Figure 2.

Trehalose decreases hepatocyte ATP and activates AMPK and autophagic flux. A. ATP quantification in hepatocytes treated with or without 100 mM trehalose for 30 minutes. Data are shown as mean fold of control (growth-media-treated) ATP ± SEM for, N= 3 independent experiments, n = 3 replicates per experiment. *, P < 0.05 by two-tailed T-Test. B. Left, immunoblot demonstrating AMPK (Thr¹⁷²) phosphorylation in trehalose-treated hepatocytes (0–4hrs). Right, immunoblot quantification of pAMPK (Thr¹⁷²) normalized to AMPKα band density. Data are shown as mean ± SEM for, N= 3 independent experiments, n = 2–3 replicates per experiment. **, P < 0.01 ***, P < 0.001 versus untreated control (“0 hour” data) (one-way ANOVA and Sidak’s post hoc analysis). C. Left, immunoblot depicting AMPK and ACC1 phosphorylation in HBSS-starved hepatocytes, followed by refeeding with unsupplemented HBSS (denoted as “−” group on blot and “control” on graph) or HBSS containing 25 mM glucose in the presence or absence of 100 mM trehalose for 30 minutes. Right, quantification of AMPK and ACC1 phosphorylation at Thr¹⁷² and Ser⁷⁹ normalized to AMPKα and ACC1 band density, respectively. Data are shown as mean ± SEM for, N = 3 independent experiments, n = 2 replicates per experiment, In the glucose-treated group * and *** represent P < 0.05 and P < 0.001 versus controls (one-way ANOVA and Sidak’s post hoc analysis). In the glucose + trehalose-treated group, *** represents P < 0.001 versus cells treated with glucose alone (one-way ANOVA and Sidak’s post hoc analysis). ## and ###, P < 0.01 and P < 0.001 versus control group (one-way ANOVA and Sidak’s post hoc analysis) D. Left, immunoblot depicting ULK1 phosphorylation (Ser³¹⁷ and Ser⁷⁵⁷) and LC3B-II accumulation after 1 hour incubation without (control) or with 100 mM trehalose. Right, quantification of AMPK and ULK1 phosphorylation, and LC3B-II band density normalized to AMPKα, ULK1, and GAPDH, respectively. Data are shown as mean ± SEM for, N = 3 independent experiments, n = 2 replicates per experiment, **, P < 0.01 versus control and ***, P < 0.001 versus control by 2-tailed T-Test. E. LC3B-I and LC3B–II immunoblotting in hepatocytes following treatment with or without 100 mM trehalose and 100 nM bafilomycin A1 for 1 hr. Control groups were treated with DMSO. Right, LC3B-II band density, normalized to GAPDH density. Data are shown as mean ± SEM for, N = 5 independent experiments, n = 1–3 replicates per experiment ***, P < 0.001 versus control groups. ###, P < 0.001 between bracketed groups by one-way ANOVA and Sidak’s post-hoc analysis for multiple comparisons.