# Enhanced Lysosomal Glycogen Breakdown is associated with Liver Tumorigenesis in Glycogen Storage Disease Type III

Valle Montalvo-Romeral, Louisa Jauze, Gwendoline Perrot, Mouna

Amaouche, Antoine Gardin, Araceli Aguilar González, Alicia Leblond, Carine

Zitoun-Ardon, Félicie Evrard, Jérémie Cosette, et al.

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Valle Montalvo-Romeral, Louisa Jauze, Gwendoline Perrot, Mouna Amaouche, Antoine Gardin, et al. Enhanced Lysosomal Glycogen Breakdown is associated with Liver Tumorigenesis in Glycogen Storage Disease Type III. JHEP Reports Innovation in Hepatology, In press, pp.101702. 10.1016/j.jhepr.2025.101702. hal-05428665

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Enhanced Lysosomal Glycogen Breakdown is associated with Liver Tumorigenesis in Glycogen Storage Disease Type III

Valle Montalvo-Romeral, Louisa Jauze, Gwendoline Perrot, Mouna Amaouche, Antoine Gardin, Araceli Aguilar González, Alicia Leblond, Carine Zitoun-Ardon, Félicie Evrard, Jérémie Cosette, Christophe Tatout, Fanny Bordier, Emilie Bertil-Froidevaux, Christophe Georger, Laetitia van Wittenberghe, Valérie Paradis, Simon Gay, Fanny Dujardin, François Maillot, Amandine Gautier-Stein, Gilles Mithieux, Edoardo Malfatti, Charlotte Mussini, Philippe Labrune, Alicia Mayeuf-Louchart, Giuseppe Ronzitti, Fabienne Rajas

PII: S2589-5559(25)00385-4

DOI: https://doi.org/10.1016/j.jhepr.2025.101702

Reference: JHEPR 101702

To appear in: *JHEP Reports*

Received Date: 26 February 2025

Revised Date: 19 November 2025

Accepted Date: 24 November 2025

Please cite this article as: Montalvo-Romeral V, Jauze L, Perrot G, Amaouche M, Gardin A, Aguilar González A, Leblond A, Zitoun-Ardon C, Evrard F, Cosette J, Tatout C, Bordier F, Bertil-Froidevaux E, Georger C, van Wittenberghe L, Paradis V, Gay S, Dujardin F, Maillot F, Gautier-Stein A, Mithieux G, Malfatti E, Mussini C, Labrune P, Mayeuf-Louchart A, Ronzitti G, Rajas F, Enhanced Lysosomal Glycogen Breakdown is associated with Liver Tumorigenesis in Glycogen Storage Disease Type III, *JHEP Reports*, https://doi.org/10.1016/j.jhepr.2025.101702.

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Enhanced Lysosomal Glycogen Breakdown is associated with Liver
Tumorigenesis in Glycogen Storage Disease Type III

Valle Montalvo-Romeral¹⁻²*, Louisa Jauze¹⁻²-³*, Gwendoline Perrot³*, Mouna Amaouche⁴⁻⁵, Antoine Gardin¹⁻², Araceli Aguilar González¹, Alicia Leblond³, Carine Zitoun-Ardon³, Félicie Evrard³, Jérémie Cosette¹, Christophe Tatout⁶,⁷, Fanny Bordier¹, Emilie Bertil-Froidevaux¹, Christophe Georger¹, Laetitia van Wittenberghe¹, Valérie Paradis⁸⁻⁹, Simon Gay¹⁰, Fanny Dujardin¹¹, François Maillot¹⁰,¹², Amandine Gautier-Stein³, Gilles Mithieux³, Edoardo Malfatti¹³⁻¹⁴, Charlotte Mussini¹⁵, Philippe Labrune¹⁶, Alicia Mayeuf-Louchart⁴⁻⁵, Giuseppe Ronzitti¹⁻²*† and Fabienne Rajas³*†

1- Généthon, 91000 Evry, France

2- Université Paris-Saclay, Université Evry, INSERM, Integrate Research Unit
UMR_S951, 91000 Evry, France

3- Université Claude Bernard Lyon 1, INSERM, UMR_S1213, Lyon Hepatology
Institute-IHU EVEREST, Villeurbanne, 69100, France

4- Université de Lille, CHU Lille, Institut Pasteur de Lille, INSERM, UMR-1011 - EGID,
59000, Lille, France

5- Université de Lille, CHU Lille, INSERM UMR-S1172, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, 59000, Lille, France.

6- Université Clermont Auvergne, CNRS, INSERM, iGReD Clermont-Ferrand, France

7- Université Claude Bernard Lyon 1, SFR Santé Lyon Est, CNRS UAR3453,
INSERM US7, Villeurbanne, 69100, France
8- INSERM UMR1149, Université Paris Cité, Paris, France

9- Service d'Anatomie et de Cytologie Pathologiques Hôpital Beaujon, AP-HP 92110, Clichy, France

10-Université de Tours, 37000, Tours France; Internal Medicine Department Tours Hospital, 37000 Tours, France

11-Pathological Department Tours Hospital, 37000 Tours, France.

12-UMR INSERM 1253, «iBraiN», 37000 Tours, France

13-Reference Center for Neuromuscular Disorders, AP-HP, Henri Mondor University Hospital, 94010, Créteil, France

14-Université Paris-Est Créteil, INSERM, UMR-955, IMRB, 94010, Créteil, France

15-Université Paris-Saclay, AP-HP, Hôpital Bicêtre, Département de Pathologie, 94270, Le Kremlin Bicêtre, France

16-Université Paris-Saclay, AP-HP, Hôpital Antoine-Béclère, Centre de référence des maladies héréditaires du métabolisme hépatique, Filière G2M, 92140, France

*Contributed equally

†Corresponding authors:

Dr. Fabienne Rajas

Inserm UMR_S1213-Université Claude Bernard Lyon 1 Laennec

7 rue Guillaume Paradin, 69372 Lyon cedex 08, France

Tel : +33 478 77 10 28

Email : fabienne.rajas@univ-lyon1.fr
Dr. Giuseppe Ronzitti

Généthon

1 rue de l'Internationale, 91000 Evry, France

Tel : +33 169 47 29 90

Email : gronzitti@genethon.fr

**Data availability statement:** all data presented in the manuscript are presented in histograms as individual points. There are no large data files to be shared via online resources.
**Short title: Liver tumorigenesis in GSDIII**

**Key words:** hepatocellular carcinoma, fibrosis, inflammation, glucose metabolism, glycophagy, Hippo/YAP pathway, rare disease

**Electronic word count:** 6633 words

**Number of figures and tables:** 5 Figures, 1 Table

**Competing interest statement:** The authors who have taken part in this study declare that they do not have anything to disclose regarding funding or there is no conflict of interest with respect to this manuscript.

