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. 2019 Sep 24;3(11):1544-1555.
doi: 10.1002/hep4.1426. eCollection 2019 Nov.

Liver Glycogen Phosphorylase Deficiency Leads to Profibrogenic Phenotype in a Murine Model of Glycogen Storage Disease Type VI

Lane H Wilson  1 , Jun-Ho Cho  1 , Ana Estrella  1 , Joan A Smyth  2 , Rong Wu  3 , Tayoot Chengsupanimit  4 , Laurie M Brown  4 , David A Weinstein  1   5 , Young Mok Lee  1
Affiliations

Liver Glycogen Phosphorylase Deficiency Leads to Profibrogenic Phenotype in a Murine Model of Glycogen Storage Disease Type VI

Lane H Wilson et al. Hepatol Commun. .

Abstract

Mutations in the liver glycogen phosphorylase (Pygl) gene are associated with the diagnosis of glycogen storage disease type VI (GSD-VI). To understand the pathogenesis of GSD-VI, we generated a mouse model with Pygl deficiency (Pygl -/-). Pygl -/- mice exhibit hepatomegaly, excessive hepatic glycogen accumulation, and low hepatic free glucose along with lower fasting blood glucose levels and elevated blood ketone bodies. Hepatic glycogen accumulation in Pygl -/- mice increases with age. Masson's trichrome and picrosirius red staining revealed minimal to mild collagen deposition in periportal, subcapsular, and/or perisinusoidal areas in the livers of old Pygl -/- mice (>40 weeks). Consistently, immunohistochemical analysis showed the number of cells positive for alpha smooth muscle actin (α-SMA), a marker of activated hepatic stellate cells, was increased in the livers of old Pygl -/- mice compared with those of age-matched wild-type (WT) mice. Furthermore, old Pygl -/- mice had inflammatory infiltrates associated with hepatic vessels in their livers along with up-regulated hepatic messenger RNA levels of C-C chemokine ligand 5 (Ccl5/Rantes) and monocyte chemoattractant protein 1 (Mcp-1), indicating inflammation, while age-matched WT mice did not. Serum levels of aspartate aminotransferase and alanine aminotransferase were elevated in old Pygl -/- mice, indicating liver damage. Conclusion: Pygl deficiency results in progressive accumulation of hepatic glycogen with age and liver damage, inflammation, and collagen deposition, which can increase the risk of liver fibrosis. Collectively, the Pygl-deficient mouse recapitulates clinical features in patients with GSD-VI and provides a model to elucidate the mechanisms underlying hepatic complications associated with defective glycogen metabolism.

