Ascorbic Acid Supplementation Improves Skeletal Muscle Growth in Pacu (Piaractus mesopotamicus) Juveniles: In Vivo and In Vitro Studies

Abstract

In fish, fasting leads to loss of muscle mass. This condition triggers oxidative stress, and therefore, antioxidants can be an alternative to muscle recovery. We investigated the effects of antioxidant ascorbic acid (AA) on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu (Piaractus mesopotamicus) following fasting, using in vitro and in vivo strategies. Isolated muscle cells of the pacu were subjected to 72 h of nutrient restriction, followed by 24 h of incubation with nutrients or nutrients and AA (200 µM). Fish were fasted for 15 days, followed by 6 h and 15 and 30 days of refeeding with 100, 200, and 400 mg/kg of AA supplementation. AA addition increased cell diameter and the expression of anabolic and cell proliferation genes in vitro. In vivo, 400 mg/kg of AA increased anabolic and proliferative genes expression at 6 h of refeeding, the fiber diameter and the expression of genes related to cell proliferation at 15 days, and the expression of catabolic and oxidative metabolism genes at 30 days. Catalase activity remained low in the higher supplementation group. In conclusion, AA directly affected the isolated muscle cells, and the higher AA supplementation positively influenced muscle growth after fasting.

Amino Acids and IGF1 Regulation of Fish Muscle Growth Revealed by Transcriptome and microRNAome Integrative Analyses of Pacu (Piaractus mesopotamicus) Myotubes

“…The pacu myotubes were well separated according to the experimental groups, showing that the treatments were effective, and the cell culture replicates were similar to each other (Supplementary Figure S1). We further confirmed the effectiveness of our protocol via the digital expression of fbxo32 (f-box protein 32), a well-known marker of muscle protein degradation and atrophy [42,[47][48][49][50], and myog (myogenin), related to myogenesis and growth [51][52][53][54]. Both AA and IGF1 groups showed decreased fbxo32 transcription (fold-change = 0.37 in AA and 0.39 in IGF1; p-adj < 0.01) and increased myog transcription (fold-change = 2.02 in AA and 1.68 in IGF1; p-adj < 0.01) compared to CTR (Figure 1).…”

“…The FBXO25 is classified as an E3 ubiquitin ligase with high homology to FBXO32 in fish [95]. We previously demonstrated the increased expression of fbxo25 during fasting and down-regulation during re-feeding in pacu muscle and cultured cells [42,96], and this same molecular scenario was observed in fasted and re-fed rainbow trout [97]. In the present work, a pronounced reduction in fbxo25 expression was observed in IGF1, indicating that the protein breakdown should be stopped, so that anabolism and muscle growth take place in the treatment.…”

“…Fish myoblast cell cultures are a very powerful tool to study all of these molecular networks and signals regulating myogenesis and muscle growth [33][34][35][36][37][38][39][40][41][42]. This in vitro model encompasses the main stages of myogenesis, especially myoblast proliferation, differentiation, and fusion into myotubes [38,39,41].…”

“…This enables a more in-depth study of regulatory molecules and the investigation of their roles at different stages of cell culture [7]. Furthermore, myoblast cell culture medium can be modified to assess the role of nutrients, growth factors or hormones in regulating the muscle growth process [12][13][14]35,38,40,[42][43][44][45], such as the amino acids and IGF1.…”

An insight on the impact of teleost whole genome duplication on the regulation of the molecular networks controlling skeletal muscle growth

“…Effectiveness of the protocol was further confirmed by determine fbxo32 expression. The FBXO32 is a muscle specific E3-ubiquitin ligase involved on muscle protein degradation and atrophy [75] whose transcription is strongly modulated by nutrition in fish, being a well-known and much characterized marker of fastingrefeeding [15,59,[76][77][78]. For both species, fbxo32 transcription increased during fasting (26-98 fold-change range; P<0.001), concomitant with weight loss, to rapidly decrease after refeeding started (>150 fold-change; P<0.001) (Fig 1B) and growth and protein synthesis were…”