A Mouse Model of Glycogen Storage Disease Type IX-Beta: A Role for Phkb in Glycogenolysis

Abstract

Glycogen storage disease type IX (GSD-IX) constitutes nearly a quarter of all GSDs. This ketotic form of GSD is caused by mutations in phosphorylase kinase (PhK), which is composed of four subunits (α, β, γ, δ). PhK is required for the activation of the liver isoform of glycogen phosphorylase (PYGL), which generates free glucose-1-phosphate monomers to be used as energy via cleavage of the α -(1,4) glycosidic linkages in glycogen chains. Mutations in any of the PhK subunits can negatively affect the regulatory and catalytic activity of PhK during glycogenolysis. To understand the pathogenesis of GSD-IX-beta, we characterized a newly created PHKB knockout (Phkb−/−) mouse model. In this study, we assessed fasting blood glucose and ketone levels, serum metabolite concentrations, glycogen phosphorylase activity, and gene expression of gluconeogenic genes and fibrotic genes. Phkb−/− mice displayed hepatomegaly with lower fasting blood glucose concentrations. Phkb−/− mice showed partial liver glycogen phosphorylase activity and increased sensitivity to pyruvate, indicative of partial glycogenolytic activity and upregulation of gluconeogenesis. Additionally, gene expression analysis demonstrated increased lipid metabolism in Phkb−/− mice. Gene expression analysis and liver histology in the livers of old Phkb−/− mice (>40 weeks) showed minimal profibrogenic features when analyzed with age-matched wild-type (WT) mice. Collectively, the Phkb−/− mouse recapitulates mild clinical features in patients with GSD-IX-beta. Metabolic and molecular analysis confirmed that Phkb−/− mice were capable of sustaining energy homeostasis during prolonged fasting by using partial glycogenolysis, increased gluconeogenesis, and potentially fatty acid oxidation in the liver.

Keywords: glucose; glycogenolysis; hepatomegaly; hypoglycemia; ketosis.

Figure 1

Generation of Phosphorylase kinase beta deficient mouse. (A) Schematic representation of the Phkb-knockout and WT alleles. Deletion of 329 bp of exon 4 and flanking sequence of the phosphorylase kinase beta (Phkb) gene generates Phkb-knockout allele without exon 4. Arrows indicate the primer set used for genotyping. (B) PCR amplification of two PCR fragments, 485 bp and 156 bp in length amplified genomic DNA of Phkb^−/− and WT (Phkb^+/+). (C) Relative mRNA expression (Fold change) of phosphorylase kinase beta subunit gene (Phkb) in wild-type (n = 5; WT, white bar) and Phkb^−/− (n = 11, grey bar) mice. (D) Representative images of livers (WT, left; Phkb^−/− right). (E) Mean Liver weight to Body weight (LW/BW) percentage in young nonfasted (WT = 38, Phkb^−/− = 22) mice. mRNA expression and LW/BW percentage data were expressed as Mean ± SEM with ** p < 0.01.

Figure 2

Phenotypical analysis of Phkb^−/− mice. (A) Fasting glucose and (B) ketone tests were performed in (WT, n = 11, Phkb^−/−, n = 9) mice. Blood glucose and ketone (Beta-hydroxybutyrate) levels were measured at 0, 2, 4, 6, and 8 h intervals; Mean blood glucose (mg/dL) in Phkb^−/− mice (black dots) showed reduced baseline (0 h) blood glucose levels and significantly lower blood glucose levels at 6 (* p < 0.05) and 8 (** p < 0.01) hour intervals. WT (white dot) maintained blood glucose levels >160 mg/dL through fasting; fasting in Phkb^−/− mice revealed significantly elevated ketone bodies (mM) at baseline (0 h, * p < 0.05) and at all fasting intervals (0 h; * p < 0.05, 2 h, 4 h, 6 h, and 8 h; ** p < 0.01 compared to WT mice. (C) Enzymatic activity of glycogen phosphorylase (PYGL) in wild-type (n = 6; WT, white bar), Pygl^−/− (n = 6, light grey bar) and Phkb^−/− (n = 6, grey bar) mice. (D) Hepatic glycogen and (E) free glucose levels at 0 h, 2 h, and 6 h of fasted WT (0 h, n = 11, 2 h, n = 13, and 6 h, n = 13), Pygl^−/− (0 h, n = 7, 2 h, n = 5, and 6 h, n = 5), and Phkb^−/− (0 h, n = 9, 2 h, n = 6, and 6 h, n = 7) mice. (F) Serum levels of ALT, AST in WT (n = 7) and Phkb^−/− (n = 7) mice. (G) Pyruvate tolerance test for WT and Phkb^−/− mice with mean blood glucose levels in mice (WT, n = 9 and Phkb^−/− n = 6) through time intervals following pyruvate bolus injection (2.0 g/kg) after 16 h of fasting (0 h). (H) Hepatic triglyceride contents in WT (n = 9) and Phkb^−/− (n = 10) mice. Fasting blood ketone, hepatic glycogen, hepatic free glucose, ALT, AST, and TG data shown as box and whisker plot, Min to Max. Fasting glucose levels and pyruvate tolerance test are expressed as the mean ± SEM. * p < 0.05 and ** p < 0.01.

Figure 3

mRNA expression profile of gluconeogenic genes and fibrotic genes in WT and Phkb^−/− mice. Quantification of hepatic mRNA for (A) gluconeogenic genes (mG6Pc, Fbp1, and Aldob), WT (0 h, n = 5, 2 h, n = 7, and 6 h, n = 5) and Phkb^−/− (0 h, n = 11, 2 h, n = 6, and 6 h, n = 5) mice. (B) fibrosis related genes (Ctgf and Col1a1), WT (0 h, n = 5, 2 h, n = 7, and 6 h, n = 7) and Phkb^−/− (0 h, n = 11, 2 h, n = 6, and 6 h, n = 7) mice. Data represent the mean ± SD. * p < 0.05 and ** p < 0.01. Abbreviations: mG6Pc, mouse Glucose-6-phosphatase-α, Fbp1, Fructose-bisphosphatase 1, Aldob, Aldolase b. Ctgf, Connective tissue growth factor. Col1a1, Type I collagen.

Figure 4

Histologic analysis of livers from WT and Phkb^−/− mice. Representative images of liver sections stained with H&E, PAS, Masson’s trichrome, and Oil Red O in old WT mice (A1,B1,C1,D1), old Phkb^−/− mice (A2,B2,C2–C4,D2–D4). With Masson’s Trichrome staining, an individual old Phkb^−/− mouse exhibited minimal collagen deposition in minimal collagen deposition in perisinusoidal areas (C2); the images in (C2) present higher magnification views of the image of (C2). Scale bars represent 100 µm.