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Figures

Figure 1
Figure 1
Generation of Pygl‐deficient mice. (A) Schematic representation of the Pygl‐knockout and WT alleles. Insertion of the FRT/loxP cassette in the intron between exon 2‐3 disrupts Pygl mRNA expression. Arrows indicate WT or lacZ primers used for genotyping. (B) PCR genotype analysis of WT (+/+), heterozygous (+/–), and Pygl −/− mice. The lacZ primer set and the WT primer set are expected to amplify a fragment of 500 bp and 800 bp, respectively. (C) Quantification of hepatic mRNA for Pygl in young WT (n = 9) and Pygl −/− (n = 24) mice and old WT (n = 16) and Pygl −/− (n = 13) mice. (D) Liver and kidney weights expressed as percentage of body weight. Numbers of mice analyzed were young WT (n = 13) and Pygl −/− (n = 24) mice for liver weight and young WT (n = 7) and Pygl −/− (n = 17) mice for kidney weight. (E) Representative image of liver and kidney in WT and Pygl −/− mouse. Scale shows length in centimeters. Data in (C,D) represent mean ± SD. ***P < 0.0001.
Figure 2
Figure 2
Pygl −/− mice exhibit excessive hepatic glycogen accumulation along with fasting ketotic hypoglycemia. (A,B) Hepatic glycogen and free glucose levels in 24‐hour fasted young and old WT (n = 5) and Pygl −/− (n = 6) mice. (C) Fasting glucose test in young WT (n = 12) and Pygl −/− (n = 17) mice. (D) Box‐and‐whisker plots of fasting blood ketone levels in young WT (n = 18) and Pygl −/− (n = 18) mice. Statistical analyses of box‐and‐whisker plots were performed using the Mann‐Whitney test and show the interquartile range (box), median (horizontal line), and maximum and minimum observations (whiskers). Hepatic glycogen and free glucose data (A,B) present the mean ± SD. Fasting glucose data (C) are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
Histologic analysis of livers from WT and Pygl −/− mice. Livers from young WT (n = 6) and Pygl −/− (n = 9) mice and old WT (n = 7) and Pygl −/− (n = 13) mice were evaluated histologically by a veterinary pathologist. Representative images of liver sections stained with H&E, Masson's trichrome, and picrosirius red in young WT mice (A1‐A4), young Pygl −/− mice (B1‐B4), old WT mice (C1‐C4), and four individual old Pygl −/− mice (D1‐D4, E1‐E4, F1‐F4, G1‐G4). With picrosirius red staining, individual old Pygl −/− mouse exhibited distinct pathological patterns, including minimal collagen deposition in subcapsular areas (D3,D4); subcapsular and sinusoidal mild collagen deposition (E3,E4); minimal collagen deposition in perisinusoidal and periportal areas (F3,F4); and regionally severe fibrosis with central to central bridging and collapse of the intervening lobular structure (G3,G4). In picrosirius red staining, the images in the fourth column (A4‐G4) present higher magnification views of the images in the third column (A3‐F3). Scale bars represent 200 µm. Arrow heads indicate immune cell infiltrations.
Figure 4
Figure 4
Elevated serum transaminases and activated HSCs in old Pygl −/− mice. (A) Box‐and‐whisker plots for quantification of hepatic hydroxyproline in both young and old WT (n = 11) and Pygl −/− (n = 13) mice. (B) Box‐and‐whisker plots showing serum levels of ALT, AST, ALP, and bilirubin in young WT (n = 5) and Pygl −/− (n = 5) mice and old WT (n = 6) and Pygl −/− (n = 6) mice. Data in (A,B) show the interquartile range (box), median (horizontal line), and maximum and minimum observations (whiskers). Statistical analyses were performed using the Mann‐Whitney test. *P < 0.05, **P < 0.01. (C) Immunohistochemical analysis of α‐SMA. Old Pygl −/− mice exhibited elevated but variable numbers of α‐SMA‐positive cells in periportal, perisinusoidal, and/or lobular areas in their liver sections. The insets show higher magnification views. Scale bars represent 200 µm.
Figure 5
Figure 5
Expression profile of fibrotic and inflammatory genes in WT and Pygl −/− mice. Quantification of hepatic mRNA for fibrogenic genes (Ctgf, Tgf‐β), inflammatory genes (Il‐6, Tnf‐a), extracellular matrix (Col1a1, Col1a2, Col4a2, Fn 1), and chemokines (Mcp‐1, Ccl5/Rantes, Cxcl1/KC, Mip‐2α/Cxcl2) in (A) young WT (n = 9) and Pygl −/− (n = 24) mice and (B) old WT (n = 16) and Pygl −/− (n = 13) mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: Fn1, fibronectin; Il‐6, interleukin‐6; KC, C‐X‐C chemokine ligand 1; Mip‐2α, macrophage inflammatory protein 2α.
Figure 6
Figure 6
Pathophysiology of Pygl‐deficient liver. In a fasting period, WT mice maintain blood glucose by breakdown of the hepatic glycogen store to glucose through glycogenolysis. However, in Pygl‐deficient mice, hepatic glycogen accumulates instead of being released as glucose into the blood stream. As a result, Pygl‐deficient mice exhibit hepatomegaly and fasting hypoglycemia. Moreover, hepatic glycogen accumulation increases with age in Pygl‐deficient mice. Thus, prolonged Pygl deficiency leads to excessive buildup of hepatic glycogen, liver damage, inflammation, and collagen deposition, all of which may increase the risk of liver fibrosis.
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