Dysregulation of multiple facets of glycogen metabolism in a murine model of Pompe disease

Abstract

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA(-/-) mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA(-/-) mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA(-/-) mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA(-/-) mice, phosphorylase activity is suppressed in the GAA(-/-) mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity.

Figure 1. Glycogen is not reduced in GAA^−/− mice lacking S6K1 and S6K2.

Glycogen was quantified in tissue lysates collected from groups (n = 10) of 3–4 month old mice of the indicated strains. Values shown are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing all groups to C57Bl/6. ***P<0.001.

Figure 2. AMP-dependent phosphorylase activity is reduced in GAA^−/− mice.

Phosphorylase activity was quantified in muscle lysates from triceps collected from groups (n = 5) of 3–4 month old mice of the indicated. Values shown are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing all groups to C57Bl/6 in the absence of AMP. ***P<0.001.

Figure 3. Muscle glycogen synthase protein is elevated in GAA^−/− mice.

Tissue lysates were prepared for the indicated tissues from each mouse strain. Lysates from 5 mice were pooled and 100 µg of protein from the pooled lysate was analyzed by Western blot analysis. All of the blots were probed with an anti-GAPDH antibody (∼37 kDa in all panels) to verify equal protein loading of all the test samples. (A) An anti-muscle glycogen synthase antibody was used to probe the blots containing samples from the heart, triceps, and quad. An anti-liver glycogen synthase antibody was used to probe the blot containing samples from the liver. (B) An anti-phospho-glycogen synthase (Ser641) antibody was used to probe the blots for phosphorylated GS. (C) Glycogen synthase transcript levels are not elevated in GAA^−/− mice compared to wild type animals. RNA was isolated from triceps and heart of C57Bl/6 and GAA^−/− mice and probed with qPCR primers as described under “Experimental Procedures.” Values shown are means ± SEM.

Figure 4. rhGAA treatment normalizes glycogen synthase protein and activity levels in heart of GAA^−/− mice.

Groups (n = 5) of 3 to 4-month old mice of the indicated strains were dosed with rhGAA (100 mg/kg) weekly for 4 weeks by tail vein injection. Muscle homogenates were prepared, pooled and analyzed by: (A) Western blot using an anti-GS antibody and (B) densitometry analysis of the western blots (with GS normalized to GAPDH)., Samples were also processed for measurements of (C) glycogen synthase activity (D) tissue glycogen levels. Values are means ± SEM. Values are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing groups. ***P<0.001.

Figure 5. rhGAA treatment reduces glycogen synthase protein and activity levels in triceps of GAA^−/− mice.

Triceps were analyzed as described in the legend to Figure 4.

Figure 6. G6P (A) and hexokinase (B) levels are elevated in GAA^−/− mice compared to wild type (C57/Bl6) and reduced by rhGAA treatment.

GAA^−/− mice were dosed with rhGAA as described in the legend to Figure 4. G6P and hexokinase was quantified in heart and triceps homogenates from the strains indicated. Values are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing groups. ***P<0.001.

Figure 7. Glycogenin levels are dysregulated in GAA^−/− mice and normalized by rhGAA treatment.

Homogenates from triceps were prepared for the indicated strains. Lysates from 5 mice for each strain were pooled and 100 µg of protein analyzed by Western blot. A monoclonal antibody to glycogenin was used to probe the blots. (A) lysates not treated with amyloglucosidase. (B), treated with amyloglucosidase. (C) GAA^−/− mice were dosed with rhGAA as described in the legend for Figure 4. Lysates from the strains indicated in this panel were not treated with amyloglucosidase.