No description available.

Understanding the regulatory mechanisms controlling utrophin A expression at the sarcolemma of dystrophic muscles will facilitate the development of therapeutic strategies to ameliorate the pathophysiological features of Duchenne Muscular Dystrophy (DMD). The main goal of this study was to characterize the regulation of utrophin A IRES activity using a transgenic mouse model expressing the utrophin A 5’UTR bicistronic reporter and to identify trans-acting factors that could mediate IRES activity and endogenous expression of utrophin A. We found that utrophin A IRES activity is specifically expressed in skeletal muscles. Moreover, we identified eEF1A2 as a muscle-specific trans-acting factor that can interact with utrophin A and mediate IRES-dependent translation of utrophin A. Finally, we showed that eEF1A2 mediates endogenous utrophin A expression and localization in skeletal muscle. Identifying pharmacological compounds that would specifically target eEF1A2 and increase endogenous levels of utrophin A expression could serve as a drug-based therapy to treat DMD.

Understanding the regulatory mechanisms controlling utrophin A expression at the sarcolemma of dystrophic muscles will facilitate the development of therapeutic strategies to ameliorate the pathophysiological features of Duchenne Muscular Dystrophy (DMD). The main goal of this study was to characterize the regulation of utrophin A IRES activity using a transgenic mouse model expressing the utrophin A 5’UTR bicistronic reporter and to identify trans-acting factors that could mediate IRES activity and endogenous expression of utrophin A. We found that utrophin A IRES activity is specifically expressed in skeletal muscles. Moreover, we identified eEF1A2 as a muscle-specific trans-acting factor that can interact with utrophin A and mediate IRES-dependent translation of utrophin A. Finally, we showed that eEF1A2 mediates endogenous utrophin A expression and localization in skeletal muscle. Identifying pharmacological compounds that would specifically target eEF1A2 and increase endogenous levels of utrophin A expression could serve as a drug-based therapy to treat DMD.

Altered apoptotic signaling in skeletal muscle has been observed in a number of disease states associated with skeletal muscle atrophy. Therefore, understanding the mechanisms that lead to increased skeletal muscle apoptosis may help to prevent the atrophy associated with various diseases. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is able to inhibit apoptosis mediated by both the death-receptor and mitochondrial pathways. In addition, ARC has a unique distribution pattern and is highly expressed in terminally differentiated tissue such as skeletal muscle. To characterize the role of ARC in skeletal muscle morphology and apoptosis, soleus and plantaris muscles of 18 week-old ARC-deficient mice were excised and compared to those of age-matched wild-type littermates. While no differences were seen in muscle weights between genotypes, in the ARC KO animals, the cross-sectional area (CSA) of the soleus was smaller, while the CSA of the plantaris was larger. With respect to fiber type distribution, both muscles demonstrated a shift towards a faster myosin heavy chain expression pattern. For example, soleus muscles of ARC KO animals had significantly less type I fibers and more IIa fibers, while plantaris muscles had significantly less type IIa fibers, and more IIb fibers. In ARC KO animals, type I and IIa fibers were significantly smaller in the soleus, while type IIb fibers were larger in the plantaris. DNA fragmentation (a hallmark of apoptosis) was increased in the soleus, but not plantaris muscles of ARC KO animals. Surprisingly, activity of the proteolytic enzymes caspase-2, -3, -8, and -9, as well as calpains, was not different in either soleus or plantaris muscles. To determine whether a lack of ARC protein affects apoptotic signaling in skeletal muscle, the total expression of pro- and anti-apoptotic proteins were also assessed. In the soleus, no changes were observed in whole tissue AIF, cytochrome c, EndoG, and Smac. In the plantaris, there was no change in total muscle AIF; however, there were trends towards decreased cytochrome c, and increased Smac, as well as a significant decrease in EndoG ARC KO animals. No changes were observed in Bcl-2 and XIAP in the soleus; however, there were significant reductions in FLIP(s) and HSP70 content. In the plantaris, no changes were observed in anti-apoptotic protein content. Subcellular fractionation of red quadriceps for ARC KO mice revealed an increased Bax:Bcl-2 ratio in the isolated mitochondrial fractions. Furthermore, in cytosolic fractions of red quadriceps, AIF protein content was significantly increased in ARC KO animals. Conversely, no changes in apoptotic-related protein content were observed in any fractions from white quadriceps between groups. In agreement with these findings, isolated mitochondria from ARC-deficient animals were more susceptible to calcium induced swelling, as well as membrane potential loss compared to controls. Taken together, these results suggest that in slow-oxidative skeletal muscle of ARC-deficient mice there is increased apoptosis due to caspase-independent, mitochondrial-mediated apoptotic signaling. Furthermore, this study is the first to show ARC plays an important role in skeletal muscle morphology, as ARC KO mice have an altered skeletal muscle phenotype and morphology.

