Elevated GLUT4 and glycogenin protein abundance correspond to increased glycogen content in the soleus muscle of mdx mice with no benefit associated with taurine supplementation

Abstract

Duchenne muscular dystrophy (DMD) patients and the dystrophic mdx mouse have an elevated demand for ATP requiring processes, including Ca²⁺ regulation and skeletal muscle regeneration. As a key substrate for cellular ATP production, altered glycogen metabolism may contribute significantly to dystrophic pathology and explain reports of mild glucose intolerance. We compare the soleus and extensor digitorum longus (EDL) muscles of the mdx mouse during active muscle necrosis (at 28 days) and at 70 days where pathology is stable. We further investigate the impact of taurine (tau) on dystrophic glycogen metabolism to identify if the benefit seen with tau in a previous study (Barker et al. ) was in part owed to altered glycogen handling. The soleus muscle of 28- and 70-day-old mdx mice had elevated glucose transporter type 4 (GLUT4), glycogenin protein abundances and glycogen content compared to WT (C57BL10/ScSn) controls. Mdx tau mice exhibited modestly reduced glycogen compared to their respective mdx group. The EDL muscle of 28 days mdx tau mice had a ~70% increase in glycogenin protein abundance compared to the mdx but 50% less glycogen content. A twofold greater phosphorylated glycogen synthase (p-GS) and glycogen phosphorylase (p-GP) protein abundance was observed in the 70-day-old mdx soleus muscle than in the 28-day-old mdx soleus muscle. Glycogen debranching enzyme (GDE) protein abundance was elevated in both 28- and 70-day-old mdx soleus muscles compared to WT controls. We identified an increase in proteins associated with glucose uptake and utilization specific to the predominantly slow-twitch soleus muscle of mdx mice regardless of age and that taurine affords no obvious benefit to glycogen metabolism in the mdx mouse.

Keywords: Duchenne muscular dystrophy; glycogen; mdx mouse; metabolism; skeletal muscle; taurine.

Figure 1

Effect of α‐amylase treatment on abundance of glycogen synthase in the EDL muscle detected by western blot. Myosin from the Stain Free gel indicative of total protein (top), representative western blot for glycogen synthase (GS, middle) and quantification of the abundance of GS from matching muscle homogenates prepared as normal (control) or with α‐amylase (amylase) treatment, expressed relative to control for a given sample pair, mean + SD, n = 5. Student's paired t‐test. GS, glycogen synthase

Figure 2

GLUT4 and glycogenin protein abundance, and glycogen content in the soleus and EDL muscles from 28 and 70 days WT, mdx, and mdx tau mice. (A) Myosin from the Stain Free gel, indicative of total protein (top), representative western blot for GLUT4 (middle), quantification of GLUT4 abundance (bottom) in the soleus muscles. (B) As for A, but for glycogenin. bottom band revealed after amylase treatment identified as glycogenin (data not shown). (C) Soleus glycogen content from 28 days (solid symbols) and 70 days (open symbols) WT (circles), mdx (triangles), and mdx tau (squares) mice. D, E, F as for A, B, C but EDL muscles. In (D) noncontiguous lanes from the same gel separated by black vertical lane. One‐way ANOVA with Holm–Sidak's post hoc analyses between relevant groups. Data presented as data points surrounding means ± SD, n indicated by number of symbols. Lines connecting different bars indicate significance at * P < 0.05, **P < 0.01 or P‐value shown.

Figure 3

Relative skeletal muscle glycogen content as visualized by PAS stain. PAS stain of 10 μm cross‐sections of 28 and 70 days WT, mdx, and mdx tau soleus (sol, left) and EDL (right) muscles shown at 400× magnification. All images shown from each muscle (EDL or soleus) were stained simultaneously (see Methods). Darker colored muscle fibers indicate greater glycogen content – see arrows indicating a darkly stained fiber next to a weakly stained fiber. Scale bar = 100 μm, n = 5. PAS, Periodic acid–Schiff

Figure 4

Anabolic glycogen associated protein abundance in the soleus muscle of 28 and 70 days WT, mdx, and mdx tau mice. Shown for each panel is the myosin from the Stain Free gel, indicative of total protein (top), the representative western blot protein (middle), and quantification of protein abundance (bottom) from 28 days (solid symbols) and 70 days (open symbols) WT (circles), mdx (triangles), and mdx tau (squares) mice. (A) Glycogen synthase (GS), (B) phosphorylated GS (p‐GS), (C) glycogen branching enzyme, each expressed relative to average of the 28 days WT run on a given gel. One–way ANOVA with Holm–Sidak's post hoc analyses between relevant groups. Data presented as data points surrounding means ± SD, n indicated by number of symbols. Lines connecting different bars indicate significance at P < 0.05 or P‐value as shown.

Figure 5

Catabolic glycogen‐associated protein abundance in the soleus muscle of 28 and 70 days WT, mdx, and mdx tau mice. Shown for each panel is the myosin from the Stain Free gel, indicative of total protein (top), the representative western blot protein (middle), and quantification of protein abundance (bottom) from 28 days (solid symbols) and 70 days (open symbols) WT (circles), mdx (triangles), and mdx tau (squares) mice. Protein abundance expressed relative to the average of the 28 days WT run on a given gel. (A) Glycogen phosphorylase (GP), (B) phosphorylated glycogen phosphorylase (p‐GP), and (C) glycogen debranching enzyme (GDE). In (C), noncontiguous lanes from the same gel separated by black vertical lane. Note the GP and p‐GP probes were after GS which is still seen as the lower band in A and B. One‐way ANOVA with Holm–Sidak's post hoc analyses between relevant groups. Data presented as data points surrounding means ± SD, n indicated by number of symbols. Lines connecting different bars indicate significance at *P < 0.05, ***P < 0.001 or P‐value as shown.

Figure 6

Abundances of glycogen‐associated proteins in the EDL muscle from 28 and 70 days WT, mdx, and mdx tau mice. Shown for each panel is the myosin from the Stain Free gel, indicative of total protein (top), the representative western blot protein (middle), and quantification of protein abundances (bottom) from 28 days (solid symbols) and 70 days (open symbols) WT (circles), mdx (triangles), and mdx tau (squares) mice. Phosphorylated proteins are expressed relative to its total protein, that is glycogen synthase (GS) or glycogen phosphorylase (GP) (A) GS, B. phosphorylated GS (p‐GS), (C) glycogen branching enzyme (GBE), (D) GP, (E) phosphorylated GP (p‐GP). and (F) glycogen debranching enzyme (GDE). Noncontiguous lanes from the same gel separated by black vertical lane. Protein abundance expressed relative to the average of 28 days WT. One‐way ANOVA with Holm–Sidak's post hoc analyses between relevant groups. Data presented as data points surrounding means ± SD, n indicated by number of symbols. Significance at P < 0.05.