cytoplasmic materials are engulfed by autophagosomes in a process where LC3\I is usually lipidated to form the autophagosome\resident anchoring protein LC3\II. This targets the material as well as LC3\II for lysosomal degradation or recycling. As reviewed, the content of LC3\II is considered a valid indicator of the content of autophagosomes under most conditions (Tanida em et?al /em . 2005; Klionsky em et?al /em . 2012). In contrast, using either the MK-2866 inhibitor database content of LC3\II or the ratio between LC3\II Rabbit polyclonal to ABCB5 and LC3\I as a direct marker of autophagic flux should be done with caution, as the content of autophagosomes depend on both the rate of formation and the rate of lysosomal degradation (Mizushima & Yoshimori, 2007; Rubinsztein em et?al /em . 2009). To illustrate this, autophagosomes may accumulate at a given time point both as a consequence of accelerated formation (increased autophagic flux) or suppressed degradation (reduced autophagic flux). Here we show that acute one\legged knee extensor exercise substantially reduces LC3 lipidation and subsequently the LC3\II/LC3\I ratio in skeletal muscle and that this effect continues at least 4?h into recovery from exercise. This adaptation to exercise has recently been observed in response to.