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

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.

The MEF2 family of transcription factors (MEF2A, MEF2C, and MEF2D) are crucial during skeletal muscle differentiation. Although the roles of MEF2D isoforms are well established, the roles of MEF2A and MEF2C are not as well understood. This thesis, we investigated the expression, localization, and function of MEF2A and MEF2C, using specific antibodies. While MEF2A is expressed in both proliferating and differentiated myoblasts, protein levels of MEF2C were only detected during differentiation. During early stages of differentiation MEF2A is expressed in both the cytoplasm and the nucleus. However during later stages of differentiation, it is localized predominately in the nucleus. MEF2C appears to be localized differently depending on which isoform is being investigated. Using an affinity purification and mass spectrometry based approach we identified PRMT1 as a unique interacting protein with MEF2A during skeletal muscle differentiation. PRMT1 is a protein arginine methyltransferase which mediates the addition of methyl groups onto various proteins including histone H4 arginine 3 (H3R4) which is associated with gene activation. Both MEF2A and PRMT1 occupy genomic targets of MEF2A. Inhibition of PRMT1 with a specific inhibitor delays C2C12 myoblast differentiation in the early stages of differentiation but no effect was observed during late stage differentiation. The MEF2 family of transcription factors show distinct but overlapping function during skeletal muscle differentiation.

CARM1 and skeletal muscle atrophy / Coactivator-associated arginine methyltransferase 1 (CARM1) is emerging as an important player in skeletal muscle biology. We sought to elucidate the role of CARM1 in mediating muscle mass and function, as well as in the induction and progression of the muscle atrophy program. To this end, we engineered CARM1 skeletal muscle-specific knockout (mKO) mice and employed distinct, but complementary models of muscle atrophy, including neurogenic muscle disuse, food deprivation, and the sarcopenia of aging. CARM1 mKO resulted in reduced muscle mass and myofiber cross-sectional area concomitant with dysregulated autophagic and atrophic signaling, which indicates the requirement of CARM1 for the maintenance of muscle biology. Interestingly, CARM1 deletion mitigated the progression of both denervation- and fasting-induced skeletal muscle atrophy as compared to wild-type (WT) mice. Key mechanistic findings revealed that CARM1 interacts with the master neuromuscular regulator AMPK and attenuates the expression and activity of its downstream autophagy and atrophy networks. Surprisingly, both male and female mKO mice have a significantly shorter lifespan versus their WT littermates, revealed by a ~50% reduction in survival at 22-months-old, which is equivalent to ~70 years-old in humans. As such, we observed significantly reduced functional outcomes of integrative physiology in old mKO mice compared to old WT animals, such as strength and motor performance. Taken together, these results indicate that skeletal muscle CARM1 is indispensable for maintaining muscle mass, function, and lifespan. Targeting the interplay between CARM1 and AMPK may offer a viable therapeutic strategy for combating life-limiting muscle wasting conditions. / Thesis / Doctor of Philosophy (PhD) / While muscle wasting and weakness remains a widespread issue, the mechanisms that control muscle atrophy are not entirely understood. Previous evidence suggests that coactivator-associated arginine methyltransferase 1 (CARM1) regulates skeletal muscle remodeling. However, the role of CARM1 during muscle atrophy is unknown. Therefore, the purpose of this work was to investigate the function of CARM1 during muscle wasting. We generated mice with CARM1 deleted in skeletal muscle and studied the impact of CARM1 deficiency on the loss of skeletal muscle mass during muscle disuse, food deprivation, and aging. We found that CARM1 is required to maintain muscle mass under basal conditions. Interestingly, knocking out CARM1 in muscle attenuated the progression of denervation- and fasting-induced atrophy. However, CARM1 deletion in muscle resulted in lower muscle strength and a reduced lifespan. CARM1 deficiency did not prevent aging-induced muscle loss. Overall, these findings advance our understanding of CARM1 in skeletal muscle biology.

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Weight loss through dietary energy restriction (ER) is an effective method to promote fat mass loss. However, a negative consequence of ER is the loss of lean body mass (LBM), particularly skeletal muscle, which is induced by an imbalance between rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Increased protein intake and resistance exercise (RE) during ER promote the retention of LBM. Currently, the relative contribution of MPS and MPB to diet-induced LBM loss, and the effect of protein intake and RE on these variables is not well characterized. In Study 1 we compared the acute (hour-to-hour) MPS response to the ingestion of whey and soy protein, before and after 14 days of ER (-750kcal/d). The results of Study 1 indicated that whey protein was superior to soy protein in stimulating MPS before and after ER. In Studies 2 and 3 we examined the effect of 10 days of a marked 40% energy restriction on acute postabsorptive MPS and MPB and integrated (day-to-day) MPS. Using unilateral RE, we examined the effects of protein (1.2g protein/kg/g or 2.4g protein/kg/d) at rest and in combination with resistance exercise. The results of Study 2 showed that there were no changes in acute MPB or markers of proteolysis with ER. The results of Study 3 indicated that acute and integrated MPS were reduced following ER at both protein levels (1.2g protein/kg/g or 2.4g protein/kg/d), but RE was able to prevent this decline. Taken together, these studies demonstrate that reductions in MPS are the likely reason for LBM loss during short-term dietary energy restriction, and strategies such as RE and high quality protein intake can help to prevent the decline in MPS. These findings provide information for the design of weight loss programs that wish to preserve skeletal muscle. / Thesis / Doctor of Philosophy (PhD) / Dietary energy restriction is commonly used to promote weight/fat loss; however, a potential negative consequence of dietary energy restriction is the loss of skeletal muscle mass. This thesis examines the impact of dietary energy restriction on the two processes that regulate skeletal muscle mass: muscle protein synthesis and muscle protein breakdown. Additionally, this thesis investigates the role of protein intake and resistance exercise as strategies to prevent diet-induced changes in muscle protein synthesis and breakdown. The studies within this thesis demonstrate that during energy restriction rates of muscle protein synthesis are reduced whilst muscle protein breakdown is unchanged. Importantly, consuming high quality protein such as whey protein and performing resistance exercise prevent the diet-induced decline in rates of muscle protein synthesis. These findings provide new and insightful information for the design of weight loss programs that aim to preserve skeletal muscle whilst also promoting the loss of body fat.

