Identification and characterization of a membrane receptor for proteolysis-inducing factor on skeletal muscle

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (Kd, in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (Kd, 1.1 × 10-7 mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [ 35S]PIF to triton solublized membranes from C2C 12 cells provided evidence for a binding protein of apparent M r of ∼40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target. ©2007 American Association for Cancer Research.

Attenuation of skeletal muscle atrophy in cancer cachexia by d-myo-inositol 1,2,6-triphosphate

PURPOSE: To determine the effectiveness of the polyanionic, metal binding agent D-myo-inositol-1,2,6-triphosphate (alpha trinositol, AT), and its hexanoyl ester (HAT), in tissue wasting in cancer cachexia. METHODS: The anti-cachexic effect was evaluated in the MAC16 tumour model. RESULTS: Both AT and HAT attenuated the loss of body weight through an increase in the nonfat carcass mass due to an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. The decrease in protein degradation was associated with a decrease in activity of the ubiquitin-proteasome proteolytic pathway and caspase-3 and -8. Protein synthesis was increased due to attenuation of the elevated autophosphorylation of double-stranded RNA-dependent protein kinase, and of eukaryotic initiation factor 2alpha together with hyperphosphorylation of eIF4E-binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2. In vitro, AT completely attenuated the protein degradation in murine myotubes induced by both proteolysis-inducing factor and angiotensin II. CONCLUSION: These results show that AT is a novel therapeutic agent with the potential to alleviate muscle wasting in cancer patients.

Investigation of potential intervention targets to improve insulin action: a therapeutic approach

Impaired insulin action (insulin resistance) is a key factor in the pathogenesis of diabetes mellitus. To investigate therapeutic targets against insulin resistance, this thesis explores the mechanism of action of pharmacological agents and exogenous peptides known or suspected to modify insulin action. These included leptin, a hormone primarily involved in the regulation of body weight; sibutramine, an antiobesity agent; plant-derived compounds (pinitol and chamaemeloside) and agents known to affect insulin sensitivity, e.g. metformin, tolbutamide, thiazolidinediones, vanadyl sulphate and thioctic acid. Models used for investigation included the L6 skeletal muscle cell line and isolated skeletal muscles. In vivo studies were undertaken to investigate glycaemia, insulinaemia, satiety and body weight in streptozotocin-induced diabetic mice and obese (ob/ob) mice. Leptin acutely altered insulin action in skeletal muscle cells via the short form of the leptin receptor. This direct action of leptin was mediated via a pathway involving PI 3-kinase but not Jak2. The active metabolites of sibutramine had antidiabetic properties in vivo and directly improved insulin sensitivity in vitro. This effect appeared to be conducted via a non-PI 3-kinase-mediated increase in protein synthesis with facilitated glucose transport, and was independent of the serotonin and noradrenaline reuptake inhibition produced by sibutramine. Pinitol (a methyl inositol) had an insulin mimetic effect and was an effective glucose-lowering agent in insulinopenic states, acting directly on skeletal muscle. Conversely chamaemeloside appeared to improve glucose tolerance without directly altering glucose transport. Metformin directly increased basal glucose uptake independently of PI 3-kinase, possibly via an increase in the intrinsic activity of glucose transporters. Neither tolbutamide nor thiazolidinediones directly altered insulin sensitivity in L6 skeletal muscle cells: however vanadyl sulphate and thioctic acid increased glucose transport but appeared to exert toxic effects at therapeutic concentrations. Examination of glucose transport in skeletal muscle in this thesis has identified various components of post-receptor insulin signalling pathways which may be targeted to ameliorate insulin resistance. Type 2 Diabetes Mellitus Obesity L6 Skeletal Muscle Cells Glucose Transport Insulin Signalling 2

The effect of cis-5,8,11,14,17-eicosapentaenoic acid upon the contractile mechanisms linked to calcium influx and the mobilisation of intracellular calcium in aortic smooth muscle

