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

Normalization of hepatic ChREBP activity does not protect against liver disease progression in a mouse model for Glycogen Storage Disease type Ia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Normalization of hepatic ChREBP activity does not protect against liver disease progression in a mouse model for Glycogen Storage Disease type Ia Martijn G.S. Rutten, Yu Lei, Joanne H. Hoogerland, Vincent W. Bloks, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2514060/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2023 Read the published version in Cancer & Metabolism → Version 1 posted 7 You are reading this latest preprint version 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. Glycogen Storage Disease type 1a Carbohydrate Response Element Binding Protein hepatomegaly Yes Associated Protein cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Glycogen Storage Disease type Ia (GSD Ia) (MIM#232200) is a rare inborn error of metabolism (IEM) caused by mutations in the gene encoding for the catalytic subunit of glucose-6-phosphatase ( G6PC1 ( G6pc in mice), G6Pase-α) [ 1 ], which is expressed in liver, kidney, and intestine, where it converts glucose-6-phosphate (G6P) into glucose. Patients primarily display severe metabolic liver disease, characterized by hepatomegaly and non-alcoholic fatty liver disease (NAFLD), while liver tumour development represents the major long-term complication of GSD Ia, affecting up to 70% of patients by the age of 30 years [ 2 ]. Carbohydrate Response Element Binding Protein (ChREBP, also known as MLXIPL, MONDOB, or WBSCR14) is the major glucose-sensitive transcription factor in hepatocytes [ 3 ], and ChREBP and its regulated pathways are activated in hepatic GSD Ia [ 4 – 7 ]. We previously showed that short-term normalization of ChREBP activity aggravates hepatomegaly and NAFLD in hepatocyte-specific GSD Ia mice [ 8 ]. These findings suggest that sustained hepatic ChREBP normalization in hepatic GSD Ia may drive advanced liver disease elements, including hepatic inflammation, liver fibrosis, hepatocellular death and/or oncogenic transformation. On the other hand, evidence that ChREBP-regulated pathways represent a typical hallmark of many cancer cells has accumulated [ 9 ]. Consistently, ChREBP has been linked to the incidence and prognosis of hepatocellular carcinoma (HCC) [ 10 – 14 ]. ChREBP deficient mice are protected against HCC development in an oncogene-specific manner, and ChREBP deficiency inhibits growth of β-catenin/YAP-driven hepatoblastomas [ 11 , 15 ]. Moreover, reduced ChREBP expression inhibits hepatocellular proliferation through oxidative stress-induced, p53-mediated cell cycle arrest in vitro [ 16 ], while ChREBP-deficient hepatocytes show impaired proliferation rates during liver repopulation in vivo [ 15 ]. Combined, these studies indicate that ChREBP serves as a competent factor for cell growth and liver tumour progression. These previous studies from our laboratory and others suggest context-specific roles of hepatic ChREBP in advanced liver disease, in particular hepatocellular tumour susceptibility, which are of critical importance to establish the therapeutic potential of ChREBP for the treatment of liver disease in GSD Ia patients. In the current study we therefore investigated the impact of prolonged ChREBP normalization on liver disease progression in hepatic GSD Ia mice. Our data show that normalization of hepatic ChREBP activity sensitizes liver-specific GSD Ia mice to advanced liver disease development, DNA damage, cellular senescence, as well as hepatocellular proliferation and dedifferentiation, suggesting increased susceptibility for hepatocarcinogenesis. Methods AAV-shRNA construction and production See Supplement. Animals Male adult G6pc- floxed Alb -Cre negative (B6. G6pc lox/lox ) and G6pc- floxed Alb -Cre positive (B6. G6pc lox/lox .SA creERT2/w mice) on a C57BL/6J background were infected with shRNAs directed against ChREBP (AAV-shChREBP) or a scrambled control (AAV-shScramble (shSCR)) (1x10 12 particles per mouse) by intravenous injection into the retro-orbital plexus under isoflurane anaesthesia [ 8 ]. At 11–12 days after AAV-shRNA administration, all mice received i.p. injections of tamoxifen for 5 consecutive days to generate liver-specific G6pc -deficient mice (L- G6pc −/− ) and wildtype littermates (L- G6pc +/+ ). Nonfasted animals were sacrificed for tissue collection at 8AM at 10 or 25–26 days after the last treatment (dpt). Two shChREBP/L- G6pc −/− mice were euthanized at 21 dpt because a humane endpoint was reached, yet were included in the analyses. Absolute liver weight of the 10-day follow-up study cohort has previously been reported [ 8 ]. For further details, see Supplementary Materials & Methods. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Groningen and are in line with the Guide for the Care and Use of Laboratory Animals. Histological and pathological analysis of the liver See Supplement. Biochemical assays See Supplement. Gene expression analysis, RNA-sequencing, gene set enrichment analysis (GSEA) and reporter transcription factors analysis See Supplement. Targeted proteomics, SDS-PAGE, and Western Blot See Supplement. Ploidy analysis For analysis of hepatocyte ploidy, ~ 20 mg of frozen powdered liver tissue was used for nuclei isolation (as described [ 17 ]) in lysis buffer (10 mM Tris-HCl (pH 8), 0.32 M Sucrose, 5 mM CaCl 2 , 3 mM Mg(Ac) 2 , and 0.1 mM EDTA, with fresh addition of 1 mM DTT and 0.1% Triton X-100). In short, a 50–100 µm filter was placed on a 50 mL Falcon tube, and liver powder was poured onto the filter. Liver powder was stepwise and gently homogenized in 1.5 mL lysis buffer and pushed through the filter using a 5 mL syringe plunger. The resulting 1.5 mL nuclear suspension was transferred to a 1.5 or 2.0 mL tube, and supplemented with ~ 25,000 control cells (diploid human GFP-positive cells). Nuclei were spun down at 500xg for 5 min at 4°C and the pellet was resuspended in 300–1000 µL PBS/BSA with Hoechst/PI DNA dyes (10 µg/mL for both). Nuclei were filtered through 35µm FACS tubes and analysed on the Canto FACS machine (BD Biosciences) for ploidy analysis. Statistics Data in figures is presented as dot plots with median ± interquartile range (IQR), unless stated otherwise. Data in tables is presented as median (range), unless stated otherwise. Data in heatmaps represent z-score normalized values. Statistical analysis was performed using BrightStat and GraphPad PRISM software. Differences between multiple groups were tested by a Kruskal Wallis H-test followed by post-hoc Conover pairwise comparisons. P values < 0.001 (***, ^^^ , or ### ), 0.001 to 0.01 (**, ^^ , or ## ), and 0.01 to 0.05 (*, ^ , or # ) were considered significant. Results Normalization of hepatic ChREBP expression in GSD Ia liver induces oxidative stress, p53 activation, and cell cycle inhibition while inducing mitosis We previously showed that short-term normalization of ChREBP activity aggravates hepatomegaly and NAFLD in a mouse model for hepatic GSD Ia [ 8 ]. Here, histopathological analysis revealed an increase in single cell death and inflammatory foci in shChREBP/L- G6pc −/− mice, while the number of γH2Ax-positive hepatocytes tended to increase (Fig. 1 A-B, S1A). This was paralleled by a significant induction of the p53-target gene p21 (Fig. 1 C). Moreover, the number of pH3- and Ki67-positive hepatocytes and mitotic figures was increased in shChREBP/L- G6pc −/− mice (Fig. 1 D, S1A). Complementary Gene Set Enrichment Analysis (GSEA) of RNA expression data (Table 1 ) revealed that normalization of hepatic ChREBP expression in GSD Ia liver induces oxidative stress, apoptosis, p53 activation, cell cycle inhibition, hepatocyte death, chromosomal instability, DNA damage, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway (cGAS-STING) activation, cellular senescence, inflammatory response, epithelial mesenchymal transition, and mitotic activity within a timeframe of two weeks. Table 1 Gene Set Enrichment Analysis on RNAseq data of short-term shSCR- and shChREBP-treated L- G6pc +/+ and L- G6pc −/− mice L- G6pc −/− shSCR vs L- G6pc +/+ shSCR L- G6pc +/+ shChREBP vs L- G6pc +/+ shSCR L- G6pc −/− shChREBP vs L- G6pc −/− shSCR Gene set (size $ ) NES Nominal p-value FDR q-value NES Nominal p-value FDR q-value NES Nominal p-value FDR q-value P53 pathway (158) 1.22 0.109 0.286 1.54 0.000 0.034 1.53 0.002 0.026 G2/M checkpoint (153) 1.66 0.000 0.018 0.91 0.704 0.813 1.93 0.000 0.001 Oxidative stress (25) -0.97 0.474 1.000 1.12 0.295 0.479 1.47 0.045 0.187 Apoptosis (122) 1.11 0.265 0.416 1.54 0.005 0.032 1.66 0.000 0.012 CIN29 (15) 2.15 0.000 0.000 1.97 0.002 0.002 2.07 0.000 0.001 cGAS-STING (39) -1.10 0.289 0.627 1.03 0.402 0.607 1.90 0.000 0.001 MMC2-senescence (133) 1.33 0.046 0.155 1.79 0.000 0.007 2.19 0.000 0.000 Inflammatory response (114) 1.03 0.408 0.488 1.94 0.000 0.002 1.98 0.000 0.001 Epithelial mesenchymal transition (123) -1.02 0.408 0.691 1.81 0.000 0.007 2.01 0.000 0.001 $ size: refers to the size of the gene set after filtering out those genes that were not in the expression data set Prolonged hepatic ChREBP normalization in L-G6pc −/− mice induces extreme hepatomegaly and sensitizes to hepatic inflammation As these early changes suggested acceleration of metabolic-associated fatty liver disease towards advanced liver disease in response to shChREBP, we next evaluated the hepatic effects of prolonged normalized ChREBP activity in L- G6pc −/− mice (Fig. S2A). Three weeks of ChREBP normalization reduced fed blood glucose levels and increased plasma ketone bodies, while body weight remained unaffected (Table 2 ). It furthermore exacerbated hepatomegaly as compared to 10-day ChREBP normalization (Fig. 2 A-B, Table 2 ), and caused a concomitant progressive increase in plasma ALT levels (Fig. 2 A) and hepatocyte vacuolization (Fig. 2 B), highlighting the progressive nature of the liver disease. Hepatic G6P and glycogen contents were further increased in shChREBP/L- G6pc −/− mice, while hepatic triglyceride contents varied, and relative hepatic protein content was reduced (Fig. 2 D, Table 2 ). Hepatic water content was not different between shSCR/L- G6pc −/− and shChREBP/L- G6pc −/− mice (Fig. 2 E). Livers of shChREBP/L- G6pc −/− mice showed a marked increase in inflammatory foci, in line with GSEA data on short-term ChREBP normalization in hepatic GSD Ia (Table 1 ), while the expression of inflammatory genes Il1β , Il6 , Tnfα , and Cd68 was minimally or not (significantly) induced (Fig. 2 F). The expression of fibrosis marker genes was increased in shChREBP/L- G6pc −/− livers (Fig. 2 G). Yet, at the histological level, this was not paralleled by enhanced hepatic collagen deposition (data not shown). These data indicate that prolonged hepatic ChREBP normalization in GSD Ia progressively exacerbates hepatomegaly and hepatocyte hypertrophy and predisposes to hepatic inflammation. Table 2 General data of prolonged shSCR- and shChREBP-treated L- G6pc +/+ and L- G6pc −/− mice Variable Median (range) L- G6pc +/+ shSCR L- G6pc +/+ shChREBP L- G6pc −/− shSCR L- G6pc −/− shChREBP Body weight (g) 29.3 (27.8–31.6) 27.4 (25.6–29.2) 29.1 (25.3–30.5) 27.6 (21.1–32.8) Liver weight (g) 1.27 (1.14–1.75) 1.89 (1.28–2.10) ** 2.06 (1.17–2.39) *** 5.72 (2.15–9.20) ***^^^### Blood glucose (mmol/L) 11.1 (7.7–14.2) 10.4 (8.8–14.2) 9.2 (4.9–13.1) 4.9 (1.1–11.5) ***^^# Liver G6P (µmol/g liver) 0.47 (0.29–0.66) 0.53 (0.31–0.63) 1.38 (0.77–3.04) ***^^^ 1.71 (1.28–3.60) ***^^^ G6P (µmol/liver) 0.65 (0.41–0.81) 1.00 (0.49–1.19) * 1.99 (1.50–6.26) ***^^^ 11.58 (2.75–19.15) ***^^^## Glycogen (mg/liver) 98 (68–136) 163 (93–224) * 190 (71–234) ** 915 (151–1926) ***^^^## Triglycerides (µmol/liver) 8.7 (0.1–23.9) 79.3 (7.6-106.1) *** 60.8 (22.3–80.7) ** 50.3 (0.6-238.9) ** Free cholesterol (µmol/g liver) 3.94 (2.70–4.54) 3.36 (3.23–3.91) * 3.46 (2.97–4.02) 2.20 (1.13–5.43) ** Free cholesterol (µmol/liver) 5.08 (3.74–6.44) 6.31 (4.88–7.01) 6.92 (4.71–8.23) ** 11.73 (10.23–14.48) ***^^^### Cholesteryl-esters (µmol/g liver) 0.68 (0.02–0.94) 1.80 (0.57–3.02) ** 1.85 (1.16–3.03) ** 1.23 (0.10–5.47) * Cholesteryl-esters (µmol/liver) 0.83 (0.03–1.65) 3.57 (0.73–4.78) ** 3.23 (2.26–5.11) *** 7.73 (0.79–11.77) *** Total cholesterol (µmol/g liver) 4.45 (2.91–5.27) 5.13 (3.98–6.51) 5.27 (4.14–6.68) 3.36 (1.33–10.90) Total cholesterol (µmol/liver) 5.87 (4.02–7.93) 10.01 (5.73–10.73) ** 9.93 (6.97–13.15) *** 20.06 (11.03–24.79) ***^^^### Protein (mg/liver) 300 (241–401) 381 (290–412) 430 (229–481) ** 656 (394–821) ***^^^## Plasma Lactate (mmol/L) 3.94 (3.46–5.83) 4.72 (3.53–6.09) 5.62 (4.02–7.17) 5.03 (2.65–6.38) Total ketone bodies (mmol/L) 0.072 (0.055–0.112) 0.087 (0.055–0.165) 0.082 (0.059–0.197) 0.123 (0.090–0.224) **^# 3HB (mmol/L) 0.069 (0.051–0.109) 0.084 (0.052–0.161) 0.079 (0.056–0.192) 0.121 (0.087–0.219) **^# ACA (mmol/L) 0.003 (0.002–0.007) 0.004 (0.003–0.007) 0.003 (0.002–0.005) 0.003 (0.000-0.005) FFA (µmol/L) 153 (96–259) 166 (129–254) 224 (69–306) 167 (97–409) Prolonged hepatic ChREBP normalization in L-G6pc −/− mice promotes the transcriptional activity of Yes Associated Protein (YAP) To further investigate the origin of the extreme liver enlargement observed in hepatic GSD Ia mice upon prolonged ChREBP normalization (Fig. 2 A), we next assessed hepatocyte proliferation. Indeed, livers of shChREBP/L- G6pc −/− mice showed an increase in mitotic figures and the number of BrdU-positive hepatocytes (Fig. 3 