**Financial support statement:** This work was supported by Genethon, the "Association Française contre la Myopathie", the "Association Francophone des Glycogénoses", the "Fondation pour la Recherche Médicale" (Equipe FRM EQU202103012572), and the National Research Agency (ANR-17-CE18-0014 ; ANR-22-PEBI-0012). The salary of AG was supported by public funding from the French National Institute of Health and Medical Research ("poste d'accueil", INSERM). The salaries of GP and AL were supported by the Gen&Zic association. The salaries of GR, AML & CZA, and FR & GM were supported by INSERM and CNRS, respectively. The salary of AGS was supported by INRAE. The salary of MA was supported by PreciDIAB (ANR-18-IBHU-0001) and Hauts-de-France Regional Council (#22005973).
**Author Contribution.** VMR, LJ, GP, FR performed or directed experimental activities, contributed significantly to experimental design and data analysis. VMR, LJ, GR and FR wrote the manuscript. AML, AGS, GM and EM provided insights into the liver metabolism, glycophagy, and TEM technology and analysis. Histological analyses were performed by VP and CM. JC and CT developed the pipeline for quantification of immunostainings. LJ, AG, MA, AAG, AL, CZA, FE, FB, EBF, CG, and LVW contributed to experimental activities. CM, PL, AG, SG, FD, and FM provided the patients' biological data and the histological images of the human biopsies. LJ, AGS, GM, GR, and FR critically edited the manuscript. All authors approved the final manuscript.

**Highlights**

* Lysosomal degradation of glycogen was activated in liver tumors of GSDIII mice.

* Glycogen may serve as fuel to promote tumor growth in GSDIII mice and patients.

* Loss of glycogen debranching enzyme causes a severe energy deficit in the liver.

* Extrahepatic gluconeogenesis sustains blood glucose levels in GSDIII mice during fasting.
## Impact and Implications

This study provides novel insights into the metabolic dysregulations driving liver tumorigenesis in Glycogen Storage Disease type III (GSDIII). Our findings reveal a potential link between abnormal glycogen accumulation and liver cancer, with the pivotal role of lysosomal glycogen degradation in supporting tumor growth. These results are particularly important for researchers and clinicians working on metabolic liver diseases, as they suggest potential glycogen-targeting therapeutic strategies for GSDIII and other related liver disorders. Practically, they could guide future interventions aimed at modulating glycogen metabolism, offering new treatment avenues for GSDIII patients at risk of hepatocellular carcinoma, while contributing to the broader understanding of metabolic dysregulation in cancer biology.
**Abstract**

**Background & Aims:** Glycogen storage disease type III (GSDIII) is a rare metabolic disorder caused by mutations in the glycogen debranching enzyme (AGL), leading to hepatic glycogen accumulation, fibrosis and increased hepatocellular carcinoma (HCC) risk. This study investigates the metabolic mechanisms driving liver tumorigenesis in an *Agl*⁻/⁻ model of GSDIII.

**Methods:** Liver and tumor samples from 14-month-old *Agl*⁻/⁻ and *Agl*⁺/⁺ mice, and liver biopsies from GSDIII patients (n=4), were analyzed using histological, biochemical and molecular approaches.

**Results:** *Agl*⁻/⁻ mice recapitulated key features of GSDIII, including a 3.5-fold hepatic glycogen overload (p<0.001), and chronic liver disease. More than 30% of the animals developed liver tumors, associated to a 2.5-fold increase in α-fetoprotein levels (p<0.005). Despite marked reductions in glucose (7.5-fold, p<0.0001), glucose-6 phosphate (266-fold, p<0.0001), lactate (8-fold, p<0.005), cholesterol (1.9-fold, p<0.001) and triglyceride levels (6.2-fold, p<0.001) in the liver, glycaemia was maintained at around 87.0±9.6 mg/dl after 6h of fasting, through activated extrahepatic, but not hepatic, gluconeogenesis. Intriguingly, most tumors exhibited lower glycogen content than surrounding tissue (3.3-fold decrease, p<0.0001), associated with increased lysosomal α-acid glucosidase activity (19.5±5.5 in tumor vs 9.9±2.0 mmol/h/mg in *Agl*⁻/⁻ liver; p<0.0005) and the presence of glycophagosomes. PAS-negative staining in GSDIII patient HCCs supported these observations. Although YAP nuclear staining varied between tumors, overall increased YAP nuclear localization and CTGF expression suggest that Hippo/YAP pathway inhibition could contribute to tumorigenesis in GSDIII hepatocytes.
**Conclusions:** In GSDIII, liver metabolism is characterized by the accumulation of structurally abnormal glycogen and a significant reduction of key energy substrates. In this metabolic context, enhanced lysosomal glycogen degradation may support tumor growth, highlighting a mechanistic link between glycogen metabolism and the development of liver cancer.
Introduction

As the central regulator of metabolism, the liver plays a crucial role in maintaining
whole-body metabolism. One of its main functions is to maintain blood glucose levels
between 0.8-1.1 g/L. This allows humans and animals to endure extended periods without
food¹, ². Glucose is indeed the main energy source for all living cells, supplying carbon
skeletons for nucleotide, protein, and lipid synthesis required for cell growth and
proliferation.

After a meal, the excess of glucose is stored as glycogen, mainly in the liver and skeletal muscles, through a process called glycogenesis. Glycogen, a highly branched polymer of glucose, constitutes a major form of energy storage. During the post-absorptive state, the breakdown of hepatic glycogen (i.e. glycogenolysis) represents the fastest process to restore blood glucose levels. Glycogen can be degraded by two different pathways. The first occurs in the cytoplasm and involves two reactions catalyzed by the glycogen phosphorylase (PYG) and the glycogen debranching enzyme (GDEamylo-alpha-1,6-glucosidase enzyme, encoded by AGL). The second pathway recycles ~10% of glycogen and is dependent on autophagy. It occurs in lysosomes and involves acid α-glucosidase (encoded by GAA)³, ⁴. In hepatocytes, glucose-6-phosphate (G6P), produced from cytosolic glycogen degradation, is hydrolyzed by glucose-6-phosphatase (G6Pase) to release glucose into bloodstream. When glycogen stores become depleted, glucose is synthetized from non-carbohydrate substrates via gluconeogenesis in the liver, kidney and intestine¹.

The loss of function of enzymes involved in glycogen metabolism leads to abnormal
glycogen accumulation observed in inherited disorders known as glycogen storage
diseases (GSDs). Recent studies have highlighted the importance of glycogen
metabolism in supplying energy and in maintaining cellular homeostasis⁵. Large amounts of glycogen have been found in various cancer cell lines, particularly those undergoing neoplastic transformation⁶. Glycogen supports cell survival under hypoxic conditions⁷⁻⁹ and has recently been identified as an oncogenic metabolite that can drive cell transformation and tumor initiation¹⁰, ¹¹. However, the specific role of glycogen in driving tumor progression and growth is still unreported.

