Expression of heparan sulfate proteoglycans in murine models of experimental colitis.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are considered important in maintaining physiological homeostasis in many systems. Their expression is altered greatly in several pathophysiological conditions. Herein, we assess the expression and cellular localization of HSPGs in two murine models of human inflammatory bowel disease (IBD). METHODS: Expression and localization of HSPGs, syndecans, and HS epitopes were examined in the colon of 129SvEv interleukin 10 knockout (IL10(-/-)), C3Bir IL10(-/-), and their genetic control (IL10(+/+)) counterparts (129SvEv; C3H/HeJ). mRNA expression of syndecans and heparan sulfate biosynthesis enzymes were evaluated by real-time polymerase chain reaction (PCR). Localization of HSPGs was determined by immunofluorescence. RESULTS: mRNA for all syndecans was detected and expression in colonic tissues altered in IL10(-/-) mice. Syndecan-1 protein was expressed in the intestinal epithelium and on lamina propria cells of IL10(-/-) and control mice but was significantly reduced on the intestinal epithelial cells of IL10(-/-), mice particularly with severe colitis. Syndecan-2 was not detected, whereas syndecan-3 immunoreactivity was localized in the lamina propria but did not differ between control and IL10(-/-) mice. Syndecan-4 was present on epithelial cells of all mice but was significantly reduced in IL10(-/-) mice. Differences in the expression of HS epitopes between control and IL10(-/-) mice were also confirmed. CONCLUSIONS: The study has revealed altered expression of syndecan-1 and -4 and HS epitopes in the gut of mice with an IBD-like gut disorder. The IL10(-/-) mouse is a useful model for further study of the functional role of HSPGs in chronic inflammation and in maintaining healthy gut barrier.

Moderate maternal vitamin A deficiency alters myogenic regulatory protein expression and perinatal organ growth in the rat.

Vitamin A deficiency is one of the most common dietary deficiencies in the developing world and is a major health concern where it is associated with increased risk of fetal and infant mortality and morbidity. Early studies in the rat demonstrated that, in addition to respiratory problems, neonates showed evidence of mobility problems in response to moderate vitamin A deficiency. This study investigated whether moderate deficiency of this vitamin plays a role in regulating key skeletal muscle regulatory pathways during development. Thirty female rats were fed vitamin A-moderate (VAM) or vitamin A-sufficient diets from weaning and throughout pregnancy. Fetal and neonatal hindlimb and muscle samples were collected on days 13.5, 15.5, 17.5, and 19.5 of pregnancy and 1 day following birth. Mothers fed the VAM diet had reduced retinol concentrations at all time points studied (P < 0.01), and neonates had reduced relative lung weights (P < 0.01). Fetal weight and survival did not differ between groups but neonatal survival was lower in the VAM group where neonates had increased relative heart weights (P < 0.05). Analysis of myogenic regulatory factor expression and calcineurin signaling in fetuses and neonates demonstrated decreased protein levels of myf5 [50% at 17.5 dg (P < 0.05)], myogenin [70% at birth (P < 0.001)], and myosin heavy chain fast [50% at birth (P < 0.05)] in response to moderate vitamin A deficiency. Overall, these changes suggest that vitamin A status during pregnancy may have important implications for fetal muscle development and subsequent muscle function in the offspring.

Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo.

The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.

Impact of manipulations of myogenesis in utero on the performance of adult skeletal muscle.

The possibility that early fetal programming affects health or disease status in adult life has been considered in relation to tissues such as the cardiovascular system but not with respect to skeletal muscle. Since muscle mass and function are important for life, it is pertinent to ask whether events during the development of muscle in utero can affect the performance of the tissue in later life. This review discusses the factors that influence muscle performance, outlines the current understanding of myogenesis and examines how manipulations alter myogenic outcome after birth. The performance of muscle is determined by the number, type and size of the muscle fibres, these in turn being affected by a number of factors, and the evidence indicates that the proportions of types of muscle fibre have a heritable component. The formation of muscle occurs early in embryogenesis and it appears that the major impacts on myogenesis are associated with extremes of treatment or embryo manipulations. The impact of extremes of treatment or embryo manipulations on myogenesis is seen in the secondary fibres whereas primary fibres appear to be insensitive or protected. Overall, the opportunities for manipulation of myogenesis in utero to improve adult performance are limited.

