Robert M. Palmer

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.

Mass and Information Feedbacks through Receptor Endocytosis Govern Insulin Signaling as Revealed Using a Parameter-free Modeling Framework

Insulin and other hormones control target cells through a network of signal-mediating molecules. Such networks are extremely complex due to multiple feedback loops in combination with redundancy, shared signal mediators, and cross-talk between signal pathways. We present a novel framework that integrates experimental work and mathematical modeling to quantitatively characterize the role and relation between co-existing submechanisms in complex signaling networks. The approach is independent of knowing or uniquely estimating model parameters because it only relies on (i) rejections and (ii) core predictions (uniquely identified properties in unidentifiable models). The power of our approach is demonstrated through numerous iterations between experiments, model-based data analyses, and theoretical predictions to characterize the relative role of co-existing feedbacks governing insulin signaling. We examined phosphorylation of the insulin receptor and insulin receptor substrate-1 and endocytosis of the receptor in response to various different experimental perturbations in primary human adipocytes. The analysis revealed that receptor endocytosis is necessary for two identified feedback mechanisms involving mass and information transfer, respectively. Experimental findings indicate that interfering with the feedback may substantially increase overall signaling strength, suggesting novel therapeutic targets for insulin resistance and type 2 diabetes. Because the central observations are present in other signaling networks, our results may indicate a general mechanism in hormonal control.

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.

A Hierarchical Whole-body Modeling Approach Elucidates the Link between in Vitro Insulin Signaling and in Vivo Glucose Homeostasis

Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.

SIGNALLING PATHWAYS REGULATING PROTEIN TURNOVER IN SKELETAL MUSCLE

The protein content of skeletal muscle is determined by the relative rates of synthesis and degradation which must be regulated coordinately to maintain equilibrium. However, in conditions such as fasting where amino acids are required for gluconeogenesis, or in cancer cachexia, this equilibrium is disrupted and a net loss of protein ensues. This review, utilising studies performed in several situations, summarizes the current state of knowledge on the possible signalling pathways regulating protein turnover in skeletal muscle and highlights areas for future work.

A framework for mapping, visualisation and automatic model creation of signal-transduction networks

Intracellular signalling systems are highly complex. This complexity makes handling, analysis and visualisation of available knowledge a major challenge in current signalling research. Here, we present a novel framework for mapping signal‐transduction networks that avoids the combinatorial explosion by breaking down the network in reaction and contingency information. It provides two new visualisation methods and automatic export to mathematical models. We use this framework to compile the presently most comprehensive map of the yeast MAP kinase network. Our method improves previous strategies by combining (I) more concise mapping adapted to empirical data, (II) individual referencing for each piece of information, (III) visualisation without simplifications or added uncertainty, (IV) automatic visualisation in multiple formats, (V) automatic export to mathematical models and (VI) compatibility with established formats. The framework is supported by an open source software tool that facilitates integration of the three levels of network analysis: definition, visualisation and mathematical modelling. The framework is species independent and we expect that it will have wider impact in signalling research on any system.

Effect of the β‐adrenoceptor agonist clenbuterol and phytohaemagglutinin on growth, protein synthesis and polyamine metabolism of tissues of the rat

1 The kidney bean lectin, phytohaemagglutinin (PHA), induced a marked atrophy of skeletal muscle which was evident from the changes in tissue composition (protein, RNA, DNA and polyamine content) and from the reduction in weight and protein synthesis of hind leg muscles of rats fed on kidney bean‐diets for four days. The β‐adrenoceptor agonist, clenbuterol, induced skeletal muscle hypertrophy by transiently stimulating protein synthesis. As a consequence, the muscle loss caused by a short exposure to PHA was, in part, ameliorated by clenbuterol treatment. 2 Cardiac muscle was affected to a lesser extent than skeletal muscle by both clenbuterol and the lectin. However, there was evidence that protein synthesis in heart was reduced by PHA. 3 PHA had opposite effects on the gut, the lectin‐induced hyperplasia of the jejunum was accompanied by a large increase in protein synthesis. Clenbuterol alone had no effect on the jejunum whereas a combination of PHA and clenbuterol appeared to exacerbate the effect of the lectin on gut. 4 Both the lectin‐induced gut growth and the hypertrophy of skeletal muscle caused by clenbuterol were preceded by the accumulation of polyamines in the respective tissues. Of particular note was the observation that a significant increase in the proportion of the intraperitoneally injected 14C‐labelled spermidine or putrescine taken up by the growing tissues could be detected by the second day. Therefore, the measurement of uptake of labelled polyamines may be used as a sensitive indicator of early alterations in tissue metabolism.

Stimulation of myofibrillar protein degradation and expression of mRNA encoding the ubiquitin‐proteasome system in C2C12 myotubes by dexamethasone: effect of the proteasome inhibitor MG‐132

Addition of the synthetic glucocorticoid, dexamethasone (Dex) to serum‐deprived C2C12 myotubes elicited time‐ and concentration‐dependent changes in Nτ‐methylhistidine (3‐MH), a marker of myofibrillar protein degradation. Within 24 h, 100 nM Dex significantly decreased the cell content of 3‐MH and increased release into the medium. Both of these responses had increased in magnitude by 48 h and then declined toward basal values by 72 h. The increase in the release of 3‐MH closely paralleled its loss from the cell protein. Furthermore, Dex also decreased the 3‐MH:total cell protein ratio, suggesting that myofibrillar proteins were being preferentially degraded. Incubation of myotubes with the peptide aldehyde, MG‐132, an inhibitor of proteolysis by the (ATP)‐ubiquitin (Ub)‐dependent proteasome, prevented both the basal release of 3‐MH (>95%) and the increased release of 3‐MH into the medium in response to Dex (>95%). Northern hybridization studies demonstrated that Dex also elicited similar time‐ and concentration‐dependent increases in the expression of mRNA encoding two components (14 kDa E2 Ub‐conjugating enzyme and Ub) of the ATP‐Ub‐dependent pathway. The data demonstrate that Dex stimulates preferential hydrolysis of myofibrillar proteins in C2C12 myotubes and suggests that the ATP‐Ub‐dependent pathway is involved in this response. J. Cell. Physiol. 181:455–461, 1999.

Stimulation of actin and myosin synthesis in rat gastrocnemius muscle by clenbuterol; evidence for translational control.

1. A transient rise in fractional rates of protein and actomyosin synthesis was observed in gastrocnemius muscles of rats fed clenbuterol for 1-2 days but the muscle RNA:protein ratio was unchanged, therefore protein synthesis per unit RNA (kRNA) also increased. 2. Myosin heavy and light chains and actin showed increased incorporation of [3H]phenylalanine at 2 days; these changes were proportional to increases in total protein synthesis. 3. The ratios actin mRNA:18S RNA and fast myosin heavy chain mRNA:18S RNA were unaffected by clenbuterol. 4. The data suggest that the clenbuterol-induced increase in muscle protein synthesis involves both translational control and increased tissue RNA.