Glycogen storage disease type III (GSDIII, OMIM 232400) is an autosomal recessive disorder caused by mutations in the *AGL* gene¹². Impaired glycogen breakdown leads to the accumulation of abnormally structured, soluble glycogen, called limit dextrin, within cells. In cardiac and skeletal muscles, glycogen accumulation results in cardiomyopathy and progressive muscle weakness with age. In the liver, this leads to hepatomegaly and hypoglycemia starting in early childhood, alongside elevated plasma transaminase levels reflecting early liver damage. Limit dextrin accumulation is associated with the development of hepatic fibrosis in childhood, which may progress to severe liver complications in adulthood, including cirrhosis, hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC). In some cases, a liver transplant may be necessary¹³⁻¹⁷. Due to the typical glycogen accumulation in GSDIII, hepatocytes from affected individuals represent a relevant model to investigate glycogen's contribution to tumorigenesis. In this study, we aimed to investigate the long-term progression of liver disease in GSDIII, focusing specifically on the relationship between hepatic metabolism and tumor development. Acknowledging the high energy demands of tumor growth and proliferation, we specifically examined the metabolic substrates, particularly glycogen, that could be utilized by GSDIII hepatocytes to sustain their proliferation and drive tumorigenesis. To this end, we used a GSDIII mouse model (Agl⁻/⁻ mice) that recapitulates the key
pathological features observed in patients, i.e. glycogen accumulation, hypoglycemia, hepatomegaly, tumor development, and muscle weakness¹⁸. Selected findings were further validated using liver biopsies from GSDIII patients who developed HCC.

## Materials and methods

### In vivo studies

**Animal model.** Agl knockout mice (Agl/-) were generated by replacing exons 6–10 of the Agl gene with a neomycin cassette, as described by Vidal et al.¹⁸, within the IMPC framework. Mice were bred on a mixed BALB/c-C57Bl/6J background and housed in groups of 4–6 males or females at 21-23°C, with a 12/12h light/dark cycle, and free access to chow diet (A04-Safe) and water, at CERFE (Généthon, Evry) or ALECS (University of Lyon 1). Blood was collected in 3.8% citrate or EDTA.

Glutamine and pyruvate tolerance tests were performed after 6h (post-absorptive) or 12h of fasting (glycogen-depleted), respectively. Mice received intraperitoneal injections of L-glutamine (1.5g/kg) or pyruvate (2g/kg), and blood glucose was monitored for 2h.

At 14 months, mice were euthanized (fed or 6h-fasted) by cervical dislocation. Livers were freeze-clamped in liquid nitrogen and stored at -80°C or fixed for histology.

**Study approval.** All procedures complied with the European directive 2010/63/EU and were approved by local ethics committees and the French Ministry of Research (Apafis: #23334, #37405 and #44716).

### Patients
Four French GSDIII patients were retrospectively reviewed after liver transplantation or partial hepatectomy. Histological analyses were performed on tumors and peritumor livers. Informed consent was obtained in accordance with French legislation.

## Histological analyses

**Staining.** Fresh liver was fixed in 10% formalin for 48h, paraffin-embedded and sectioned (4µm). Sections were stained with HPS, PAS, Sirius red, or Oil-Red O (ORO). Immunohistochemical analyses were performed, after heat-mediated antigen retrieval (TintoRetriever Pressure Cooker, Diagnostics), peroxidases inhibition (3% H₂O₂, 5 min), blocking, and overnight antibody incubation at 4°C (CTAT Table). Detection was performed HRP-conjugated anti-rabbit IgG and DAB chromogen, followed by hematoxylin counterstaining. Quantification methods are described in **Supplemental Data**.

**Transmission electron microscopy.** Liver samples (5 mice/group) were fixed in 2.5% glutaraldehyde, postfixed in 2% osmium tetroxide, dehydrated, and embedded in an epoxy resin (EMBed-812, Electron Microscopy Sciences). Ultrathin sections were stained with uranyl acetate and lead citrate, observed at 80 kV (CM120, Philips Electronics) and photographed (Morada, Olympus). Ten fields per sample were analyzed and the number of autophagosomes was averaged per group.

## Biochemical parameters

**Blood parameters.** Plasma Alpha-fetoprotein (AFP) levels were determined by ELISA (R&D Systems). Aspartate aminotransferase (AST)/alanine aminotransferase (ALT) activities were determined by colorimetric assays (Abcam). Blood glucose was measured with an Accu-Check Go glucometer (Roche Diagnostic).
**Liver metabolites.** Glucose, G6P and glycogen were measured as previously described from a fresh liver piece homogenized in 6% perchloric acid (FastPrep, MP Biomedicals)¹⁸,¹⁹. Lactate levels were measured in liver homogenate using a colorimetric kit (Abcam). Hepatic lipids were extracted (Folch method) from 50-100 mg of liver in chloroform/methanol (2:1). Triglycerides and cholesterol were quantified using colorimetric kits (DiaSys Diagnostic Systems GmbH). NADPH levels were measured from liver homogenate after heating at 60°C for 30 min, using a colorimetric kit (Abcam).

**Glucose-6 phosphatase and acid α-glucosidase activities.** G6Pase activity (U/g tissue) was assayed at maximal velocity (with 20 mmol/l G6P) at 30°C, as previously described²⁰. GAA activity (nmol/h/mg protein) was measured as previously described²¹, and normalized to protein concentration, quantified by BCA method (Thermo Fisher Scientific).

### Gene expression analyses

Total RNA was extracted using Trizol (Thermo Fisher Scientific). DNA contaminants were removed (Free DNA kit, Thermo Fisher Scientific), and total RNA was reverse transcribed (RevertAid, Thermo Fisher Scientific). Expression of mRNA was assessed by RT-qPCR using a 7900 Fast Real Time thermocycler and Fast SYBR Green assays (Applied Biosystems). Primer sequences are listed in **CTAT Table**. Data are expressed as -ΔΔCt (the delta between the gene of interest Ct value and the Ct value of housekeeping gene, normalized to the Agl⁺/⁺ group).

### Protein expression analysis
Whole-cell lysates were obtained by liver homogenization (Fast Prep system, MP Biomedicals). Immunoblots were performed with specific antibodies (CTAT Table). Detection of eIF2α, Casp3/7, ACC, CPT1, STBD1, vinculin was based on immunofluorescence, visualized with Odyssey (LI-COR Biosciences), and densitometric quantification was performed with Image Studio Lite 5.2 (LI-COR Biosciences). The detection of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-c) and G6PC1 proteins was performed by chemiluminescence (Chemidoc™ imager, Bio-Rad) and densitometric quantification was performed using Image Lab™ software (Bio-Rad). Blots were normalized to a housekeeping protein, to the total form for phosphorylated/cleaved proteins, or to total protein for stain-free gels (Bio-Rad)²² (see figure legend).

**Statistical analysis**

Data are presented as mean ± S.D. (Standard Deviation). GraphPad Prism 7.0 was used for statistical analyses. *p*-value<0.05 was considered significant. The statistical tests performed are indicated in **Supplementary Table 1**.