A, Fig. S2B). They also exhibited an induction of YAP target genes (Fig. 3 B) in parallel to increased nuclear YAP protein levels, while p-YAP/YAP ratios remained unaffected as compared to shSCR-treated controls (Fig. 3 C). Interestingly, within shChREBP/L- G6pc −/− mice, relative liver weights, mRNA levels of YAP-target gene Ctgf ( Ccn2 ), and hepatic glycogen contents were positively correlated (Fig. 3 D, S2F). In line with our previous work [ 18 ], ChREBP normalization in L- G6pc −/− mice suppressed the expression of Cyp8b1 (Fig. 3 E). It furthermore reduced the hepatic expression of bile acid transporters Ntcp ( Slc10a1 ) and Bsep ( Abcb11 ) (Fig. 3 E) while increasing plasma bile acid levels (Fig. 3 F). Interestingly, relative liver weight correlated significantly yet moderately with total plasma bile acid (r = 0.3000, p < 0.05) across different study cohorts (n = 64). In the current study cohort, plasma bile acids levels also positively correlated with Ctgf mRNA levels (r = 0.6884, p < 0.0001 (n = 32)). Combined, these data indicate that prolonged hepatic ChREBP normalization in hepatic GSD Ia mice enhances YAP activity, which may be mediated by hepatocyte-autonomous effects, such as cellular glycogen accumulation, and/or by hepatic bile acid sensing. Prolonged ChREBP knockdown in L-G6pc −/− mice induces hepatocyte DNA damage, cellular senescence, and hepatocyte dedifferentiation As short-term ChREBP-normalized hepatic GSD Ia mice suggested induction of chromosomal instability, DNA damage, cGAS-STING, and cellular senescence (Fig. 1 F, Table 1 ), we also investigated these parameters upon prolonged ChREBP normalization. Prolonged ChREBP knockdown in L- G6pc −/− mice induced chromosomal instability (CIN) marker genes (Fig. 4 A). In parallel, histopathological analysis revealed an incidence of chromosome bridges, a hallmark of CIN [ 19 ], in these animals (Fig. 4 A). Hepatic ChREBP knockdown also tended to further increase nuclear ploidy in L- G6pc −/− mice (Fig. 4 B). This was paralleled by a strong increase in γH2Ax positivity (Fig. 4 C, Fig. S3A). PARP cleavage was not different between shChREBP- and shSCR/L- G6pc −/− mice (Fig. 4 C, Fig. S3B). shChREBP/L- G6pc −/− mice showed increased mRNA levels of Cgas as well as the cellular senescence marker genes p16 INK4a , p19 ARF (both encoded from the Cdkn2a locus), and p21 ( Cdkn1a ), and a massive increase in hepatic p21 protein levels (Fig. 4 D). Strikingly, hepatic ChREBP knockdown in L- G6pc −/− mice reduced long non-coding Hnf4aos and total Hnf4a mRNA and HNF4A peptide levels, while increasing Hnf4a-P2 / Hnf4a-P1 ratio (Fig. 4 E). As reduced Hnf4aos (HNF4A-AS1) and HNF4A expression are associated with hepatocyte dedifferentiation and advanced liver disease including liver cancer [ 20 – 27 ], these changes likely reflect hepatocellular dedifferentiation in shChREBP/L- G6pc −/− mice. Reporter transcription factors analysis revealed that ChREBP- and HNF4A-targeted transcriptomes showed parallel responses to hepatic G6pc deficiency and combined G6pc deficiency/ Chrebp normalization (Fig. 4 F). In parallel, hepatic Alb and Hgfac mRNA levels were reduced and Afp expression was induced (Fig. 4 G), while Krt19 and Sox9 remained unchanged (Fig. 4 H). Taken together, these data indicate that prolonged ChREBP normalization in GSD Ia hepatocytes aggravates CIN while inducing DNA damage, cGAS-STING pathway activation, cellular senescence, and hepatocellular dedifferentiation. Discussion The current study shows that normalization of hepatic ChREBP activity in GSD Ia liver induces progressive and extreme dysplastic liver growth, hepatocyte hypertrophy and -proliferation, YAP activation, cholestasis, CIN, DNA damage, cGAS-STING pathway activation, inflammation, cellular senescence, and hepatocellular dedifferentiation. Altogether, our data indicate that constitutive ChREBP activation in hepatic GSD Ia protects against advanced liver disease development, and disqualifies ChREBP as a therapeutic target for treatment of liver disease in GSD Ia. A key finding in this study is that aggravation of hepatomegaly upon hepatic ChREBP knockdown in GSD Ia liver associates with enhanced nuclear levels and activity of YAP, a transcription factor that is critical for homeostatic control of liver size [ 28 – 32 ]. It was previously shown that hepatic YAP cooperates with ChREBP to regulate glycolytic and lipogenic gene expression [ 33 ], while our current work indicates that YAP is activated when ChREBP activity is reduced in hepatic GSD Ia. As we did not observe altered YAP activity upon hepatic ChREBP knockdown in wildtype mice, we propose that its activation is triggered by ChREBP-dependent physiological changes that occur within the context of hepatic GSD Ia. Among these, modulated bile acid metabolism was of primary interest to us, as we have previously implicated hepatic ChREBP in regulation of bile acid metabolism in GSD Ia [ 18 ], while hepatocyte YAP is activated upon high bile acid exposure [ 34 , 35 ]. In agreement with these studies, plasma bile acids levels were increased upon hepatic ChREBP knockdown in GSD Ia liver. Moreover, the massive hepatocyte hypertrophy observed in ChREBP-normalized GSD Ia mice severely perturbed the cellular architecture of the liver, thereby likely distorting the bile canalicular system and impairing hepatic bile acid secretion. This may in turn have caused intrahepatic accumulation of bile acids and consequent YAP activation [ 35 ]. It was recently reported that accumulation of hepatic glycogen after 3 months of hepatocyte G6pc deletion induces hepatocyte phase separation and formation of glycogen-Mst1/2 aggregates. As this aggregation relieves the inhibitory phosphorylation of hepatic YAP by Mst1/2 signalling, it contributes to hepatomegaly in progressed GSD Ia [ 36 ]. Previous work [ 8 , 37 ] and our current study indicate that attenuation of hepatic ChREBP activity aggravates hepatic glycogen storage in hepatic GSD Ia, while in the current study we show that ChREBP silencing activates hepatocyte YAP. However, as shSCR/L- G6pc −/− mice did not exhibit hepatic YAP activation, and ChREBP normalization did not decrease YAP phosphorylation, glycogen-dependent Mst1/2 sequestration most likely contributes to YAP activation during advanced hepatic GSD Ia. As we primarily aimed to evaluate the role of ChREBP in liver disease progression in GSD Ia, the mechanisms underlying the observed YAP activation were not addressed and warrant follow-up studies. An increased presence of chromosome bridges, induction of CIN marker genes, and enhanced DNA damage and hepatocyte death in shChREBP/L- G6pc −/− mice indicate that ChREBP activation protects against chromosomal instability in hepatic GSD Ia. These changes likely reflect a high degree of hepatocellular stress and damage which may occur as a consequence of activated YAP [ 38 ]. On the other hand, DNA damage may trigger hepatocyte renewal through liver regeneration and YAP activation [ 39 , 40 ]. However, the enrichment of CIN genes, enhanced PARP cleavage, and presence of chromosome bridges that occur in absence of YAP activation in shSCR/L- G6pc −/− mice suggest that CIN/DNA damage occurs prior to YAP activation in early hepatic GSD Ia. Our data also indicate that ChREBP normalization in hepatic GSD Ia activates the cytosolic DNA-sensing cGAS-STING pathway [ 41 , 42 ]. Enhanced cGAS-STING signalling, in turn, likely contributes to the observed induction of cellular senescence [ 41 ] in shChREBP/L- G6pc −/− mice. Increased YAP activity, CIN, and aberrant cell division in ChREBP-normalized L- G6pc −/− mice associated with increased hepatocyte dedifferentiation and trends towards increases in hepatocyte ploidy, in agreement with previous studies in non-GSD Ia contexts [ 29 , 35 , 37 , 43 – 46 ]. Interestingly, the hepatic expression of Hnf4aos , a non-coding RNA which is associated with hepatocyte differentiation and, when decreased, has been linked to advanced liver disease in humans [ 25 – 27 ], was lower in these animals. Consistently, ChREBP normalization in L- G6pc −/− mice increased the ratio of the Hnf4α isoforms Hnf4αP2/P1 , halved HNF4A protein expression levels, and suppressed HNF4α-regulated genes, which was consistently paralleled by induction of dedifferentiation- and proliferation-related gene expression [ 47 ]. Our finding that ChREBP controls the degree of hepatomegaly and the progression to non-alcoholic steatohepatitis (NASH) is in line with previous studies [ 37 , 48 , 49 ]. Importantly, our current work indicates that ChREBP activation in hepatic GSD Ia protects against hepatocellular dedifferentiation, and suggests that it may decelerate tumorigenesis. This is the first study that attributes a potential protective role for ChREBP in liver tumour development, and our findings are in line with published work showing that YAP expression induces or associates with liver tumour formation [ 28 , 30 , 34 ], that reduced HNF4A expression is linked to liver tumour risk in mice and humans [ 20 – 24 ], and that YAP represses HNF4A target genes [ 46 ]. The animal discomfort associated with extreme hepatomegaly that we observed upon attenuation of hepatic ChREBP activity in GSD Ia, however, prevented us from performing longer follow-up studies and thus assessment of liver tumour formation. Interestingly, although attenuation of ChREBP expression in GSD Ia mouse liver induced p53 activation and cell death, this was paralleled by increased proliferation, oncogenic YAP activation, and hepatocyte dedifferentiation. This is likely partly explained by the hepatic regenerative response induced in vivo , in which the consequence of hepatocyte death is not limited to single cells but impacts on the liver as a whole. Moreover, when comparing our current findings on hepatic GSD Ia to published work on hepatic ChREBP in liver tumour development [ 11 , 15 ], its role appears to be disease-specific. Altogether, these insights underline the importance of establishing the context-specific roles of ChREBP to define its therapeutic potential for prevention and/or treatment of liver disease and tumour development. Conclusions In summary, we show that ChREBP normalization in hepatic GSD Ia induces hepatocellular stress, chromosomal instability, DNA damage, and cGAS-STING pathway activation and provokes hepatocyte damage and inflammation, cellular senescence, and hepatocyte dedifferentiation. We hypothesize that hepatic YAP is induced to remove the damaged cells and to stimulate hepatocyte regeneration in order maintain liver function [ 40 ]. However, persistent metabolic stress, chromosomal instability, and DNA damage induced upon long-term ChREBP suppression in hepatic GSD Ia result in constitutive YAP activation, hence likely predisposing to liver tumorigenesis. Altogether, we propose that by sensing and balancing intracellular glucose levels [ 50 ], hepatic ChREBP decelerates hepatomegaly induction, liver disease progression, and hepatocellular tumour formation in GSD Ia. Abbreviations GSD Ia: Glycogen Storage Disease type 1a; G6PC/G6Pase-α: glucose-6-phosphatase, catalytic subunit; G6P: glucose-6-phosphate; ChREBPα/β (MLXIPL): Carbohydrate Response Element Binding Protein alpha/beta; L- G6pc -/- mice: hepatocyte-specific G6pc knockout mice; cGAS-STING: cyclic GMP-AMP synthase-stimulator of interferon genes; IEM: inborn error of metabolism; NAFLD: non-alcoholic fatty liver disease; HCC: hepatocellular carcinoma; dpt: days post treatment (= days post final tamoxifen injection); GSEA: gene set enrichment analysis; IQR: interquartile range; shRNA: small hairpin RNA; siRNA: small interfering RNA; YAP: Yes Associated Protein; CIN: chromosomal instability; HNF4A: Hepatocyte Nuclear Factor 4 Alpha; Hnf4aos /HNF4A-AS1: HNF4A, opposite strand / HNF4A antisense RNA 1; Alb : albumin; Hgfac : hepatocyte growth factor activator; Afp : alpha-fetoprotein; Krt19 : keratin 19; Sox9 : SRY-box transcription factor 9; NASH: non-alcoholic steatohepatitis; H&E: Hematoxylin&Eosin; PRCF: percent relative cumulative frequency; IHC: immunohistochemistry. Declarations Acknowledgements We thank N.L. Mulder, Y.T. van der Veen, R. Havinga, N.J. Kloosterhuis, K. Tholen, M. Koehorst, A.J.C. Tol, and A.H. Heida for excellent technical assistance and F. Kuipers, B.M. Bakker, and F. Foijer for scientific discussion. Ethics approval and consent to participate Human participants: Not applicable. Animal studies: All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Groningen and are in line with the Guide for the Care and Use of Laboratory Animals. Consent for publication Not applicable. Availability of data and materials RNA-sequencing data has previously been submitted to GEO (Gene Expression Omnibus) under GSE143357, which is yet to be made publicly available. Data generated or analysed during this study are included in this published article and its supplementary information files. Additional raw datasets and/or data files used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by a VIDI grant from the Dutch Scientific Organization, a grant from the Stichting Vrienden Beatrix Kinderziekenhuis (Foundation Friends Beatrix Children’s Hospital), and a grant from the De Cock-Hadders Foundation. In addition, this work is supported by European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement PoLiMeR, No 812616. M.H.O holds a Rosalind Franklin Fellowship from the University of Groningen. Authors’ contributions Designing research studies: M.G.S.R., Y.L, J.H.H., B.S., and M.H.O., conducting