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Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis

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Science Signaling  23 Feb 2016:
Vol. 9, Issue 416, pp. ra21
DOI: 10.1126/scisignal.aac5472
  • Fig. 1 Trehalose inhibits glucose transport through SLC2A transporters.

    (A) Common evolutionary ancestry for trehalase, Tre1, and SLC2A family members by ClustalW2 phylogenic analysis. (B) Tre1 pairwise alignment with the substrate-selecting seventh transmembrane domains in SLC2A1 and SLC2A8. (C) 2-DG uptake in response to increasing trehalose concentrations in 293 cells expressing the indicated GLUT protein. Data are expressed as the means percent uptake relative to control cultures not treated with trehalose ± SEM from n = 3 independent experiments, n = 3 replicates per experiment. (D) [3H]2-DG and [14C]fructose uptake in primary hepatocytes. Data are expressed as the means uptake relative to trehalose-untreated control groups ± SEM from n = 3 independent experiments, n = 3 replicates per experiment. (E) [3H]2-DG and [14C]fructose uptake in HepG2 cells. Data are shown as means uptake relative to trehalose-untreated control groups ± SEM from n = 3 independent experiments, n = 3 replicates per experiment. ***P < 0.001 versus vehicle-treated by two-tailed t test. ns, not significant versus vehicle-treated.

  • Fig. 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 min. Data are shown as means 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 to 4 hours). Right: Immunoblot quantification of phosphorylated AMPK (pAMPK) (Thr172) normalized to AMPKα band density. Data are shown as means ± SEM for n = 3 independent experiments, n = 2 to 3 replicates per experiment. **P < 0.01 and ***P < 0.001 versus untreated control (“0 hour” data) [one-way analysis of variance (ANOVA) and Sidak’s post hoc analysis]. pACC1, phosphorylated ACC1. A.U., arbitrary units. (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 min. Right: Quantification of AMPK and ACC1 phosphorylation at Thr172 and Ser79 normalized to AMPKα and ACC1 band density, respectively. Data are shown as means ± SEM for n = 3 independent experiments, n = 2 replicates per experiment. In the glucose-treated group, *P < 0.05 and ***P < 0.001 versus controls (one-way ANOVA and Sidak’s post hoc analysis). In the glucose + trehalose–treated group, ***P < 0.001 versus cells treated with glucose alone (one-way ANOVA and Sidak’s post hoc analysis). ##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 (glyceraldehyde-3-phosphate dehydrogenase), respectively. Data are shown as means ± SEM for n = 3 independent experiments, n = 2 replicates per experiment; **P < 0.01 versus control and ***P < 0.001 versus control by two-tailed t test. pULK1, phosphorylated ULK1. (E) LC3B-I and LC3B-II immunoblotting in hepatocytes after treatment with or without 100 mM trehalose and 100 nM bafA1 for 1 hour. Control groups were treated with dimethyl sulfoxide (DMSO). Right: LC3B-II band density, normalized to GAPDH density. Data are shown as means ± SEM for n = 5 independent experiments, n = 1 to 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.

  • Fig. 3 Orally administered trehalose rapidly accumulates in peripheral circulation and induces hepatic autophagy.

    (A) Liquid chromatography–mass spectrometry analysis of serum isolated from mice administered trehalose (3 g/kg) by gavage and analyzed after 0.5, 1, 2, or 4 hours. Serum analyzed at the “0 hour” trehalose treatment time point was derived from mice 30 min after gavage with 0.9% NaCl. n = 5 mice per treatment group. (B) Left: Immunoblot analysis of crude liver lysates from mice administered trehalose (3 g/kg) and analyzed after 0.5, 1, 2, or 4 hours. Liver lysates analyzed at the “0 hour” trehalose treatment time point were derived from mice 30 min after gavage with 0.9% NaCl. Right: Quantification of pAMPK, pULK1, and LC3B-II, normalized to AMPKα, ULK1, and GAPDH, respectively. Data are shown as means ± SEM for n = 5 independent mice per treatment group. ***P < 0.001 versus 0-hour treatment group (one-way ANOVA and Sidak’s post hoc analysis). (C) Left: Immunoblot analysis of LC3B-II in liver lysates from mice treated with or without leupeptin (40 mg/kg) 1 hour before oral gavage with trehalose (3 g/kg). Right: Quantification of LC3B-II/actin band density ratio. Data are shown as means ± SEM for n = 3 to 4 independent mice per treatment group; ***P < 0.001 and ****P < 0.0001 versus saline-treated controls (two-tailed t test). ###P < 0.001 between trehalose-treated groups treated with leupeptin versus leupeptin-untreated group (denoted by brackets) by two-tailed t test.

