Evaluation of novel dairy fatty acids in inflammatory muscle-wasting conditions

This work examined the effects of a novel dairy fatty acid conjugated linoleic acid (CLA) and its effects on muscle wasting in advanced cancer. Results showed a positive anti-inflammatory role of CLA on the supression of tumour growth and established a model for studying the action of CLA in human muscle-wasting conditions.

Eicosapentaenoic acid and oxypurinol in the treatment of muscle wasting in a mouse model of cancer cachexia

Cancer cachexia is a wasting condition, driven by systemic inflammation and oxidative stress. This study investigated eicosapentaenoic acid (EPA) in combination with oxypurinol as a treatment in a mouse model of cancer cachexia. Mice with cancer cachexia were randomized into 4 treatment groups (EPA (0.4 g/kg/day), oxypurinol (1 mmol/L ad-lib), combination, or control), and euthanized after 29 days. Analysis of oxidative damage to DNA, mRNA analysis of pro-oxidant, antioxidant and proteolytic pathway components, along with enzyme activity of pro- and antioxidants were completed on gastrocnemius muscle. The control group displayed earlier onset of tumor compared to EPA and oxypurinol groups (P<0.001). The EPA group maintained body weight for an extended duration (20 days) compared to the oxypurinol (5 days) and combination (8 days) groups (P<0.05). EPA (18.2±3.2 pg/ml) and combination (18.4±3.7 pg/ml) groups had significantly higher 8-OH-dG levels than the control group (12.9±1.4 pg/ml, P≤0.05) indicating increased oxidative damage to DNA. mRNA levels of GPx1, MURF1 and MAFbx were higher following EPA treatment compared to control (P≤0.05). Whereas oxypurinol was associated with higher GPx1, MnSOD, CAT, XDH, MURF1, MAFbx and UbB mRNA compared to control (P≤0.05). Activity of total SOD was higher in the oxypurinol group (32.2±1.5 U/ml) compared to control (27.0±1.3 U/ml, P<0.01), GPx activity was lower in the EPA group (8.76±2.0 U/ml) compared to control (14.0±1.9 U/ml, P<0.05), and catalase activity was lower in the combination group (14.4±2.8 U/ml) compared to control (20.9±2.0 U/ml, P<0.01). There was no change in XO activity. The increased rate of weight decline in mice treated with oxypurinol indicates that XO may play a protective role during the progression of cancer cachexia, and its inhibition is detrimental to outcomes. In combination with EPA, there was little significant improvement from control, indicating oxypurinol is unlikely to be a viable treatment compound in cancer cachexia.

Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease

Rationale: The molecular mechanisms of muscle atrophy in chronic obstructive pulmonary disease (COPD) are poorly understood. In wasted animals, muscle mass is regulated by several AKT-related signaling pathways.
Objectives: To measure the protein expression of AKT, forkhead box class O (FoxO)-1 and -3, atrogin-1, the phosphophrylated form of AKT, p70^S6K glycogen synthase kinase-3ß (GSK-3ß), eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1), and the mRNA expression of atrogin-1, muscle ring finger (MuRF) protein 1, and FoxO-1 and -3 in the quadriceps of 12 patients with COPD with muscle atrophy and 10 healthy control subjects. Five patients with COPD with preserved muscle mass were subsequently recruited and were compared with six patients with low muscle mass.
Methods: Protein contents and mRNA expression were measured by Western blot and quantitative polymerase chain reaction, respectively.
Measurements and Main Results: The levels of atrogin-1 and MuRF1 mRNA, and of phosphorylated AKT and 4E-BP1 and FoxO-1 proteins, were increased in patients with COPD with muscle atrophy compared with healthy control subjects, whereas atrogin-1, p70^S6K, GSK-3ß, and FoxO-3 protein levels were similar. Patients with COPD with muscle atrophy showed an increased expression of p70^S6K, GSK-3ß, and 4E-BP1 compared with patients with COPD with preserved muscle mass.
Conclusions: An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The transcriptional regulation of atrogin-1 and MuRF1 may occur via FoxO-1, but independently of AKT. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

Conjugated linoleic acid suppresses myogenic gene expression in a model of human muscle cell inflammation

Proinflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha}, contribute to muscle wasting in inflammatory disorders, where TNF{alpha} acts to regulate myogenic genes. Conjugated linoleic acid (CLA) has shown promise as an antiproliferative and antiinflammatory agent, leading to its potential as a therapeutic agent in muscle-wasting disorders. To evaluate the effect of CLA on myogenesis during inflammation, human primary muscle cells were grown in culture and exposed to varying concentrations of TNF{alpha} and the cis-9, trans-11 and trans-10, cis-12 CLA isomers. Expression of myogenic genes (Myf5, MyoD, myogenin, and myostatin) and the functional genes creatine kinase (CK) and myosin heavy chain (MHC IIx) were measured by real-time PCR. TNF{alpha} significantly downregulated MyoD and myogenin expression, whereas it increased Myf5 expression. These changes corresponded with a decrease in both CK and MHC IIx expression. Both isomers of CLA mimicked the inhibitory effect of TNF{alpha} treatment on MyoD and myogenin expression, whereas myostatin expression was diminished in the presence of both isomers of CLA either alone or in combination with TNF{alpha}. Both isomers of CLA decreased CK and MHC IIx expression. These findings demonstrate that TNF{alpha} can have specific regulatory effects on myogenic genes in primary human muscle cells. A postulated antiinflammatory role of CLA in myogenesis appears more complex, with an indication that CLA may have a negative effect on this process.