experiments: M.G.S.R., Y.L., J.H.H., T.B., K.A.K., A. Bl., M.H.K., J.C.W., and H.B., analysing data: M.G.S.R., Y.L., J.H.H., H.Y., V.W.B., T.B., K.A.K., A.Bl., R.E.T., J.C.W., H.B., D.C.J.S., A.Br., B.S., and M.H.O., writing the first draft of the manuscript: M.G.S.R., B.S., and M.H.O., critical revisions of the manuscript: Y.L., J.H.H., H.Y., V.W.B., K.A.K., R.E.T., G.M., F.R., A.M., D.C.J.S., A.Br., B.S., and M.H.O. All authors read and approved the final manuscript. Authors’ information N/A References Chou JY, Jun HS, Mansfield BC. Type I glycogen storage diseases: disorders of the glucose-6-phosphatase/glucose-6-phosphate transporter complexes. J Inherit Metab Dis 2015;38:511–9. https://doi.org/10.1007/s10545-014-9772-x. Rake J, Visser G, Labrune P, Leonard J, Ullrich K, Smit P. Glycogen storage disease type I: diagnosis, management, clinical course and outcome. Results of the European Study on Glycogen Storage Disease Type I (ESGSD I). Eur J Pediatr 2002;161 Suppl:S20–34. https://doi.org/10.1007/S00431-002-0999-4. Abdul-Wahed A, Guilmeau S, Postic C. Sweet Sixteenth for ChREBP: Established Roles and Future Goals. Cell Metab 2017;26:324–41. https://doi.org/10.1016/j.cmet.2017.07.004. Calderaro J, Labrune P, Morcrette G, Rebouissou S, Franco D, Prévot S, et al. Molecular characterization of hepatocellular adenomas developed in patients with glycogen storage disease type i. J Hepatol 2013;58:350–7. https://doi.org/10.1016/j.jhep.2012.09.030. Kim GY, Lee YM, Cho JH, Pan CJ, Jun HS, Springer DA, et al. Mice expressing reduced levels of hepatic glucose-6-phosphatase-a activity do not develop age-related insulin resistance or obesity. Hum Mol Genet 2015;24:5115–25. https://doi.org/10.1093/hmg/ddv230. Iizuka K. The transcription factor carbohydrate-response element-binding protein (ChREBP): A possible link between metabolic disease and cancer. Biochim Biophys Acta - Mol Basis Dis 2017;1863:474–85. https://doi.org/10.1016/j.bbadis.2016.11.029. Abdul-Wahed A, Gautier-Stein A, Casteras S, Soty M, Roussel D, Romestaing C, et al. A link between hepatic glucose production and peripheral energy metabolism via hepatokines. Mol Metab 2014;3:531–43. https://doi.org/10.1016/j.molmet.2014.05.005. Lei Y, Hoogerland JA, Bloks VW, Bos T, Bleeker A, Wolters H, et al. Hepatic ChREBP activation limits NAFLD development in a mouse model for Glycogen Storage Disease type Ia. Hepatology 2020. https://doi.org/10.1002/hep.31198. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell 2011;144:646–74. https://doi.org/10.1016/j.cell.2011.02.013. Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, et al. Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma. Gastroenterology 2011;140:1071-1083.e5. https://doi.org/10.1053/j.gastro.2010.12.006. Ribback S, Che L, Pilo MG, Cigliano A, Latte G, Pes GM, et al. Oncogene-dependent addiction to carbohydrate-responsive element binding protein in hepatocellular carcinoma. Cell Cycle 2018;17:1496–512. https://doi.org/10.1080/15384101.2018.1489182. Evert M, Calvisi DF, Evert K, De Murtas V, Gasparetti G, Mattu S, et al. V-AKT murine thymoma viral oncogene homolog/mammalian target of rapamycin activation induces a module of metabolic changes contributing to growth in insulin-induced hepatocarcinogenesis. Hepatology 2012;55:1473–84. https://doi.org/10.1002/hep.25600. Lei Y, Hu Q, Gu J. Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and Clinical Significance. Pathol Oncol Res 2020;26:1331–40. https://doi.org/10.1007/s12253-019-00708-y. Dong X, Wang F, Liu C, Ling J, Jia X, Shen F, et al. Single-cell analysis reveals the intra-tumor heterogeneity and identifies MLXIPL as a biomarker in the cellular trajectory of hepatocellular carcinoma. Cell Death Discov 2021;7. https://doi.org/10.1038/s41420-021-00403-5. Wang H, Dolezal JM, Kulkarni S, Lu J, Mandel J, Jackson LE, et al. Myc and ChREBP transcription factors cooperatively regulate normal and neoplastic hepatocyte proliferation in mice. J Biol Chem 2018;293:14740–57. https://doi.org/10.1074/jbc.RA118.004099. Tong X, Zhao F, Mancuso A, Gruber JJ, Thompson CB. The glucose-responsive transcription factor ChREBP contributes to glucose-dependent anabolic synthesis and cell proliferation. Proc Natl Acad Sci U S A 2009;106:21660–5. https://doi.org/10.1073/pnas.0911316106. van den Bos H, Bakker B, Taudt A, Guryev V, Colomé-Tatché M, Lansdorp PM, et al. Quantification of aneuploidy in mammalian systems. Methods Mol. Biol., vol. 1896, Methods Mol Biol; 2019, p. 159–90. https://doi.org/10.1007/978-1-4939-8931-7_15. Hoogerland JA, Lei Y, Wolters JC, de Boer JF, Bos T, Bleeker A, et al. Glucose‐6‐phosphate regulates hepatic bile acid synthesis in mice. Hepatology 2019;0:1–14. https://doi.org/10.1002/hep.30778. Gisselsson D. Chromosome instability in cancer: How, when, and why? Adv Cancer Res 2003;87:1–29. https://doi.org/10.1016/S0065-230X(03)87164-6. Bonzo JA, Ferry CH, Matsubara T, Kim JH, Gonzalez FJ. Suppression of hepatocyte proliferation by hepatocyte nuclear factor 4α in adult mice. J Biol Chem 2012;287:7345–56. https://doi.org/10.1074/jbc.M111.334599. Dubois V, Staels B, Lefebvre P, Verzi MP, Eeckhoute J. Control of Cell Identity by the Nuclear Receptor HNF4 in Organ Pathophysiology. Cells 2020;9. https://doi.org/10.3390/CELLS9102185. Kalkuhl A, Kaestner K, Buchmann A, Schwarz M. Expression of hepatocyte-enriched nuclear transcription factors in mouse liver tumours. Carcinogenesis 1996;17:609–12. https://doi.org/10.1093/carcin/17.3.609. Lazarevich NL, Cheremnova OA, Varga E V., Ovchinnikov DA, Kudrjavtseva EI, Morozova O V., et al. Progression of HCC in Mice Is Associated with a Downregulation in the Expression of Hepatocyte Nuclear Factors. Hepatology 2004;39:1038–47. https://doi.org/10.1002/hep.20155. Ning BF, Ding J, Yin C, Zhong W, Wu K, Zeng X, et al. Hepatocyte nuclear factor 4α suppresses the development of hepatocellular carcinoma. Cancer Res 2010;70:7640–51. https://doi.org/10.1158/0008-5472.CAN-10-0824. Esposti DD, Hernandez-Vargas H, Voegele C, Fernandez-Jimenez N, Forey N, Bancel B, et al. Identification of novel long non-coding RNAs deregulated in hepatocellular carcinoma using RNA-sequencing. Oncotarget 2016;7:31862–77. https://doi.org/10.18632/oncotarget.7364. Atanasovska B, Rensen SS, Marsman G, Shiri-Sverdlov R, Withoff S, Kuipers F, et al. Long non-coding rnas involved in progression of non-alcoholic fatty liver disease to steatohepatitis. Cells 2021;10. https://doi.org/10.3390/cells10081883. Guo S, Lu H. Novel mechanisms of regulation of the expression and transcriptional activity of hepatocyte nuclear factor 4α. J Cell Biochem 2019;120:519–32. https://doi.org/10.1002/jcb.27407. Cai J, Zhang N, Zheng Y, De Wilde RF, Maitra A, Pan D. The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev 2010;24:2383–8. https://doi.org/10.1101/gad.1978810. Camargo FD, Gokhale S, Johnnidis JB, Fu D, Bell GW, Jaenisch R, et al. YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells. Curr Biol 2007;17:2054–60. https://doi.org/10.1016/j.cub.2007.10.039. Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA, et al. Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals. Cell 2007;130:1120–33. https://doi.org/10.1016/j.cell.2007.07.019. Lee KP, Lee JH, Kim TS, Kim TH, Park HD, Byun JS, et al. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc Natl Acad Sci U S A 2010;107:8248–53. https://doi.org/10.1073/pnas.0912203107. Song H, Mak KK, Topol L, Yun K, Hu J, Garrett L, et al. Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression. Proc Natl Acad Sci U S A 2010;107:1431–6. https://doi.org/10.1073/pnas.0911409107. Shu Z, Yi G, Deng S, Huang K, Wang Y. Hippo pathway cooperates with ChREBP to regulate hepatic glucose utilization. Biochem Biophys Res Commun 2020;530:115–21. https://doi.org/10.1016/j.bbrc.2020.06.105. Anakk S, Bhosale M, Schmidt VA, Johnson RL, Finegold MJ, Moore DD. Bile Acids Activate YAP to Promote Liver Carcinogenesis. Cell Rep 2013;5:1060–9. https://doi.org/10.1016/j.celrep.2013.10.030. Pepe-Mooney BJ, Dill MT, Alemany A, Ordovas-Montanes J, Matsushita Y, Rao A, et al. Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration. Cell Stem Cell 2019;25:23-38.e8. https://doi.org/10.1016/j.stem.2019.04.004. Liu Q, Li J, Zhang W, Xiao C, Zhang S, Nian C, et al. Glycogen accumulation and phase separation drives liver tumor initiation. Cell 2021. https://doi.org/10.1016/j.cell.2021.10.001. Rajas F, Dentin R, Cannella Miliano A, Silva M, Raffin M, Levavasseur F, et al. The absence of hepatic glucose-6 phosphatase/ChREBP couple is incompatible with survival in mice. Mol Metab 2021;43. https://doi.org/10.1016/j.molmet.2020.101108. Weiler SME, Pinna F, Wolf T, Lutz T, Geldiyev A, Sticht C, et al. Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer. Gastroenterology 2017;152:2037-2051.e22. https://doi.org/10.1053/j.gastro.2017.02.018. Pefani DE, O’Neill E. Hippo pathway and protection of genome stability in response to DNA damage. FEBS J 2016;283:1392–403. https://doi.org/10.1111/febs.13604. Michalopoulos GK. Hepatostat: Liver regeneration and normal liver tissue maintenance. Hepatology 2017;65:1384–92. https://doi.org/10.1002/hep.28988. Loo TM, Miyata K, Tanaka Y, Takahashi A. Cellular senescence and senescence-associated secretory phenotype via the cGAS-STING signaling pathway in cancer. Cancer Sci 2020;111:304–11. https://doi.org/10.1111/cas.14266. Li T, Chen ZJ. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer. J Exp Med 2018;215:1287–99. https://doi.org/10.1084/jem.20180139. Zhang S, Chen Q, Liu Q, Li Y, Sun X, Hong L, et al. Hippo Signaling Suppresses Cell Ploidy and Tumorigenesis through Skp2. Cancer Cell 2017;31:669-684.e7. https://doi.org/10.1016/j.ccell.2017.04.004. Pandit SK, Westendorp B, De Bruin A. Physiological significance of polyploidization in mammalian cells. Trends Cell Biol 2013;23:556–66. https://doi.org/10.1016/j.tcb.2013.06.002. Sargsyan A, Doridot L, Hannou SA, Tong W, Srinivasan H, Ivison R, et al. HGFAC is a ChREBP regulated hepatokine that enhances glucose and lipid homeostasis. JCI Insight 2022. https://doi.org/10.1172/jci.insight.153740. Biagioni F, Croci O, Sberna S, Donato E, Sabò A, Bisso A, et al. Decoding YAP dependent transcription in the liver. Nucleic Acids Res 2022;50:7959–71. https://doi.org/10.1093/nar/gkac624. Tanaka T, Jiang S, Hotta H, Takano K, Iwanari H, Sumi K, et al. Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4α in the pathogenesis of human cancer. J Pathol 2006;208:662–72. https://doi.org/10.1002/path.1928. Bricambert J, Alves-Guerra MC, Esteves P, Prip-Buus C, Bertrand-Michel J, Guillou H, et al. The histone demethylase Phf2 acts as a molecular checkpoint to prevent NAFLD progression during obesity. Nat Commun 2018;9. https://doi.org/10.1038/s41467-018-04361-y. Shi JH, Lu JY, Chen HY, Wei CC, Xu X, Li H, et al. Liver ChREBP protects against fructose-induced glycogenic hepatotoxicity by regulating L-type pyruvate kinase. Diabetes 2020;69:591–602. https://doi.org/10.2337/db19-0388. Agius L, Chachra SS, Ford BE. The Protective Role of the Carbohydrate Response Element Binding Protein in the Liver: The Metabolite Perspective. Front Endocrinol (Lausanne) 2020;11. https://doi.org/10.3389/fendo.2020.594041. Additional Declarations No competing interests reported. Supplementary Files shChREBPCancerandMetabolismSupplementFINAL.docx shChREBPCancerandMetabolismSupplementRawblots.docx Graphicalabstract.jpg Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2023 Read the published version in Cancer & Metabolism → Version 1 posted Editorial decision: Accepted 21 Mar, 2023 Reviews received at journal 16 Feb, 2023 Reviewers agreed at journal 06 Feb, 2023 Reviewers invited by journal 05 Feb, 2023 Editor assigned by journal 30 Jan, 2023 Submission checks completed at journal 30 Jan, 2023 First submitted to journal 25 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2514060","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":171765421,"identity":"ca4a0f29-7208-4125-8780-fcf3889cf0d7","order_by":0,"name":"Martijn G.S. Rutten","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYNCDDxCK8QBRqnnYgEpnQDnEa2HmIUaLbgP7BcYfDPcS98s3H3ts27ZNXt6B9wBeLWYHeAqAhhcn9rCxpRvntt023HiAL4GQlgRmBoYEoBYeM2mglgTDBh4DglqADgNp4f8mbUmcFvYDDDwQW9ikGYFa5BkIaTnMw3CYxyDBuOdYmplkz7nbhhuYCWk53v7w4Y+KBNn25sPPJH6U3ZaXb+8xfIBPCwPYTAMkAYPDeNWDADuakfINBLWMglEwCkbBCAMA3RdE/Ce6dpoAAAAASUVORK5CYII=","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":true,"prefix":"","firstName":"Martijn","middleName":"G.S.","lastName":"Rutten","suffix":""},{"id":171765422,"identity":"af267454-40b4-4e3e-a62d-7db4232d938b","order_by":1,"name":"Yu Lei","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Lei","suffix":""},{"id":171765423,"identity":"5929cd28-ec9c-48a6-856c-1946f2538d1f","order_by":2,"name":"Joanne H. Hoogerland","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Joanne","middleName":"H.","lastName":"Hoogerland","suffix":""},{"id":171765424,"identity":"8af0e134-6dda-4cd4-9628-e0a12cfed434","order_by":3,"name":"Vincent W. Bloks","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"W.","lastName":"Bloks","suffix":""},{"id":171765425,"identity":"c7a414cf-dec8-48e2-9ea9-a902a317b7cb","order_by":4,"name":"Hong Yang","email":"","orcid":"","institution":"KTH - Royal Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Yang","suffix":""},{"id":171765426,"identity":"1e7d7b8a-d46e-47c1-a010-83753b9d34a3","order_by":5,"name":"Trijnie Bos","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Trijnie","middleName":"","lastName":"Bos","suffix":""},{"id":171765427,"identity":"014cbd76-4f1e-4f60-971d-968f72734201","order_by":6,"name":"Kishore