Elevated IGF-II mRNA and phosphorylation of 4E-BP1 and p70(S6k) in muscle showing clenbuterol-induced anabolism.

Muscle wasting affects large numbers of people, but few therapeutic approaches exist to treat and/or reverse this condition. The beta(2)-adrenoceptor agonist clenbuterol produces a muscle-specific protein anabolism in both normal and catabolic muscle and has been used to limit muscle wasting in humans. Because clenbuterol appears to interact with or mimic innervation, its effect on the expression of the neurotrophic agents insulin-like growth factor (IGF)-II and H19 and their putative pathways was examined in normal rat plantaris muscle. The results showed that the well-documented early effects of clenbuterol on protein metabolism were preceded by elevated levels of IGF-II and H19 transcripts together with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1) and p70(S6k). By 3 days, transcript levels for IGF-II and H19 and 4E-BP1 and p70(S6k) phosphorylation had returned to control values. These novel findings indicate that clenbuterol-induced muscle anabolism is potentially mediated, at least in part, by an IGF-II-induced activation of 4E-BP1 and p70(S6k).

Amelioration of denervation-induced atrophy by clenbuterol is associated with increased PKC-alpha activity.

Rat soleus muscle was denervated for 3 or 7 days, and total membrane protein kinase C (PKC) activity and translocation and immunocytochemical localization of PKC isoforms were examined. Dietary administration of clenbuterol concomitant with denervation ameliorated the atrophic response and was associated with increased membrane PKC activity at both 3 (140%) and 7 (190%) days. Of the five PKC isoforms (alpha, epsilon, theta, zeta, and mu) detected in soleus muscle by Western immunoblotting, clenbuterol treatment affected only the PKC-alpha and PKC-theta forms. PKC-alpha was translocated to the membrane fraction upon denervation, and the presence of clenbuterol increased membrane-bound PKC-alpha and active PKC-alpha as assayed by Ser(657) phosphorylation. PKC-theta protein was downregulated upon denervation, and treatment with clenbuterol further decreased both cytosolic and membrane levels. Immunolocalization of PKC-theta showed differences for regulatory and catalytic domains, with the latter showing fast-fiber type specificity. The results suggest potential roles of PKC-alpha and PKC-theta in the mechanism of action of clenbuterol in alleviating denervation-induced atrophy.

Masseteric hypertrophy?: preliminary report.

We report radiological and histological investigations of a patient who presented with the masseteric hypertrophy. Sections of the patient's masseter muscles were also investigated using a series of histological techniques. The histological and morphometric analysis of the patient's masseter muscle showed numerous small fibres, which indicated that the masseteric enlargement was not the result of classic fibre hypertrophy. We suggest that the use of the term 'hypertrophy' in this condition may be misleading.

Clenbuterol increases the expression of myogenin but not myoD in immobilized rat muscles.

Immobilization of one hindlimb of young rats in plantar flexion for 3 days led to changes in the plantaris muscles. These comprised a loss of muscle mass and a reduction in protein and RNA content, but no change in the transcript levels of the myogenic regulatory factors myogenin and myoD. Dietary administration of the beta-adrenoceptor agonist clenbuterol (2 mg/kg diet), which has been shown to ameliorate muscle wasting in a wide range of atrophic conditions, also limited muscle wasting in terms of weight, protein, and RNA in the immobilized plantaris muscles. In addition, drug treatment in immobilized plantaris muscles was associated with a marked increase in the steady-state levels of mRNA for myogenin (approximately 360% increase over control) but not myoD. These data provide the first evidence for independent changes in these two myogenic regulatory factors in immobilized muscle and suggest that the action of clenbuterol on these factors may depend on the mechanistic basis for the atrophic response.