Effects of conjugated linoleic acid on myogenic and inflammatory responses in a human primary muscle and tumor coculture model

The antiproliferative and anti-inflammatory properties of conjugated linoleic acid (CLA) make it a potentially novel treatment in chronic inflammatory muscle wasting disease, particularly cancer cachexia. Human primary muscle cells were grown in coculture with MIA PaCa-2 pancreatic tumor cells and exposed to varying concentrations of c9,t11 and t10,c12 CLA. Expression of myogenic (Myf5, MyoD, myogenin, and myostatin) and inflammatory genes (CCL-2, COX-2, IL-8, and TNF-) were measured by real-time PCR. The t10,c12 CLA isomer, but not the c9,t11 isomer, significantly decreased MIA PaCa-2 proliferation by between 15% and 19%. There was a marked decrease in muscle MyoD and myogenin expression (78% and 62%, respectively), but no change in either Myf5 or myostatin, in myotubes grown in coculture with MIA PaCa-2 cells. CLA had limited influence on these responses. A similar pattern of myogenic gene expression changes was observed in myotubes treated with TNF- alone. Several-fold significant increases in CCL-2, COX-2, IL-8, and TNF- expression in myotubes were observed with MIA PaCa-2 coculture. The c9,t11 CLA isomer significantly decreased basal expression of TNF- in myotubes and could ameliorate its tumor-induced rise. The study provides insight into the anti-inflammatory and antiproliferative actions of CLA and its application as a therapeutic agent in inflammatory disease states.

AMPK-mediated transcription in skeletal muscle

Skeletal muscle phenotype plays a critical role in human performance and health, and skeletal muscle oxidative capacity is a key determinant of exercise tolerance. More recently, defective muscle oxidative metabolism has been implicated in a number of conditions associated with the metabolic syndrome, cardiovascular disease and muscle-wasting disorders. AMPK (AMP-activated protein kinase) is a critical regulator of cellular and organismal energy balance. AMPK has also emerged as a key regulator of skeletal muscle oxidative function, including metabolic enzyme expression, mitochondrial biogenesis and angiogenesis. AMPK mediates these processes primarily through alterations in gene expression. The present review examines the role of AMPK in skeletal muscle transcription and provides an overview of the known transcriptional substrates mediating the effects of AMPK on skeletal muscle phenotype.

HSP72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin¹, ², ³. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca²⁺, which activates inflammatory and muscle degenerative pathways⁴, ⁵, ⁶. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death⁷, ⁸, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca²⁺) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders.

Identification of MicroRNAs linked to regulators of muscle protein synthesis and regeneration in young and old skeletal muscle

Over the course of ageing there is a natural and progressive loss of skeletal muscle mass. The onset and progression of age-related muscle wasting is associated with an attenuated activation of Akt-mTOR signalling and muscle protein synthesis in response to anabolic stimuli such as resistance exercise. MicroRNAs (miRNAs) are novel and important post-transcriptional regulators of numerous cellular processes. The role of miRNAs in the regulation of muscle protein synthesis following resistance exercise is poorly understood. This study investigated the changes in skeletal muscle miRNA expression following an acute bout of resistance exercise in young and old subjects with a focus on the miRNA species predicted to target Akt-mTOR signalling.

The effects of a protein enriched diet with lean red meat combined with a multi-modal exercise program on muscle and cognitive health and function in older adults: study protocol for a randomised controlled trial

BACKGROUND: Age-related muscle wasting has been strongly implicated with falls and fractures in the elderly, but it has also been associated with cognitive decline and dementia. Progressive resistance training (PRT) and adequate dietary protein are recognised as important contributors to the maintenance of muscle health and function in older adults. However, both factors also have the potential to improve brain function and prevent cognitive decline via several pathways, including the regulation of various growth and neurotrophic factors [insulin-like growth factor-1 (IGF-1)]; brain-derived growth factor (BDNF)] and/or the modulation of systemic inflammation. The primary aim of this study is to investigate whether a modest increase in dietary protein achieved through the consumption of lean red meat three days per week, when combined with PRT, can enhance muscle mass, size and strength and cognitive function in community-dwelling older people. METHODS/DESIGN: The study design is a 48-week randomised controlled trial consisting of a 24-week intervention with a 24-week follow-up. Men and women (n=152) aged 65 years and over residing in the community will be randomly allocated to: 1) PRT and provided with 220 g (raw weight) of lean red meat to be cooked and divided into two 80 g servings on each of the three days that they complete their exercise session, or 2) control PRT in which participants will be provided with and advised to consume ≥1 serving (~1/2 cup) of rice and/or pasta or 1 medium potato on each of the three training days. The primary outcome measures will be muscle mass, size and strength and cognitive function. Secondary outcomes will include changes in: muscle function, neural health (corticospinal excitability and inhibition and voluntary activation), serum IGF-1 and BDNF, adipokines and inflammatory markers, fat mass and inter-/intra-muscular fat, blood pressure, lipids and health-related quality of life. All outcome measures will be assessed at baseline and 24 weeks, with the exception of cognitive function and the various neurobiological and inflammatory markers which will also be assessed at week 12. DISCUSSION: The findings from this study will provide important new information on whether a modest increase in dietary protein achieved through the ingestion of lean red meat can enhance the effects of PRT on muscle mass, size and strength as well as cognitive function in community-dwelling older adults. If successful, the findings will form the basis for more precise exercise and nutrition guidelines for the management and prevention of age-related changes in muscle and neural health and cognitive function in the elderly. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12613001153707 . Date registered 16(th) October, 2013.

