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. 2017 May 30;13(5):e1006819.
doi: 10.1371/journal.pgen.1006819. eCollection 2017 May.

Downregulation of SIRT1 Signaling Underlies Hepatic Autophagy Impairment in Glycogen Storage Disease Type Ia

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Free PMC article

Downregulation of SIRT1 Signaling Underlies Hepatic Autophagy Impairment in Glycogen Storage Disease Type Ia

Jun-Ho Cho et al. PLoS Genet. .
Free PMC article

Abstract

A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid β-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Impaired hepatic autophagy in L-G6pc-/- mice.
(A) Electron micrographs of hepatocytes in the livers of control and L-G6pc-/- mice. Arrowheads indicate deformed mitochondria. N: nucleus, L: lipid droplet. Scale bar, 5 μm. (B) Quantification of mRNA for hepatic autophagy components by real-time RT-PCR (n = 8). (C) Western blots of hepatic ATG101, Beclin-1, ATG14, LC3B, ATG3, BNIP3, p62 and β-actin, and densitometry analysis (n = 8). (D) Immunofluorescence analysis of autophagic vacuoles (green) and DAPI-stained nuclei (blue) in hepatocytes isolated from control and L-G6pc-/- mice after 24 hours of fast, and quantification of autophagy vacuoles by flow cytometry (n = 4). Scale bar, 25 μm. (E) Western blots for hepatic LC3B-II and β-actin in mice treated with either saline (-) or leupeptin (+) and determination of autophagic flux (n = 4). Data represent the mean ± SEM. *P < 0.05, **P < 0.005.
Fig 2
Fig 2. Impaired hepatic SIRT1-FoxO signaling in L-G6pc-/- mice.
(A) Western blots and densitometry analysis (n = 5), and quantification of mRNA for hepatic SIRT1 and FoxO3a (n = 8). (B) Hepatic NAD+ levels (n = 9). (C) Western blots and densitometry analysis of PPAR-γ, PPAR-α and β-actin (n = 5). (D) Immunohistochemical analysis of hepatic ChREBP and quantification of nuclear ChREBP-translocated cells (n = 4). Scale bar, 25 μm. (E) Quantification of mRNA for hepatic Acaca, Fasn and Elovl6 by real-time RT-PCR (n = 6). (F) Western blots of acetylated and total FoxO3a after immunoprecipitation of nuclear extracts using anti-FoxO3a, and quantification of the acetylated FoxO3a/total FoxO3a (n = 5). Data represent the mean ± SEM. *P < 0.05, **P < 0.005.
Fig 3
Fig 3. Increased acetylated ATG proteins and decreased ATG5-ATG12 conjugation in the livers of L-G6pc-/- mice.
(A) Western blots of acetylated and total ATG proteins after immunoprecipitation of liver lysates using anti-acetylated lysine, and quantification of the acetylated ATG proteins/total ATG proteins (n = 4). (B) Quantification of mRNA for hepatic Atg5 and Atg12 by real-time RT-PCR (n = 8). (C) Western blots and densitometry analysis of ATG12-ATG5 conjugate and β-actin (n = 4). Data represent the mean ± SEM. *P < 0.05, **P < 0.005.
Fig 4
Fig 4. Ad-SIRT1 treatment corrects hepatic autophagy impairment in L-G6pc-/- mice.
Control and L-G6pc-/- mice at 12 WP were treated with 1 x 108 pfu/mice of Ad-GFP or Ad-SIRT1 and analyzed at 13 WP. (A) Western blots for autophagy-related proteins and densitometry analysis (n = 4). (B) Western blots for hepatic LC3B and β-actin in mice that were treated with either saline (-) or leupeptin (+) and determination of autophagic flux (n = 3). (C) The levels of hepatic metabolites in control and L-G6pc-/- mice treated with Ad-GFP or Ad-SIRT1 (n = 4). Data represent the mean ± SEM. *P < 0.05, **P < 0.005.
Fig 5
Fig 5. Rapamycin fails to correct impaired hepatic autophagy in L-G6pc-/- mice.
(A) Western blots for hepatic p-mTOR, mTOR and β-actin, and densitometry analysis (n = 6). (B) Western blot analysis of transcriptional factor EB (TFEB) and Poly (ADP-ribose) polymerase (PARP) in liver nuclear extracts. (C) Western blots and densitometry analysis (n = 4) for autophagy-related proteins in mice treated with either vehicle (-) or 5 mg/kg body weight of rapamycin (+) for 8 consecutive days. (D) Western blots for hepatic LC3B and β-actin in mice treated with rapamycin and leupeptin as indicated, and determination of autophagic flux (n = 3). Data represent the mean ± SEM. *P < 0.05, **P < 0.005.
Fig 6
Fig 6. Correction of hepatic G6Pase-α deficiency normalizes autophagy.
L-G6pc-/- mice were treated with 1 x 1012 vp/kg of rAAV-G6PC at 4 WP and analyzed at 12 WP. (A) Hepatic G6Pase-α activity in control (n = 7), L-G6pc-/- (n = 7), and rAAV-treated L-G6pc-/- (AAV/ L-G6pc-/-, n = 8) mice. (B) Liver weights in control (n = 10), L-G6pc-/- (n = 5), and AAV/ L-G6pc-/- (n = 8) mice. (C) The levels of hepatic metabolites in control, L-G6pc-/- and AAV/ L-G6pc-/- (n = 8) mice. (D) Fasting glucose test (FGT) profile of control (n = 13), L-G6pc-/- (n = 6) and AAV/ L-G6pc-/- (n = 8) mice. (E) Western blots of hepatic SIRT1, FoxO3a, LC3B, p62 and β-actin and densitometry analysis (n = 8). (F) Hematoxylin and eosin (H&E) stained liver sections, and immunohistochemical analysis of hepatic ChREBP and quantification of nuclear ChREBP-translocated cells in control, L-G6pc-/-, and rAAV-treated L-G6pc-/- (AAV/ L-G6pc-/-) mice (n = 4). The insets present higher magnification views. Scale bar, 25 μm. Data represent the mean ± SEM. *P < 0.05, **P < 0.005.
Fig 7
Fig 7. The mechanism underlying autophagy impairment in hepatic G6Pase-α-deficiency.
Hepatic G6Pase-α-deficiency leads to metabolic alterations including G6P accumulation and suppressed expression of PPAR-α, a master regulator of fatty acid β-oxidation. The G6P-mediated activation of ChREBP signaling induces lipogenesis, leading to hepatic steatosis which increases the expression of PPAR-γ, another lipogenic factor. Moreover, aberrant PPAR-γ overexpression aggravates hepatic steatosis. The net outcome is downregulation of hepatic SIRT1 signaling. Impaired SIRT1 signaling increases ATG acetylation and decreases ATG12-ATG5 conjugation along with downregulation of FoxO signaling that induces autophagy genes. Accordingly, hepatic G6Pase-α-deficiency-mediated autophagy impairment is characterized by decreased expression of ATG proteins, defective autophagic vesicle elongation, impaired autophagosome formation, marked p62 accumulation and attenuated autophagic flux.

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Grant support

This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (https://intramural.nih.gov/search/searchview.taf?ipid=95507&ts=1493040277, HD000912, JYC) and by the Children's Fund for Glycogen Storage Disease Research (https://www.curegsd.org/portal/research-supported-by-our-foundation, JYC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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