Glycolytic-to-oxidative fiber-type switch and mTOR signaling activation are early-onset features of SBMA muscle modified by high-fat diet

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The mechanism by which expansion of polyglutamine in AR causes muscle atrophy is unknown. Here, we investigated pathological pathways underlying muscle atrophy in SBMA knock-in mice and patients. We show that glycolytic muscles were more severely affected than oxidative muscles in SBMA knock-in mice. Muscle atrophy was associated with early-onset, progressive glycolytic-to-oxidative fiber-type switch. Whole genome microarray and untargeted lipidomic analyses revealed enhanced lipid metabolism and impaired glycolysis selectively in muscle. These metabolic changes occurred before denervation and were associated with a concurrent enhancement of mechanistic target of rapamycin (mTOR) signaling, which induced peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC1α) expression. At later stages of disease, we detected mitochondrial membrane depolarization, enhanced transcription factor EB (TFEB) expression and autophagy, and mTOR-induced protein synthesis. Several of these abnormalities were detected in the muscle of SBMA patients. Feeding knock-in mice a high-fat diet (HFD) restored mTOR activation, decreased the expression of PGC1α, TFEB, and genes involved in oxidative metabolism, reduced mitochondrial abnormalities, ameliorated muscle pathology, and extended survival. These findings show early-onset and intrinsic metabolic alterations in SBMA muscle and link lipid/glucose metabolism to pathogenesis. Moreover, our results highlight an HFD regime as a promising approach to support SBMA patients. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1550-4) contains supplementary material, which is available to authorized users.

Top KEGG pathways (gene set) identified based on the genes differentially expressed in AR97Q and CTR mice fed a normal chow diet (FDR q-val ≤ 0.25) identified in a previous microarray analysis [3]. Pathways dysregulated in both AR113Q and AR97Q are highlighted in blue. ES = enrichment score, NES = normalized enrichment score, NOM p-val = nominal p value, FDR q-val = False Discovery Rate, FWER p-val = family-wise error rate, RANK AT MAX = position in the ranked list at which the maximum enrichment score occurred.

Supplementary Figures
Supplementary Figure 1 Supplementary Figure 1. The mass and force of glycolytic muscles is decreased in SBMA knock-in mice. a) Muscle weight analysis in wild type (WT, C57Bl6J) and AR21Q mice. The wet weight of quadriceps (quadri), gastrocnemius (gastro), tibialis anterior (TA), and soleus was similar in 180-day-old WT and AR21Q mice.
Graph shows mean ± sem, n = 5-11 mice. b) Wet weight of the indicated skeletal muscles of AR113Q and control (CTR, wild type) mice analyzed at different ages. Graphs show mean ± sem, n = 3-10 mice. c) In vivo force generation of gastrocnemius muscle measured in live 90-day-old AR113Q and CTR (wild type) mice. Tetanic force was decreased in AR113Q mice compared to CTR mice. Graph shows mean ± sem, n = 4 mice. d) Ex vivo force generation of soleus muscle measured in 180-day-old AR113Q and CTR (wild type) mice.
Tetanic force was similar in AR113Q mice compared to CTR mice. Graph shows mean ± sem, n = 5 mice.

