Susan M. Hay

Stimulation of muscle growth by clenbuterol: lack of effect on muscle protein biosynthesis

1. Young rats were offered to appetite a semi-synthetic diet either alone or containing the beta 2-selective agonist clenbuterol (4-amino-alpha[t-butylamino)methyl]-3,5-dichlorobenzyl alcohol). 2. In female rats (starting weight 116g) the presence of the drug at daily doses greater than 10 micrograms/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 micrograms/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 effects of maternal protein restriction on the growth of the rat fetus and its amino acid supply

Maternal protein deficiency causes fetal growth retardation which has been associated with the programming of adult disease. The growth of the rat fetus was examined when the mothers were fed on diets containing 180, 90 and 60 g protein/kg. The numbers of fetuses were similar in animals fed on the 180 and 90 g protein/kg diets but the number was significantly reduced in the animals fed on the 60 g protein/kg diet. The fetuses carried by the mothers fed on the 90 g protein/kg diet were 7.5% heavier than those of mothers fed on 180 g protein/kg diet on day 19 of gestation, but by day 21 the situation was reversed and the fetuses in the protein-deficient mothers were 14% smaller. Analysis of the free amino acids in the maternal serum showed that on day 19 the diets containing 90 and 60 g protein/kg led to threonine concentrations that were reduced to 46 and 20% of those found in animals fed on the control (180 g/kg) diet. The other essential amino acids were unchanged, except for a small decrease in the branched-chain amino acids in animals fed on the 60 g protein/kg diet. Both low-protein diets significantly increased the concentrations of glutamic acid+glutamine and glycine in the maternal serum. On day 21 the maternal serum threonine levels were still reduced by about one third in the group fed on the 90 g protein/kg diet. Dietary protein content had no effect on serum threonine concentrations in nonpregnant animals. Analysis of the total free amino acids in the fetuses on day 19 showed that feeding the mother on a low-protein diet did not change amino acid concentrations apart from a decrease in threonine concentrations to 45 and 26% of the control values at 90 and 60 g protein/ kg respectively. The results suggest that threonine is of particular importance to the protein-deficient mother and her fetuses. Possible mechanisms for the decrease in free threonine in both mother and fetuses and the consequences of the change in amino acid metabolism are discussed.

The effect of β-agonists and antagonists on muscle growth and body composition of young rats (Rattus sp.)

1. The addition of the beta-selective adrenergic agonist clenbuterol to the diet was associated with an increase in the protein and RNA of skeletal and cardiac muscle, a reduction in fat deposition and an increase in energy expenditure. 2. Neither propranolol nor atenolol blocked the effect of clenbuterol on muscle protein but both reduced its effect on cardiac and fat mass and energy expenditure. 3. Five other beta-agonists were tested. All increased the interscapular brown fat mass and lowered body fat but only two increased skeletal muscle protein. 4. It is concluded that the anabolic and anti-lipogenic actions of certain beta-agonists are mechanistically distinct.

Folate deficiency during pregnancy impacts on methyl metabolism without affecting global DNA methylation in the rat fetus.

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.

A Methyl-Deficient Diet Fed to Rat Dams during the Peri-Conception Period Programs Glucose Homeostasis in Adult Male but Not Female Offspring

Methyl deficiencies have been implicated in metabolic programming during the periods of oocyte and embryo development. Semisynthetic methyl-deficient diets (MD) with no folic acid, 0.05% choline, and approximately one-half the recommended content of methionine were fed to female rats for 3 wk prior to mating and for the first 5 d of gestation. During the period of MD feeding, plasma homocysteine concentrations were approximately twice those of rats fed the complete (CON) diet. From d 5, both groups received a complete semipurified AIN diet until birth. On d 8, plasma homocysteine concentrations did not differ between the 2 groups. Thereafter, dams and offspring were fed a nonpurified diet for the remainder of the experiment. At 6 mo of age, the homeostatic model assessment (HOMA) index of the male MD offspring tended to be 32% higher (P = 0.053) and peak insulin during an oral glucose tolerance test (oGTT) was 39% higher (P < 0.05) compared with the male CON offspring. There was no difference in the response to an oGTT in the female offspring at 6 mo of age. The increased HOMA index of male MD offspring persisted to 12 mo of age. The peak glucose concentration during oGTT was 23% higher (P < 0.05) in MD compared with the CON males despite 39% greater (P < 0.05) peak insulin concentrations. This study shows that in rats, a physiologically relevant methyl-deficient diet fed during the period of oocyte maturation and preimplantation development programs gender-specific changes in glucose handling by the offspring.

Effects of maternal vitamin A status on fetal heart and lung: changes in expression of key developmental genes

Vitamin A is required during pregnancy for fetal lung development. These experiments monitored fetal lung morphology in normal and vitamin A-deficient rats. The expression of elastin and the growth arrest-specific gene 6 ( gas6) in fetal and neonatal hearts and lungs was assessed by Northern blotting. In normal-fed rats, elastin and gas6 were expressed in the fetal lung and heart from day 19 of gestation up to day 2 postnatally. Maternal vitamin A deficiency altered fetal lung development. On day 20, the bronchial passageways were less developed and showed reduced staining for elastic fibers, and in the neonates, the relative air space and the size of the sacculi were reduced. In the fetal lung, the mRNAs for elastin and gas6 were reduced to 56 and 68% of the control values, respectively. In the fetal heart, the mRNA for elastin was reduced to 64% of the control value, whereas gas6 was increased twofold. In the neonate, there was no change in elastin expression in the lung or heart, but gas6 expression in the heart was increased twofold. These results suggest that, in the pregnant rat, vitamin A deficiency may retard fetal lung development or influence the differentiation of critical cell lines. The changes in elastin and gas6 expression may be used to identify the cell types affected.