No description available.

<i>Trichinella spiralis</i> is a parasitic nematode of mammalian skeletal muscle.  Its life cycle includes two stages where developmental progression appears to be inhibited until a specific host niche is encountered.  The newborn larva, released within the host intestine depends upon entry to skeletal muscle for continued development.  The muscle larva encapsulates within skeletal muscle and further reproductive development is dependent upon ingestion by a new host.  Developmental arrest has been extensively characterised in <i>Caenorhabditis</i> <i>elegans</i>, where an alternative L3 larva, the dauer larva, is formed in response to environmental conditions refractive to continued reproductive development.  Using the wealth of genetic information regarding <i>C. elegans</i> dauer formation, putative periods of arrest were examined in <i>T. spiralis.</i> TGF-<span lang=EN-GB style='font-family:Symbol'>b-like and insulin-like signalling pathways are critical mediators of <i>C. elegans </i>dauer formation.  A <i>T. spiralis</i> TGF-<span lang=EN-GB style='font-family: Symbol'>b-ligand was identified and designated <i>ts-tll-1</i>.  Sequencing and analysis revealed <i>ts-tll-1</i> to be similar to vertebrate bone morphogenetic proteins and <i>C. elegans </i>DBL-1, is involved in body size regulation.  EST mining identified putative type I and II TGF-<span lang=EN-GB style='font-family:Symbol'>b receptors and a subtilsin-like proprotein convertase, suggesting conservation of TGF-<span lang=EN-GB style='font-family:Symbol'>b-like signalling in <i>T. spiralis.  </i>A partial <i>Trichinella </i>gene encoding an orthologue of the <i>C. elegans</i> insulin-like, tyrosine kinase receptor, DAF-2, was identified by degenerate PCR and designated <i>ts-tkr.  ts-tkr</i> is most similar to <i>C. elegans daf-2</i> within the highly conserved tyrosine kinase domain.  Two alternative transcripts of <i>ts-tkr</i> were identified by 3’ RACE, which differed in their 3’ UTRs.  Semi-quantitative RT-PCR analysis suggested <i>ts-tkr </i>expression was greatest in adult worms, implying a role in promoting reproductive development. Semi-quantitative RT-PCR was also to assess the expression of selected housekeeping and ES protein encoding genes during the <i>T. spiralis </i>life cycle.  While transcription in the <i>C. elegans</i> dauer is depressed, there was no obvious transcriptional repression in <i>Trichinella</i> newborn or muscle larva.

