Evaluation of the mechanism by which animal growth promotants operating through B2-adrenoceptors induce muscle growth: Methodological development, and a study of clenbuterol

There were two main objectives for this study. The first of these was to develop a research methodology with which both the affinity and efficacy of B-adrenoceptor agonists on protein degradation in skeletal muscle could be studied.

B2-Adrenoceptors were isolated from toad skeletal muscle and were characterised in radioligand binding experiments. The resultant affinity constants for 14 drugs were compared with values obtained from bovine Bradrenoceptors, and paired t-test analysis indicated that no significant difference existed between these two populations of B2-adrenoceptors. Thus, toad skeletal muscle B2-adrenoceptors provide a suitable model for screening B-ligand affinities, and values may be extrapolated to the corresponding receptors in cattle (target species).

Recombinant Escherichia coli expressing human B1,-and human B2-adrenoceptors were also characterised in radioligand binding experiments. The data obtained verified that each clone provided a source of homogeneous receptors, which displayed the ligand binding affinities appropriate for the respective receptor subtype. Thus, recombinant bacteria are a useful tool for screening the subtype-selectivity of ligands.

The bacterial clone expressing human B2-adrenoceptors displayed ligand binding affinities that were not significantly different to drug affinity determinations performed with bovine B2-adrenoceptors. Thus, the B2-strain is a useful model of the Bradrenoceptor expressed in the skeletal muscle of cattle. Additionally, recombinant B2-adrenoceptors exhibited differential binding behaviour for agonists and antagonists: all antagonists tested displayed monophasic displacement curves, whereas agonist molecules having molecular weight of less than 250 g/mol and an octanol/water partition coefficient (log P) less than zero, displayed biphasic binding behaviour. From these experiments, the cloned Escherichia coli cell lines were selected as the preferred source of B2-adrenoceptors for radioligand binding screens.

The B-adrenoceptor agonist efficacy assay selected for this study was the isolated perfused rat hemicorpus. In the first experiment, the rate of 3-methylhistidine (3-MH) release from perfused hemicorpora was examined: the 3 MH efflux rate of 0.227 ± 0.009 Amo1/30 g/3 hours corresponded to a protein degradation rate of 8.7 ± 0.3 %/day. While this value was comparable to other estimates reported in the literature, the HPLC analytical technique used to quantify 3-MH was at the lower limit of detection sensitivity. This author was not confident that the small changes in 3-MH release that may result from B-agonist treatment would be detected using this method. Hence, an alternative approach of measuring proteolysis as a function of other essential amino acids was examined. With this second method, reincorporation of amino acid markers into protein was prevented by the protein synthesis inhibitor, cycloheximide. In the presence of radiolabelled phenylalanine and leucine, the addition of 40 Amo1/1 cycloheximide to the perfusion medium was found to inhibit protein synthesis by 78 to 90%, without influencing the composition of the free amino acid pools within muscle. Furthermore, this radiolabel incorporation data (for rat hindlimbs perfused in the absence of cycloheximide) enabled the protein synthetic rate of the muscle to be estimated at between 2.0 and 3.06 %/day.

When proteolysis is measured as a function of the release of essential amino acids, the rate of myofibrillar protein breakdown is indistinguishable from non-myofibrillar breakdown. Thus, specific increases in myofibrillar degradation may potentially be undetected. However, the probability of detecting significant changes in degradation may be improved by co-analysing a total of eight essential and semi-essential amino acids. This approach was endorsed when the amino acid efflux rate from rat hemicorpora perfused in the presence and absence of cycloheximide were examined: logically, the blockade of protein synthesis would be expected to result in elevated rates of amino acid efflux. Indeed, the amino acid efflux rates from cycloheximide treated tissues were consistently, though not necessarily significantly, higher than the rates determined in cycloheximide-free tissues. However, when a paired t-test analysis was performed with all eight amino acids, the level of statistical significance improved dramatically.

