Enhanced lysosomal glycogen breakdown is associated with liver tumorigenesis in glycogen storage disease type III

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 patients with GSDIII (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 with a 2.5-fold increase in alpha-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 6 h 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), which was associated with increased lysosomal alpha-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 HCCs from patients with GSDIII supported these observations. Although YAP nuclear staining varied among tumors, the overall increase in YAP nuclear localization and CTGF expression suggests that inhibition of the Hippo/YAP pathway may 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.

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, highlighting 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 patients with GSDIII at risk of hepatocellular carcinoma, while contributing to the broader understanding of metabolic dysregulation in cancer biology.

Keywords: Hippo/YAP pathway; fibrosis; glucose metabolism; glycophagy; hepatocellular carcinoma; inflammation; rare disease.

Graphical abstract

Fig. 1

Liver pathology shows inflammation and fibrosis in Agl^-/- mice. (A) Liver weight normalized by body weight, (B) hepatic glycogen content, and (C) blood glucose in 14-month-old fed male and female Agl^+/+ (black) and Agl^-/- (red) mice. (D-E) Representative images of HPS, PAS and Sirius red staining of liver sections. Scale bars = 200 μm (main), or 100 μm (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 MPO, F4/80, and CD3. Scale bar = 100 μm. Statistical analyses are detailed in Table S1. ∗p <0.05, ∗∗p <0.01 and ∗∗∗p <0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; HPS, hematoxylin–phloxine–saffron; PAS, periodic acid–Schiff.

Fig. 2

Hepatic tumorigenesis in Agl^-/- mice. (A) Representative images of liver and nodules and table showing the number of macroscopic nodules (>1 mm) in 14-month-old Agl^+/+ and Agl^-/- mice at necropsy. Scale bar = 1 cm. (B) Plasma AFP in Agl^+/+ mice (black bars) and Agl^-/- 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 Agl^-/- mice (1 and 2). Scale bars = 500 μm (main), and 50 μm (magnification). (D) Representative images and quantification of Ki67 immunohistochemistry in livers. Tumor is outlined with hatched line. NT = peritumor tissue. Scale bar = 200 μm. (E) TEM images from non-tumor livers of Agl^+/+ and Agl^-/- 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 and 3 protein expression in livers. (I) Representative images of YAP IHC in tumor (T) and peritumor (NT) liver tissue. Scale bars = 100 μm (main), and 50 μm (magnification). (J) Hepatic Ccn2 mRNA expression. Statistical analyses are detailed in Table S1. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. ER, endoplasmic reticulum; HPS, hematoxylin–phloxine–saffron; IHC, immunohistochemistry; PAS, periodic acid–Schiff; TEM, transmission electron microscopy.

Fig. 3

Energy deficiency in GSDIII livers and induction of extrahepatic gluconeogenesis to maintain glycemia in GSDIII mice. (A) G6P and glucose levels in 14-month-old Agl^+/+ and Agl^-/- 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 Fig. S3A). (D) Pyruvate (12 h fast, left panel) and glutamine (6 h fast, right panel) tolerance tests. (E) Quantitative analysis of G6PC1 and PEPCK-c protein expression (normalization to total protein or tubulin shown in Fig. S3B and 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 ORO staining in liver. Scale bar = 200 μm. (I) Quantitative analysis of G6PD protein expression in liver (normalization to total protein levels shown in Fig. S3D). (J) Hepatic NADPH content. All the analyzed mice were female. Statistical analyses are detailed in Table S1. ∗p <0.05, ∗∗p <0.01 or ∗∗∗p <0.001. G6P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; G6pc1, glucose-6-phosphatase catalytic subunit 1; G6PD, glucose-6-phosphate dehydrogenase; GSDIII, glycogen storage disease type III; ORO, Oil Red O; Pck1, phosphoenolpyruvate carboxykinase 1; PEPCK, phosphoenolpyruvate carboxykinase; PEPCK-c, cytosolic phosphoenolpyruvate carboxykinase.

Fig. 4

Glycogen is depleted in liver tumors from both GSDIII mice and patients. (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 = 200 μm. PAS staining from the same sample as in Fig. 2C is included (1: nodule type 1; 2: nodule type 2); scale bars = 500 μm. (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 Table S1. ∗∗∗p <0.001. GSDIII, glycogen storage disease type III; HCC, hepatocellular carcinoma; HES, hematoxylin–eosin–saffron; HPS, hematoxylin–phloxine–saffron; PAS, periodic acid–Schiff.

Fig. 5

Glycophagy is associated with tumor development in GSDIII hepatocytes. (A) 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 GABARAPL1 and STBD1 proteins in mouse livers. Arrows indicate glycophagosomes (D) Representative TEM images of glycophagosomes in 14-month-old Agl^-/- livers. Scale bars = 500 μm. (E-F) STBD1, p62, and GABARAPL1 protein expression in livers. Statistical analyses are detailed in Table S1. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. GAA, acid alpha-glucosidase; GABARAPL1, GABA type A receptor–associated protein like 1; GSDIII, glycogen storage disease type III; STBD1, starch binding domain containing 1; TEM, transmission electron microscopy.