Although much work has been carried out to identify the mechanisms by which muscle is formed, many of the regulatory pathways involved have yet to be fully elucidated. In creating perturbations during the embryonic period, either nutritionally (with a marginal vitamin A deficiency model) or pharmacologically (with the b₂-adrenerguic agonist clenbuterol), a comparison with 'normal' muscle development may be attained. Differences in the temporal expression of specific regulatory proteins may then enhance the existing knowledge of their function in regulating muscle development. Prior to studying changes in muscle regulatory proteins due to perturbations, it was first necessary to illustrate their temporal pattern in "normal" muscle development. The results indicated that a complex regulatory system operates in myogenesis with a number of proteins appearing to be involved in the process of muscle development. A marginal vitamin A deficiency model was established in which maternal retinol levels were clearly reduced in treatment animals in comparison with controls. This resulted in offspring that showed clearly symptoms of marginal vitamin A deficiency. Changes in the abundance of five proteins were observed in response to marginal vitamin A deficiency. Overall, these changes suggested a potential reduction in secondary myogenesis, based on reduced levels of MHCfast, associated with secondary fibres, following birth. Analysis of RNA, DNA and protein values suggested that neonates from clenbuterol fed dams may have reduced hyperplasia and/or increased hypertrophy. Biochemical analysis revealed that proteins such as GATA-2, PKC and Shh, which have previously been associated with hypertrophy, were altered in response to clenbuterol. Further evidence in support of hypertrophy was indicated in an apparent increase in fibre size of neonates detected by MHC immunolocalisation. In conclusion, it has been demonstrated that both nutritional and pharmacological manipulations throughout are gestation capable of altering myogeneiss <i>in utero</i> by two different mechanisms.

Understanding the regulatory mechanisms controlling utrophin A expression at the sarcolemma of dystrophic muscles will facilitate the development of therapeutic strategies to ameliorate the pathophysiological features of Duchenne Muscular Dystrophy (DMD). The main goal of this study was to characterize the regulation of utrophin A IRES activity using a transgenic mouse model expressing the utrophin A 5’UTR bicistronic reporter and to identify trans-acting factors that could mediate IRES activity and endogenous expression of utrophin A. We found that utrophin A IRES activity is specifically expressed in skeletal muscles. Moreover, we identified eEF1A2 as a muscle-specific trans-acting factor that can interact with utrophin A and mediate IRES-dependent translation of utrophin A. Finally, we showed that eEF1A2 mediates endogenous utrophin A expression and localization in skeletal muscle. Identifying pharmacological compounds that would specifically target eEF1A2 and increase endogenous levels of utrophin A expression could serve as a drug-based therapy to treat DMD.

Altered apoptotic signaling in skeletal muscle has been observed in a number of disease states associated with skeletal muscle atrophy. Therefore, understanding the mechanisms that lead to increased skeletal muscle apoptosis may help to prevent the atrophy associated with various diseases. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is able to inhibit apoptosis mediated by both the death-receptor and mitochondrial pathways. In addition, ARC has a unique distribution pattern and is highly expressed in terminally differentiated tissue such as skeletal muscle. To characterize the role of ARC in skeletal muscle morphology and apoptosis, soleus and plantaris muscles of 18 week-old ARC-deficient mice were excised and compared to those of age-matched wild-type littermates. While no differences were seen in muscle weights between genotypes, in the ARC KO animals, the cross-sectional area (CSA) of the soleus was smaller, while the CSA of the plantaris was larger. With respect to fiber type distribution, both muscles demonstrated a shift towards a faster myosin heavy chain expression pattern. For example, soleus muscles of ARC KO animals had significantly less type I fibers and more IIa fibers, while plantaris muscles had significantly less type IIa fibers, and more IIb fibers. In ARC KO animals, type I and IIa fibers were significantly smaller in the soleus, while type IIb fibers were larger in the plantaris. DNA fragmentation (a hallmark of apoptosis) was increased in the soleus, but not plantaris muscles of ARC KO animals. Surprisingly, activity of the proteolytic enzymes caspase-2, -3, -8, and -9, as well as calpains, was not different in either soleus or plantaris muscles. To determine whether a lack of ARC protein affects apoptotic signaling in skeletal muscle, the total expression of pro- and anti-apoptotic proteins were also assessed. In the soleus, no changes were observed in whole tissue AIF, cytochrome c, EndoG, and Smac. In the plantaris, there was no change in total muscle AIF; however, there were trends towards decreased cytochrome c, and increased Smac, as well as a significant decrease in EndoG ARC KO animals. No changes were observed in Bcl-2 and XIAP in the soleus; however, there were significant reductions in FLIP(s) and HSP70 content. In the plantaris, no changes were observed in anti-apoptotic protein content. Subcellular fractionation of red quadriceps for ARC KO mice revealed an increased Bax:Bcl-2 ratio in the isolated mitochondrial fractions. Furthermore, in cytosolic fractions of red quadriceps, AIF protein content was significantly increased in ARC KO animals. Conversely, no changes in apoptotic-related protein content were observed in any fractions from white quadriceps between groups. In agreement with these findings, isolated mitochondria from ARC-deficient animals were more susceptible to calcium induced swelling, as well as membrane potential loss compared to controls. Taken together, these results suggest that in slow-oxidative skeletal muscle of ARC-deficient mice there is increased apoptosis due to caspase-independent, mitochondrial-mediated apoptotic signaling. Furthermore, this study is the first to show ARC plays an important role in skeletal muscle morphology, as ARC KO mice have an altered skeletal muscle phenotype and morphology.