  • Fig. 4 Trehalose mitigates triglyceride accumulation in multiple in vitro models of steatosis.

    (A) Triglyceride (TG) quantification in hepatocytes treated with or without 5 mM fructose in the presence or absence of 100 mM trehalose. Data are shown as means ± SEM for n = 3 independent experiments, n = 3 replicates per treatment group per experiment. ***P < 0.001 (one-way ANOVA and Sidak’s post hoc analysis). (B) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of ChREBP and GPAT mRNA in hepatocytes treated with fructose in the presence or absence of 100 mM trehalose. Target abundances are normalized to α-actin expression within each sample. Data are shown as means ± SEM for n = 3 independent experiments, n = 3 replicates per treatment group per experiment. ****P < 0.0001, ***P < 0.001, and *P < 0.05 (one-way ANOVA and Sidak’s post hoc analysis). (C) TG quantification in hepatocytes treated with or without BSA-conjugated fatty acids (500 μM) in the presence or absence of 100 mM trehalose (48 hours). Data are shown as means ± SEM for n = 3 independent experiments, n = 2 to 3 replicates per treatment group per experiment. ***P < 0.001 (one-way ANOVA and Sidak’s post hoc analysis). (D) TG quantification in MTTP-deficient primary hepatocytes treated with or without 100 mM trehalose (48 hours). ****P < 0.0001 by two-tailed t test. Data are shown as means ± SEM for n = 3 independent experiments, n = 3 replicates per treatment group per experiment. MTTPKO, MTTP-knockout. (E) TG quantification in wild-type (WT) and ATG16L1HM hepatocytes treated with BSA alone (control) or with BSA-conjugated fatty acids in the presence or absence of trehalose (100 mM; 48 hours). Data are shown as means ± SEM for n = 3 independent experiments, n = 3 replicates per treatment group per experiment. ****P < 0.0001 by one-way ANOVA and Sidak’s post hoc test. ns, not significant.

  • Fig. 5 Trehalose reduces HFrD-induced hepatic steatosis in vivo.

    (A) Eight-week-old mice were fed chow, 10-day 60% fructose diet, or 10-day 60% fructose diet initiated 2 days after initiating 3% trehalose fed ad libitum in drinking water. Fasting plasma triglycerides, cholesterol, and free fatty acids (FFAs) were quantified. Data are shown as means ± SEM for n = 6 to 12 independent mice per treatment group. ***P < 0.001 (one-way ANOVA and Sidak’s post hoc analysis). (B) Left: Oil Red O staining in frozen liver sections from mice described above. Scale bar, 200 μm. Right: Blinded-observer quantification of staining red staining density (minimum three random fields from three cryosections obtained from three different mice per group) by ImageJ (version 1.47) densitometry software. Data are shown as means ± SEM for n = 9 to 12 mice per treatment group. ****P < 0.0001 (one-way ANOVA and Sidak’s post hoc analysis). (C) Hepatic TG, cholesterol, and FFA quantification in 8-week-old mice fed chow, 10-day 60% fructose diet, or 10-day 60% fructose diet initiated 2 days after initiating 3% trehalose fed ad libitum in drinking water. Data are shown as means ± SEM for n = 9 to 12 mice per treatment group. ****P < 0.0001, ***P < 0.001, and **P < 0.01 (one-way ANOVA and Sidak’s post hoc analysis). (D) Left to right: qRT-PCR analysis of ACC1, SCD1, GPAT, and PPARγ mRNA in liver tissue mRNA extracted from mice fed 10-day HFrD with or without 3% trehalose water. Target abundances are normalized to α-actin expression within each sample. Data are shown as means ± SEM for n = 9 to 12 mice per treatment group. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 (one-way ANOVA and Sidak’s post hoc analysis).