The results from perfusion experiments indicated that all subsequent studies of protein degradation should be performed in the presence of 40/Imola cycloheximide; and the essential amino acids, valine, methionine, tryptophan, phenylalanine, isoleucine, lysine, leucine, and the semi-essential amino acid tyrosine should be used as co-indices of the rate of protein degradation.

When rat hemicorpora were perfused in the presence of 40 Amo1/1 cycloheximide, the mean ± SEM rate of potassium released from 30 grams of skeletal muscle was 740 ± 60 Amo1/3 hours. Likewise, the mean ± SEM rate of glucose consumed by 30 grams of skeletal muscle was 330 ± 20 Amo1/3 hours. HPLC analysis of muscle amino acids derived from both intact rats, and from hemicorpora which had been perfused for three hours, revealed that the intracellular free amino acid content of muscle was not significantly altered by the perfusion process. Thus, the rate of protein degradation in perfused muscle tissue was estimated directly from the rate of amino acid efflux for eight essential/semi-essential amino acids.

The second major objective of this study was to use the rat hemicorpus preparation to determine whether the B2-adrenoceptor agonist, clenbuterol, mediates its growth promoting effects on skeletal muscle through a reduction in the rate of myofibrillar protein degradation.

Clenbuterol treatment (4 mg/kg diet, 265 μg/kg BWT/day) resulted in net protein accretion, and significantly enhanced the growth rate of treated animals. In mature male rats, clenbuterol treatment in the diet increased the rate of weight gain to 260% of the untreated level. However, the magnitude of the growth response declined after six days of treatment, which is consistent with B-adrenoceptor desensitisation. Clenbuterol-induced changes in blood flow to different tissues was evident from the change in the colour of adipose tissue in abdominal fat depots.

Clenbuterol treatment had no significant effect on either the perfusate pressure range or the rate of glucose uptake in perfused rat hemicorpora. In contrast, potassium efflux rates in chronically treated hindlimb preparations were elevated by 95%. The increase in potassium efflux may have been an artefact of the cycloheximide-induced inhibition of protein synthesis. However, more recent evidence of elevated plasma potassium levels in cows chronically treated with clenbuterol indicate that this may be a real effect of long-term B-agonist treatment.

When the rate of protein degradation was evaluated in terms of the sum of essential amino acids effluxed from perfused rat hemicorpora, acute (three hours) clenbuterol treatment caused a significant, but transient increase in proteolysis (11%). In contrast, longer-term clenbuterol treatment (6 and 12 days) effected a non-significant decrease in the rate of protein degradation (11.8 and 7.6%, respectively), and this effect was diminished over time, in a manner consistent with B-adrenoceptor desensitisation. Furthermore, the withdrawal of clenbuterol from chronically-treated tissues resulted in an immediate and significant increase (25.1%) in the rate of protein degradation, which indicates that the continued presence of clenbuterol is required to maintain the effect of reduced proteolysis.

Clearly, the reduction in the rate of protein degradation observed with chronic clenbuterol treatment is insufficient to account for the 160% increase seen in the growth rate of treated rats. Consequently, it can be concluded that protein synthetic rates must have also been elevated. Indeed, it has been estimated that the 160% increase in the growth rate of clenbuterol-treated rats could be accounted for by a net 16.4% change in the component processes of protein turnover (ie an 11.8% reduction in protein breakdown, and a 4.6% increase in protein synthesis). This is equivalent to a 10.25% change in protein turnover for every 100% increase in growth rate, and compares exceptionally well with the 10 to 13% change estimated by Kim and Sainz (1992).

From these studies, it was concluded that the mechanism of clenbuterol-induced muscle accretion includes components of both increased protein synthesis and decreased protein degradation. In tropical cattle, this saving in metabolic energy resulting from decreased proteolysis may offer an advantage to the animal during the dry winter months where seasonal weight losses are often observed.