Effects of insulin on apoB100 degradation and VLDL-apoB100 secretion are blunted in autophagy-deficient mouse primary hepatocytes.

<p>Primary hepatocytes were isolated from mice with floxed alleles of <i>Atg5</i> (<i>Atg5^+/+</i>) or <i>Atg5^f/f x Alb-Cre</i> (<i>Atg5^−/−</i>) mice (i.e., mice with hepatic deficiency of Atg5), and cultured in serum-free conditions for 16 h before insulin addition. A) A western-blot for LC3 was performed with lysates of primary hepatocytes from <i>Atg5^+/+</i> or <i>Atg5^−/−</i> mice; “(+)” represents the condition in which lysosomal degradation has been blocked (20 mM NH₄Cl + 10 µM E64D) to increase LC3 recovery and “(-)” represents untreated cells. When autophagy is active, LC3 (“LC3-I”) is lipidated to form LC3-II. GAPDH was used as the loading control. B) Primary hepatocytes from <i>Atg5^−/−</i> or <i>Atg5^+/+</i> mice were incubated in media with (INS) or without insulin (CONT) and pulse-labeled for 15 min with [³⁵S]-protein labeling mix, and were then chased for 30 and 120 min in non-radioactive medium with the treatments maintained. Total apoB100 recovery and quantification were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057590#pone-0057590-g001" target="_blank">Figure 1</a>. The histogram (mean±SEM) represents the results from 2 independent experiments, each one performed in triplicate; ** indicates P<0.01. C) Primary hepatocytes from <i>Apobec1^−/−</i> mice were labeled with [³⁵S]-protein labeling mix for 4 h in the presence (+ Insulin) or absence (− Insulin) of 100 nM insulin. Conditional media samples were collected and lipoproteins were separated by density gradient ultracentrifugation. ApoB100 in individual fractions was immunoprecipitated, resolved by SDS-PAGE, and quantified by densitometry after bands were detected by a phosphorImager. The statistical significance of the comparisons between the density profiles is based on 3 independent replicate experiments. D) An experiment similar to the one in panel C was performed, but using Atg5-deficent primary hepatocytes prepared from <i>Apobec1^−/−</i> mice.