Epidemiological studies previously identified cis-5,8,11,14,17-eicosapentaenoic acid (EPA) as the biologically active component of fish oil of benefit to the cardiovascular system. Although clinical investigations demonstrated its usefulness in surgical procedures, its mechanism of action still remained unclear. It was shown in this thesis, that EPA partially blocked the contraction of aortic smooth muscle cells to the vasoactive agents KCl and noradrenaline. The latter effect was likely caused by reducing calcium influx through receptor-operated channels, supporting a recent suggestion by Asano et al (1997). Consistently, EPA decreased noradrenaline-induced contractures in aortic tissue, in support of previous reports (Engler, 1992b). The observed effect of EPA on cell contractions to KCl was not simple due to blocking calcium influx through L-type channels, consistent with a previous suggestion by Hallaq et al (1992). Moreover, EPA caused a transient increase in [Ca2+]i in the absence of extracellular calcium. To resolve this it was shown that EPA increased inositol phosphate formation which, it is suggested, caused the release of calcium from an inositol phosphate-dependent internal binding site, possibly that of an intracellular membrane or superficial sarcoplasmic reticulum, producing the transient increase in [Ca2+]i. As it was shown that the cellular contractile filaments were not desensitised to calcium by EPA, it is suggested that the transient increase in [Ca2+]i subsequently blocks further cell contraction to KCl by activating membrane-associated potassium channels. Activation of potassium channels induces the cellular efflux of potassium ions, thereby hyperpolarising the plasma membrane and moving the membrane potential farther from the activation range for calcium channels. This would prevent calcium influx in the longer term and could explain the initial observed effect of EPA to block cell contraction to KCl.

Cellular signalling pathways involved in muscle atrophy in cancer cachexia

Cancer cachexia encompases severe weight loss, characterised by the debilitating atrophy of adipose and skeletal muscle mass. Skeletal muscle proteolysis in cancer cachexia is mediated by a sulphated glycoprotein with a relative molecular mass of 24kDa, termed Proteolysis-Inducing Factor (PIF). PIF induced a significant increase in protein degradation, peaking at 4.2nM PIF (p<0.001), ‘chymotrypsin-like’ activity of the proteasome (p<0.001) and increased expression of components of the ATP-ubiquitin dependent proteolytic pathway. This was attenuated in vitro by pre-incubation with the PKC inhibitor calphostin C (100µM) and NF-kB the inhibitors SN50 (18µM), curcumin (50µM) and resveratrol (30µM), 2 hours prior to the addition of PIF. In vivo studies found the IKK inhibitor resveratrol (1mg/kg) to be successful in attenuating protein degradation (p<0.001) and upregulation of ubiquitin-dependent proteolysis in MAC16 tumour bearing mice. C2C12 myoblasts transfected with mutant IkBα and PKCα inserts did not elicit a PIF-induced response, suggesting the importance of the transcription factor NF-kB and PKC  involvement in PIF signal transduction. 15(S)-HETE acts as an intracellular mediator of PIF and exerts an effect through the activation of PKC and subsequently IKK, which phosphorylates IkBα and allows NF-kB to migrate to the nucleus. This effect was negated with the PKC inhibitor calphostin C (300nM). A commercially produced PIF receptor antibody was raised in rabbits immunised with a peptide containing the partial N-terminal sequence of the PIF receptor. The PIF receptor antibody was successful in attenuating the PIF-induced increase in skeletal muscle catabolism and protein degradation in vitro at 10µg/ml (p<0.001) and 3.47mg/kg in vivo (p<0.001). The data suggest great potential in the development of this antibody as a therapy against cancer cachexia.

Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers

Aim - The aim of the study was to determine the potential for KV1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia. Methods and results - Blood vessels were obtained from patients or mice and studied in culture. Reverse transcriptasepolymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function.  KV1.3 was unique among the  KV1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of  KV1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia. Conclusion - KV1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events. © 2010 The Author.

Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of NF-κB activation

The potential for inhibitors of nuclear factor-κB (NF-κB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-κB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-κB inhibitor SN50 (18 μM) attenuated the expression of 205 proteasome α-subunits, two subunits of the 195 regulator MSSI and p42, and the ubiquitin-conjugating enzyme, E214k, as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-κB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 μM) and resveratrol (30 μM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k. However, curcumin (150 and 300 mg kg-1) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg-1) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-κB DNA-binding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-κB may prove useful for the treatment of muscle wasting in cancer cachexia.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Glucosamine-induced insulin resistance in L6 muscle cells

