* Manuscript Text

Amyloid Toxicity In Skeletal Myoblasts: Implications For Inclusion -Body Myositis

Murali Jayaraman, Gomathi Kannayiram and Jayakumar Rajadas* Bioorganic and Neurochemistry laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India.

Corresponding Author’s Present Address: Dr. Jayakumar Rajadas Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305-5025. Phone: (650) 724-1581 Fax: (650) 723-9780

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SUMMARY

Skeletal muscle disorder, Inclusion-body myositis (IBM) has been known for accumulation of amyloid characteristic proteins in muscle. To understand the biophysical basis of IBM, the interaction of amyloid fibrils with skeletal myoblast cells (SMC) has been studied in vitro. Synthetic insulin fibrils and $ȕ25-35 fibrils were used for this investigation. From the saturation binding analysis, the calculated dissociation constant (Kd) for insulin fibril and $ȕ25-35 fibrils were 69.37 r 11.17 nM and 115.60 r 12.17 nM respectively. The fibrillar insulin comparatively has higher affinity binding to SMC than Abeta fibrils. The competitive binding studies with native insulin showed that the amount of bound insulin fibril was significantly decreased due to displacement of native insulin. However, the presence of native insulin is not altered the binding of beta amyloid fibril. The cytotoxicity of insulin amyloid intermediates was measured. The pre-fibrillar intermediates of insulin showed significant toxicity (35%) as compared to matured fibrils. Myoblast treated with beta amyloid fibrils showed more oxidative damage than the insulin fibril. Cell differentiating action of amyloidic insulin was assayed by creatine kinase activity. The insulin fibril treated cells differentiated more slowly compared to native insulin. However, beta amyloid fibrils do not show cell differentiation property. These findings reinforce the hypothesis that accumulation of amyloid related proteins is significant for the pathological events that could lead to muscle degeneration and weakness in IBM.

Keywords: Amyloid fibril; Inclusion-body myositis; Cytotoxicity; Oxidative stress; Insulin fibril; Abeta fibril

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INTRODUCTION

Aggregation of proteins into insoluble intracellular complexes and inclusion bodies is the basis of several amyloid diseases, including Alzheimer’s disease (AD), Amyotropic lateral sclerosis, Huntington disease, Diabetes mellitus and Parkinson disease [1]. E-Amyloid peptide (AE) is the primary constituent of senile plaques present in AD. Peptide aggregates are toxic to neurons, myocytes, endothelial cells and erythrocytes, although the mechanism of toxicity is uncertain [2-6]. AD related abnormalities also persists in cultured cells such as fibroblasts, suggesting that the AD related changes may lead to secondary pathology to non- neuronal cells. Peripheral cells were accompanied with AD are showing multiple changes in cell signaling pathways [7]. There are number of pathologic similarities exists between Alzheimer’s disease brain and inclusion-body myositis (IBM) muscle, such as accumulation of E amyloid and phosphorylated tau [8-10]. In general, more than 20 unrelated proteins, including E-amyloid, prion, tau, insulin and transthyretin can abnormally unfold and self – aggregate to form E-pleated-sheet amyloid [11]. Although matured amyloid fibrils were previously considered to be cytotoxic, current experimental evidence suggest that pre-amyloid oligomeric complexes and/or protofibrillar aggregates are more cytotoxic [12, 13]. The possibility that a molecular species other than the amyloid fibril could be pathogenic arose when oligomeric species rich in E-structure (protofibrils) were found to be discrete intermediates in the fibrillization of E-amyloid (AE), Dsynuclein and insulin in vitro [14-16]. In IBM muscle, variations in the activities of antioxidant enzymes associated with free radical injury have been reported [9]. In this work, the possible roles of amyloid fibrils in cell membrane interaction, oxidative pathways, cell differentiation and

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proliferation were studied. Insulin has been reported to protect neurons against Abeta-induced cell-death [17]. In the present investigation, we found the nature of binding and toxicity of amyloid fibrils with myoblast. AE25-35 peptide is present in senile plaques and degenerating hippocampal neurons in AD brains however not in age-matched control subjects, and certain forms of AE1-40 can be converted to AE25-35 peptide by brain proteases [18]. In addition, the proteolytic fragments of AE1-42 peptides were also observed in IBM muscle. Because of its potential toxicity of 25-35 region, we have used fibrils derived from the AE25-35 peptide for the present study. We have found the existence of binding sites for insulin fibril on myoblasts. The results showed that insulin fibril has cytotoxic effect similar to E-amyloid. The oxidative stress induced cytotoxic mechanism was also observed in amyloid treated myoblast.