Supplementary Figure 2. The cross-sectional area of glycolytic muscles is decreased in SBMA knock-in mice.
a) Hematoxylin and eosin cross-sectional area (CSA) analysis of transversal sections of the indicated muscles of 90-day-old AR113Q and CTR (wild type) mice revealed that the CSA of these glycolytic muscles is decreased in AR113Q compared to CTR mice. Graphs show mean ± sem, n = 3 mice, n fibers = 2,125 CTR, 2,377 AR113Q (quadriceps); 1,398 CTR, 1,975 AR113Q (gastrocnemius); 1,263 CTR, 1,579 AR113Q (tibialis anterior). b) Hematoxylin and eosin CSA analysis of transversal sections of the indicated muscles of 90-day-old AR113Q and AR21Q mice confirmed that the CSA of quadriceps, gastrocnemius, and TA muscles is decreased in AR113Q mice compared to AR21Q mice. Graphs show mean ± sem, n = 3 mice, n fibers = 1,836 AR21Q, 2,377 AR113Q (quadriceps); 1,706 AR21Q, 1,975 AR113Q (gastrocnemius); 1,059 AR21Q, 1,579 AR113Q (TA). c) Hematoxylin and eosin CSA analysis of transversal sections of the indicated muscles of 180-day-old AR113Q and CTR (wild type) mice showed that the CSA of soleus is increased, whereas that of gastrocnemius is decreased in AR113Q mice. Graphs show mean ± sem, n = 3 mice, n fibers = 2,356 CTR, 2,409 AR113Q (gastrocnemius); 3,322 CTR, 2,282 AR113Q (soleus). a) Immunofluorescence analysis of type IIa (green) and IIb (red) myosin heavy chain-positive fibers in the quadriceps muscle of 180-day-old AR113Q and CTR (wild type) mice showed that the number of type IIa fibers is increased in SBMA muscle. Shown are representative images of quadriceps muscle transversal sections (n = 3 mice). b) NADH staining of transversal sections of gastrocnemius muscle from 180-day-old AR113Q and CTR (wild type) mice revealed that the number of oxidative fibers is increased in AR113Q mice. Graph shows mean ± sem, n mice = 3-5, n fibers = 1,298 AR113Q and 1,273 CTR. Figure 4. Atrophy of glycolytic fibers exceeds that of oxidative fibers in AR113Q mice. a) CSA distribution of oxidative and glycolytic fibers of quadriceps in 40-, 90-, and 180-day-old AR113Q and CTR (wild type) mice. Graphs show mean ± sem; 40-day-old mice: n mice = 5 AR113Q and CTR, n fibers = 1,490 AR113Q and 1,129 CTR; 90-day-old mice: n mice = 8 AR113Q and CTR, n fibers = 1,674 AR113Q and 1,401 CTR; 180-day-old mice: n mice = 3 AR113Q and 5 CTR, n fibers = 880 AR113Q and 771 CTR. b) CSA distribution of oxidative and glycolytic fibers of gastrocnemius in 180-day-old AR113Q and CTR (wild type) mice. Graphs show mean ± sem, n mice = 3-5 for each group, n fibers = 1,298 AR113Q and 1,273 CTR. a) Steady-state serum glucose and insulin levels were measured upon 4 hour fasting in AR113Q and CTR (wild type) mice fed a normal chow diet. Steady-state glucose and insulin levels were normal in AR113Q mice.
b) Intraperitoneal glucose tolerance test (IPGTT) performed in 180-day-old AR113Q and CTR (wild type) mice fed either a normal chow diet (NCD) or a high-fat diet (HFD) upon 18 hour fasting. IPGTT revealed glucose intolerance in CTR-HFD mice, but not AR113Q-HFD mice. Graphs show mean ± sem, n = 4 mice, *0.05. CTR (wild type) mice showed that protein synthesis is increased at late stages of disease in AR113Q mice.
Puromycin incorporation was detected with anti-puromycin (anti-Pur) antibody, and Red Ponceau (Red P) was used as loading control. Graph shows mean ± sem, n = 4-5 mice.
b) Western blotting as in (a) in mice upon 4 hours fasting showed that protein synthesis is increased in AR113Q mice even upon fasting. Graph shows mean ± sem, n = 6-8 mice.
c) Western blotting analysis of protein ubiquitination in the quadriceps of 270-day-old mice showed that protein degradation is increased in AR113Q mice. Ubiquitinated proteins were detected with anti-ubiquitin (anti-Ub) antibody. Graph shows mean ± sem, n = 4-5 mice.
d) Proteasome activity was measured in muscle lysates in the presence of the indicated concentrations of the proteasome inhibitor, MG132. As expected, MG132 inhibited proteasome activity in a dose-dependent manner, indicating that the signal measured in tissues is specific. e) Total protein content was decreased in the quadriceps of 270-day-old AR113Q mice compared to CTR mice.
Graph shows mean ± sem, n = 4-5 mice. b) Body weight analysis revealed that rapamycin treatment causes body weight loss in AR113Q, but not CTR (wild type), mice. Graph shows mean ± sem, n = 7-11 mice.

