Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Anabolic effects of feeding β2-adrenergic agonists on rainbow trout muscle proteases and proteins
Introduction
Protein turnover is the net effect of two dynamic cellular processes, protein synthesis and degradation. Protein turnover limits growth efficiency of animals. Young et al. (1975) reported that 15% to 25% of the energy consumed by growing animals is used for protein breakdown and re-synthesis. Consequently the reduction of the cost of protein turnover is a strategic goal towards enhancing animal growth efficiency.
β2-Adrenergic agonists (BAAs) have pharmacological actions similar to those of the catecholamines epinephrine and norepinephrine (Mills, 2001). BAAs bind to the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor, transduce cellular signals and produce tissue-specific responses that assist animals in dealing with different stress conditions (Fabbri and Moon, 1994). Oral administration of BAAs is shown to increase muscle protein and decrease muscle fat in growing poultry, pigs, sheep and cattle (Moloney et al., 1991, NRC, 1994). The mechanism of this effect involves altering rates of protein degradation and synthesis (Mersmann, 1998). The Food and Drug Administration (FDA) in 1999 approved the use of RACT for finishing swine (last 7 weeks) in swine farming. Administration of BAAs has been conducted on several fish species including rohu fish, Labeo rohita (Satpathy et al., 2001), channel catfish, Ictalurus punctatus (Mustin and Lovell, 1993), blue catfish, Ictalurus furcatus (Webster et al., 1995) and rainbow trout (RBT), Oncorhynchus mykiss (Vandenberg and Moccia, 1998, Vandenberg et al., 1998). These studies demonstrated that BAAs had less anabolic effects in fish than in mammals. Before BAAs are employed in the aquaculture industry, a better understanding of the precise mechanism by which BAAs modulate fish muscle metabolism is needed. The major objective of the present study was to identify the chronic effect of feeding BAAs to RBT on protein degradation by measuring the expression of genes involved in the major proteolytic systems of muscle including cathepsins, calpains and the multi-catalytic proteasome. In addition, the expression of genes encoding myofibrillar proteins, f-MHC and s-MHC, and the cytoskeletal protein, β-actin, was measured to monitor the changes in muscle protein synthesis (Gupta et al., 1996, Overturf and Hardy, 2001). Since CLEN treatment in mammals has been reported to cause a shift from the slow-type to the fast-type MHC isoform within slow-twitch muscles (Rajab et al., 2000, Oishi et al., 2002), the relative expression of s-MHC versus f-MHC was also measured to determine if muscle fiber-type transformation occurred.
Recently, Lortie and Moon (2003) reported that the BAAs clenbuterol (CLEN) and ractopamine (RACT) bind to the β2-AR and stimulate the activity of adenylyl cyclase (ACase) in RBT muscle membranes. Therefore, the second objective of the present study was to determine the chronic effects of BAAs on the expression of the β2-AR and ACase genes in addition to the c-AMP-dependent transcription factor, activating transcription factor 1 (ATF-1) gene.
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Animals, diets and experimental design
Juvenile rainbow trout were obtained from Linwood Acres Trout Farm (Campbellcroft, ON). Fish were transported to the University of Ottawa Aquatic Care Facility and held in 110–115 L fiberglass tanks supplied with flowing, aerated and dechloraminated city of Ottawa tap water at a temperature of 13 °C. Fish (approximately 6 g per fish) were divided into three groups of 100 fish per tank and held on a 12L:12D photoperiod. Fish were hand-fed to satiation daily a commercial trout diet (Purina Trout
Expression and activity of proteolytic enzymes
RACT feeding significantly increased mRNA levels of the calpain catalytic subunit, Capn1, the regulatory subunit, cpns (p < 0.01) and the calpastatin large isoform, CAST-L (p < 0.05) but did not affect the expression of the calpain catalytic subunit, Capn2, and the calpastatin small isoform, CAST-S (p > 0.05). The changes at the mRNA levels of Capn1, cpns and CAST-L genes did not lead to a corresponding change in calpain catalytic activities (Fig. 1). Feeding the BAA CLEN did not affect the
Discussion
Our results showed no significant change in body weight, length, or condition factor after feeding RBT 10 ppm of either RACT or CLEN for 80 days (data not shown). These results are consistent with several reports in fish such as blue catfish (20–100 ppm RACT; Webster et al., 1995) and RBT (5–40 ppm RACT; Vandenberg and Moccia, 1998, Vandenberg et al., 1998, Lortie et al., 2004). However, Satpathy et al. (2001) reported a 6–12% increase of growth in rohu fish fed 3–6 ppm salbutamol. In addition,
Acknowledgments
This investigation was supported by Hatch Funds (Project No. 427) of the West Virginia Agricultural Experiment Station, a USDA Cooperative Agreement No. 58-1930-8-039, and a grant to TWM from the Natural Sciences and Engineering Research Council of Canada. It is published with the approval of the Director of the West Virginia Agriculture and Forestry Experiment Station as scientific paper No. 2949.
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