Figure 7.

Addition of leucine and insulin to whole blood reduces autophagic flux. (A-B) Whole blood was taken and incubated without or with a mixture of leucine and insulin for three h. Whole blood was then processed for measurement of autophagic flux. (C) LC3B-II normalized to ACTB was determined for blood taken from N = 10 subjects. CQ increased LC3B-II in both vehicle and [leucine + insulin] treatments (p < 0.0001 control – vehicle vs CQ – vehicle, p < 0001 control – [leucine + insulin] vs CQ – [leucine + insulin]; p = 0.0455 CQ-vehicle vs CQ – [leucine + insulin]; repeated measures two-way ANOVA followed by Fisher’s LSD test; N = 10). Error bars are SEM. (D) Western blots showing analysis of LC3B in blood cultured without or with leucine and insulin and then treated without or with CQ (2 subjects displayed). (E) ΔLC3B-II = (LC3B-II:ACTB with CQ) – (LC3B-II:ACTB without CQ) was determined for blood taken from N = 10 subjects. The effect of leucine and insulin on the autophagic flux was statistically significant (p = 0.0280; paired t-test; N = 10). * p < 0.05, **** p < 0.0001. Mr: molecular weight marker