A. Krishnamurthy","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Kishore","middleName":"A.","lastName":"Krishnamurthy","suffix":""},{"id":171765428,"identity":"0f0b76ca-8ac9-4b03-a3a5-f6b53fa12f8b","order_by":7,"name":"Aycha Bleeker","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Aycha","middleName":"","lastName":"Bleeker","suffix":""},{"id":171765429,"identity":"a87d2666-2404-445c-883e-e86030d06f56","order_by":8,"name":"Mirjam H. Koster","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Mirjam","middleName":"H.","lastName":"Koster","suffix":""},{"id":171765430,"identity":"6a5a68f6-0186-4605-8ab9-778f553e48df","order_by":9,"name":"Rachel E. Thomas","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Rachel","middleName":"E.","lastName":"Thomas","suffix":""},{"id":171765431,"identity":"dcc8c3da-289d-426e-b7af-089214aa863f","order_by":10,"name":"Justina C. Wolters","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Justina","middleName":"C.","lastName":"Wolters","suffix":""},{"id":171765432,"identity":"ba27e627-2bc5-4d48-810c-3423ed5ef8e6","order_by":11,"name":"Hilda van den Bos","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Hilda","middleName":"van den","lastName":"Bos","suffix":""},{"id":171765433,"identity":"059e2886-35f1-47f0-a2f6-0826dd2849b3","order_by":12,"name":"Gilles Mithieux","email":"","orcid":"","institution":"Institut National de la Santé et de la Recherche Médicale","correspondingAuthor":false,"prefix":"","firstName":"Gilles","middleName":"","lastName":"Mithieux","suffix":""},{"id":171765434,"identity":"b7e7b8bf-b162-48ee-ba3e-cd9c6d324064","order_by":13,"name":"Fabienne Rajas","email":"","orcid":"","institution":"Institut National de la Santé et de la Recherche Médicale","correspondingAuthor":false,"prefix":"","firstName":"Fabienne","middleName":"","lastName":"Rajas","suffix":""},{"id":171765435,"identity":"f1dbd604-1942-47cb-8389-95dd35f10a4f","order_by":14,"name":"Adil Mardinoglu","email":"","orcid":"","institution":"KTH - Royal Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Adil","middleName":"","lastName":"Mardinoglu","suffix":""},{"id":171765436,"identity":"ac868c8d-eccb-4de0-bf55-5cc592a09132","order_by":15,"name":"Diana C.J. Spierings","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Diana","middleName":"C.J.","lastName":"Spierings","suffix":""},{"id":171765437,"identity":"12d98eb4-6afa-4b29-9ff5-323917b6f7bd","order_by":16,"name":"Alain de Bruin","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Alain","middleName":"","lastName":"de Bruin","suffix":""},{"id":171765438,"identity":"a48e7de9-7756-472c-9b28-0ec9ca5b9fcf","order_by":17,"name":"Bart van de Sluis","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Bart","middleName":"van","lastName":"de Sluis","suffix":""},{"id":171765440,"identity":"2eb7fff0-1f0d-4387-8000-149552d53db9","order_by":18,"name":"Maaike H. Oosterveer","email":"","orcid":"","institution":"University of Groningen, University Medical Center Groningen","correspondingAuthor":false,"prefix":"","firstName":"Maaike","middleName":"H.","lastName":"Oosterveer","suffix":""}],"badges":[],"createdAt":"2023-01-25 13:29:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2514060/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2514060/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40170-023-00305-3","type":"published","date":"2023-04-21T20:33:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":32337198,"identity":"27697d92-f552-4b83-8df9-a0ff33dd8f01","added_by":"auto","created_at":"2023-02-01 16:29:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":937667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChREBP knockdown in hepatic GSD Ia mice\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecauses hepatocyte death, inflammation, DNA damage, and proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Single cell death and inflammatory foci, (B) γH2Ax positivity, (C) \u003cem\u003ep21 \u003c/em\u003e(\u003cem\u003eCdkn1a\u003c/em\u003e) expression, and (D) pH3 and Ki67 positivity and mitotic figures in livers after 10 days of shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e. A-D: median ± interquartile range; Kruskal Wallis H-test, post-hoc Conover pairwise comparisons, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; ^ vs shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; # vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e (n=7-9).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/4148ee651898e515c450070d.png"},{"id":32337197,"identity":"fbafae5f-a171-4b8f-a741-5391f18be8c5","added_by":"auto","created_at":"2023-02-01 16:29:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":990295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProlonged hepatic ChREBP normalization in L-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG6pc\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice progressively induces hepatomegaly and sensitizes to hepatic inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Liver weight and plasma ALT levels in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice (n=8). (B) Representative macroscopic liver photos and photos of H\u0026amp;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-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice (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 \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; ^ vs shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; # vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/1844f0cdcaa63c6be995ad00.png"},{"id":32338450,"identity":"06ec0b93-fabe-4f05-949b-ba59af44b252","added_by":"auto","created_at":"2023-02-01 16:37:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1542459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProlonged hepatic ChREBP normalization in L-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG6pc\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice promotes Yes Associated Protein (YAP) transcriptional activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData after 21-26 days of shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e and n=8, unless stated otherwise. (A) Mitotic figures and BrdU positivity (n=4-6, 20-21 days). (B) YAP-target genes and \u003cem\u003eShp\u003c/em\u003e. (C) YAP nuclear protein and whole liver lysate pYAP/YAP ratio (Blots/Ponceau S: Fig. S2C-E). (D) Correlations between liver weight and \u003cem\u003eCtgf \u003c/em\u003eexpression in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. (F) Expression of bile acid synthesis enzymes and transporters, and (G) 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 \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; ^ vs shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; # vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/2e2ae7f91be454d050dab69c.png"},{"id":32337186,"identity":"b515c38e-d035-4b8c-ae60-bcd64a1af90f","added_by":"auto","created_at":"2023-02-01 16:29:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":822319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProlonged ChREBP knockdown in L-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG6pc\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice induces DNA damage, cellular senescence, and hepatocyte dedifferentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData after 21-26 days of shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003eand 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) \u003cem\u003eCgas \u003c/em\u003eand senescence-associated genes and p21 protein, and (E) HNF4A-related genes. (F) Reporter transcription factors analysis (after 10 days of shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e). (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 \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; ^ vs shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e; # vs shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/88318ff1416126a971b3fc10.png"},{"id":44725914,"identity":"6b2cba42-99eb-42d8-81c2-24a7e6c2c005","added_by":"auto","created_at":"2023-10-16 20:44:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1866680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/9f8e6f8d-70af-4738-91a4-15828de8aa07.pdf"},{"id":32337210,"identity":"b8771e11-e599-4ebd-8ece-f08eccbbc309","added_by":"auto","created_at":"2023-02-01 16:29:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5929889,"visible":true,"origin":"","legend":"","description":"","filename":"shChREBPCancerandMetabolismSupplementFINAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/f0bb6a6c024dc9b0e408e57e.docx"},{"id":32337200,"identity":"d5e74c81-56a6-4df7-86cd-b021a8acf47b","added_by":"auto","created_at":"2023-02-01 16:29:43","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17923273,"visible":true,"origin":"","legend":"","description":"","filename":"shChREBPCancerandMetabolismSupplementRawblots.docx","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/d71bb905bc72ff3c7dc1220a.docx"},{"id":32337201,"identity":"fc72f655-edd4-4974-bb06-a71fd6e1ad72","added_by":"auto","created_at":"2023-02-01 16:29:43","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":67258,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2514060/v1/30a031302d7242e897995415.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Normalization of hepatic ChREBP activity does not protect against liver disease progression in a mouse model for Glycogen Storage Disease type Ia","fulltext":[{"header":"Background","content":"\u003cp\u003eGlycogen Storage Disease type Ia (GSD Ia) (MIM#232200) is a rare inborn error of metabolism (IEM) caused by mutations in the gene encoding for the catalytic subunit of glucose-6-phosphatase (\u003cem\u003eG6PC1\u003c/em\u003e (\u003cem\u003eG6pc\u003c/em\u003e in mice), G6Pase-α) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which is expressed in liver, kidney, and intestine, where it converts glucose-6-phosphate (G6P) into glucose. Patients primarily display severe metabolic liver disease, characterized by hepatomegaly and non-alcoholic fatty liver disease (NAFLD), while liver tumour development represents the major long-term complication of GSD Ia, affecting up to 70% of patients by the age of 30 years [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCarbohydrate Response Element Binding Protein (ChREBP, also known as MLXIPL, MONDOB, or WBSCR14) is the major glucose-sensitive transcription factor in hepatocytes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and ChREBP and its regulated pathways are activated in hepatic GSD Ia [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. We previously showed that short-term normalization of ChREBP activity aggravates hepatomegaly and NAFLD in hepatocyte-specific GSD Ia mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings suggest that sustained hepatic ChREBP normalization in hepatic GSD Ia may drive advanced liver disease elements, including hepatic inflammation, liver fibrosis, hepatocellular death and/or oncogenic transformation. On the other hand, evidence that ChREBP-regulated pathways represent a typical hallmark of many cancer cells has accumulated [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consistently, ChREBP has been linked to the incidence and prognosis of hepatocellular carcinoma (HCC) [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. ChREBP deficient mice are protected against HCC development in an oncogene-specific manner, and ChREBP deficiency inhibits growth of β-catenin/YAP-driven hepatoblastomas [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, reduced ChREBP expression inhibits hepatocellular proliferation through oxidative stress-induced, p53-mediated cell cycle arrest \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], while ChREBP-deficient hepatocytes show impaired proliferation rates during liver repopulation \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Combined, these studies indicate that ChREBP serves as a competent factor for cell growth and liver tumour progression.\u003c/p\u003e \u003cp\u003eThese previous studies from our laboratory and others suggest context-specific roles of hepatic ChREBP in advanced liver disease, in particular hepatocellular tumour susceptibility, which are of critical importance to establish the therapeutic potential of ChREBP for the treatment of liver disease in GSD Ia patients. In the current study we therefore investigated the impact of prolonged ChREBP normalization on liver disease progression in hepatic GSD Ia mice. Our data show that normalization of hepatic ChREBP activity sensitizes liver-specific GSD Ia mice to advanced liver disease development, DNA damage, cellular senescence, as well as hepatocellular proliferation and dedifferentiation, suggesting increased susceptibility for hepatocarcinogenesis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAAV-shRNA construction and production\u003c/h2\u003e \u003cp\u003eSee Supplement.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale adult \u003cem\u003eG6pc-\u003c/em\u003efloxed \u003cem\u003eAlb\u003c/em\u003e-Cre negative (B6.\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003elox/lox\u003c/sup\u003e) and \u003cem\u003eG6pc-\u003c/em\u003efloxed \u003cem\u003eAlb\u003c/em\u003e-Cre positive (B6.