  • Fig. 6 GLUT8 and AMPK mediate trehalose-induced autophagy and protection from triglyceride accumulation.

    (A) LC3B-II immunoblots in hepatocyte lysates after adenoviral GFP, GLUT8, or GLUT2 expression. (B) Quantitative triglyceride measurements in hepatocytes treated with or without 5 mM fructose in the presence or absence of 100 mM trehalose after adenoviral expression of GFP, GLUT8, or GLUT2. Data are shown as means ± 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) Top: Immunoblot depicting pULK1 (Ser317) in trehalose-treated hepatocytes expressing a kinase-dead AMPK mutant (KD-AMPK). Bottom: pULK1 band density quantification, normalized to ULK1 band density. Data are shown as means ± SEM for n = 3 independent experiments, n = 1 to 3 replicates per experiment. ****P < 0.0001 between bracketed groups (two-tailed t test). (D) Top: Immunoblot of LC3B-II accumulation in 100 mM trehalose–treated hepatocytes (1 hour) expressing KD-AMPK. Bottom: Quantification of LC3B-II band normalized to GAPDH. *P < 0.05; ns, not significantly different versus growth media group (two-tailed t test). (E) Quantitative triglyceride measurements in hepatocytes treated with or without 5 mM fructose in the presence or absence of 100 mM trehalose (48 hours) after adenoviral expression of GFP or KD-AMPK. Data are shown as means ± SEM for n = 3 independent experiments, n = 3 replicates per experiment. ****P < 0.0001, ***P < 0.001, and *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. Glc, glucose; AAs, amino acids.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/416/ra21/DC1

    Fig. S1. The effect of the HIV protease inhibitor lopinavir on 2-DG uptake, AMPK activation, and autophagic flux.

    Fig. S2. The effect of sucrose on primary hepatocyte autophagy and AMPK signaling.

    Fig. S3. The effect of trehalose on LC3B-II accumulation and triglyceride accumulation in HepG2 cells.

    Table S1. Absolute mRNA copy numbers in the 293 GLUT expression system.

  • Supplementary Materials for:

    Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis

    Brian J. DeBosch,* Monique R. Heitmeier, Allyson L. Mayer, Cassandra B. Higgins, Jan R. Crowley, Thomas E. Kraft, Maggie Chi, Elizabeth P. Newberry, Zhouji Chen, Brian N. Finck, Nicholas O. Davidson, Kevin E. Yarasheski, Paul W. Hruz, Kelle H. Moley

    *Corresponding author. E-mail: debosch_b{at}kids.wustl.edu

    This PDF file includes:

    • Fig. S1. The effect of the HIV protease inhibitor lopinavir on 2-DG uptake, AMPK activation, and autophagic flux.
    • Fig. S2. The effect of sucrose on primary hepatocyte autophagy and AMPK signaling.
    • Fig. S3. The effect of trehalose on LC3B-II accumulation and triglyceride accumulation in HepG2 cells.
    • Table S1. Absolute mRNA copy numbers in the 293 GLUT expression system.

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    Citation: B. J. DeBosch, M. R. Heitmeier, A. L. Mayer, C. B. Higgins, J. R. Crowley, T. E. Kraft, M. Chi, E. P. Newberry, Z. Chen, B. N. Finck, N. O. Davidson, K. E. Yarasheski, P. W. Hruz, K. H. Moley, Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis. Sci. Signal. 9, ra21 (2016).

    © 2016 American Association for the Advancement of Science

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