Supplementary
c) Food intake analysis showed that rapamycin does not alter the feeding behavior of 170-day-old AR113Q and CTR (wild type) mice. Graph, mean ± sem, n = 5-9 mice. d) Testis weight in AR113Q and CTR (wild type) mice. Rapamycin caused testicular atrophy in both AR113Q and CTR mice. Graph, mean ± sem, n = 7-11 mice.
e) Quadriceps and gastrocnemius muscle wet weight. Rapamycin did not modify the mass of quadriceps and gastrocnemius muscles of AR113Q mice. Graph, mean ± sem, n = 7-11 mice. Locomotor activity was recorded by a video tracking system with computer interface and video camera (ANYmaze; Stoelting). Parameters were set within the ANY-maze program to ensure continuous movement tracking.
Distance traveled within the cage was analyzed as a measure of activity. Mouse locomotor activity was assessed every hour for 48 hours. Graph, mean ± sem, n = 5-8 mice.
f) Force of gastrocnemius muscle measured in living 180-day-old AR113Q and CTR (wild type) mice. Graph shows mean ± sem, n = 8.

Animals and treatments
Animal care and experimental procedures were conducted in accordance with the Italian Institute of Technology and the University of Trento ethical committees and were approved by the Italian Ministry of Health.
Generation and genotyping of knock-in AR21Q and AR113Q mice were previously described [5]. Male mice were used in this study, and AR113Q female mice were used as indicated (Supplementary Figure 12).

Human samples
Anonymized control and patient biopsy sample collection was approved by the ethics committee of the University of Padova (Italy). Written informed consent was obtained from each patient. Confidentiality was guaranteed by assigning a study code to each patient. All patients who underwent muscle biopsy were clinically affected and showed weakness and/or fasciculation and/or muscle atrophy. Myopathic changes together with neurogenic atrophy were observed in muscle biopsies.

Histological analysis
Muscles were collected immediately after euthanasia, flash-frozen in isopentane precooled in liquid nitrogen, and stored at -80°C until further processing. Frozen muscles were embedded in optimal cutting temperature (OCT) compound (Tissue Tek, Sakura), and cross-sections (10 µm thick) were cut with a cryostat For analysis of enzymatic activity, lactate dehydrogenase (LDH) activity was measured in total muscle homogenate by following the rate of NADH oxidation at 340 nm for 3 minutes at 25°C, as previously described [2]. For analysis of enzymatic activity, lactate dehydrogenase (LDH) activity was measured in total muscle homogenate by following the rate of NADH oxidation at 340 nm for 3 minutes at 25°C, as previously described [2]. Citrate synthase activity was assessed in total muscle homogenate by following the rate of 5-thio-2-nitrobenzoic acid (TNB) formation at 412 nm for 5 minutes at 37°C, as previously described [4]. For proteasome activity assay, muscles were lysed The proteasome inhibitor MG132 (Calbiochem) was used as a control for the assay.

