A Cell-Based Assessment of the Muscle Anabolic Potential of Blue Whiting (Micromesistius poutassou) Protein Hydrolysates

Abstract

Blue whiting (BW) represents an underutilised fish species containing a high-quality protein and amino acid (AA) profile with numerous potentially bioactive peptide sequences, making BW an economic and sustainable alternative source of protein. This study investigated the impact of three different BW protein hydrolysates (BWPH-X, Y and Z) on growth, proliferation and muscle protein synthesis (MPS) in skeletal muscle (C2C12) myotubes. BWPHs were hydrolysed using different enzymatic and heat exposures and underwent simulated gastrointestinal digestion (SGID), each resulting in a high degree of hydrolysis (33.41-37.29%) and high quantities of low molecular mass peptides (86.17-97.12% <1 kDa). C2C12 myotubes were treated with 1 mg protein equivalent/mL of SGID-BWPHs for 4 h. Muscle growth and myotube thickness were analysed using an xCelligence™ platform. Anabolic signalling (phosphorylation of mTOR, rpS6 and 4E-BP1) and MPS measured by puromycin incorporation were assessed using immunoblotting. BWPH-X significantly increased muscle growth (p < 0.01) and myotube thickness (p < 0.0001) compared to the negative control (amino acid and serum free media). Muscle protein synthesis (MPS), as measured by puromycin incorporation, was significantly higher after incubation with BWPH-X compared with the negative control, but did not significantly change in response to BWPH-Y and Z treatments. Taken together, these preliminary findings demonstrate the anabolic potential of some but not all BWPHs on muscle enhancement, thus providing justification for human dietary intervention studies to confirm and translate the results of such investigations to dietary recommendations and practices.

Keywords: C2C12 cells; blue whiting protein hydrolysates; muscle growth; muscle protein synthesis.

Figure 1

Viability of muscle myotube (C2C12) cells treated with 0.1, 0.5 and 1.0 mg·mL⁻¹ protein equivalent blue whiting (Micromesistius poutassou) protein hydrolysates (BWPHs). The negative control was amino acid and serum free media without BWPHs. Treatment of cells was for 4 h after 1 h of nutrient deprivation. The results represent mean ± SD (n = 3) and are expressed relative to the negative control.

Figure 2

Effect of blue whiting (Micromesistius poutassou) protein hydrolysate (BWPH) treatment on AUC and myotube diameter in skeletal muscle cells. C2C12 myotubes were nutrient deprived for 1 h followed by 4 h treatment with 1 mg·mL⁻¹ protein equivalent of BWPH-X, Y and Z. Myotube growth was monitored every 2 min over 4 h. (A) Representative graph comparing myotube growth (AUC) in the presence of different samples relative to the negative control. (B) Representative graph comparing myotube diameter in the presence of different samples relative to the negative control (C) Quantification of myotube diameter taken 4 h post treatment as measured by microscopy. Images of myotubes treated with samples were taken at 4× magnification following 4 h treatment. All values are expressed as mean ± SD (n = 6) for 3 plates (duplicate in each plate). p < 0.05 * compared to the negative control (amino acid and serum free media). Ctl−: negative control (amino acid and serum free media), Ctl+: positive control (100 ng·mL⁻¹ IGF-1), X: SGID-BWPH-X, Y: SGID-BWPH-Y, Z: SGID-BWPH-Z.

Figure 3

Phosphorylation of mTOR, 4EBP1 and ribosomal S6 incubated with 1 mg·mL⁻¹ protein equivalent BWPH-X, Y and Z (n = 4). C2C12 myotubes were nutrient deprived for 1 h followed by treatment with SGID-BWPHs plus 1 µM puromycin for 4 h. Data reported as the ratio of phosphoproteins relative to the total protein. All values were expressed as a percent of the negative control within each assay. Phosphorylation of mTOR (A), rpS6 (B) and 4EB-P1 (C) following SGID-BWPH treatment and their corresponding representative immunoblot. (D) Muscle protein synthesis (MPS) after treatment with SGID-BWPHs and their representative immunoblot of MPS (measured by puromycin incorporation) relative to total protein (loading control). Data reported as mean ± SEM, * compared to negative control, p < 0.01. Ctl−: negative control (amino acid and serum free media), Ctl+: positive control (100 ng·mL⁻¹ IGF-1), X: SGID-BWPH-X, Y: SGID-BWPH-Y, Z: SGID-BWPH-Z.

Figure 3

Phosphorylation of mTOR, 4EBP1 and ribosomal S6 incubated with 1 mg·mL⁻¹ protein equivalent BWPH-X, Y and Z (n = 4). C2C12 myotubes were nutrient deprived for 1 h followed by treatment with SGID-BWPHs plus 1 µM puromycin for 4 h. Data reported as the ratio of phosphoproteins relative to the total protein. All values were expressed as a percent of the negative control within each assay. Phosphorylation of mTOR (A), rpS6 (B) and 4EB-P1 (C) following SGID-BWPH treatment and their corresponding representative immunoblot. (D) Muscle protein synthesis (MPS) after treatment with SGID-BWPHs and their representative immunoblot of MPS (measured by puromycin incorporation) relative to total protein (loading control). Data reported as mean ± SEM, * compared to negative control, p < 0.01. Ctl−: negative control (amino acid and serum free media), Ctl+: positive control (100 ng·mL⁻¹ IGF-1), X: SGID-BWPH-X, Y: SGID-BWPH-Y, Z: SGID-BWPH-Z.