\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003elox/lox\u003c/sup\u003e.SA\u003csup\u003ecreERT2/w\u003c/sup\u003e mice) on a C57BL/6J background were infected with shRNAs directed against ChREBP (AAV-shChREBP) or a scrambled control (AAV-shScramble (shSCR)) (1x10\u003csup\u003e12\u003c/sup\u003e particles per mouse) by intravenous injection into the retro-orbital plexus under isoflurane anaesthesia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. At 11\u0026ndash;12 days after AAV-shRNA administration, all mice received i.p. injections of tamoxifen for 5 consecutive days to generate liver-specific \u003cem\u003eG6pc\u003c/em\u003e-deficient mice (L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) and wildtype littermates (L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e). Nonfasted animals were sacrificed for tissue collection at 8AM at 10 or 25\u0026ndash;26 days after the last treatment (dpt). Two shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were euthanized at 21 dpt because a humane endpoint was reached, yet were included in the analyses. Absolute liver weight of the 10-day follow-up study cohort has previously been reported [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For further details, see Supplementary Materials \u0026amp; Methods. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Groningen and are in line with the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eHistological and pathological analysis of the liver\u003c/h2\u003e \u003cp\u003eSee Supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eBiochemical assays\u003c/h2\u003e \u003cp\u003eSee Supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eGene expression analysis, RNA-sequencing, gene set enrichment analysis (GSEA) and reporter transcription factors analysis\u003c/h2\u003e \u003cp\u003eSee Supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eTargeted proteomics, SDS-PAGE, and Western Blot\u003c/h2\u003e \u003cp\u003eSee Supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003ePloidy analysis\u003c/h2\u003e \u003cp\u003eFor analysis of hepatocyte ploidy, ~\u0026thinsp;20 mg of frozen powdered liver tissue was used for nuclei isolation (as described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]) in lysis buffer (10 mM Tris-HCl (pH 8), 0.32 M Sucrose, 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 3 mM Mg(Ac)\u003csub\u003e2\u003c/sub\u003e, and 0.1 mM EDTA, with fresh addition of 1 mM DTT and 0.1% Triton X-100). In short, a 50\u0026ndash;100 \u0026micro;m filter was placed on a 50 mL Falcon tube, and liver powder was poured onto the filter. Liver powder was stepwise and gently homogenized in 1.5 mL lysis buffer and pushed through the filter using a 5 mL syringe plunger. The resulting 1.5 mL nuclear suspension was transferred to a 1.5 or 2.0 mL tube, and supplemented with ~\u0026thinsp;25,000 control cells (diploid human GFP-positive cells). Nuclei were spun down at 500xg for 5 min at 4\u0026deg;C and the pellet was resuspended in 300\u0026ndash;1000 \u0026micro;L PBS/BSA with Hoechst/PI DNA dyes (10 \u0026micro;g/mL for both). Nuclei were filtered through 35\u0026micro;m FACS tubes and analysed on the Canto FACS machine (BD Biosciences) for ploidy analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eData in figures is presented as dot plots with median\u0026thinsp;\u0026plusmn;\u0026thinsp;interquartile range (IQR), unless stated otherwise. Data in tables is presented as median (range), unless stated otherwise. Data in heatmaps represent z-score normalized values. Statistical analysis was performed using BrightStat and GraphPad PRISM software. Differences between multiple groups were tested by a Kruskal Wallis H-test followed by post-hoc Conover pairwise comparisons. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***, \u003csup\u003e^^^\u003c/sup\u003e, or \u003csup\u003e###\u003c/sup\u003e), 0.001 to 0.01 (**, \u003csup\u003e^^\u003c/sup\u003e, or \u003csup\u003e##\u003c/sup\u003e), and 0.01 to 0.05 (*, \u003csup\u003e^\u003c/sup\u003e, or \u003csup\u003e#\u003c/sup\u003e) were considered significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eNormalization of hepatic ChREBP expression in GSD Ia liver induces oxidative stress, p53 activation, and cell cycle inhibition while inducing mitosis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe previously showed that short-term normalization of ChREBP activity aggravates hepatomegaly and NAFLD in a mouse model for hepatic GSD Ia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Here, histopathological analysis revealed an increase in single cell death and inflammatory foci in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, while the number of γH2Ax-positive hepatocytes tended to increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, S1A). This was paralleled by a significant induction of the p53-target gene \u003cem\u003ep21\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, the number of pH3- and Ki67-positive hepatocytes and mitotic figures was increased in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, S1A). Complementary Gene Set Enrichment Analysis (GSEA) of RNA expression data (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) revealed that normalization of hepatic ChREBP expression in GSD Ia liver induces oxidative stress, apoptosis, p53 activation, cell cycle inhibition, hepatocyte death, chromosomal instability, DNA damage, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway (cGAS-STING) activation, cellular senescence, inflammatory response, epithelial mesenchymal transition, and mitotic activity within a timeframe of two weeks.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene Set Enrichment Analysis on RNAseq data of short-term shSCR- and shChREBP-treated L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e shSCR vs \u003c/p\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e shSCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e shChREBP vs \u003c/p\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e shSCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e shChREBP vs \u003c/p\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e shSCR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene set (size\u003csup\u003e$\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNominal p-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFDR \u003c/p\u003e \u003cp\u003eq-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNominal p-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFDR \u003c/p\u003e \u003cp\u003eq-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNominal p-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFDR \u003c/p\u003e \u003cp\u003eq-value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP53 pathway (158)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.54\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.034\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.53\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.026\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG2/M checkpoint (153)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1.66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.018\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.93\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidative stress (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.47\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.045\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.187\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApoptosis (122)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.54\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.005\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.032\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.012\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCIN29 (15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e2.15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.97\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2.07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecGAS-STING (39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.627\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.90\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMC2-senescence (133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.046\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.155\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.79\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2.19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInflammatory response (114)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.94\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.98\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpithelial mesenchymal transition (123)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.691\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.81\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003e$\u003c/sup\u003esize: refers to the size of the gene set after filtering out those genes that were not in the expression data set\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProlonged hepatic ChREBP normalization in L-G6pc\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice induces extreme hepatomegaly and sensitizes to hepatic inflammation\u003c/h2\u003e \u003cp\u003eAs these early changes suggested acceleration of metabolic-associated fatty liver disease towards advanced liver disease in response to shChREBP, we next evaluated the hepatic effects of prolonged normalized ChREBP activity in L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig. S2A). Three weeks of ChREBP normalization reduced fed blood glucose levels and increased plasma ketone bodies, while body weight remained unaffected (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It furthermore exacerbated hepatomegaly as compared to 10-day ChREBP normalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and caused a concomitant progressive increase in plasma ALT levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and hepatocyte vacuolization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), highlighting the progressive nature of the liver disease. Hepatic G6P and glycogen contents were further increased in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, while hepatic triglyceride contents varied, and relative hepatic protein content was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Hepatic water content was not different between shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Livers of shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed a marked increase in inflammatory foci, in line with GSEA data on short-term ChREBP normalization in hepatic GSD Ia (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), while the expression of inflammatory genes \u003cem\u003eIl1β\u003c/em\u003e, \u003cem\u003eIl6\u003c/em\u003e, \u003cem\u003eTnfα\u003c/em\u003e, and \u003cem\u003eCd68\u003c/em\u003e was minimally or not (significantly) induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The expression of fibrosis marker genes was increased in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Yet, at the histological level, this was not paralleled by enhanced hepatic collagen deposition (data not shown). These data indicate that prolonged hepatic ChREBP normalization in GSD Ia progressively exacerbates hepatomegaly and hepatocyte hypertrophy and predisposes to hepatic inflammation.\u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneral data of prolonged shSCR- and shChREBP-treated L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003cp\u003eMedian (range)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e shSCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e shChREBP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e shSCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eL-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e shChREBP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.3 (27.8\u0026ndash;31.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.4 (25.6\u0026ndash;29.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.1 (25.3\u0026ndash;30.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.6 (21.1\u0026ndash;32.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.27 (1.14\u0026ndash;1.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.89 (1.28\u0026ndash;2.10)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.06 (1.17\u0026ndash;2.39)\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.72 (2.15\u0026ndash;9.20)\u003csup\u003e***^^^###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood glucose (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.1 (7.7\u0026ndash;14.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.4 (8.8\u0026ndash;14.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.2 (4.9\u0026ndash;13.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.9 (1.1\u0026ndash;11.5)\u003csup\u003e***^^#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLiver\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG6P (\u0026micro;mol/g liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.47 (0.29\u0026ndash;0.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.53 (0.31\u0026ndash;0.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.38 (0.77\u0026ndash;3.04)\u003csup\u003e***^^^\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.71 (1.28\u0026ndash;3.60)\u003csup\u003e***^^^\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG6P (\u0026micro;mol/liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.65 (0.41\u0026ndash;0.