The incidence of nodules between males and females was calculated using a two-sided Fisher's exact test (RStudio 2025.05.0+496).

**Results**

**Liver analysis revealed chronic liver damage and liver fibrosis in 14-month-old Agf/- mice.**

As previously reported¹⁸, Agf/- mice developed hepatomegaly due to glycogen accumulation (Fig. 1A-B) and showed decreased blood glucose level compared to Agf/+ mice, even when fed (Fig. 1C). However, only two 14-month-old Agf/- mice exhibited
glycemia below 70 mg/dl. HPS staining revealed enlarged, clear ballooning hepatocytes homogeneously distributed throughout all hepatic lobules, and PAS staining confirmed glycogen accumulation in *Agl*⁻/⁻ hepatocytes (**Fig. 1D**). In addition, Sirius red staining revealed enhanced centrilobular perisinusoidal fibrosis (METAVIR scale F2/F3; **Fig. 1E-F**). Accordingly, mRNA levels of activated hepatic stellate cell markers, such as actin alpha 2 (*Acta2*) and type 1 collagen (*Col1a1*), were increased, while plasminogen activator inhibitor 1 (*Pai1*) expression remained unchanged (**Fig. 1G**). In accordance with the presence of fibrosis and hepatocyte damage, plasma ALT/AST levels were elevated in 14-month-old *Agl*⁻/⁻ mice compared to *Agl*⁺/⁺ mice (**Fig. 1H**).

Since hepatic inflammation drives liver disease progression and sustains fibrosis²³, we explored the hepatic inflammatory response in *Agl*⁻/⁻ mice. Monocyte chemoattractant protein-1 (*Ccl2*) mRNA expression was higher in *Agl*⁻/⁻ compared to *Agl*⁺/⁺ livers (**Fig. 1I**). Immunohistochemical analyses confirmed that the number of F4/80 positive cells (Kupffer cells and macrophages) was greatly increased in *Agl*⁻/⁻ livers, compared with controls (**Fig. 1J**), supporting macrophage recruitment. CD3⁺ and MPO⁺ cells tended to be slightly more abundant in *Agl*⁻/⁻ mice, suggesting increased hepatic T lymphocyte and neutrophil populations (**Fig. 1J**).

In conclusion, loss of GDE results in extensive glycogen accumulation in the liver which is associated with chronic liver damage, fibrosis and inflammation.

## Hepatic tumorigenesis in *Agl*⁻/⁻ mice

Liver fibrosis can progress to cirrhosis in approximately 40-45% of GSDIII patients at a median age of 40 years, and development of liver neoplasms (HCA/HCC) can occur in approximately 15% of GSDIII patients at a median age of 65 years¹⁷. In *Agl*⁻/⁻ mice,
about one third of 14-month-old mice developed at least one macroscopic tumor (>1 mm), ranging from 1 mm to 10 mm (Fig. 2A). Surprisingly, females showed markedly higher tumor incidence than males (55% vs 8%) (Fig. 2A), a difference statistically confirmed by a two-sided Fisher's exact test (p<0.0001, odds ratio=13.57). Importantly, elevated levels of plasma AFP, a biomarker of HCC²⁴, were observed in Agl⁻/⁻ mice with tumors (Fig. 2B), but not significantly in age-matched Agl⁻/⁻ mice without macroscopic nodules. A complete histological characterization of several tumors isolated from Agl⁻/⁻ mice revealed non-encapsulated tumors of two types (Fig. 2C). The first type was composed of large cells, with atypical nuclear-cytoplasmic ratio, clonal appearance, eosinophilic infiltration, and no vascularization (nodule 1, Fig. 2C). The second type was composed of small and clarified cells, with the aspect of progenitor cells (nodule 2, Fig. 2C).

Consistent with tumor development, high cell proliferation was detected by immunohistochemistry of Ki67 marker in the tumors, whereas proliferation was only moderated in non-tumoral regions of Agl⁻/⁻ liver (Fig. 2D). Specifically, non-tumor Agl⁻/⁻ livers exhibited only a modest increase in mRNA expression of the proliferative markers cyclin E1 (Ccne1), Ki67 and DNA topoisomerase 2 α (Topo2α) compared to Agl⁺/⁺ liver (Supplementary Fig. 1). On the contrary, the expression of cyclin B1 (Ccnb1) was slightly reduced in the liver of Agl⁻/⁻ mice, while the expression of cyclin D1 (Ccnd1) remained unchanged (Supplementary Fig. 1).

Transmission electron microscopy (TEM) images revealed dilated endoplasmic reticulum (ER) in Agl⁻/⁻ non-tumor livers, a sign of ER stress²⁵ (Fig. 2E). ER stress activates the unfolded protein response (UPR) to restore cell homeostasis; however, sustained activation of ER stress drives cell death. Furthermore, ER stress and the UPR have been clearly implicated in the pathogenesis of several liver diseases²⁵. In Agl⁻/⁻ mice,
the phosphorylation at serine 51 of p-eIF2α, a known marker of ER stress induction²⁵, was
increased compared to control mice, confirming the activation of the stress response in
*Agl*⁻/⁻ non-tumor livers (**Fig. 2F**). In addition, we observed an increased mRNA expression
of C/EBP Homologous Protein (CHOP), a proapoptotic transcription factor triggered by
ER stress²⁵ (**Fig. 2G**). The increase in the apoptotic signaling was confirmed by the
significant increase in the downstream effectors cleaved-caspase 7 in the non-tumor *Agl*⁻/
⁻/⁻ liver, as well as by the trend towards increased levels of cleaved-caspase 3 (**Fig. 2H**).

To further investigate the mechanisms underlying tumorigenesis in *Agl*⁻/⁻ livers, we
analyzed the Hippo/YAP signaling pathway. Recent studies have demonstrated that
excessive glycogen accumulation can inhibit this pathway and activate the oncogenic
transcriptional co-activator YAP, promoting tumor initiation¹¹. Consistently, YAP
immunostaining revealed positive nuclear staining in some tumor and adjacent non-tumor
tissues (**Figure 2I**). Moreover, the YAP downstream target CTGF (*Ccn2*) was upregulated
in both tumor and non-tumor livers (**Figure 2J**).

In conclusion, chronic ER stress in *Agl*⁻/⁻ liver is associated with increased
hepatocyte apoptosis and compensatory proliferation, mechanisms known to support
tumorigenesis. This ER stress is likely driven by accumulation of abnormally structured
glycogen, given the well-established link between glycogen and ER²⁶. Moreover,
Hippo/YAP pathway inhibition may participate in the early events driving tumorigenesis in
GSDIII livers.

