LEUCINE AND METHIONINE DEFICIENCY INDUCE CATABOLISM THROUGH NON-OVERLAPPING MECHANISMS IN RAINBOW TROUT Oncorhynchus mykiss PRIMARY MYOBLASTS

Amino acids (AA) have anabolic effects on protein accretion in muscle.  In fish it is unknown if this anabolic response is directly attributed to a single AA or a specific AA profile.  Therefore, our experimental objective was to determine if AAs or AA profiles regulate protein turnover and growth-related mechanisms in rainbow trout myocyte cultures.  The overall goal of this research is to identify the functional roles of AAs and identify nutrient-gene interactions that may affect growth performance in rainbow trout.  

Myogenic precursor cells were isolated from rainbow trout skeletal muscle and incubated in media containing 10% fetal bovine serum for 7 days, thereby differentiating into myoblasts. On day 7 cells were incubated with treatment media varying in AA concentrations, and the proteolytic or gene expression response was analyzed after 6 or 24 hours, respectively.  Incubation of cells in minimal essential media (MEM) lacking essential AAs (EAA) increased protein degradation by 1.5-fold (P<0.05) compared to MEM containing a complete AA profile.  This response was attenuated 50% and 75% by addition of leucine and branch chain amino acids (BCAA), respectively (P<0.05).  Further addition of lysine decreased protein degradation to levels comparable to cells in complete media, suggesting that BCAAs and lysine are central regulators of protein degradation in muscle.  Therefore, dietary deficiencies of these amino acids, particularly leucine, may be most detrimental to muscle accretion in salmonids.  Further supporting this observation was that cells treated with leucine-less media exhibited protein degradation rates equivalent to cells treated with media lacking all EAA, indicating that a leucine deficiency mimics the effects of an EAA deficiency on muscle proteolysis.

While media lacking methionine did not affect protein degradation rates, it did affect expression of several genes associated with muscle growth, reducing expression of follistatin (fst), myogenin (myog), growth hormone receptor (ghr2), insulin-like growth factor binding protein-5b1 (igfbp5b1), and the signaling protein, smad7 (p<0.05).  In contrast, media lacking leucine reduced expression of fst only (P<0.05).  Collectively, these findings suggest that a dietary deficiency of leucine and methionine produce catabolic effects directly in muscle, although through different mechanisms (Figure 1).