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00 (0.49\u0026ndash;1.19)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.99 (1.50\u0026ndash;6.26)\u003csup\u003e***^^^\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.58 (2.75\u0026ndash;19.15)\u003csup\u003e***^^^##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycogen (mg/liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98 (68\u0026ndash;136)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e163 (93\u0026ndash;224)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e190 (71\u0026ndash;234)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e915 (151\u0026ndash;1926)\u003csup\u003e***^^^##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriglycerides (\u0026micro;mol/liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.7 (0.1\u0026ndash;23.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.3 (7.6-106.1)\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.8 (22.3\u0026ndash;80.7)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.3 (0.6-238.9)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFree cholesterol (\u0026micro;mol/g liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.94 (2.70\u0026ndash;4.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.36 (3.23\u0026ndash;3.91)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.46 (2.97\u0026ndash;4.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.20 (1.13\u0026ndash;5.43)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFree cholesterol (\u0026micro;mol/liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.08 (3.74\u0026ndash;6.44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.31 (4.88\u0026ndash;7.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.92 (4.71\u0026ndash;8.23)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.73 (10.23\u0026ndash;14.48)\u003csup\u003e***^^^###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholesteryl-esters (\u0026micro;mol/g liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.68 (0.02\u0026ndash;0.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.80 (0.57\u0026ndash;3.02)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.85 (1.16\u0026ndash;3.03)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.23 (0.10\u0026ndash;5.47)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholesteryl-esters (\u0026micro;mol/liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.83 (0.03\u0026ndash;1.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.57 (0.73\u0026ndash;4.78)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.23 (2.26\u0026ndash;5.11)\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.73 (0.79\u0026ndash;11.77)\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal cholesterol (\u0026micro;mol/g liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.45 (2.91\u0026ndash;5.27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.13 (3.98\u0026ndash;6.51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.27 (4.14\u0026ndash;6.68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.36 (1.33\u0026ndash;10.90)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal cholesterol (\u0026micro;mol/liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.87 (4.02\u0026ndash;7.93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.01 (5.73\u0026ndash;10.73)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.93 (6.97\u0026ndash;13.15)\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.06 (11.03\u0026ndash;24.79)\u003csup\u003e***^^^###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein (mg/liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 (241\u0026ndash;401)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e381 (290\u0026ndash;412)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e430 (229\u0026ndash;481)\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e656 (394\u0026ndash;821)\u003csup\u003e***^^^##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePlasma\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactate (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.94 (3.46\u0026ndash;5.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.72 (3.53\u0026ndash;6.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.62 (4.02\u0026ndash;7.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.03 (2.65\u0026ndash;6.38)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal ketone bodies (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.072 (0.055\u0026ndash;0.112)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.087 (0.055\u0026ndash;0.165)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.082 (0.059\u0026ndash;0.197)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.123 (0.090\u0026ndash;0.224)\u003csup\u003e**^#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3HB (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.069 (0.051\u0026ndash;0.109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.084 (0.052\u0026ndash;0.161)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.079 (0.056\u0026ndash;0.192)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.121 (0.087\u0026ndash;0.219)\u003csup\u003e**^#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003 (0.002\u0026ndash;0.007)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.004 (0.003\u0026ndash;0.007)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.003 (0.002\u0026ndash;0.005)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.003 (0.000-0.005)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFFA (\u0026micro;mol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e153 (96\u0026ndash;259)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e166 (129\u0026ndash;254)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e224 (69\u0026ndash;306)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e167 (97\u0026ndash;409)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eProlonged hepatic ChREBP normalization in L-G6pc\u003c/em\u003e \u003csup\u003e \u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e \u003c/sup\u003e \u003cem\u003emice promotes the transcriptional activity of Yes Associated Protein (YAP)\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the origin of the extreme liver enlargement observed in hepatic GSD Ia mice upon prolonged ChREBP normalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), we next assessed hepatocyte proliferation. Indeed, livers of shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed an increase in mitotic figures and the number of BrdU-positive hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Fig. S2B). They also exhibited an induction of YAP target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) in parallel to increased nuclear YAP protein levels, while p-YAP/YAP ratios remained unaffected as compared to shSCR-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Interestingly, within shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, relative liver weights, mRNA levels of YAP-target gene \u003cem\u003eCtgf\u003c/em\u003e (\u003cem\u003eCcn2\u003c/em\u003e), and hepatic glycogen contents were positively correlated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, S2F). In line with our previous work [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], ChREBP normalization in L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice suppressed the expression of \u003cem\u003eCyp8b1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). It furthermore reduced the hepatic expression of bile acid transporters \u003cem\u003eNtcp\u003c/em\u003e (\u003cem\u003eSlc10a1\u003c/em\u003e) and \u003cem\u003eBsep\u003c/em\u003e (\u003cem\u003eAbcb11\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) while increasing plasma bile acid levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Interestingly, relative liver weight correlated significantly yet moderately with total plasma bile acid (r\u0026thinsp;=\u0026thinsp;0.3000, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) across different study cohorts (n\u0026thinsp;=\u0026thinsp;64). In the current study cohort, plasma bile acids levels also positively correlated with \u003cem\u003eCtgf\u003c/em\u003e mRNA levels (r\u0026thinsp;=\u0026thinsp;0.6884, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (n\u0026thinsp;=\u0026thinsp;32)). Combined, these data indicate that prolonged hepatic ChREBP normalization in hepatic GSD Ia mice enhances YAP activity, which may be mediated by hepatocyte-autonomous effects, such as cellular glycogen accumulation, and/or by hepatic bile acid sensing.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eProlonged ChREBP knockdown in L-G6pc\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice induces hepatocyte DNA damage, cellular senescence, and hepatocyte dedifferentiation\u003c/h2\u003e \u003cp\u003eAs short-term ChREBP-normalized hepatic GSD Ia mice suggested induction of chromosomal instability, DNA damage, cGAS-STING, and cellular senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we also investigated these parameters upon prolonged ChREBP normalization. Prolonged ChREBP knockdown in L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice induced chromosomal instability (CIN) marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In parallel, histopathological analysis revealed an incidence of chromosome bridges, a hallmark of CIN [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], in these animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Hepatic ChREBP knockdown also tended to further increase nuclear ploidy in L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This was paralleled by a strong increase in γH2Ax positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Fig. S3A). PARP cleavage was not different between shChREBP- and shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Fig. S3B). shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed increased mRNA levels of \u003cem\u003eCgas\u003c/em\u003e as well as the cellular senescence marker genes \u003cem\u003ep16\u003c/em\u003e\u003csup\u003e\u003cem\u003eINK4a\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ep19\u003c/em\u003e\u003csup\u003e\u003cem\u003eARF\u003c/em\u003e\u003c/sup\u003e (both encoded from the \u003cem\u003eCdkn2a\u003c/em\u003e locus), and \u003cem\u003ep21\u003c/em\u003e (\u003cem\u003eCdkn1a\u003c/em\u003e), and a massive increase in hepatic p21 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Strikingly, hepatic ChREBP knockdown in L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice reduced long non-coding \u003cem\u003eHnf4aos\u003c/em\u003e and total \u003cem\u003eHnf4a\u003c/em\u003e mRNA and HNF4A peptide levels, while increasing \u003cem\u003eHnf4a-P2\u003c/em\u003e/\u003cem\u003eHnf4a-P1\u003c/em\u003e ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). As reduced \u003cem\u003eHnf4aos\u003c/em\u003e (HNF4A-AS1) and HNF4A expression are associated with hepatocyte dedifferentiation and advanced liver disease including liver cancer [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], these changes likely reflect hepatocellular dedifferentiation in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Reporter transcription factors analysis revealed that ChREBP- and HNF4A-targeted transcriptomes showed parallel responses to hepatic \u003cem\u003eG6pc\u003c/em\u003e deficiency and combined \u003cem\u003eG6pc\u003c/em\u003e deficiency/\u003cem\u003eChrebp\u003c/em\u003e normalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In parallel, hepatic \u003cem\u003eAlb\u003c/em\u003e and \u003cem\u003eHgfac\u003c/em\u003e mRNA levels were reduced and \u003cem\u003eAfp\u003c/em\u003e expression was induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), while \u003cem\u003eKrt19\u003c/em\u003e and \u003cem\u003eSox9\u003c/em\u003e remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Taken together, these data indicate that prolonged ChREBP normalization in GSD Ia hepatocytes aggravates CIN while inducing DNA damage, cGAS-STING pathway activation, cellular senescence, and hepatocellular dedifferentiation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study shows that normalization of hepatic ChREBP activity in GSD Ia liver induces progressive and extreme dysplastic liver growth, hepatocyte hypertrophy and -proliferation, YAP activation, cholestasis, CIN, DNA damage, cGAS-STING pathway activation, inflammation, cellular senescence, and hepatocellular dedifferentiation. Altogether, our data indicate that constitutive ChREBP activation in hepatic GSD Ia protects against advanced liver disease development, and disqualifies ChREBP as a therapeutic target for treatment of liver disease in GSD Ia.