Pig muscle fibre characteristics as a source of variation in eating quality.

Despite the application of the MLC Blueprint specifications there is still unacceptable variation in meat eating quality. Evidence from the literature suggests that the intrinsic characteristics of the muscle may be an important source of variation, but there is no indication as to what extent these characteristics may explain the residual variation in eating quality. The purpose of the present study was to quantify the role of muscle fibre characteristics in accounting for eating quality variability. In the study, evaluation of samples from 125 pigs from eight breeding company populations indicated that fibre characteristics, particularly the diameter of the fast twitch oxidative glycolytic fibres, contributed to variation in instrumental texture of meat. In addition, the data suggest that there are genetic differences in fibre type distribution which can be used to segregate populations.

Effect of prednisone on protease activities and structural protein levels in rat muscles in vivo.

To further elucidate the biochemical mechanism by which the corticosteroid prednisone induces differential changes in muscle mass (via altered protein synthesis/degradation rates) in normal or degenerating muscle tissues, we have determined the activity of a range of proteolytic enzyme types, together with levels of muscle structural proteins, in five innervated and denervated muscle types from control and drug treated rats. In both normal and wasting muscles, the activity of many protease types was substantially down-regulated following treatment with prednisone; however, accompanying net decreases in muscle mass were observed (although the structural protein composition of muscles was unaltered following drug treatment). We conclude that whilst overall rates of protein degradation in both normal and degenerating muscle may be reduced (via protease down-regulation) following prednisone treatment, the effect of the latter in reducing protein synthesis rates must be proportionately greater (even in actively degenerating tissue). Thus, the data do not support the hypothesis that the beneficial effect of prednisone in maintaining muscle mass in pathological tissues (e.g., Duchenne muscular dystrophy (DMD)) operates principally via down-regulation of protease action/protein catabolism.

Clenbuterol mimics effects of innervation on myogenic regulatory factor expression.

The amelioration of denervation atrophy by the beta-adrenoceptor agonist clenbuterol has led to the suggestion that the drug mimics or stimulates production of neurotrophic factors. Neurotrophic factors have profound effects on muscle growth, but the precise mechanisms through which this influence is exerted are unknown. The expression of myoD and myogenin, proteins that in turn regulate the transcription of tissue-specific genes during skeletal muscle differentiation, is controlled by innervation. In muscle undergoing denervation-induced atrophy, myoD and myogenin mRNAs increase. However, this is only partially reversed by electrical activity, thus implicating neurotrophic factors in regulation of these genes. Here we demonstrate that clenbuterol represses myoD and myogenin expression and decreases the levels of acetylcholine receptors in denervated muscles. The data provide the first evidence that the action of clenbuterol is directed through the neural axis.

Clenbuterol, a beta-adrenoceptor agonist, increases relative muscle strength in orthopaedic patients.

1. The sympathomimetic agent clenbuterol has a muscle-specific anabolic effect in normal and wasted muscles from animals. This trial was designed to examine the effect of the drug on the recovery of muscle strength and area after open medial meniscectomy. 2. A double-blind, completely randomized, placebo-controlled study was carried out on 20 healthy male patients. Muscle strength and cross-sectional area were determined before and after surgery. Patients were treated with drug or placebo for 4 weeks postoperatively and there was a 2 week washout period. 3. The results suggest that, in the operated leg, clenbuterol treatment is associated with a more rapid rehabilitation of strength in knee extensor muscles; in the unoperated leg, knee extensor strength increased above the initial values after 6 weeks (P = 0.01). However, in terms of absolute strength the differences were not significant between the two groups. 4. It is concluded that the data lend support to the proposition that clenbuterol has therapeutic potential in the treatment of muscle-wasting conditions.

Tissue specific responses to clenbuterol; temporal changes in protein metabolism of striated muscle and visceral tissues from rats.

