Normalization of hepatic ChREBP activity does not protect against liver disease progression in a mouse model for Glycogen Storage Disease type Ia

Abstract

Background: Glycogen storage disease type 1a (GSD Ia) is an inborn error of metabolism caused by a defect in glucose-6-phosphatase (G6PC1) activity, which induces severe hepatomegaly and increases the risk for liver cancer. Hepatic GSD Ia is characterized by constitutive activation of Carbohydrate Response Element Binding Protein (ChREBP), a glucose-sensitive transcription factor. Previously, we showed that ChREBP activation limits non-alcoholic fatty liver disease (NAFLD) in hepatic GSD Ia. As ChREBP has been proposed as a pro-oncogenic molecular switch that supports tumour progression, we hypothesized that ChREBP normalization protects against liver disease progression in hepatic GSD Ia.

Methods: Hepatocyte-specific G6pc knockout (L-G6pc^-/-) mice were treated with AAV-shChREBP to normalize hepatic ChREBP activity.

Results: Hepatic ChREBP normalization in GSD Ia mice induced dysplastic liver growth, massively increased hepatocyte size, and was associated with increased hepatic inflammation. Furthermore, nuclear levels of the oncoprotein Yes Associated Protein (YAP) were increased and its transcriptional targets were induced in ChREBP-normalized GSD Ia mice. Hepatic ChREBP normalization furthermore induced DNA damage and mitotic activity in GSD Ia mice, while gene signatures of chromosomal instability, the cytosolic DNA-sensing cGAS-STING pathway, senescence, and hepatocyte dedifferentiation emerged.

Conclusions: In conclusion, our findings indicate that ChREBP activity limits hepatomegaly while decelerating liver disease progression and protecting against chromosomal instability in hepatic GSD Ia. These results disqualify ChREBP as a therapeutic target for treatment of liver disease in GSD Ia. In addition, they underline the importance of establishing the context-specific roles of hepatic ChREBP to define its therapeutic potential to prevent or treat advanced liver disease.

Keywords: Carbohydrate Response Element Binding Protein; Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING); Glycogen Storage Disease type 1a; Hepatomegaly; Yes Associated Protein.

Fig. 1

ChREBP knockdown in hepatic GSD Ia mice causes hepatocyte death, inflammation, DNA damage, and proliferation. (A) Single cell death and inflammatory foci, (B) γH2Ax positivity, (C) p21 (Cdkn1a) expression, and (D) pH3 and Ki67 positivity and mitotic figures in livers after 10 days of shChREBP/L-G6pc^−/−. A-D: median ± interquartile range; Kruskal Wallis H-test, post-hoc Conover pairwise comparisons, *p < 0.05, **p < 0.01, ***p < 0.001 vs shSCR/L-G6pc^+/+; ^ vs shChREBP/L-G6pc^+/+; # vs shSCR/L-G6pc.^−/− (n = 7–9)

Fig. 2

Prolonged hepatic ChREBP normalization in L-G6pc^−/− mice progressively induces hepatomegaly and sensitizes to hepatic inflammation. (A) Liver weight and plasma ALT levels in shChREBP/L-G6pc^−/− mice (n = 8). (B) Representative macroscopic liver photos and photos of H&E stainings of livers, and (C) Percent relative cumulative frequency (PRCF) of hepatocyte size. (D) Hepatic glycogen, triglyceride, and protein content (n = 8), (E) hepatic water content, (F) number of inflammatory foci and inflammatory gene expression, and (G) fibrosis marker gene expression in livers of shChREBP/L-G6pc^−/− mice (n = 6–9). A, D-G: median ± interquartile range. C: box-and-whisker plots. A-G: Kruskal Wallis H-test, post-hoc Conover pairwise comparisons, *p < 0.05, **p < 0.01, ***p < 0.001 vs shSCR/L-G6pc^+/+; ^ vs shChREBP/L-G6pc^+/+; # vs shSCR/L-G6pc^−/−

Fig. 3

Prolonged hepatic ChREBP normalization in L-G6pc^−/− mice promotes Yes Associated Protein (YAP) transcriptional activity. Data after 21–26 days of shChREBP/L-G6pc^−/− and n = 8, unless stated otherwise. (A) Mitotic figures and BrdU positivity (n = 4–6, 20–21 days). (B) YAP-target genes and Shp. (C) YAP nuclear protein and whole liver lysate pYAP/YAP ratio (Blots/Ponceau S: Fig. S2C-E). (D) Correlations between liver weight and Ctgf expression in shChREBP/L-G6pc^−/− mice. (E) Expression of bile acid synthesis enzymes and transporters, and (F) Total plasma bile acid levels. A/C/F-G: median ± interquartile range. E: box-and-whisker plots. A/C/F-G: Kruskal Wallis H-test, post-hoc Conover pairwise comparisons, *p < 0.05, **p < 0.01, ***p < 0.001 vs shSCR/L-G6pc^+/+; ^ vs shChREBP/L-G6pc^+/+; # vs shSCR/L-G6pc^−/−

Fig. 4

Prolonged ChREBP knockdown in L-G6pc^−/− mice induces DNA damage, cellular senescence, and hepatocyte dedifferentiation. Data after 21–26 days of shChREBP/L-G6pc^−/− and n = 8/group, unless stated otherwise. (A) CIN marker genes, spontaneous chromosome bridge incidence (with representative image), (B) Hepatocyte ploidy, (C) γH2Ax positivity (n = 4–8/group) and PARP protein expression, (D) Cgas and senescence-associated genes and p21 protein, and (E) HNF4A-related genes. (F) Reporter transcription factors analysis (after 10 days of shChREBP/L-G6pc^−/−). (G-H) Hepatocyte differentiation marker genes (n = 7–8). Blots/Ponceau S: Fig. S4B-C. A/C-E/G-H: median ± interquartile range. B: box-and-whisker plots. A-E/G-H: Kruskal Wallis H-test, post-hoc Conover pairwise comparisons, *p < 0.05, **p < 0.01, ***p < 0.001 vs shSCR/L-G6pc^+/+; ^ vs shChREBP/L-G6pc^+/+; # vs shSCR/L-G6pc^−/−