\u003c/p\u003e \u003cp\u003eA key finding in this study is that aggravation of hepatomegaly upon hepatic ChREBP knockdown in GSD Ia liver associates with enhanced nuclear levels and activity of YAP, a transcription factor that is critical for homeostatic control of liver size [\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It was previously shown that hepatic YAP cooperates with ChREBP to regulate glycolytic and lipogenic gene expression [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while our current work indicates that YAP is activated when ChREBP activity is reduced in hepatic GSD Ia. As we did not observe altered YAP activity upon hepatic ChREBP knockdown in wildtype mice, we propose that its activation is triggered by ChREBP-dependent physiological changes that occur within the context of hepatic GSD Ia. Among these, modulated bile acid metabolism was of primary interest to us, as we have previously implicated hepatic ChREBP in regulation of bile acid metabolism in GSD Ia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while hepatocyte YAP is activated upon high bile acid exposure [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In agreement with these studies, plasma bile acids levels were increased upon hepatic ChREBP knockdown in GSD Ia liver. Moreover, the massive hepatocyte hypertrophy observed in ChREBP-normalized GSD Ia mice severely perturbed the cellular architecture of the liver, thereby likely distorting the bile canalicular system and impairing hepatic bile acid secretion. This may in turn have caused intrahepatic accumulation of bile acids and consequent YAP activation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It was recently reported that accumulation of hepatic glycogen after 3 months of hepatocyte \u003cem\u003eG6pc\u003c/em\u003e deletion induces hepatocyte phase separation and formation of glycogen-Mst1/2 aggregates. As this aggregation relieves the inhibitory phosphorylation of hepatic YAP by Mst1/2 signalling, it contributes to hepatomegaly in progressed GSD Ia [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Previous work [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and our current study indicate that attenuation of hepatic ChREBP activity aggravates hepatic glycogen storage in hepatic GSD Ia, while in the current study we show that ChREBP silencing activates hepatocyte YAP. However, as shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice did not exhibit hepatic YAP activation, and ChREBP normalization did not decrease YAP phosphorylation, glycogen-dependent Mst1/2 sequestration most likely contributes to YAP activation during advanced hepatic GSD Ia. As we primarily aimed to evaluate the role of ChREBP in liver disease progression in GSD Ia, the mechanisms underlying the observed YAP activation were not addressed and warrant follow-up studies.\u003c/p\u003e \u003cp\u003eAn increased presence of chromosome bridges, induction of CIN marker genes, and enhanced DNA damage and hepatocyte death in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice indicate that ChREBP activation protects against chromosomal instability in hepatic GSD Ia. These changes likely reflect a high degree of hepatocellular stress and damage which may occur as a consequence of activated YAP [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. On the other hand, DNA damage may trigger hepatocyte renewal through liver regeneration and YAP activation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, the enrichment of CIN genes, enhanced PARP cleavage, and presence of chromosome bridges that occur in absence of YAP activation in shSCR/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice suggest that CIN/DNA damage occurs prior to YAP activation in early hepatic GSD Ia. Our data also indicate that ChREBP normalization in hepatic GSD Ia activates the cytosolic DNA-sensing cGAS-STING pathway [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Enhanced cGAS-STING signalling, in turn, likely contributes to the observed induction of cellular senescence [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] in shChREBP/L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Increased YAP activity, CIN, and aberrant cell division in ChREBP-normalized L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice associated with increased hepatocyte dedifferentiation and trends towards increases in hepatocyte ploidy, in agreement with previous studies in non-GSD Ia contexts [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Interestingly, the hepatic expression of \u003cem\u003eHnf4aos\u003c/em\u003e, a non-coding RNA which is associated with hepatocyte differentiation and, when decreased, has been linked to advanced liver disease in humans [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], was lower in these animals. Consistently, ChREBP normalization in L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice increased the ratio of the \u003cem\u003eHnf4α\u003c/em\u003e isoforms \u003cem\u003eHnf4αP2/P1\u003c/em\u003e, halved HNF4A protein expression levels, and suppressed HNF4α-regulated genes, which was consistently paralleled by induction of dedifferentiation- and proliferation-related gene expression [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur finding that ChREBP controls the degree of hepatomegaly and the progression to non-alcoholic steatohepatitis (NASH) is in line with previous studies [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Importantly, our current work indicates that ChREBP activation in hepatic GSD Ia protects against hepatocellular dedifferentiation, and suggests that it may decelerate tumorigenesis. This is the first study that attributes a potential protective role for ChREBP in liver tumour development, and our findings are in line with published work showing that YAP expression induces or associates with liver tumour formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], that reduced HNF4A expression is linked to liver tumour risk in mice and humans [\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and that YAP represses HNF4A target genes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The animal discomfort associated with extreme hepatomegaly that we observed upon attenuation of hepatic ChREBP activity in GSD Ia, however, prevented us from performing longer follow-up studies and thus assessment of liver tumour formation. Interestingly, although attenuation of ChREBP expression in GSD Ia mouse liver induced p53 activation and cell death, this was paralleled by increased proliferation, oncogenic YAP activation, and hepatocyte dedifferentiation. This is likely partly explained by the hepatic regenerative response induced \u003cem\u003ein vivo\u003c/em\u003e, in which the consequence of hepatocyte death is not limited to single cells but impacts on the liver as a whole. Moreover, when comparing our current findings on hepatic GSD Ia to published work on hepatic ChREBP in liver tumour development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], its role appears to be disease-specific. Altogether, these insights underline the importance of establishing the context-specific roles of ChREBP to define its therapeutic potential for prevention and/or treatment of liver disease and tumour development.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we show that ChREBP normalization in hepatic GSD Ia induces hepatocellular stress, chromosomal instability, DNA damage, and cGAS-STING pathway activation and provokes hepatocyte damage and inflammation, cellular senescence, and hepatocyte dedifferentiation. We hypothesize that hepatic YAP is induced to remove the damaged cells and to stimulate hepatocyte regeneration in order maintain liver function [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, persistent metabolic stress, chromosomal instability, and DNA damage induced upon long-term ChREBP suppression in hepatic GSD Ia result in constitutive YAP activation, hence likely predisposing to liver tumorigenesis. Altogether, we propose that by sensing and balancing intracellular glucose levels [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], hepatic ChREBP decelerates hepatomegaly induction, liver disease progression, and hepatocellular tumour formation in GSD Ia.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGSD Ia: Glycogen Storage Disease type 1a; G6PC/G6Pase-\u0026alpha;: glucose-6-phosphatase, catalytic subunit; G6P: glucose-6-phosphate; ChREBP\u0026alpha;/\u0026beta; (MLXIPL): Carbohydrate Response Element Binding Protein alpha/beta; L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice: hepatocyte-specific \u003cem\u003eG6pc\u0026nbsp;\u003c/em\u003eknockout mice; cGAS-STING: cyclic GMP-AMP synthase-stimulator of interferon genes; IEM: inborn error of metabolism; NAFLD: non-alcoholic fatty liver disease; HCC: hepatocellular carcinoma; dpt: days post treatment (= days post final tamoxifen injection); GSEA: gene set enrichment analysis; IQR: interquartile range; shRNA: small hairpin RNA; siRNA: small interfering RNA; YAP: Yes Associated Protein; CIN: chromosomal instability; HNF4A: Hepatocyte Nuclear Factor 4 Alpha; \u003cem\u003eHnf4aos\u003c/em\u003e/HNF4A-AS1: HNF4A, opposite strand / HNF4A antisense RNA 1; \u003cem\u003eAlb\u003c/em\u003e: albumin; \u003cem\u003eHgfac\u003c/em\u003e: hepatocyte growth factor activator; \u003cem\u003eAfp\u003c/em\u003e: alpha-fetoprotein; \u003cem\u003eKrt19\u003c/em\u003e: keratin 19; \u003cem\u003eSox9\u003c/em\u003e: SRY-box transcription factor 9; NASH: non-alcoholic steatohepatitis; H\u0026amp;E: Hematoxylin\u0026amp;Eosin; PRCF: percent relative cumulative frequency; IHC: immunohistochemistry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank N.L. Mulder, Y.T. van der Veen, R. Havinga, N.J. Kloosterhuis, K. Tholen, M. Koehorst, A.J.C. Tol, and A.H. Heida for excellent technical assistance and F. Kuipers, B.M. Bakker, and F. Foijer for scientific discussion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHuman participants: Not applicable.\u003c/p\u003e\n\u003cp\u003eAnimal studies: All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Groningen and are in line with the Guide for the Care and Use of Laboratory Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRNA-sequencing data has previously been submitted to GEO (Gene Expression Omnibus) under GSE143357, which is yet to be made publicly available.\u003c/p\u003e\n\u003cp\u003eData generated or analysed during this study are included in this published article and its supplementary information files. Additional raw datasets and/or data files used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a VIDI grant from the Dutch Scientific Organization, a grant from the Stichting Vrienden Beatrix Kinderziekenhuis (Foundation Friends Beatrix Children\u0026rsquo;s Hospital), and a grant from the De Cock-Hadders Foundation. In addition, this work is supported by European Union\u0026rsquo;s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement PoLiMeR, No 812616. M.H.O holds a Rosalind Franklin Fellowship from the University of Groningen. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDesigning research studies: M.G.S.R., Y.L, J.H.H., B.S., and M.H.O., conducting experiments: M.G.S.R., Y.L., J.H.H., T.B., K.A.K., A. Bl., M.H.K., J.C.W., and H.B., analysing data: M.G.S.R., Y.L., J.H.H., H.Y., V.W.B., T.B., K.A.K., A.Bl., R.E.T., J.C.W., H.B., D.C.J.S., A.Br., B.S., and M.H.O., writing the first draft of the manuscript: M.G.S.R., B.S., and M.H.O., critical revisions of the manuscript: Y.L., J.H.H., H.Y., V.W.B., K.A.K., R.E.T., G.M., F.R., A.M., D.C.J.S., A.Br., B.S., and M.H.O. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026rsquo; information\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChou JY, Jun HS, Mansfield BC. Type I glycogen storage diseases: disorders of the glucose-6-phosphatase/glucose-6-phosphate transporter complexes. J Inherit Metab Dis 2015;38:511\u0026ndash;9. https://doi.org/10.1007/s10545-014-9772-x.\u003c/li\u003e\n\u003cli\u003eRake J, Visser G, Labrune P, Leonard J, Ullrich K, Smit P. Glycogen storage disease type I: diagnosis, management, clinical course and outcome. Results of the European Study on Glycogen Storage Disease Type I (ESGSD I). Eur J Pediatr 2002;161 Suppl:S20\u0026ndash;34. https://doi.org/10.1007/S00431-002-0999-4.\u003c/li\u003e\n\u003cli\u003eAbdul-Wahed A, Guilmeau S, Postic C. Sweet Sixteenth for ChREBP: Established Roles and Future Goals. Cell Metab 2017;26:324\u0026ndash;41. https://doi.org/10.1016/j.cmet.2017.07.004.\u003c/li\u003e\n\u003cli\u003eCalderaro J, Labrune P, Morcrette G, Rebouissou S, Franco D, Pr\u0026eacute;vot S, et al. Molecular characterization of hepatocellular adenomas developed in patients with glycogen storage disease type i. J Hepatol 2013;58:350\u0026ndash;7. https://doi.org/10.1016/j.jhep.2012.09.030.\u003c/li\u003e\n\u003cli\u003eKim GY, Lee YM, Cho JH, Pan CJ, Jun HS, Springer DA, et al. Mice expressing reduced levels of hepatic glucose-6-phosphatase-a activity do not develop age-related insulin resistance or obesity. Hum Mol Genet 2015;24:5115\u0026ndash;25. https://doi.org/10.1093/hmg/ddv230.\u003c/li\u003e\n\u003cli\u003eIizuka K. The transcription factor carbohydrate-response element-binding protein (ChREBP): A possible link between metabolic disease and cancer. Biochim Biophys Acta - Mol Basis Dis 2017;1863:474\u0026ndash;85. https://doi.org/10.1016/j.bbadis.2016.11.029.\u003c/li\u003e\n\u003cli\u003eAbdul-Wahed A, Gautier-Stein A, Casteras S, Soty M, Roussel D, Romestaing C, et al. A link between hepatic glucose production and peripheral energy metabolism via hepatokines. Mol Metab 2014;3:531\u0026ndash;43. https://doi.org/10.1016/j.molmet.2014.05.005.\u003c/li\u003e\n\u003cli\u003eLei Y, Hoogerland JA, Bloks VW, Bos T, Bleeker A, Wolters H, et al. Hepatic ChREBP activation limits NAFLD development in a mouse model for Glycogen Storage Disease type Ia. Hepatology 2020. https://doi.org/10.1002/hep.31198.\u003c/li\u003e\n\u003cli\u003eHanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell 2011;144:646\u0026ndash;74. https://doi.org/10.1016/j.cell.2011.02.013.\u003c/li\u003e\n\u003cli\u003eCalvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, et al. Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma. Gastroenterology 2011;140:1071-1083.e5. https://doi.org/10.1053/j.gastro.2010.12.006.\u003c/li\u003e\n\u003cli\u003eRibback S, Che L, Pilo MG, Cigliano A, Latte G, Pes GM, et al. Oncogene-dependent addiction to carbohydrate-responsive element binding protein in hepatocellular carcinoma. Cell Cycle 2018;17:1496\u0026ndash;512. https://doi.org/10.1080/15384101.2018.1489182.\u003c/li\u003e\n\u003cli\u003eEvert M, Calvisi DF, Evert K, De Murtas V, Gasparetti G, Mattu S, et al. V-AKT murine thymoma viral oncogene homolog/mammalian target of rapamycin activation induces a module of metabolic changes contributing to growth in insulin-induced hepatocarcinogenesis. Hepatology 2012;55:1473\u0026ndash;84. https://doi.org/10.1002/hep.25600.\u003c/li\u003e\n\u003cli\u003eLei Y, Hu Q, Gu J. Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and Clinical Significance. Pathol Oncol Res 2020;26:1331\u0026ndash;40. https://doi.org/10.1007/s12253-019-00708-y.\u003c/li\u003e\n\u003cli\u003eDong X, Wang F, Liu C, Ling J, Jia X, Shen F, et al. Single-cell analysis reveals the intra-tumor heterogeneity and identifies MLXIPL as a biomarker in the cellular trajectory of hepatocellular carcinoma. Cell Death Discov 2021;7. https://doi.org/10.1038/s41420-021-00403-5.\u003c/li\u003e\n\u003cli\u003eWang H, Dolezal JM, Kulkarni S, Lu J, Mandel J, Jackson LE, et al. Myc and ChREBP transcription factors cooperatively regulate normal and neoplastic hepatocyte proliferation in mice. J Biol Chem 2018;293:14740\u0026ndash;57. https://doi.org/10.1074/jbc.RA118.004099.\u003c/li\u003e\n\u003cli\u003eTong X, Zhao F, Mancuso A, Gruber JJ, Thompson CB. The glucose-responsive transcription factor ChREBP contributes to glucose-dependent anabolic synthesis and cell proliferation. Proc Natl Acad Sci U S A 2009;106:21660\u0026ndash;5. https://doi.org/10.1073/pnas.0911316106.\u003c/li\u003e\n\u003cli\u003evan den Bos H, Bakker B, Taudt A, Guryev V, Colom\u0026eacute;-Tatch\u0026eacute; M, Lansdorp PM, et al. Quantification of aneuploidy in mammalian systems. Methods Mol. Biol., vol. 1896, Methods Mol Biol; 2019, p. 159\u0026ndash;90. https://doi.org/10.1007/978-1-4939-8931-7_15.\u003c/li\u003e\n\u003cli\u003eHoogerland JA, Lei Y, Wolters JC, de Boer JF, Bos T, Bleeker A, et al. Glucose‐6‐phosphate regulates hepatic bile acid synthesis in mice. Hepatology 2019;0:1\u0026ndash;14. https://doi.org/10.1002/hep.30778.\u003c/li\u003e\n\u003cli\u003eGisselsson D. Chromosome instability in cancer: How, when, and why? Adv Cancer Res 2003;87:1\u0026ndash;29. https://doi.org/10.1016/S0065-230X(03)87164-6.\u003c/li\u003e\n\u003cli\u003eBonzo JA, Ferry CH, Matsubara T, Kim JH, Gonzalez FJ. Suppression of hepatocyte proliferation by hepatocyte nuclear factor 4\u0026alpha; in adult mice. J Biol Chem 2012;287:7345\u0026ndash;56. https://doi.org/10.1074/jbc.M111.334599.\u003c/li\u003e\n\u003cli\u003eDubois V, Staels B, Lefebvre P, Verzi MP, Eeckhoute J. Control of Cell Identity by the Nuclear Receptor HNF4 in Organ Pathophysiology. Cells 2020;9. https://doi.org/10.3390/CELLS9102185.\u003c/li\u003e\n\u003cli\u003eKalkuhl A, Kaestner K, Buchmann A, Schwarz M. Expression of hepatocyte-enriched nuclear transcription factors in mouse liver tumours. Carcinogenesis 1996;17:609\u0026ndash;12. https://doi.org/10.1093/carcin/17.3.609.\u003c/li\u003e\n\u003cli\u003eLazarevich NL, Cheremnova OA, Varga E V., Ovchinnikov DA, Kudrjavtseva EI, Morozova O V., et al. Progression of HCC in Mice Is Associated with a Downregulation in the Expression of Hepatocyte Nuclear Factors. Hepatology 2004;39:1038\u0026ndash;47. https://doi.org/10.1002/hep.20155.\u003c/li\u003e\n\u003cli\u003eNing BF, Ding J, Yin C, Zhong W, Wu K, Zeng X, et al. Hepatocyte nuclear factor 4\u0026alpha; suppresses the development of hepatocellular carcinoma. Cancer Res 2010;70:7640\u0026ndash;51. https://doi.org/10.1158/0008-5472.CAN-10-0824.\u003c/li\u003e\n\u003cli\u003eEsposti DD, Hernandez-Vargas H, Voegele C, Fernandez-Jimenez N, Forey N, Bancel B, et al. Identification of novel long non-coding RNAs deregulated in hepatocellular carcinoma using RNA-sequencing. Oncotarget 2016;7:31862\u0026ndash;77. https://doi.org/10.18632/oncotarget.7364.\u003c/li\u003e\n\u003cli\u003eAtanasovska B, Rensen SS, Marsman G, Shiri-Sverdlov R, Withoff S, Kuipers F, et al. Long non-coding rnas involved in progression of non-alcoholic fatty liver disease to steatohepatitis. Cells 2021;10. https://doi.org/10.3390/cells10081883.\u003c/li\u003e\n\u003cli\u003eGuo S, Lu H. Novel mechanisms of regulation of the expression and transcriptional activity of hepatocyte nuclear factor 4\u0026alpha;. J Cell Biochem 2019;120:519\u0026ndash;32. https://doi.org/10.1002/jcb.27407.\u003c/li\u003e\n\u003cli\u003eCai J, Zhang N, Zheng Y, De Wilde RF, Maitra A, Pan D. The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev 2010;24:2383\u0026ndash;8. https://doi.org/10.1101/gad.1978810.\u003c/li\u003e\n\u003cli\u003eCamargo FD, Gokhale S, Johnnidis JB, Fu D, Bell GW, Jaenisch R, et al. YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells. Curr Biol 2007;17:2054\u0026ndash;60. https://doi.org/10.1016/j.cub.2007.10.039.\u003c/li\u003e\n\u003cli\u003eDong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA, et al. Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals. Cell 2007;130:1120\u0026ndash;33. https://doi.org/10.1016/j.cell.2007.07.019.\u003c/li\u003e\n\u003cli\u003eLee KP, Lee JH, Kim TS, Kim TH, Park HD, Byun JS, et al. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc Natl Acad Sci U S A 2010;107:8248\u0026ndash;53. https://doi.org/10.1073/pnas.0912203107.\u003c/li\u003e\n\u003cli\u003eSong H, Mak KK, Topol L, Yun K, Hu J, Garrett L, et al. Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression. Proc Natl Acad Sci U S A 2010;107:1431\u0026ndash;6. https://doi.org/10.1073/pnas.0911409107.\u003c/li\u003e\n\u003cli\u003eShu Z, Yi G, Deng S, Huang K, Wang Y. Hippo pathway cooperates with ChREBP to regulate hepatic glucose utilization. Biochem Biophys Res Commun 2020;530:115\u0026ndash;21. https://doi.org/10.1016/j.bbrc.2020.06.105.\u003c/li\u003e\n\u003cli\u003eAnakk S, Bhosale M, Schmidt VA, Johnson RL, Finegold MJ, Moore DD. Bile Acids Activate YAP to Promote Liver Carcinogenesis. Cell Rep 2013;5:1060\u0026ndash;9. https://doi.org/10.1016/j.celrep.2013.10.030.\u003c/li\u003e\n\u003cli\u003ePepe-Mooney BJ, Dill MT, Alemany A, Ordovas-Montanes J, Matsushita Y, Rao A, et al. Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration. Cell Stem Cell 2019;25:23-38.e8. https://doi.org/10.1016/j.stem.2019.04.004.\u003c/li\u003e\n\u003cli\u003eLiu Q, Li J, Zhang W, Xiao C, Zhang S, Nian C, et al. Glycogen accumulation and phase separation drives liver tumor initiation. Cell 2021. https://doi.org/10.1016/j.cell.2021.10.001.\u003c/li\u003e\n\u003cli\u003eRajas F, Dentin R, Cannella Miliano A, Silva M, Raffin M, Levavasseur F, et al. The absence of hepatic glucose-6 phosphatase/ChREBP couple is incompatible with survival in mice. Mol Metab 2021;43. https://doi.org/10.1016/j.molmet.2020.101108.\u003c/li\u003e\n\u003cli\u003eWeiler SME, Pinna F, Wolf T, Lutz T, Geldiyev A, Sticht C, et al. Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer. Gastroenterology 2017;152:2037-2051.e22. https://doi.org/10.1053/j.gastro.2017.02.018.\u003c/li\u003e\n\u003cli\u003ePefani DE, O\u0026rsquo;Neill E. Hippo pathway and protection of genome stability in response to DNA damage. FEBS J 2016;283:1392\u0026ndash;403. https://doi.org/10.1111/febs.13604.\u003c/li\u003e\n\u003cli\u003eMichalopoulos GK. Hepatostat: Liver regeneration and normal liver tissue maintenance. Hepatology 2017;65:1384\u0026ndash;92. https://doi.org/10.1002/hep.28988.\u003c/li\u003e\n\u003cli\u003eLoo TM, Miyata K, Tanaka Y, Takahashi A. Cellular senescence and senescence-associated secretory phenotype via the cGAS-STING signaling pathway in cancer. Cancer Sci 2020;111:304\u0026ndash;11. https://doi.org/10.1111/cas.14266.\u003c/li\u003e\n\u003cli\u003eLi T, Chen ZJ. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer. J Exp Med 2018;215:1287\u0026ndash;99. https://doi.org/10.1084/jem.20180139.\u003c/li\u003e\n\u003cli\u003eZhang S, Chen Q, Liu Q, Li Y, Sun X, Hong L, et al. Hippo Signaling Suppresses Cell Ploidy and Tumorigenesis through Skp2. Cancer Cell 2017;31:669-684.e7. https://doi.org/10.1016/j.ccell.2017.04.004.\u003c/li\u003e\n\u003cli\u003ePandit SK, Westendorp B, De Bruin A. Physiological significance of polyploidization in mammalian cells. Trends Cell Biol 2013;23:556\u0026ndash;66. https://doi.org/10.1016/j.tcb.2013.06.002.\u003c/li\u003e\n\u003cli\u003eSargsyan A, Doridot L, Hannou SA, Tong W, Srinivasan H, Ivison R, et al. HGFAC is a ChREBP regulated hepatokine that enhances glucose and lipid homeostasis. JCI Insight 2022. https://doi.org/10.1172/jci.insight.153740.\u003c/li\u003e\n\u003cli\u003eBiagioni F, Croci O, Sberna S, Donato E, Sab\u0026ograve; A, Bisso A, et al. Decoding YAP dependent transcription in the liver. Nucleic Acids Res 2022;50:7959\u0026ndash;71. https://doi.org/10.1093/nar/gkac624.\u003c/li\u003e\n\u003cli\u003eTanaka T, Jiang S, Hotta H, Takano K, Iwanari H, Sumi K, et al. Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4\u0026alpha; in the pathogenesis of human cancer. J Pathol 2006;208:662\u0026ndash;72. https://doi.org/10.1002/path.1928.\u003c/li\u003e\n\u003cli\u003eBricambert J, Alves-Guerra MC, Esteves P, Prip-Buus C, Bertrand-Michel J, Guillou H, et al. The histone demethylase Phf2 acts as a molecular checkpoint to prevent NAFLD progression during obesity. Nat Commun 2018;9. https://doi.org/10.1038/s41467-018-04361-y.\u003c/li\u003e\n\u003cli\u003eShi JH, Lu JY, Chen HY, Wei CC, Xu X, Li H, et al. Liver ChREBP protects against fructose-induced glycogenic hepatotoxicity by regulating L-type pyruvate kinase. Diabetes 2020;69:591\u0026ndash;602. https://doi.org/10.2337/db19-0388.\u003c/li\u003e\n\u003cli\u003eAgius L, Chachra SS, Ford BE. The Protective Role of the Carbohydrate Response Element Binding Protein in the Liver: The Metabolite Perspective. Front Endocrinol (Lausanne) 2020;11. https://doi.org/10.3389/fendo.2020.594041.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-and-metabolism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmet","sideBox":"Learn more about [Cancer \u0026 Metabolism](http://cancerandmetabolism.biomedcentral.com/)","snPcode":"40170","submissionUrl":"https://submission.nature.com/new-submission/40170/3","title":"Cancer \u0026 Metabolism","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Glycogen Storage Disease type 1a, Carbohydrate Response Element Binding Protein, hepatomegaly, Yes Associated Protein, cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING)","lastPublishedDoi":"10.21203/rs.3.rs-2514060/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2514060/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBackground\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGlycogen 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethods\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHepatocyte-specific \u003cem\u003eG6pc\u003c/em\u003e knockout (L-\u003cem\u003eG6pc\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e) mice were treated with AAV-shChREBP to normalize hepatic ChREBP activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHepatic ChREBP normalization in GSD Ia mice induced dysplastic liver growth, massively increased hepatocyte size, and was associated with increased hepatic inflammation. \u0026nbsp;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e","manuscriptTitle":"Normalization of hepatic ChREBP activity does not protect against liver disease progression in a mouse model for Glycogen Storage Disease type Ia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-01 16:29:31","doi":"10.21203/rs.3.rs-2514060/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2023-03-21T04:31:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-02-16T18:19:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94ce23aa-e4a2-43cd-9a6b-0714715c2226","date":"2023-02-06T20:49:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-02-06T00:36:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-01-30T12:17:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-01-30T12:17:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer \u0026 Metabolism","date":"2023-01-25T13:29:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-and-metabolism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmet","sideBox":"Learn more about [Cancer \u0026 Metabolism](http://cancerandmetabolism.biomedcentral.com/)","snPcode":"40170","submissionUrl":"https://submission.nature.com/new-submission/40170/3","title":"Cancer \u0026 Metabolism","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4f082c7b-8655-4a39-9695-2a1ff925fbe1","owner":[],"postedDate":"February 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:37:52+00:00","versionOfRecord":{"articleIdentity":"rs-2514060","link":"https://doi.org/10.1186/s40170-023-00305-3","journal":{"identity":"cancer-and-metabolism","isVorOnly":false,"title":"Cancer \u0026 Metabolism"},"publishedOn":"2023-04-21 20:33:09","publishedOnDateReadable":"April 21st, 2023"},"versionCreatedAt":"2023-02-01 16:29:31","video":"","vorDoi":"10.1186/s40170-023-00305-3","vorDoiUrl":"https://doi.org/10.1186/s40170-023-00305-3","workflowStages":[]},"version":"v1","identity":"rs-2514060","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2514060","identity":"rs-2514060","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}