Amyotrophic lateral sclerosis associated disturbance of iron metabolism is blunted by swim training-role of AKT signaling pathway.

Swim training has increased the life span of the transgenic animal model of amyotrophic lateral sclerosis (ALS). Conversely, the progress of the disease is associated with the impairment of iron metabolism and insulin signaling. We used transgenic hmSOD1 G93A (ALS model) and non-transgenic mice in the present study. The study was performed on the muscles taken from trained (ONSET and TERMINAL) and untrained animals at three stages of the disease: BEFORE, ONSET, and TERMINAL. In order to study the molecular mechanism of changes in iron metabolism, we used SH-SY5Y and C2C12 cell lines expression vector pcDNA3.1 and transiently transfected with specific siRNAs. The progress of ALS resulted in decreased P-Akt/Akt ratio, which is associated with increased proteins responsible for iron storage ferritin L, ferritin H, PCBP1, and skeletal muscle iron at ONSET. Conversely, proteins responsible for iron export- TAU significantly decrease. The training partially reverses changes in proteins responsible for iron metabolism. AKT silencing in the SH-SY5Y cell line decreased PCBP2 and ferroportin and increased ferritin L, H, PCBP1, TAU, transferrin receptor 1, and APP. Moreover, silencing APP led to an increase in ferritin L and H. Our data suggest that swim training in the mice ALS model is associated with significant changes in iron metabolism related to AKT activity. Down-regulation of AKT mainly upregulates proteins involved in iron import and storage but decreases proteins involved in iron export.

Swim Training Affects on Muscle Lactate Metabolism, Nicotinamide Adenine Dinucleotides Concentration, and the Activity of NADH Shuttle Enzymes in a Mouse Model of Amyotrophic Lateral Sclerosis.

In this study, we aim to verify whether swim training can improve lactate metabolism, NAD+ and NADH levels, as well as modify the activity of glycolytic and NADH shuttle enzymes and monocarboxylate transporters (MCTs) in skeletal muscle of amyotrophic lateral sclerosis (ALS) mice. ALS mice (SOD1G93A) (n = 7 per group) were analyzed before the onset of ALS, at first disease symptoms (trained and untrained), and the last stage of disease (trained and untrained), and then compared with a wild-type (WT) group of mice. The blood lactate and the skeletal muscle concentration of lactate, NAD+ and NADH, MCT1 and MCT4 protein levels, as well as lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in skeletal muscle were determined by fluorometric, Western blotting, liquid chromatography-MS3 spectrometry, and spectrometric methods. In the untrained terminal ALS group, there were decreased blood lactate levels (p < 0.001) and increased skeletal muscle lactate levels (p < 0.05) as compared with a WT group of mice. The amount of nicotinamide adenine dinucleotides in the ALS groups were also significantly reduced as well as LDH activity and the level of MCT1. Swim training increased lactate levels in the blood (p < 0.05 vs. ALS TERMINAL untrained). In addition, cytosolic MDH activity and the cMDH/LDH 2.1 ratio were significantly higher in trained vs. untrained mice (p < 0.05). The data indicate significant dysfunction of lactate metabolism in ALS mice, associated with a reduction in muscle anaerobic metabolism and NADH transporting enzymes, as well as swim-induced compensation of energy demands in the ALS mice.

Swim training affects Akt signaling and ameliorates loss of skeletal muscle mass in a mouse model of amyotrophic lateral sclerosis.

We tested the hypothesis that swim training reverses the impairment of Akt/FOXO3a signaling, ameliorating muscle atrophy in ALS mice. Transgenic male mice B6SJL-Tg (SOD1G93A) 1Gur/J were used as the ALS model (n = 35), with wild-type B6SJL (WT) mice as controls (n = 7). ALS mice were analyzed before ALS onset, at ALS onset, and at terminal ALS. Levels of insulin/Akt signaling pathway proteins were determined, and the body and tibialis anterior muscle mass and plasma creatine kinase. Significantly increased levels of FOXO3a in ALS groups (from about 13 to 21-fold) compared to WT mice were observed. MuRF1 levels in the ONSET untrained group (12.0 ± 1.7 AU) were significantly higher than in WT mice (1.12 ± 0.2 AU) and in the BEFORE ALS group (3.7 ± 0.9 AU). This was associated with body mass and skeletal muscle mass reduction. Swim training significantly ameliorated the reduction of skeletal muscle mass in both TERMINAL groups (p < 0.001) and partially reversed changes in the levels of Akt signaling pathway proteins. These findings shed light on the swimming-induced attenuation of skeletal muscle atrophy in ALS with possible practical implications for anti-cachexia approaches.