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MATERIALS AND METHODS

Cell culture:

Skeletal myoblast cultures were prepared from adult wistar rats [19]. Briefly, after removing the hair, muscle tissue was dissected out and subjected to collagenase treatment for 30 min and trypsinized at 37qC for 20 min. The resultant cell suspension was filtered through Nitex mesh (40 PM), planted in 75 cm2 flask and cultured in Dulbecco's modified Eagle's medium (Gibco-BRL, USA) medium containing 10% fetal bovine serum, 2 mM glutamine and 100kU/L penicillin. Cells were trypsinized after 11 days in culture and transferred into flasks at a density of 6 u 105 / flask and grown until confluent. This procedure produced highly purified culture of myoblasts consisting of more than 95 % myoblast, as determined by creatine kinase (CK) activity and S100 protein immunostaining.

Amyloid binding studies:

Myoblast membranes were prepared from primary cells by the method reported earlier [20]. Insulin was obtained from Sigma and AE25-35 peptide was synthesized by Fmoc solid phase method [21]. Insulin and AE25-35 were biotinylated as described in the procedure [22]. The pH of buffer was kept 6.0 in order to specifically biotinylate D-amino group. The excess unreacted biotin was removed using microcentricon tubes by centrifugation. Biotinylated insulin and AE2535

peptide were later converted to amyloidic fibril as described in the procedure [23, 24].

Membrane samples (100 Pg of protein) were incubated overnight at 4 qC with biotinylated

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insulin fibril or AE25-35 fibril (2 PM final concentrations) in a final volume of 500 Pl binding buffer containing 0.2 % bovine serum albumin. Nonspecific binding was determined by adding an excess of insulin (non-biotinylated; 50Pg/ ml final concentration). They were washed with 500 Pl of phosphate buffered saline, pH 7.4 and resuspended in 500Pl of the same buffer. Samples were counter labeled with FITC-Streptavidin for 30 min in the dark at 4 qC. Membrane – bound and free FITC-Streptavidin were separated by adding 1.0 ml of ice-cold binding buffer and centrifugation at 5000 u g for 30 min at 4 qC. The supernatant was removed and membrane bound amyloid was determined by measuring the fluorescence intensity in a Cary Bio-50 spectrofluorimeter.

Treatment of myoblast with amyloids:

Confluent myoblasts were trypsinized and plated into 6-well tissue culture plates at a cell density of 2 u 105 / well (for GSH, GPx assay) or into 96- well plates at a density of 1 u 104 / well (for MTT assay, H2DCF-DA fluorescence measurement). After 20 hr, insulin, insulin fibril, denatured insulin, and AE25-35 fibril was added to the culture medium at various concentrations as indicated. The heat treated insulin fibril samples were cooled to room temperature and adjusted to physiological pH before incubating with cells.

MTT assay: Cell viability was measured by the colorimetric MTT tetrazolium salt assay [25]. MTT tetrazolium salt was dissolved in serum-free culture medium to a final concentration of 0.5 mg/ml and added to the cells for 1h at 37 qC. The medium was then removed, isopropanol added

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and the absorbance was measured at O592nm in a spectrophotometer. The optical density values of the treated cells were normalized against the untreated cells.

Dichlorofluorescein diacetate (H2DCF-DA) oxidation assay:

Over expression of catalase or Cu/Zn–SOD in the amyloid treated cell lines is reported to alter the steady-state levels of intracellular H2O2. The cellular H2O2 levels were determined through the oxidation of the dye 2, 7-dichlorodihydro fluorescein diacetate (H2DCF-DA) using Cary Bio 50 spectrofluorimeter [26]. After incubation with amyloid fibrils, myoblasts (1 mg/mL) ZHUH ZDVKHG ZLWK 3%6 DQG LQFXEDWHG ZLWK  ȝ0 RI QRQ-fluorescent DCFH-DA for 30 min. Cells were spun at 2800 u g in a tabletop Eppendorf centrifuge for 5 min at 4 °C and UHVXVSHQGHG LQ  ȝ/ RI 3%6 Fluorescent measurements were made with excitation and emission filters set at 485 ± 10 nm and 530 ± 12.5 nm respectively. All initial fluorescent values (time 0) were found to differ from each other by less than 5%. Results were expressed as percentage increase in fluorescence calculated using the following equation: [(Ft30 – Ft0) / Ft0 X 100)] where Ft0 and Ft30 are fluorescence intensities at 0 and 30 min respectively. This dye allows determination of intracellular ROS levels, predominantly detecting peroxides as expected, myoblast had reduced levels of H2O2, while insulin amyloid and beta amyloid treated cells expected to have increased ROS levels compared to control cells. Myoblasts were allowed to adhere to wells on a 96-well plate precoated with polylysine. After three washes in adhesion buffer, cells were incubated with amyloids were fixed in 2 % Para formaldehyde, and viewed on Olympus inverted fluorescence microscope.