Obesity is a condition in which fat accumulation in adipose tissue is in excess to an extent that health may be impaired. Insulin resistance is integral to the pathophysiology of obesity-related metabolic complications. Central adiposity and skeletal muscle mass and function determine insulin sensitivity and metabolic risk. A high visceral fat-to-skeletal muscle mass-ratio contributes to an unfavourable metabolic profile. Epidemiological and experimental studies suggest that high dietary saturated fat intake is deleterious while polyunsaturated fatty acids (PUFAs), in particular n-3 PUFAs of marine origin, may be advantageous to metabolic health. The aim was to determine the effect of subcutaneous (SC) and visceral (IAB) fat, and long-chain saturated, n-3 and n-6 PUFAs, and the interactions between them, on insulin sensitivity and the pathways regulating energy metabolism in skeletal muscle. Thereby an adipose tissue-conditioned media-skeletal muscle myotube co-culture system was developed. Adipose tissue-conditioned medium (CM) was generated from SC and IAB fat biopsy of obese humans. Viability of the tissue explants was confirmed by the measurement of lactate dehydrogenase activity in the CM and nuclear DNA fragmentation of tissue explants. The concentrations of cytokines (leptin, adiponectin, interleukin (IL)-1β, IL 6, IL-8, tumor necrosis factor-α, resistin and plasminogen activator inhibitor-1) and long-chain fatty acids were determined in CM. CM from IAB but not SC fat reduced insulin-stimulated glucose uptake. The effect of IAB fat was predominantly mediated by IL-6 via the activation of a nuclear factor kappa B/mammalian target of rapamycin complex 1 (NFκB/mTORC1)-dependent pathway. Palmitic acid (PA; 16:0) reduced insulin-stimulated glucose uptake, an effect mediated by intramuscular accumulation of ceramide and the activation of NFκB and mTORC1. The effects of fatty acids were similar in the presence of CM from either fat depot, where the effect of PA was partially reversed by docosahexaenoic acid (DHA; 22:6n-3) and completely by linoleic acid (LA; 18:2n-6). The effect of each fatty acid in the presence or absence of CM from each fat depot on mRNA expression of key genes regulating muscle energy metabolism was determined. Protein phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)-α and acetyl-coenzyme A carboxylase (ACC)-β were also determined. PA increased SCD1 mRNA. DHA and LA increased AMPKα2 mRNA and AMPKα and ACCβ protein phosphorylation. Microarray analysis was used to determine the global gene expression changes in PAand DHA-treated L6 myotubes. DHA down-regulated lipogenic genes and upregulated genes which were involved in β-oxidation and mitochondrial function. When compared to PA, DHA down-regulated genes which were involved in lipid synthesis, endoplasmic reticulum metabolism and mitogen-activated protein kinase activity. Taken together, pro-inflammatory cytokines from IAB fat and PA induced insulin resistance in skeletal muscle and both were at least partly mediated by a NFκB/mTORC1-dependent pathway. In contrast, DHA and LA may improve insulin sensitivity by diverting fatty acids towards oxidation and subsequently reducing substrate availability for the formation of lipid metabolites including ceramide. A reduction in PA intake and substitution (rather than addition) of DHA and LA, together with a reduction in overall energy intake and increase in physical activity, is optimal for metabolic health. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1523054 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010