High-resolution LC-MS/MS analysis for lipidomic profiling
Solvents and chemicals were purchased from Sigma and were used without further purification. Analyses were conducted on a UPLC Acquity system coupled to a Synapt G2 QToF mass spectrometer (Waters).
Samples were analyzed using a reversed-phase C18 T3 column (2.1 x 100 mm) kept at 55°C at a flow rate of 400 μl/min. The following gradient conditions were utilized: eluent A was 10 mM ammonium formate in 60:40 ACN/water, eluent B was 10 mM ammonium formate in 90 / 10 isopropyl alcohol / acetonitrile; after 1 minute at 30%, solvent B was brought to 35% in 3 minutes, then to 50% in 1 minute, and then to 100% in 13 minutes, followed by a 1-minute 100% B isocratic step and reconditioning to 30% B. Total run time was 22 minutes.
Injection volume was set at 3 ml. The capillary voltages were set at 3 kV and 2 kV for ESI+ and ESI-, respectively.
The cone voltages were set at 30V for ESI+ and 35V for ESI-. The source temperature was 120°C. Desolvation gas and cone gas (N2) flow were 800 and 20 l/h, respectively. Desolvation temperature was 400°C. Data were acquired in MSe mode2 with MS/MS fragmentation performed in the trap region. Low-energy scans were acquired at fixed 4eV potential and high-energy scans were acquired with an energy ramp from 25 to 45 eV. Scan rate was set to 0.3 seconds per spectrum. Scan range was set to 50 to 1200 m/z. Leucine enkephalin (2 ng/ml) was infused as lock mass for spectra recalibration. Raw data from high resolution LC-MS/MS runs were subjected to principal component analysis (PCA) using MarkerLynx software (Waters). Metabolite accurate mass and retention time values were included in the multivariate analysis and assigned as X-variables. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) and scatter plots were used to identify metabolites whose abundance was significantly altered between the experimental groups (P < 0.01 was set as minimum threshold for significance). To evaluate the over/under expression value, the peak area of each metabolite was normalized to the total metabolite intensity to generate a normalized peak area. Detected features were exported in a Microsoft Excel datasheet and subjected to two-tailed t-test for significance. Analytes were identified by interrogating the METLIN, HMDB, and LipidMaps databases. Tolerance on m/z values was set to 5 ppm. Identification was based on both accurate mass and calculated brute formula matching and, whenever possible, confirmed with tandem mass data. analyzed using the limma package from R and false discovery rate (FDR) control for statistical assessment of the microarray data (corrected P < 0.05 were considered significant). Gene set enrichment analysis (GSEA) was performed to identify sets of related genes altered in each experimental group (http://www.broad.mit.edu/gsea). Heat maps were generated using MeV TM4 software V 4.9 (http://www.tm4.org). Microarray data are available at the Gene Expression Omnibus database under accession number GSE68441.

Mitochondrial membrane potential and complex activity analyses
Mitochondrial membrane potential was measured in isolated fibers from flexor digitorum brevis muscles.
Mitochondrial membrane potential was measured by epifluorescence microscopy based on the accumulation of were added to the cell culture medium. TMRM staining was monitored in at least 10 fibers per group. Images were acquired, stored, and analysis of TMRM fluorescence over mitochondrial regions of interest was performed using ImageJ. For mitochondrial complex activity, total muscle lysates were extracted and the enzymatic activities of the respiratory chain complexes I-IV were assayed in duplicate or triplicate with a single-wavelength, temperaturecontrolled spectrophotometer at 37°C, as previously described [4]. The enzymatic activities for each mitochondrial enzyme was calculated as nmol min−1 mg−1 of protein and normalized to the activity of CS where indicated, which was used as a marker of the abundance of mitochondria.

Analysis of muscle force
To measure muscle force in living animals, the contractile performance of gastrocnemius muscle in vivo was measured as previously described [1]. Briefly, anesthetized mice were placed on a thermostatically controlled table, keeping the knee stationary, and the foot firmly fixed to a footplate, which was connected to the shaft of the motor of a muscle-lever system (305B, Aurora Scientific). Contraction was elicited by electrical stimulation of sciatic nerve. Teflon-coated seven-stranded steel wires (AS 632, Cooner Sales) were implanted with sutures on either side of the sciatic nerve proximal to the knee before its branching. At the distal ends of the two wires, the insulation was removed, and the proximal ends were connected to a stimulator (S88, Grass). To avoid recruitment of the dorsal flexor muscles, the common peroneal nerve was cut. For in vitro muscle mechanics, force measurements