Energy deficiency in *Agl*⁻/⁻ non-tumor livers and induction of extrahepatic
gluconeogenesis to maintain glycemia in *Agl*⁻/⁻ mice
We next investigated the metabolic dysregulations associated with the loss of GDE activity in *Agl*⁻/⁻ hepatocytes. As expected, intracellular G6P levels, but also glucose levels, were reduced in 6h-fasted non-tumor *Agl*⁻/⁻ livers (**Fig. 3A**). This resulted in lower blood glucose levels in *Agl*⁻/⁻ mice compared to *Agl*⁺/⁺ mice, but without the development of symptomatic hypoglycemic events. After 12h of fasting, *Agl*⁻/⁻ mice even showed similar blood glucose levels to *Agl*⁺/⁺ mice, reaching 100 mg/dl (**Fig. 3B**). This normalization is likely due to the progressive induction of gluconeogenesis by prolonged fasting. However, there was no evidence of gluconeogenesis activation in the liver of 6h-fasted *Agl*⁻/⁻ mice. Indeed, G6Pase activity decreased and the expression of key gluconeogenic enzymes (*G6pc1* mRNA, PEPCK-c protein) remained unchanged (**Fig. 3C**).

To further assess hepatic gluconeogenesis in GSDIII, we performed a pyruvate tolerance test after a 12-hour fast, which is sufficient to deplete glycogen stores in mice²⁷. While pyruvate is a key substrate for hepatic gluconeogenesis¹, but not entirely liver-specific, this test remains widely used to estimate hepatic gluconeogenic capacity in mice. In 12-hour fasted *Agl*⁺/⁺ mice, pyruvate injection markedly increased blood glucose, confirming the induction of hepatic gluconeogenesis, whereas *Agl*⁻/⁻ mice showed only a marginal rise, suggesting a reduced hepatic gluconeogenic flux (**Fig. 3D**). Conversely, following a 6-hour fast, *Agl*⁻/⁻ mice showed increased blood glucose 60 min after injection of glutamine, the main substrate of renal and intestinal gluconeogenesis¹ (**Fig. 3D**). In *Agl*⁺/⁺ mice, blood glucose levels remained unaffected following glutamine injection, as they were able to maintain glycemia by utilizing glycogen stores. These data support that extrahepatic gluconeogenesis likely compensates for impaired hepatic glycogenolysis and gluconeogenesis in *Agl*⁻/⁻ mice. Supporting this, the expression of gluconeogenic enzymes (PEPCK-c and G6PC1) was increased in the kidney of 6h-fasted *Agl*⁻/⁻ mice,
compared to *Agl*<sup>+/+</sup> mice (Fig. 3E). The expression of PEPCK-c was also increased in the intestine of *Agl*<sup>-/-</sup> mice, whereas G6PC1 expression remained unchanged (Fig. 3E).

Given the limited availability of glucose in *Agl*<sup>-/-</sup> hepatocytes, glycolysis flux was also reduced, as evidenced by lower lactate in non-tumor livers (Fig. 3F). Regarding lipid metabolism, triglyceride and cholesterol levels were significantly reduced in *Agl*<sup>-/-</sup> livers compared to controls (Fig. 3G). There was no change in the phosphorylation at Ser79 of acetyl-CoA carboxylase (ACC), which controls lipid synthesis, between fasted *Agl*<sup>-/-</sup> and control mice, nor in the protein levels of CPT1α, a key protein involved in the mitochondrial fatty acid β-oxidation (Supplementary Fig. 2A). Similarly, mRNA expression of key lipid regulating transcription factors (*Ppara*, *Chrebp*, *Srebp1c*) was unchanged (Supplementary Fig. 2B) and no steatosis was observed in *Agl*<sup>-/-</sup> livers (Fig. 3H). Plasma triglyceride and cholesterol levels were also comparable to controls (Supplementary Fig. 2C).

Finally, in line with the low levels of intracellular G6P in *Agl*<sup>-/-</sup> livers, we also observed a reduced expression of G6P dehydrogenase (G6PD), the limiting enzyme of the pentose phosphate pathway (Fig. 3I). However, the decrease in this pathway did not induce a change in NADPH levels in the liver of *Agl*<sup>-/-</sup> mice compared to *Agl*<sup>+/+</sup> mice, suggesting that NADPH is produced by a different biochemical pathway in the *Agl*<sup>-/-</sup> liver (Fig. 3J).

In conclusion, our data indicates that impaired glycogenolysis in the absence of GDE leads to a metabolic rewiring, resulting in greatly reduced glucose, lactate and lipid levels in G6DIII non-tumor hepatocytes. In addition, the induction of extrahepatic gluconeogenesis may help compensate for reduced hepatic glucose production in *Agl*<sup>-/-</sup> mice, potentially contributing to the partial maintenance of glycemia during fasting.
Glycophagy is associated with tumor development in GSDIII hepatocytes

Given the substantial energy requirement for tumor proliferation, we investigated which energy substrates GSDIII hepatocytes use to sustain growth and drive tumorigenesis. Tumor cells often adapt their metabolism through the Warburg effect, switching from mitochondrial respiration to glycolysis. In *Agl*⁻/⁻ mice, however, limited glucose and G6P availability and correspondingly low lactate production (Fig. 3F) suggest that a Warburg effect is unlikely. This was confirmed by similar lactate levels in *Agl*⁻/⁻ tumors compared to non-tumor livers (Supplementary Fig. 4A). Additionally, G6Pase activity was decreased in both non-tumor livers and tumors (Supplementary Fig. 4B).

To compensate for limited energy substrates, GSDIII tumor cells may use glycogen stores as an energy source. In *Agl*⁻/⁻ mice, we observed a significant reduction in glycogen levels in most tumors compared to non-tumor tissue, with heterogeneity among nodules, confirmed by variable PAS staining, particularly in the two nodule types described in Figure 2C (Fig. 4A-4B). We also assessed the glycogen level in human GSDIII livers from four patients with GSDIII. These patients had a mean age of 55.4±8.5 years old at the time of liver transplant or hepatectomy (Table 1). An analysis of native liver histology revealed: in patient 1, cirrhosis with multiple regenerative nodules, but no malignancy; in patient 2, portal fibrosis, one regenerative nodule, and two HCCs with a mixed pattern (trabecular and pseudoacinar); in patient 3, three HCCs and early staged cirrhosis. Patient 4 underwent a hepatectomy for malignant nodules and a liver transplantation few years later. Histological analyses showed one large HCC and one large HCA at the time of hepatectomy, and analysis of the native liver identified fibrosis but no cirrhosis, one HCA and two HCCs (Table 1; Figure 4C). The presence of HCCs was associated with elevated
serum AFP levels in patients 2 and 3, but not in patient 4 (Table 1). Interestingly, PAS staining was negative in HCCs (patients 2, 3 and 4), but positive in non-tumor liver and regenerative nodules (patient 1), confirming dramatic glycogen loss with some heterogeneity among nodules (Figure 4C and Supplementary Fig. 4C).