Evaluation of follistatin as a therapeutic in models of skeletal muscle atrophy associated with denervation and tenotomy

Follistatin is an inhibitor of TGF-β superfamily ligands that repress skeletal muscle growth and promote muscle wasting. Accordingly, follistatin has emerged as a potential therapeutic to ameliorate the deleterious effects of muscle atrophy. However, it remains unclear whether the anabolic effects of follistatin are conserved across different modes of non-degenerative muscle wasting. In this study, the delivery of a recombinant adeno-associated viral vector expressing follistatin (rAAV:Fst) to the hind-limb musculature of mice two weeks prior to denervation or tenotomy promoted muscle hypertrophy that was sufficient to preserve muscle mass comparable to that of untreated sham-operated muscles. However, administration of rAAV:Fst to muscles at the time of denervation or tenotomy did not prevent subsequent muscle wasting. Administration of rAAV:Fst to innervated or denervated muscles increased protein synthesis, but markedly reduced protein degradation only in innervated muscles. Phosphorylation of the signalling proteins mTOR and S6RP, which are associated with protein synthesis, was increased in innervated muscles administered rAAV:Fst, but not in treated denervated muscles. These results demonstrate that the anabolic effects of follistatin are influenced by the interaction between muscle fibres and motor nerves. These findings have important implications for understanding the potential efficacy of follistatin-based therapies for non-degenerative muscle wasting.

Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program

Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.

The role of microRNAs in regulating muscle protein synthesis

 The focus of Evelyn’s PhD was to improve the current body of knowledge in the area of age-related muscle wasting. Evelyn identified two new molecules that are potentially important in the maintenance of skeletal muscle mass. These regulators may one day serve as therapeutic targets for age-related muscle wasting.

Hospital malnutrition : prevalence, identification and impact on patients and the healthcare system

Malnutrition is a debilitating and highly prevalent condition in the acute hospital setting, with Australian and international studies reporting rates of approximately 40%. Malnutrition is associated with many adverse outcomes including depression of the immune system, impaired wound healing, muscle wasting, longer lengths of hospital stay, higher treatment costs and increased mortality. Referral rates for dietetic assessment and treatment of malnourished patients have proven to be suboptimal, thereby increasing the likelihood of developing such aforementioned complications. Nutrition risk screening using a validated tool is a simple technique to rapidly identify patients at risk of malnutrition, and provides a basis for prompt dietetic referrals. In Australia, nutrition screening upon hospital admission is not mandatory, which is of concern knowing that malnutrition remains under-reported and often poorly documented. Unidentified malnutrition not only heightens the risk of adverse complications for patients, but can potentially result in foregone reimbursements to the hospital through casemix-based funding schemes. It is strongly recommended that mandatory nutrition screening be widely adopted in line with published best-practice guidelines to effectively target and reduce the incidence of hospital malnutrition.

Total and Appendicular Lean Mass Reference Ranges for Australian Men and Women: The Geelong Osteoporosis Study

The aim of this study was to develop reference ranges for total and appendicular lean mass measured using dual-energy X-ray absorptiometry (DXA) from a randomly selected population-based sample of men and women residing in southeastern Australia. Men (n = 1,411) and women (n = 960) aged 20–93 years, enrolled in the Geelong Osteoporosis Study, were randomly selected from the Barwon Statistical Division using the electoral roll as a sampling frame in 2001–2006 (67 % participation) and 1993–1997 (77 % participation), respectively. Using DXA (Lunar DPX-L or Prodigy Pro) at baseline for men and at the 10-year follow-up for women (2004–2008), total and appendicular lean mass were measured. Means and standard deviations for each lean mass measure (absolute and relative to height squared) were generated for each age decade, and cutpoints equivalent to T scores of −2.0 and −1.0 were calculated using data from young adult men and women aged 20–39 years. Young adult reference data were derived from 374 men and 308 women. Cutpoints for relative appendicular lean mass equal to T scores of −2.0 and −1.0 were 6.94 and 7.87 kg/m2 for men and 5.30 and 6.07 kg/m2 for women. The proportions of men and women aged ≥80 years with a T score less than −2.0 were 16.0 and 6.2 %, respectively. These reference ranges may be useful for identifying lean mass deficits in the assessment of muscle wasting and sarcopenia.