1. Young rats were offered to appetite a semi-synthetic diet either alone or containing the /&-selective agonist clenbuterol (4-amino-cc[t-butylamino) methyl]-3,S-dichlorobenzyl alcohol).2. In female rats (starting weight 116g) the presence of the drug at daily doses greater than 10 pg/kg body-weight per d increased the growth of skeletal and cardiac muscle but had no stimulatory effect on the growth of the liver, gastrointestinal tract and kidney.3. Male rats (starting weight 53 g) received clenbuterol at a daily oral dose of 200 pg/kg body-weight per d.Animals were slaughtered after 0, 4, 8, 11, 18, 21 and 25 d of treatment. At 4, 11, 21 and 25 d muscle protein synthesis was measured by the method of Garlick et al. (1980). Although clenbuterol increased the rate of protein and RNA accretion in gastrocnemius and soleus muscles, protein synthesis was not increased. 4.The results suggested that the drug had a rapid, perhaps direct, inhibitory effect on protein degradation. It is concluded that the growth-promoting effect of clenbuterol may be specific to muscle and that the drug may act in a novel manner which circumvents the physiological mechanisms responsible for the control of muscle growth .The use of selective (Rothwell et al. 1 9 8 3~; Dulloo & Miller, 1984) and non-selective (Rothwell et al. 1983b) sympathomimetic agents in the manipulation of growth and body composition, and particularly in effecting a reduction in body fat mass, has received a great deal of attention in recent years. Agonists with a or / 3 receptor specificity have proved effective in increasing daily energy expenditure and in lowering the rate of fat deposition. Although the effects of these agents on body protein mass have been variable, in general they reduce the rate of protein deposition (Dulloo & Miller, 1984). Recent work, however, has shown that some compounds with a marked degree of specificity for p2 receptors, as defined by Lands et al. (1967), not only reduce body fat gain but also promote the deposition of body protein Dalrymple et al. 1983; Rothwell et al. 1 9 8 3~; Emery et al. 1984;Ricks et al. 1984). In this respect the action of these drugs and in particular clenbuterol is similar to that of some anabolic steroids (Vernon & Buttery, 1976Lobley et al. 1982Lobley et al. , 1983 but the growth-promoting effects of this drug show less species or sex specificity (Ricks et al. 1984) than steroidal growth-stimulating agents.One characteristic of previous results of experiments on the action of clenbuterol has been an increase in muscle growth. It is likely that any significant increase in body protein will also involve some degree of muscle hypertrophy and it is not certain whether the effect of the drug is confined to muscle. Furthermore, with the exception of a single report (Emery et al. 1984), the changes in protein synthesis and degradation, which must underlie any change in protein deposition, have not been investigated. The measurement of these changes is a necessary first step in the investigation of the mechanism o...

Maternal protein deficiency during pregnancy is associated with changes in glucose tolerance and hypertension in the offspring of rats. In this study the growth of rat fetuses was examined when the dams were fed diets containing 18% casein, 9% casein or 8% casein supplemented with threonine. The extra threonine was added to reverse the decrease in circulating threonine concentrations that occurs when pregnant rats are fed protein-deficient diets. The fetuses of the group fed the low protein diet supplemented with threonine were significantly smaller than those of the control group and not significantly different from those fed low protein. Homogenates prepared from the livers of dams fed the diet containing 9% casein oxidized threonine at approximately twice the rate of homogenates prepared from dams fed the diet containing 18% casein. We conclude that circulating levels of threonine fall as a consequence of an increase in the activity of the pathway that metabolizes homocysteine produced by the transulfuration of methionine. Serum homocysteine was unaffected in the dams fed low protein diets compared with controls, but was significantly greater in dams fed the low protein diet supplemented with threonine. Elevated levels of homocysteine are associated with changes in the methylation of DNA. The endogenous methylation of DNA was greater than that of controls in the livers of fetuses from dams fed the 9% protein diets and increased further when the diet was supplemented with threonine. Our results suggest that changes in methionine metabolism increase homocysteine production, which leads to changes in DNA methylation in the fetus. An increase in maternal homocysteine may compromise fetal development, leading to the onset of glucose intolerance and hypertension in adult life.