Since the cytosolic glycogen degradation pathway is impaired in *Agl*⁻/⁻ mice, glycogen degradation may be redirected through the lysosomal glycogen degradation pathway. Consistently, GAA activity was reduced in non-tumor *Agl*⁻/⁻ livers compared to controls but was restored in tumors from *Agl*⁻/⁻ mice (Fig. 5A). Furthermore, TEM revealed an increased number of autophagic vesicles (and a tendency towards an increased number of lysosome) in tumor tissue compared to non-tumor liver tissue in *Agl*⁻/⁻ mice (Fig. 5B), suggesting enhanced autophagic flux in tumors. On the contrary, the number of autophagic vesicles and lysosomes tended to decrease in non-tumor tissue compared to *Agl*⁺/⁺ liver (Fig. 5B), concomitantly with decreased lysosome number. Glycophagy is a specialized form of autophagy that enables cells to target glycogen for conversion into glucose in lysosomes²⁸. To evaluate the glycophagy pathway in *Agl*⁻/⁻ hepatic tumors, we performed GABAA receptor-associated protein like 1 (GABARAPL1) and starch-binding domain-containing protein 1 (STBD1) immunolabelling, two proteins associated with glycophagy²⁹, to directly quantify the number of glycophagosomes. Importantly, we observed an increased number of glycophagosomes in non-tumor liver and tumors of *Agl*⁻/⁻ mice compared to *Agl*⁺/⁺ mice (Fig. 5C). Additionally, glycophagosomes were visualized by TEM in *Agl*⁻/⁻ livers (Fig. 5D), supporting the involvement of glycophagy in glycogen breakdown. On the other hand, our data indicate that autophagy was impaired in *Agl*⁻/⁻ tumors, as evidenced by elevated expression of p62 relative to non-tumor liver (Fig. 5E). Moreover, total STBD1 expression was reduced in both non-tumor and tumor tissues of
Agt/- mice compared to control liver, while GABARAPL1 expression was increased (Fig. 5E-F), suggesting a shift from general autophagy towards specific glycophagy. Of note, elevated levels of Galectin-3, a marker of lysosomal damage, were recently reported in GSDIII mice³⁰. These results suggest that while GABARALP1 is retained and potentially recycled within phagolysosomes, STBD1 is likely degraded within the glycophagosome lumen. Interestingly, the observed accumulation of p62/autophagosomes, together with decrease STBD1 levels, implies that glycophagy may be upregulated at the expense of general autophagy in GSDIII-associated tumors.

Taken together, these results indicate that in the absence of GDE, tumor cells in both mice and humans likely enhance their capacity for glycogen degradation *via* the lysosomal autophagic pathway, to provide glucose to sustain cell proliferation and growth.

Discussion

Liver pathology in GSDIII is characterized by the accumulation of structurally abnormal glycogen (limit dextrin), and early-onset hepatic fibrosis. In 40-45% of adults, fibrosis can progress to cirrhosis and, in approximately 15%, to HCA/HCC, typically after 65 years of age¹⁷. Advances in dietary management aimed at preventing hypoglycemia and glycogen accumulation have improved life expectancy¹², making cirrhosis and liver tumors an emerging concern.

The Agt/- mouse represents a robust GSDIII model that faithfully recapitulates all hepatic and muscular disease features¹⁸. About 30% of Agt/- mice develop liver tumors by 14 months, a much higher incidence than currently reported in patients. However, the true prevalence of liver tumors in GSDIII is likely underestimated and may approach that seen in mice, given the limited clinical data on patients over 40–50 years old and the
typically late onset of tumor development<sup>14, 16, 31</sup>. This highlights the urgent need to
investigate the molecular pathways driving tumorigenesis in GSDIII.

Abnormal soluble glycogen accumulation in GSDIII liver may drive cellular stress, inflammation, and fibrosis. Limit dextrin likely induces ER stress<sup>10</sup>, which promotes fibrosis and HCC<sup>25</sup>. Consistently, Agl<sup>-/-</sup> livers exhibited ER dilation and chronic ER stress with increased eIF2α phosphorylation at Ser51, which may exert a cytoprotective effects under low glucose<sup>32</sup>. ER stress may be worsened by impaired protein glycosylation due to reduced intracellular glucose<sup>33</sup>. The inflammation microenvironment further contributes tumor initiation and progression<sup>34</sup>. Although detailed immune profiling was not performed, Agl<sup>-/-</sup> livers showed increased T lymphocytes and macrophages than to Agl<sup>+/-</sup> mice. These macrophages likely promote hepatic stellate cells activation<sup>35</sup>. Thus, as seen in patients, Agl<sup>-/-</sup> mice developed HCC with fibrosis, but without cirrhosis, indicating that HCC can develop independently of cirrhosis, also observed in patients. This finding suggests that cirrhosis is not a prerequisite for tumorigenesis in GSDIII, and that alternative mechanisms may drive malignant transformation in this context.

Loss of GDE activity in Agl<sup>-/-</sup> mice caused a significant decrease in blood glucose under both fed or 6h-fasted conditions versus controls. However, only a few Agl<sup>-/-</sup> mice developed mild hypoglycemia (blood glucose between 50 and 70 mg/dL), less severe than in Glycogen Storage Disease Type Ia (GSDIa) models<sup>20</sup>. Similarly, GSDIII patients GSDIa/b show milder hypoglycemia than GSDIa/b patients with, due to preserved gluconeogenesis. In the absence of glycogenolysis, gluconeogenesis can occur in the liver, kidney, or intestine, to maintain euglycemia. While hepatic gluconeogenesis was expected to be preserved, hepatic glucose and G6P levels were reduced in Agl<sup>-/-</sup> compared to Agl<sup>+/-</sup> mice and expression of G6PC1 and PEPCK-c was not upregulated.
Thus, *Agl*⁻/⁻ mice likely maintain euglycemia via extrahepatic gluconeogenesis, supported by increased kidney G6PC1 and PEPCK-c expression. Similar mechanisms occur during the anhepatic phase of liver transplantation⁴⁰, ⁴¹ and in liver-specific *G6pc1* knockout mice (hepatic GSDla model)²⁰, ³², ⁴². Interestingly, amino acids, particularly glutamine, are the main substrates for extrahepatic gluconeogenesis⁴³. Therefore, a high protein diet can help sustain glucose production¹². Substituting part of the dietary carbohydrate content with protein also help to reduce the accumulation of glycogen in the liver and muscle¹².