Twenty-five patients with a mean age of 59.7 ± 11.8 (SD) years who were hemiparetic due to a cerebrovascular lesion of the cortex were assessed to determine the degree of neuromuscular dysfunction produced in the affected lower limb. Dysfunction was postulated to be the result of a secondary lower motoneuron lesion precipitated by the primary upper motoneuron lesion. The effects of cerebrovascular disease on skeletal muscle were assessed through an evaluation of the motor unit which involved assessment of excitable muscle mass (M-wave amplitudes), motor unit counts, peripheral nerve conduction velocities, evoked contractile properties of the dorsiflexor muscles (tibialis anterior) of the lower limb and degree of motor dysfunction expressed as a function of motor unit activation and maximum voluntary contraction (twitch interpolation method). Results showed preservation of the skeletal muscle with normal contraction times (108 ± 33 ms and 106 ± 35 ms, affected limb versus unaffected limb) and half relaxation times (119.3 ± 41 ms and 114 ± 32 ms respectively). Twitch torque was maintained and did not show significant differences between limbs (2.3 ± 1.6 N.m and 2.4 + 1.5 N.m., paretic vs. non-paretic limb). Voluntary force production of the affected limb, (10 ± 12.1 N.m) however, was 38% of that produced by the unaffected limb (26 + 1.4 N.m.) and measures of mean percent motor unit activation of the paretic limb were 58% of that produced by the unaffected limb. Interpolated twitch results showed that mean percent motor unit activation was significantly different in the affected limb (46 ± 36%) than the unaffected limb (79 + 19.6%). These results indicate that some motoneurone in hemiplegic patients were healthy but not readily activated. No effect was seen for age, sex of the subject and time post stroke. No significant difference in the pattern of results was observed between initial and final test results for subjects examined more than once. Conclusions were that skeletal muscle integrity was preserved probably due to spinal reflex activity and force production was depressed due, in part, to an inability to fully activate motor units. The inability to activate motoneurone may occur because some motoneurone are in a dysfunctional state. The following data from the present experimental work revealed several trends suggesting the possibility of a sick motoneuron hypothesis due to transynaptic motoneuron degeneration and the existence of a secondary lower motoneuron lesion in stroke syndrome. These trends are: 1) decreased motor unit counts of a sub-group of the total sample consisting of subjects under 60 years of age approached conventional levels of significance. Mean values for the affected limb were 73.8 ± 52 and 130.0 ± 61 for the unaffected limb (P < 0.05, F =5.05, critical F =5.59) In addition, M-wave amplitudes showed significant differences between limbs in the sub-group (4.0 + 2.3 mV and 5.7 ± 2.2 mV affected vs unaffected limb p<0.05), indicating that transynaptic motoneuron loss may have occurred; 2) decreased nerve conduction velocities and prolonged terminal latencies in the motor nerves of the paretic limbs also suggest sick motoneurone and the possibility of a dying back phenomenon of the terminal nerve endings; 4) normal M-wave amplitudes and twitch torque values of the tibialis anterior muscle coupled with the prolonged terminal latencies may be indicative of collateral sprouting of terminal axons which have taken over previously denervated muscle fibres. Future studies are needed to confirm or refute these observations. / Thesis / Master of Science (MS)

Skeletal dysplasias and dysostoses are a genotypically and phenotypically diverse group of disorders that affect the growth, development and maintenance of cartilage and bone. General disorders of bone affecting bones and cartilage throughout the body have been referred to as skeletal dysplasias, whereas defects that selectively affect certain bones or bone groups are called skeletal dysostoses. Despite this distinction, modern molecular techniques are showing that this division is somewhat superficial, given the similarity in their underlying causes. Although the rate of disease gene discovery has grown substantially since the advent of next-generation sequencing technologies, most of the disorders have unknown molecular defects. Skeletal dysostoses are rarely observed, occurring at such low incidence levels that no comprehensive study has ascertained their frequency. The effects range from mild growth inhibition to complete absence of entire bone groups. The axial skeleton is most often involved in skeletal dysostoses with common symptoms including poorly formed cranial bones, mandible, ribs and vertebrae. Several important signaling pathways control the migration and formation of mesodermal cells, which eventually differentiate into many elements of the vertebral column. The importance of these pathways, namely the T-box transcription factors, Wnt, Notch, and Smad pathways are integrally involved in the very early stages of vertebral development. Currently, the most cost-effective method of pathogenic gene discovery for rare genetic diseases is exome sequencing. Utilizing this technology, as well as SNP arrays for identity-by-descent loci mapping, two independent skeletal dysostosis cases with similar phenotypes were studied to determine pathogenic candidate genes. Next-generation sequencing and identity-by-descent analysis revealed a possible candidate gene, PM20D2, in one proband. The gene includes peptidase dimerization, peptidase M20/M25/M40, and N-myristolylation domains based on predicted functional analysis. It is implicated in various metabolic activities, having hydrolase, protein binding, and metallopeptidase molecular functions. Further investigation into this gene, as well as further studies of these probands is needed to understand the role, if any, the defect plays in the disease.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1960. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.