Figure 6.

GLUT8 and AMPK mediate trehalose-induced autophagy and protection from triglyceride accumulation. A. LC3B-II immunoblots in hepatocyte lysates following adenoviral GFP, GLUT8 or GLUT2 expression. B. Quantitative triglyceride measurements in hepatocytes treated with or without 5mM fructose in the presence or absence of 100mM trehalose after adenoviral expression of GFP, GLUT8 or GLUT2. Data are shown as mean ± SEM for, N = 3 independent experiments, n = 2 replicates per experiment. ****, P < 0.0001, ***, P < 0.001, and *, P < 0.05 (one-way ANOVA and Sidak’s post hoc analysis). C. Above, immunoblot depicting phosphorylated ULK1 (Ser³¹⁷) in trehalose-treated hepatocytes expressing a kinase-dead AMPK mutant (KD-AMPK). Below, pULK1 band density quantification, normalized to ULK1 band density. Data are shown as mean ± SEM for, N = 3 independent experiments, n = 1–3 replicates per experiment. ****, P < 0.0001 between bracketed groups (2-tailed T-Test). D. Above, immunoblot of LC3B-II accumulation in 100mM trehalose-treated hepatocytes (1hr) expressing KD-AMPK. Below, quantification of LC3B-II band normalized to GAPDH. *, P < 0.05 and N.S., not significantly different versus growth media group (2-tailed T-Test). E. Quantitative triglyceride measurements in hepatocytes treated with or without 5 mM fructose in the presence or absence of 100mM trehalose (48hr) after adenoviral expression of GFP or KD-AMPK. Data are shown as mean ± SEM for, N = 3 independent experiments, n = 3 replicates per experiment. ****, P < 0.0001, ***, P < 0.001 *, P < 0.05 (one-way ANOVA and Sidak’s post hoc analysis). F. Proposed model depicting trehalose blockade of GLUT2 and GLUT8, resulting in AMPK and ULK1 activation, and enhanced autophagic flux.