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GSH assay:

Reduced-GSH content was measured by the method described elsewhere [27], with minor modification. Myoblast treated with native insulin, denatured insulin, insulin fibril and AE25-35 fibril for 18 h. Briefly cells were washed with cold PBS and scraped down by scraper in sodium phosphate buffer 100 mmol/ L containing edetic acid 6.3 mmol/ L (pH 7.5). After sonication, 20PL was taken for protein assay, then 1 % trichloroacetic acid was added to the lysates, and the mixture was allowed to precipitate for 2 h at 4qC. After centrifugation at 10, 000 u g for 15 min, protein free lysates were obtained. The reaction mixture for determination of GSH content consisted of lysates and 5, 5’– dithiobis - (2-nitrobenzoic acid) (DTNB) 6mmol/ L. The absorbance at 412 nm was monitored using Perkin Elmer spectrophotometer. The content of GSH was calculated from the change in the rate of absorbance on the basis of a standard curve.

GPx assay:

Glutathione Peroxidase activity was measured using the method described earlier with minor modification [28]. Briefly, to 0.1 ml of cell lysate, 0.4 ml of 0.4 M phosphate buffer pH 7.0, and 0.1ml of 10mM sodium azide, 0.2 ml of 4 mM reduced glutathione GSH, 0.1 ml of 2.5mM H2O2 and distilled water were taken in a final incubation volume of 2.0 ml. The tubes were incubated at 37qC for 3 minutes. To this 0.5 ml of 5% TCA was added and the supernatant was removed by centrifugation. 3.0 ml of disodium hydrogen phosphate and 0.1 ml of DTNB reagent added before monitoring the absorbance at 412nm. The activity was expressed as Units/mg of protein.

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Creatine Kinase assay:

Myoblast differentiation during amyloid treatment in 1-2% serum was assessed by Creatine kinase activity using CK-MB assay reagent (Pointe Scientific, Inc. lincoln park, MI) [29].

Data Analysis:

Statistical analysis was performed by using the two-way analysis of variance (ANOVA) and value of P < 0.05 was considered significant.

RESULTS

Amyloid binding studies:

Amyloid binding was measured as a function of concentration of amyloidogenic proteins, insulin fibril and AE25-35 fibril. The binding isotherm for the biotinylated insulin amyloid was non-linear for the measured concentration range (Fig 1). E-sheet structured Fibrillar insulin and AE25-35 fibril, bind significantly to myoblast membrane with dissociation constant of 69.37 r 11.17 nM and 115.60 r 12.17 nM per mg of membrane protein, respectively (Fig 1a and 1c). The result suggests that insulin fibril possess higher affinity towards the myoblast membrane compared to AE25-35 fibril. Binding studies of these fibrils were performed together with monomeric insulin, and the isotherm was given in figure 1b and 1d. The dissociation constant

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(Kd) determined for insulin fibril and AE25-35 fibril in the presence of super-physiological concentration of cold native insulin were 130.80 r 15.3 and 132.0 r 10.23 nM / mg of membrane protein respectively.

MTT assay:

Myoblasts were incubated with AE25-35 fibril and insulin fibril; the changes in cell viability were measured. The heat-denatured insulin was used as a control. The cell viability was significantly decreased upon incubation with AE25-35 fibril (42 % r 1.3; P ” 0.01) as compared to control cells (Fig. 2, curve a). Co-treatment of AE25-35 fibril with insulin fibril or native insulin induced a significant cell death (Fig 2, curve b, c). In order to measure the cytotoxic property, insulin fibril intermediates produced at different time points were incubated with myoblasts. Treatment of insulin under fibril forming conditions for 1, 2, 3, 4 and 5 h at 80qC resulted in 81.5%, 75.14%, 65.83 %, 72.78 %, and 71.39 % of viable cells respectively (Fig. 2, curve c-h). Denatured insulin treated cells showed 93 r 2.5% viability (Fig. 2, curve i). Insulin fibril intermediates produced at various time points caused different percentage of cell death and all the observed values were lower than AE25-35 fibril. Nonetheless, 3 hrs of heat treated insulin fibril showed to some extent increased cell death (35%; P ” 0.05) than control.

Dichlorofluorescein Assay:

In an attempt to elucidate the involvement of oxidative stress in AE induced cytotoxicity, we measured the reactive oxygen species (ROS) content in the amyloid treated myoblasts.