In contrast to GSDla, where tumorigenesis involves Warburg-like metabolic
reprogramming⁴⁰, tumors in GSDIII arise under energy depletion. In *Agl*⁻/⁻ hepatocytes,
impaired gluconeogenesis and blocked glycogenolysis cause a severe energy deficit, with
reduced G6P, glycolysis, lactate, triglyceride and cholesterol levels. These metabolic
changes were not associated with any change in lipogenic gene expression, even on high
fat/high sucrose diet (data not shown). Despite this, *Agl*⁻/⁻ hepatocytes displayed increased
proliferation and tumor formation, implying alternative energy sources support cell growth.
Our results suggest that glycogen may play a dual role in tumor initiation and progression.
Excess glycogen may activate YAP by disturbing the Hippo/YAP signaling, thereby
promoting tumor initiation¹¹. Since cytosolic degradation is impaired, lysosomal glycogen
breakdown *via* glycophagy could act as a compensatory mechanism. Tumor tissues from
*Agl*⁻/⁻ showed increased GAA activity, reduced glycogen content, and glycophagosomes
enriched in GABARAPL1 and STBD, indicating active glycogen degradation. Glycophagy,
is increasingly recognized as a key physiological pathway in energy homeostasis. While
initially recognized for its role in neonatal glycogen degradation to sustain blood glucose
levels after birth⁴¹, glycogen accumulation has recently been identified as an early
oncogenic event during liver tumorigenesis¹¹, ⁴²⁻⁴⁵. Interestingly, experimental depletion of
glycogen stores has been shown to reduce liver cancer incidence, whereas enhanced glycogen storage accelerates tumor development¹⁰, ¹¹. Consistently, human GSDIII HCCs exhibited reduced glycogen, a finding not observed in regenerative nodules. Despite the heterogeneity in nodule phenotypes and the limited sample size, this suggests that glycogen could be an energy source for tumor growth in both mice and patients with GSDIII. Inhibiting glycogen synthase II by RNAi reduced tumor growth in GSDIII models⁴⁶ and repressing GAA expression could validate this metabolic model. Thus, the Agl⁻/⁻ mouse model could be a valuable tool for identifying anti-tumor drugs targeting glycophagy.

In conclusion, this study provides new insights into the metabolic rewiring of the liver in GSDIII, which differs markedly from the one observed in GSDIa¹⁹, ⁴⁵. By characterizing glucose and lipid dysregulations, we uncover two distinct molecular mechanisms that contribute to liver tumorigenesis. Our findings support the emerging concept that, in GSDIII, glycogen likely plays dual roles, serving not only as a metabolic substrate but could also act as a signaling molecule that could drive tumorigenesis. However, tumor heterogeneity suggests that additional pathways are likely to be contributing to liver tumorigenesis. Further research could provide compelling data to shape the current gene therapy strategies for GSDIII under clinical development⁴⁷, ⁴⁸ and open new avenues for therapeutic interventions in GSDIII-related hepatocarcinogenesis and potentially other GSDs.

**Acknowledgements.** This work was supported by Genethon, the "Association Française contre la Myopathie", the "Association Francophone des Glycogénoses", the Gen&Zic association, and the National Research Agency (ANR-17-CE18-0014; ANR-22-PEBI-
0012). VMR, LJ, AG, AA, JC, FB, EBF, CG, LVW, and GR are Genopole's members, first French biocluster dedicated to genetic, biotechnologies and biotherapies. We are grateful to the "Imaging and Cytometry Core Facility" of Genethon for technical support, to the CiQle platform for TEM, histology and imaging analysis (SFR Santé Lyon-Est, Université Claude Bernard Lyon 1), to Ile-de-France Region, to Conseil Départemental de l'Essonne (ASTRE), to "Institut National de la Santé et de la Recherche Médicale" (INSERM) and GIP Genopole, Evry for the purchase of the equipment. We also thank Dr. Monika Gjorgjieva and Dr Marco Costa for their critical reading of the manuscript. FR, PL, and FM are members of the G2M 'Rare Hereditary Metabolic Diseases' health network.
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TABLE

<table><caption>Table 1. Characteristics of patients with GSDIII at the time of transplantation/hepatectomy.</caption><thead><tr><th>ID</th><th>Sex</th><th>Age (years)</th><th>Liver weight</th><th>Liver pathology</th><th>ALT (U/L)</th><th>AST (U/L)</th><th>AFP (µg/L)</th></tr></thead><tbody><tr><td>1</td><td>M</td><td>43</td><td>Native liver (2270 g)</td><td>Cirrhosis<br>Multiple regenerative macronodules in all segments</td><td>142</td><td>158</td><td>4.1</td></tr><tr><td>2</td><td>F</td><td>63</td><td>Lobectomy of segment II (178g)</td><td>Fibrosis<br>2 HCCs (4 cm and 0.8 cm) and 1 regenerative nodule (4 cm)</td><td>50</td><td>69</td><td>183.1</td></tr><tr><td>3</td><td>M</td><td>63</td><td>Lobectomy of segment IV and VIII</td><td>Early-stage cirrhosis with septal fibrosis<br>3 HCCs (1.5-2.2 cm)</td><td>94</td><td>90</td><td>73</td></tr><tr><td rowspan="2">4</td><td rowspan="2">M</td><td>51</td><td rowspan="2">Right hepatectomy</td><td>Fibrosis without cirrhosis<br>1 HCC (10 cm)<br>1 HCA (11 cm)</td><td rowspan="2">nd</td><td rowspan="2">nd</td><td rowspan="2">nd</td></tr><tr><td>57</td><td>Fibrosis without cirrhosis<br>1 HCA (7 cm)<br>2 HCCs (3.1 cm and 2.2 cm)</td></tr></tbody></table>

ALT: Alanine aminotransferase: N<50U/L; AST: Aspartate aminotransferase: N<50U/L; AFP: a-fetoprotein: N<7 µg/; HCC: hepatocellular carcinoma; HCA: hepatocellular adenoma; LT: liver transplant, nd: not determined.
LEGENDS OF FIGURES

**Figure 1. Liver pathology shows inflammation and fibrosis in Agt<sup>-/-</sup> mice**

(A) Liver weight normalized by body weight, (B) hepatic glycogen content, and (C) blood glucose in 14-month-old fed male and female Agt<sup>+/+</sup> (black) and Agt<sup>-/-</sup> (red) mice. (D-E) Representative images of HPS, PAS and Sirius red staining of liver sections. Scale bars = 200µm (main), or 100µm (magnification). (F) Fibrosis quantification (% Sirius red-positive surface/total surface). (G) Hepatic mRNA expression of fibrotic genes. (H) Plasma ALT and AST activities. (I) Hepatic *Ccl2* mRNA expression. (J) Representative images and quantification of hepatic immunohistochemistry for myeloperoxidase (MPO), F4/80, and CD3. Scale bar=100µm. Statistical analyses are detailed in **Supplementary Table 1**.

\*p<0.05, \*\*p<0.01 and \*\*\*p<0.001.