The methionine cycle and methyl group metabolism are implicated in the long-term programming of metabolism. Diets deficient in folic acid, methionine and choline have been fed to pregnant rats to examine the effects on amino acid metabolism, choline reserves and DNA methylation in dam and fetuses. Animals were fed folate-deficient, folate-deficient with low methionine, folate-deficient with low choline and folate-deficient, low-methionine, low-choline diets starting 2 weeks before mating. The dams and their fetuses were subsequently killed on day 21 of gestation for analysis. Diets low in methionine reduced fetal and maternal weight. Folate deficiency increased the concentrations of homocysteine, glycine, serine and threonine in the maternal plasma, and this was exacerbated by the low-methionine diets. The changes in the amino acid profile in the fetal serum were similar but less pronounced. This result suggests that fetal metabolism was less perturbed. Folate deficiency increased free choline in the maternal liver at the expense of phosphocholine stores. It has been suggested that a deficiency in methyl donors in the diet during pregnancy may impact on key methylation reactions, including the methylation of DNA. Despite widespread changes in the metabolism of choline and amino acids, there was no change in the global methylation of cytosine in DNA from either maternal or fetal livers. This suggests a more indirect mechanism in which gene -nutrient interactions modify the process of differential methylation during development (Jackson et al. 2002;Brawley et al. 2004) reverses the adverse effects of protein restriction. As both folic acid and glycine are essential factors in the methionine cycle, these experiments suggest that this pathway has a central role in early-life programming.A variety of molecules can donate methyl groups to the methionine cycle (Fig. 1), enabling the animal to respond to fluctuations in the diet. Thus, the availability of methionine, glycine, serine and choline must be taken into account when assessing the role of folic acid. In the rat, dietary choline is the major source of methyl groups (Zeisel, 2006). Although choline metabolism is intimately linked with the methionine cycle, little is known about how the balance of methyl donors in the diet impacts on choline (and its main storage form phosphocholine) in the pregnant animal. Furthermore, there is little information on how the availability of folic acid and choline in the diet affects circulating amino acids in the pregnant animal.A perturbation of the methionine cycle is associated with elevated plasma levels of homocysteine. This is believed to be secondary to changes in intracellular concentrations of S-adenosyl methionine and S-adenosyl homocysteine. High-affinity binding of S-adenosyl homocysteine to the active site of DNA methyltransferase results in product inhibition of the enzyme (James et al.

Human intestinal bacteria were grown in a 3-stage continuous culture system on a medium containing complex polysaccharides and proteins as carbon and nitrogen sources. Selected bacterial populations were enumerated and glycosidase, protease and arylamidase activities measured. Comparison of arylamidase and glycosidase activities in the multichamber system (MCS) and faeces showed that the predominant faecal enzymes were also produced by bacteria growing in the MCS. After 48 d operation, porcine gastric mucin (5.8 g/d) was independently fed to vessel 1. Elevated levels of volatile fatty acid (VFA) formation showed that the glycoprotein was actively fermented. The increase in carbohydrate availability as a result of breakdown of the mucin oligosaccharides stimulated bacterial growth and activities. The enzymological measurements showed that mucin increased production of both cell-bound and extracellular glycosidases, such as beta-galactosidase, alpha-glucosidase and N-acetyl-beta-glucosaminidase. Protease activities were profoundly influenced by mucin. These were largely cell-bound in non-mucin cultures but were predominantly extracellular and collagenolytic when mucin was present. Experiments with protease inhibitors showed that cysteine proteases were the major cell-bound and extracellular enzymes in both mucin and non-mucin cultures, but that serine and metalloproteases were also present. The effect of mucin on arylamidase formation was less marked, although there was increased production of these enzymes in vessels 1 and 2 of the MCS. These results suggest that host-produced substances such as mucin glycoprotein may play a role in modulating the growth and activity of bacteria growing in the human large intestine.

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