Disruption of lipid metabolism in the liver of the pregnant rat fed folate-deficient and methyl donor-deficient diets.

The importance of folic acid and the methionine cycle in fetal development is well recognised even though the mechanism has not been established. Since the cycle is active in the maternal liver, poor folate status may modify hepatic metabolism. Pregnant rats were fed diets deficient in folic acid (-F) or in three key methyl donors, folic acid, choline and methionine (-FLMLC) and the maternal liver was analysed on day 21 of gestation. Two-dimensional gel electrophoresis of soluble proteins identified differentially abundant proteins, which could be allocated into nine functional groups. Five involved in metabolic processes, namely, folate/methionine cycle, tyrosine metabolism, protein metabolism, energy metabolism and lipid metabolism, and three in cellular processes, namely, endoplasmic reticulum function, bile production and antioxidant defence. The mRNA for sterol regulatory element-binding protein-1c and acetyl-CoA carboxylase-1 (fatty acid synthesis) were decreased by both -F and -FLMLC diets. The mRNA for PPARalpha and PPARgamma and carnitine palmitoyl transferase (fatty acid oxidation) were increased in the animals fed the -FLMLC diets. Changes in the abundance of proteins associated with intracellular lipid transport suggest that folate deficiency interferes with lipid export. Reduced fatty acid synthesis appeared to prevent steatosis in animals fed the -F diet. Even with increased oxidation, TAG concentrations were approximately three-fold higher in animals fed the -FLMLC diet and were associated with an increase in the relative abundance of proteins associated with oxidative stress. Fetal development may be indirectly affected by these changes in hepatic lipid metabolism.

Upregulation of CHOP-10 (gadd153) expression in the mouse blastocyst as a response to stress.

CHOP‐10 (also known as gadd153 or Ddit3) is one of the genes overexpressed by mammalian cells exposed to cytotoxic agents or to nutrient stress. The response of this gene to stress was studied in the mouse blastocyst and in F9 embryonal carcinoma cells. When mouse blastocysts were exposed to the alkylating agent MMS, the metabolic inhibitor sodium arsenite or an inhibitor of protein glycosylation tunicamycin, levels of the CHOP‐10 mRNA were increased by two‐ to threefold relative to the mRNA for β‐actin. There was no increase in gene expression when blastocysts were treated with the inhibitor of nucleotide synthesis PALA. These results show that the response of CHOP‐10 is dependent on the type of stress applied to the embryo. When F9 embryonal carcinoma cells were treated with MMS or sodium arsenite, CHOP‐10 expression was induced by fourfold within 4 hr of treatment. The induction following tunicamycin treatment was slower requiring at least 24 hr. The response to tunicamycin was greater in cells treated with retinoic acid to induce differentiation. The results suggest that there is a link between the extent of glycoprotein synthesis and the sensitivity of CHOP‐10 to tunicamycin. The inhibitor PALA did not change CHOP‐10 expression in the presence or absence of retinoic acid. In F9 cells an increase in the expression of CHOP‐10 was followed by cell death due to apoptosis. The overexpression of CHOP‐10 may be a marker for one of the pathways that lead to apoptosis in the blastocyst. These results suggest that there is more than one control system regulating growth arrest in the blastocyst and the fetal outcome may differ depending on the type of stress encountered in culture. Mol. Reprod. Dev. 54:326–332, 1999.

Protein synthesis in skeletal muscle measured at different times during a 24 hour period

Six groups of 5 male rats (starting body weight 109 g) were allowed free access to a conventional rat diet. At 4 hourly intervals, starting at 10.00 h muscle protein synthesis was measured. By relating the weights of the gastrocnemius and soleus muscles to the initial body weights of the animals (i.e., at 09.30, day 1), a linear increase in muscle weight throughout the day was demonstrated. The fractional rate of muscle protein synthesis varied from 16.8% per day to 20.3% per day in gastrocnemius muscle and from 17.9% per day and 22.1% per day in the soleus. It was calculated that the maximum error incurred in estimating daily muscle protein synthesis by extrapolation of the value at any one time was 6% in gastrocnemius and 9% in soleus. It is concluded that calculations of the average rate of muscle protein degradation based on the difference between the rates of synthesis and deposition are generally valid in rats allowed free access to an adequate diet.

The expression of growth-arrest genes in the liver and kidney of the protein-restricted rat fetus.

During fetal life, there are periods of rapid cell proliferation, which are uniquely sensitive to nutritional perturbation. Feeding the pregnant rat a protein-restricted diet alters the growth trajectory of major fetal organs such as the kidney. By day 21 of gestation, the ratio of kidney weight to total body weight is reduced in the fetuses of dams fed a protein-deficient diet. In contrast, the ratio of fetal liver weight to total body weight is unchanged. To investigate the mechanisms underlying this disproportionate change in organ growth in the low-protein group, cell proliferation and differentiation have been assessed in the liver and kidney. The steady-state levels of mRNA for the growth-arrest and DNA-damage gene gadd153/CHOP-10, CCAAT enhancer-binding proteins alpha and beta were unaffected by maternal diet in both fetal liver and kidney. The mRNA for alpha-fetoprotein, albumin and hepatic glucokinase were unchanged in the liver, suggesting that maternal protein deficiency does not alter the state of differentiation. The steady-state levels of the mRNA coding for the cyclin-dependent protein kinase inhibitors (p15(INK4a), p19(INK4d), p21(CIP1), p27(KIP1) and p57(KIP2)) were unchanged in the fetal livers but were significantly increased in the kidneys of fetuses from dams fed the low-protein diet. These results show that the asymmetrical growth of the kidney is associated with increases in mRNA for the Cip/Kip cyclin-dependent kinase inhibitors and that these may reflect specific lesions in organ development.