Impaired muscle uptake of creatine in spinal and bulbar muscular atrophy

Abstract

Objective: The aim of this study was to explore the pathomechanism underlying the reduction of serum creatinine (Cr) concentrations in spinal and bulbar muscular atrophy (SBMA).

Methods: We evaluated blood chemistries, motor function, and muscle mass measured by dual-energy X-ray absorptiometry in male subjects with SBMA (n = 65), amyotrophic lateral sclerosis (ALS; n = 27), and healthy controls (n = 25). We also examined the intramuscular concentrations of creatine, a precursor of Cr, as well as the protein and mRNA expression levels of the creatine transporter (SLC6A8) in autopsy specimens derived from subjects who had SBMA and ALS and disease controls. Furthermore, we measured the mRNA expression levels of SLC6A8 in cultured muscle cells (C2C12) transfected with the polyglutamine-expanded androgen receptor (AR-97Q).

Results: Serum Cr concentrations were significantly lower in subjects with SBMA than in those with ALS (P < 0.001), despite similar muscle mass values. Intramuscular creatine concentrations were also lower in with the autopsied specimen of SBMA subjects than in those with ALS subjects (P = 0.018). Moreover, the protein and mRNA expression levels of muscle SLC6A8 were suppressed in subjects with SBMA. The mRNA levels of SLC6A8 were also suppressed in C2C12 cells bearing AR-97Q.

Interpretation: These results suggest that low serum Cr concentration in subjects with SBMA is caused by impaired muscle uptake of creatine in addition to being caused by neurogenic atrophy. Given that creatine serves as an energy source in skeletal muscle, increasing muscle creatine uptake is a possible therapeutic approach for treating SBMA.

Figure 1

Relationship between serum creatinine and muscle mass. (A, B) Comparison of the relationship between serum Cr concentration and ALST mass in SBMA, ALS, and healthy controls. N.S., not significant, ***P < 0.001 by analysis of covariance. ALS, amyotrophic lateral sclerosis; ALST, appendicular lean soft tissue; Cr, creatinine; SBMA, spinal and bulbar muscular atrophy; HC, healthy controls.

Figure 2

Muscle mass and creatine–creatinine metabolism in subjects with SBMA, ALS, and controls. The mean value of ALST mass (A), serum Cr (B), and serum creatine (C) in subjects with SBMA, ALS, and in healthy controls. (D) The intramuscular creatine in subjects with SBMA, ALS, and in disease controls. (E) The intramuscular creatine in the wild‐type mice and AR‐97Q mice, a transgenic mouse model of SBMA. *P < 0.05, **P < 0.01, and ***P < 0.001 by Tukey's multiple comparison test. Data depict the mean ± SE values. ALS, amyotrophic lateral sclerosis; AR, androgen receptor; ALST, appendicular lean soft tissue; Cr, creatinine; DC, disease controls; SBMA, spinal and bulbar muscular atrophy; HC, healthy controls.

Figure 3

SLC6A8 expression in human subjects and muscular cells. (A) Representative microphotographs of autopsied iliopsoas muscle specimens immunostained for SLC6A8. (B) Quantitative analysis of anti‐SLC6A8 immunohistochemistry in SBMA, ALS, and other disease controls (n = 3 in each group). (C, D) Immunoblotting with quantitative analysis of SLC6A8 protein expression obtained from autopsied muscle specimens of controls (lanes 1, 2, and 3), ALS (lanes 4, 5 and 6), and SBMA (lanes 7, 8, and 9). (E) Quantitative RT‐PCR analysis of muscle SLC6A8 gene expression levels in subjects with SBMA, ALS, and DC (n = 3 in each group). (F) Immunoblotting of SLC6A8 protein expression in C2C12 cells stably expressing full‐length AR‐24Q (lanes 1, 2, and 3) or AR‐97Q (lanes 4, 5 and 6). (G) Quantitative analysis of SLC6A8 protein expression in C2C12 cells (n = 6). (H) SLC6A8 gene expression levels of full‐length AR‐24Q or AR‐97Q‐expressing C2C12 cells (n = 3 in each group). Data depict the mean ± SE values. ALS, amyotrophic lateral sclerosis; ALST, appendicular lean soft tissue; AR, androgen receptor; Cr, creatinine; DC, disease controls; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SBMA, spinal and bulbar muscular atrophy. Scale bar: 25 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 by Tukey's multiple comparison test.