**Figure 2. Hepatic tumorigenesis in Agt<sup>-/-</sup> mice**

(A) Representative images of liver and nodules and table showing the number of macroscopic nodules (> 1 mm) in 14-month-old Agt<sup>+/+</sup> and Agt<sup>-/-</sup> mice at necropsy. Scale bar = 1 cm. (B) Plasma alpha-fetoprotein (AFP) in Agt<sup>+/+</sup> mice (black bars) and Agt<sup>-/-</sup> mice without (NT, red bars) and with tumors (T, blue bars). (C) Representative images of HPS and Sirius red staining of two hepatic nodules from Agt<sup>-/-</sup> mice (1 and 2). Scale bars = 500µm (main), and 50µm (magnification). (D) Representative images and quantification of Ki67 immunohistochemistry in livers. Tumor is outlined with hatched line. NT = peritumor tissue. Scale bar = 200µm. (E) TEM images from non-tumor livers of Agt<sup>+/+</sup> and Agt<sup>-/-</sup> mice. Arrows: dilated ER; circles: glycogen. (F) Quantitative analysis of phosphorylated and total eIF2-alpha protein expression in liver. (G) Hepatic *Chop* mRNA
expression. (H) Quantitative analysis of cleaved and total Caspase 7 (Casp7) and Caspase 3 (Casp3) protein expressions in livers. (I) Representative images of YAP IHC in tumor (T) and peritumor (NT) liver tissue. Scale bars = 100µm (main), and 50 µm (magnification). (J) Hepatic *Ccn2* mRNA expression. Statistical analyses are detailed in **Supplementary Table 1.** \*p<0.05, \*\*p<0.01, \*\*\*p<0.001.

<caption>**Figure 3. Energy deficiency in GSDIII livers and induction of extrahepatic gluconeogenesis to maintain glycemia in GSDIII mice**</caption>

(A) G6P and glucose levels in 14-month-old *Agtfl/+* and *Agtf/-* mice livers. (B) Fasting blood glucose level over time. (C) Quantification of hepatic G6Pase activity, *G6pc1* mRNA expression level, and PEPCK protein expression (normalization to total protein shown in **Supplementary Figure 3A**). (D) Pyruvate (12h fast, left panel) and glutamine (6h fast, right panel) tolerance tests. (E) Quantitative analysis of G6PC1 and PEPCK-c protein expression (normalization to total protein or tubulin shown in **Supplementary Figure 3B-C**), and *G6pc1* and *Pck1* mRNA expression in the kidney (left panel) and intestine (right panel). (F-G) Hepatic lactate (fasted) (F) and cholesterol and triglyceride (G) contents. (H) Representative images of Oil red O (ORO) staining in liver. Scale bar = 200µm. (I) Quantitative analysis of G6PD protein expression in liver (normalization to total protein levels shown in **Supplementary Figure 3D**). (J) Hepatic NADPH content. All the analyzed mice were female. Statistical analyses are detailed in **Supplementary Table 1.** \*p<0.05, \*\*p<0.01 or \*\*\*p<0.001.

<caption>**Figure 4. Glycogen is depleted in liver tumors from both GSDIII mice and patients.**</caption>
(A) Hepatic glycogen content in 14-month-old *Agl+/+* mice (black bars) or *Agl/-* mice in non-tumor livers (NT; red bars) and tumors (T; blue bars). (B) Representative images of HPS and PAS staining in the liver of 14-month-old *Agl/-* mice (41 tumors from 17 mice). Tumors were outlined with hatched line. NT=peritumor tissue. Scale bars = 200µm. PAS staining from the same sample as in Figure 2C is included (1: nodule type 1; 2: nodule type 2); scale bars=500µm. (C) Representative images of PAS and HES staining on human GSDIII livers from patient 1 with cirrhosis and regenerative nodules and from patients 2-4 with HCCs. NT= peritumor liver. Statistical analyses are detailed in **Supplementary Table 1. ***p<0.001.

**Figure 5. Glycophagy is associated with tumor development in GSDIII hepatocytes**

(A) Acid alpha-glucosidase (GAA) activity in non-tumor/peritumoral (NT; red bar) and tumor (T; blue bar) tissues of 14-month-old *Agl/-* and *Agl+/+* (black bar) mice. (B) Representative TEM images and quantification of lysosomes and autophagic features. Magenta arrows: lysosomes, yellow arrows: autophagosomes. (C) Immunofluorescence of GABA Type A Receptor Associated Protein Like 1 (GABARAPL1) and Starch Binding Domain 1 (STBD1) proteins in mouse livers. Arrows indicate glycophagosomes (D) Representative TEM images of glycophagosomes in 14-month-old *Agl/-* livers. Scale bars=500µm. (E-F) STBD1, p62, and GABARAPL1 protein expressions in livers. Statistical analyses are detailed in **Supplementary Table 1.** \*p<0.05, \*\*p<0.01, \*\*\*p<0.001.
<table><caption>**Table 1. Characteristics of patients with GSDIII at the time of transplantation/hepatectomy.**</caption><thead><tr><th>ID</th><th>Sex</th><th>Age (years)</th><th>Liver weight</th><th>Liver pathology</th><th>ALT (U/L)</th><th>AST (U/L)</th><th>AFP (µg/L)</th></tr></thead><tbody><tr><td>1</td><td>M</td><td>43</td><td>Native liver (2270 g)</td><td>Cirrhosis<br>Multiple regenerative macronodules in all segments</td><td>142</td><td>158</td><td>4.1</td></tr><tr><td>2</td><td>F</td><td>63</td><td>Lobectomy of segment II (178g)</td><td>Fibrosis<br>2 HCCs (4 cm and 0.8 cm) and 1 regenerative nodule (4 cm)</td><td>50</td><td>69</td><td>183.1</td></tr><tr><td>3</td><td>M</td><td>63</td><td>Lobectomy of segment IV and VIII</td><td>Early-stage cirrhosis with septal fibrosis<br>3 HCCs (1.5-2.2 cm)</td><td>94</td><td>90</td><td>73</td></tr><tr><td>4</td><td>M</td><td>51</td><td>Right hepatectomy</td><td>Fibrosis without cirrhosis<br>1 HCC (10 cm)<br>1 HCA (11 cm)</td><td>nd</td><td>nd</td><td>nd</td></tr><tr><td></td><td></td><td>57</td><td>Native liver</td><td>Fibrosis without cirrhosis<br>1 HCA (7 cm)<br>2 HCCs (3.1 cm and 2.2 cm)</td><td>60</td><td>60</td><td>&lt;5</td></tr></tbody></table>

ALT: Alanine aminotransferase; N<50U/L; AST: Aspartate aminotransferase; N<50U/L; AFP: a-fetoprotein; N<7 µg/; HCC: hepatocellular carcinoma; HCA: hepatocellular adenoma; LT: liver transplant, nd: not determined.
<figure><img src="image_8.png" /><figcaption>FIGURE 1</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>FIGURE 2</figcaption></figure>
<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>FIGURE 3</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>FIGURE 4</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>FIGURE 5</figcaption></figure>
## Highlights

* Lysosomal degradation of glycogen was activated in liver tumors of GSDIII mice.

* Glycogen may serve as fuel to promote tumor growth in GSDIII mice and patients.

* Loss of glycogen debranching enzyme causes a severe energy deficit in the liver.

* Extrahepatic gluconeogenesis sustains blood glucose levels in GSDIII mice during fasting.