The beta-adrenoceptor agonist, clenbuterol, was administered orally to male weanling rats for a period of up to 10 days. The composition and fractional rate of protein synthesis (Ks) in skeletal and cardiac muscle, gut and liver were determined. There were few changes in the visceral tissues, but there was marked protein accretion in the muscles. The results suggested that in skeletal muscles there is an increase in both Ks and the amount of protein synthesised per unit RNA. In cardiac muscle, the results indicated that there was only a very transient increase in Ks and that changes in translational capacity (RNA/prot) may account in part for the increase in protein content. It is concluded that the mechanistic basis for the increased protein gain may be different between skeletal and cardiac muscles.

Satellite cells in innervated and denervated muscles treated with clenbuterol.

The sympathomimetic agent, clenbuterol, induces a muscle-specific hypertrophy in both normal and catabolic muscle. Drug-induced hypertrophy is not generally associated with an increase in DNA content, thus the role of satellite cells in the response of soleus muscles from weanling rats is questioned. Following simultaneous sciatic section and administration of clenbuterol, responses are similar in innervated and denervated muscles after 4 days. Increased protein accretion in treated muscles is associated with evidence of satellite cell activation, but with little evidence of division. It is speculated that satellite cell production of growth factors may play an important role in the hypertrophic action of clenbuterol, and the clinical implications of the findings are discussed.

Effect of clenbuterol on protease activities and protein levels in rat muscle.

To elucidate the biochemical mechanism by which the sympathomimetic agent clenbuterol promotes skeletal muscle growth, we have determined the activity of a range of proteolytic enzyme types (acid, neutral and alkaline proteinases and peptidases), together with the levels of soluble and structural proteins (via SDS-polyacrylamide gel electrophoresis) in 5 innervated and denervated muscle types from control and drug-treated rats. No gross change in activity was found for any enzyme type in any muscle (innervated or denervated) following clenbuterol treatment; however, one enzyme, arginyl aminopeptidase, showed a small but consistent decrease in activity in all of the innervated muscles investigated. Similar fractionation profiles were obtained for structural or soluble proteins from corresponding muscle types (innervated or denervated) in control or clenbuterol-treated animals, with the exception of cardiac muscle, which showed a 50% increase in staining intensity of one band (subunit molecular mass 18 kD). We conclude that the anabolic action of clenbuterol in promoting skeletal muscle growth does not occur via downregulation of protease activity, or increase in levels of individual muscle proteins.

Denervation increases clenbuterol sensitivity in muscle from young rats.

Clenbuterol has been shown to ameliorate denervation-induced atrophy and, therefore, clearly has therapeutic potential in the treatment of muscle wasting conditions in man. Previous studies have used dosages in rats which would be unacceptable in clinical practice, but the present results show that denervated muscle has a greater sensitivity to the drug than innervated or cardiac muscle. Fiber hypertrophy and an increase in protein and RNA content could be observed in denervated muscles but not in innervated muscles at a dose of 10 micrograms/kg body weight. When considered on a metabolic body weight basis, the effective dose in rats and the "safe" dose in man are surprisingly comparable. The observations imply that there is good reason to suppose that clenbuterol could be effective in ameliorating similar wasting conditions in man.

Effects of the cyclo-oxygenase inhibitor, fenbufen, on clenbuterol-induced hypertrophy of cardiac and skeletal muscle of rats.

1. When rats were fed with clenbuterol for 7 days skeletal muscle mass increased by 21% in the tonic soleus and phasic plantaris muscles and a 16% hypertrophy of the heart was also induced. Fenbufen, fed to rats for the same period, blocked the hypertrophy of the heart but not that of the skeletal muscles. 2. When feeding of fenbufen commenced 3 days before the administration of clenbuterol, plasma prosta-glandin F2 alpha (PGF2 alpha) was reduced by 79%; there was again no effect of fenbufen on clenbuterol-induced increases in the RNA or protein content of plantaris, nor in the increased area of fast or slow twitch fibres in the soleus. In the heart the clenbuterol-induced increases in the RNA (+21%) and protein content (+20%) were totally inhibited. 3. The effects of clenbuterol on heart muscle appear to be mediated by a cyclo-oxygenase metabolite of arachidonic acid whilst the effects on skeletal muscle are not.