Swim Training Modulates Mouse Skeletal Muscle Energy Metabolism and Ameliorates Reduction in Grip Strength in a Mouse Model of Amyotrophic Lateral Sclerosis.

Metabolic reprogramming in skeletal muscles in the human and animal models of amyotrophic lateral sclerosis (ALS) may be an important factor in the diseases progression. We hypothesized that swim training, a modulator of cellular metabolism via changes in muscle bioenergetics and oxidative stress, ameliorates the reduction in muscle strength in ALS mice. In this study, we used transgenic male mice with the G93A human SOD1 mutation B6SJL-Tg (SOD1G93A) 1Gur/J and wild type B6SJL (WT) mice. Mice were subjected to a grip strength test and isolated skeletal muscle mitochondria were used to perform high-resolution respirometry. Moreover, the activities of enzymes involved in the oxidative energy metabolism and total sulfhydryl groups (as an oxidative stress marker) were evaluated in skeletal muscle. ALS reduces muscle strength (-70% between 11 and 15 weeks, p < 0.05), modulates muscle metabolism through lowering citrate synthase (CS) (-30% vs. WT, p = 0.0007) and increasing cytochrome c oxidase and malate dehydrogenase activities, and elevates oxidative stress markers in skeletal muscle. Swim training slows the reduction in muscle strength (-5% between 11 and 15 weeks) and increases CS activity (+26% vs. ALS I, p = 0.0048). Our findings indicate that swim training is a modulator of skeletal muscle energy metabolism with concomitant improvement of skeletal muscle function in ALS mice.

Swim Training Modulates Skeletal Muscle Energy Metabolism, Oxidative Stress, and Mitochondrial Cholesterol Content in Amyotrophic Lateral Sclerosis Mice.

Recently, in terms of amyotrophic lateral sclerosis (ALS), much attention has been paid to the cell structures formed by the mitochondria and the endoplasmic reticulum membranes (MAMs) that are involved in the regulation of Ca2+ signaling, mitochondrial bioenergetics, apoptosis, and oxidative stress. We assumed that remodeling of these structures via swim training may accompany the prolongation of the ALS lifespan. In the present study, we used transgenic mice with the G93A hmSOD1 gene mutation. We examined muscle energy metabolism, oxidative stress parameters, and markers of MAMs (Caveolin-1 protein level and cholesterol content in crude mitochondrial fraction) in groups of mice divided according to disease progression and training status. The progression of ALS was related to the lowering of Caveolin-1 protein levels and the accumulation of cholesterol in a crude mitochondrial fraction. These changes were associated with aerobic and anaerobic energy metabolism dysfunction and higher oxidative stress. Our data indicated that swim training prolonged the lifespan of ALS mice with accompanying changes in MAM components. Swim training also maintained mitochondrial function and lowered oxidative stress. These data suggest that modification of MAMs might play a crucial role in the exercise-induced deceleration of ALS development.

Methyl-beta-cyclodextrin induces mitochondrial cholesterol depletion and alters the mitochondrial structure and bioenergetics.

There is growing evidence of mitochondrial membrane raft-like microdomains that are involved in the apoptotic pathway. The aim of this study was to investigate the effect of methyl-beta-cyclodextrin (MßCD), being a well-known lipid microdomain disrupting agent and cholesterol chelator, on the structure and bioenergetics of rat liver mitochondria (RLM). We observed that MßCD decreases the function of RLM, induces changes in the mitochondrial configuration state and decreases the calcium chloride-induced swelling. These data suggest that disruption of mitochondrial raft-like microdomains by cholesterol efflux on one hand impairs mitochondrial bioenergetics, but on the other hand it protects the mitochondria from swelling.