Background: Glucosamine increases flux through the hexosamine pathway, causing insulin resistance and disturbances similar to diabetic glucose toxicity. Aim: This study examines the effect of glucosamine on glucose uptake by cultured L6 muscle cells as a model of insulin resistance. Methods: Glucose uptake by L6 myotubes was measured using the non-metabolized glucose analogue 2-deoxy-D-glucose after incubation with glucosamine for 4 and 24 h, with and without insulin and several other agents (metformin, peroxovanadium and D-pinitol) that improve glucose uptake in diabetic states. Results: After 4 h, high concentrations of glucosamine (5 × 10-3 and 10-2 M) reduced basal and insulin-stimulated glucose uptake by up to 50%. After 24 h, the effect of insulin was completely abolished by 10-2 M glucosamine and reduced over 50% by 5 × 10-3 M glucosamine. Lower concentrations of glucosamine did not significantly alter glucose uptake. The effect of glucosamine could not be attributed to cytotoxicity assessed by the Trypan Blue test. Metformin, peroxovanadium and D-pinitol, each of which increased glucose uptake by L6 cells, did not prevent the decrease in glucose uptake with glucosamine. Conclusion: Glucosamine decreased insulin-stimulated glucose uptake by L6 muscle cells, providing a potential model of insulin resistance with similarities to glucose toxicity. Insulin resistance induced by glucosamine was not reversed by three agents (metformin, peroxovanadium and D-pinitol) known to enhance or partially mimic the effects of insulin. © 2004 Blackwell Publishing Ltd.

Attenuation of muscle wasting in murine C2C12myotubes by epigallocatechin-3-gallate

Background: Loss of muscle protein is a common feature of wasting diseases where currently treatment is limited. This study investigates the potential of epigallocatechin-3-gallate (EGCg), the most abundant catechin in green tea, to reverse the increased protein degradation and rescue the decreased protein synthesis which leads to muscle atrophy. Methods: Studies were conducted in vitro using murine C2C12myotubes. Increased protein degradation and reduced rates of protein synthesis were induced by serum starvation and tumour necrosis factor-α (TNF-α). Results: EGCg effectively attenuated the depression of protein synthesis and increase in protein degradation in murine myotubes at concentrations as low as 10 μM. Serum starvation increased expression of the proteasome 20S and 19S subunits, as well as the proteasome ‘chymotrypsin-like’ enzyme activity, and these were all attenuated down to basal values in the presence of EGCg. Serum starvation did not increase expression of the ubiquitin ligases MuRF1 and MAFbx, but EGCg reduced their expression below basal levels, possibly due to an increased expression of phospho Akt (pAkt) and phospho forkhead box O3a (pFoxO3a). Attenuation of protein degradation by EGCg was increased in the presence of ZnSO4, suggesting an EGCg-Zn2+complex may be the active species. Conclusion: The ability of EGCg to attenuate depressed protein synthesis and increase protein degradation in the myotubule model system suggests that it may be effective in preserving skeletal muscle mass in catabolic conditions.

Muscle movement and electrodes motion artifact during vibration treatment

Vibration treatment by oscillating platforms is more and more employed in the fields of exercise physiology and bone research. The rationale of this treatment is based on the neuromuscular system response elicited by vibration loads. surface Electromyography (EMG) is largely utilized to assess muscular response elicited by vibrations and Root Mean Square of the electromyography signals is often used as a concise quantitative index of muscle activity; in general, EMG envelope or RMS is expected to increase during vibration. However, it is well known that during surface bio-potential recording, motion artifacts may arise from relative motion between electrodes and skin and between skin layers. Also the only skin stretch, modifying the internal charge distribution, results in a variation of electrode potential. The aim of this study is to highlight the movements of muscles, and the succeeding relevance of motion artifacts on electrodes, in subjects undergoing vibration treatments. EMGs from quadriceps of fifteen subjects were recorded during vibration at different frequencies (15-40 Hz); Triaxial accelerometers were placed onto quadriceps, as close as possible to muscle belly, to monitor motion. The computed muscle belly displacements showed a peculiar behavior reflecting the mechanical properties of the structures involved. Motion artifact related to the impressed vibration have been recognized and related to movement of the soft tissues. In fact large artifacts are visible on EMGs and patellar electrodes recordings during vibration. Signals spectra also revealed sharp peaks corresponding to vibration frequency and its harmonics, in accordance with accelerometers data. © 2008 Springer-Verlag.