Effects of bovine pituitary growth hormone alone or in combination with the beta-agonist clenbuterol on muscle growth and composition in veal calves.

Twenty-three British Friesian bull calves at approximately 7 d of age were allocated to one of four treatments: controls untreated (five calves), a group (Clen) given 1 mg clenbuterol/kg diet (five calves), a group (GH) given a daily subcutaneous injection of 3.5 mg bovine pituitary growth hormone (GH) (five calves) and a group (Clen + GH) given a combination of clenbuterol as in the Clen group with GH as in the GH group (seven calves). All calves were given milk-substitute at levels adjusted weekly according to metabolic live weight. The animals were slaughtered over the weight range 150-170 kg. Samples of semimembranosus and triceps muscles were excised at slaughter. Treatment with GH produced approximately a threefold increase in mean daily serum GH concentration. Calves given Clen + GH were heaviest at slaughter and the combined treatment produced a significantly higher (P less than 0.01) feed conversion ratio. Administration of clenbuterol either alone or in combination with GH increased the cross-sectional area of both fast twitch glycolytic (FG), and fast twitch oxidative glycolytic (FOG) fibres in both muscles. In contrast GH produced little change in fibre size in semimembranosus muscle, although FOG fibres in triceps were slightly larger than in controls. Neither Clen nor GH resulted in any change in fibre percentage frequency in either muscle. Treatments involving clenbuterol produced a significant decrease in muscle glycogen concentration. Muscles from all three treatment groups tended to show small increases in protein and RNA concentration compared with the controls. Muscles from animals treated with GH alone exhibited an increase in DNA concentration not seen in muscles from the two other treatment groups. Overall, the differential response to the two agents suggested that clenbuterol does not mediate its effects via the GH axis, and that an additive response in terms of protein anabolism may be achieved from the use of a combination of clenbuterol plus GH.

The effect of clenbuterol administration in utero and throughout lactation on pre- and post-natal muscle development in the rat.

The beta-adrenergic agonist, clenbuterol was fed to pregnant rats during gestation and throughout lactation. Changes in muscle morphology and composition were studied in foetal and weanling rats. Drug treatment did not affect mean foetal number, but mean foetal body weight was significantly reduced. Heart weights were increased and both muscle weight and secondary to primary fibre ratios were decreased in foetuses exposed to clenbuterol in utero. In animals exposed to clenbuterol throughout gestation and lactation, muscle weights, and protein, RNA and DNA content and total fibre numbers were reduced. In addition a drug induced anabolism was observed in the muscles of the dams. The data are discussed in terms of a direct drug effect on immature and differentiated muscle together with a possible indirect action through the repartitioning action of clenbuterol.

Fiber-type composition of nine rat muscles. I. Changes during the first year of life.

There are few data available that describe the changes in fiber-type profiles with age in a range of muscles; thus fiber-type profiles and areas were determined in nine muscles of female rats from weaning to 1 yr. The results not only demonstrated the difference in fiber-type composition, size, and hence functional area in the various muscles examined but also illustrated the changes in these parameters with age. In particular, it was clear that two ends of a spectrum of fiber compositions are represented in the muscles studied: tensor fasciae latae, mainly fast-twitch glycolytic at one end and soleus (and adductor longus), mainly slow-twitch oxidative at the other; the remaining muscles were mostly dominated by fast-twitch fibers of a variety of metabolic types. Aging resulted in an increase in fiber area that was most dramatic in fast-twitch glycolytic fibers. Changes in fiber type with age were noted in all muscles, but the exact nature of the developmental changes appeared muscle specific.