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Intracellular ROS concentration was estimated from the conversion of the fluorescence probe DCFH2-DA in to DCF. Figure 3A shows the measured ROS levels in amyloid treated myoblasts. Beta amyloid treatment produced a two fold increase in DCF fluorescence (Fig 3A). However, less than half fold increase in DCF fluorescence was observed upon incubation with insulin fibril. Co-incubation of AE25-35 fibril and insulin fibrils produced a two-fold increase in DCF fluorescence (Fig 3A) as compared with the control (P < 0.001). Fluorescence microscopic pictures of AE25-35 fibril treated myoblast are shown in Fig 3B.

Amyloid induces oxidative stress in myoblast:

To further measure the extent of cellular oxidative stress in amyloid treatment, GSH content and glutathione Peroxidase (GPx) activities were determined in myoblast. Fig 4 shows changes in the GSH content in amyloid treated myoblast. Treatment with AE25-35 fibril resulted in 42.3% decrease in GSH content as compared with control. Exposure of myoblast to insulin fibril resulted in 23 % decrease in GSH content. Co-incubation of AE25-35 fibril and insulin amyloid resulted in 38.5 % decrease GSH content. Control experiments with denatured and native insulin did not change the intracellular GSH content. The changes in GPx activities in AE25-35 fibril and insulin fibril treated myoblast are shown in Figure 5. The GPx activity was significantly decreased (53 %) in AE25-35 fibril treated myoblast compared to control. However, insulin amyloid treatment resulted in moderate decrease the GPx activity (24%) in myoblasts. Insulin amyloid co-treatment with AE25-35 fibril resulted in 43 % decrease in GPx activity.

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Creatine Kinase activity in cells:

We have investigated the effects of AE25-35 fibril and insulin fibril treatment on rat skeletal myoblast differentiation. Native insulin is used as a positive control and its treatment resulted in three fold increase CK activity (Fig 6). Insulin fibril treatment showed one fold increase in CK activity. However, AE25-35 fibril treated myoblasts showed decreased CK activity than control and it remained in the base level over seven day period. The CK activity in insulin fibril and AE25-35 fibril co-treated cells was similar to untreated control cells. Whereas, the CK activity in AE25-35 fibril and native insulin co-treated cells showed moderate increase than untreated control cells.

DISCUSSION

The mechanism of amyloid mediated disturbance of cellular function is not well understood. A growing body of evidence suggests that amyloid aggregates damage cells as a result of increase in reactive oxygen species (ROS) [30, 31]. Such an increase in ROS would disturb the endogenous antioxidant systems; including glutathione (GSH), glutathione peroxidase (GPx) [6]. Amyloid induced oxidative stress can either target the cellular apoptotic/necrotic death or in turn may impair the expression of cellular growth/differentiation factors [32]. Nonetheless, some of the other mechanisms have been proposed for amyloid activated cellular damage, includes pore formation on cell membrane, inactivation of functional proteins and components proteasome system [33, 34].

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Cellular degeneration in amyloid diseases is reconciled by toxic mechanism involving the interaction of the toxic aggregated species with plasma membrane of the affected cells [35]. We hypothesize that in the IBM muscle, elevated amyloid peptide levels results in deposition of amyloid on the myoblast membrane. Similar amyloid membrane binding studies were performed earlier with gangliosides and human placental plasma membrane (HPPM) [36, 37]. Therefore, in the present investigation, we have studied the potential interaction of AE25-35 fibril and insulin fibrils with myoblast membrane. The overall result indicates that amyloid fibril of abeta and insulin binds with myoblast membrane and insulin fibril has higher affinity for myoblast membrane. Insulin receptors are present on rat myocytes [38]. Insulin in native form is reported to inhibit the beta amyloid induced cell death [17]. To assess the involvement of insulin receptor in amyloid binding, experiments were carried out with native insulin. Earlier we have shown that fibrillar insulin and abeta non-specifically binds to human erythrocytes [5, 22]. Present findings with myoblast membrane suggest that native insulin significantly decreased the binding of fibrillar insulin, whereas, AE25-35 fibril binding did not altered by addition of native insulin. The amyloid toxicity profiles were determined to understand the relationship between the interaction of amyloids with cell membrane and its lethal effect on myoblasts. When incubated at high temperature, insulin initially forms small spherical aggregates (oligomers), then converted into protofibrils and mature fibrils [39, 40]. The significant toxicity of the early intermediates of insulin fibril toward myoblasts suggests that the small oligomeric species rather than the large fibrillar aggregates are the toxic morphologies, consistent with numerous other recent studies [41, 42]. Incubation of AE25-35 fibril with insulin fibril produced similar toxic effect to that of the AE25-35 fibril alone treatment. Several mechanisms have been proposed for AE peptide-mediated cytotoxicity. One of the mechanisms suggested being involved in AE peptides-induced

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neurotoxicity is the generation of reactive oxygen species (ROS), which seems to be responsible for membrane lipid peroxidation [4, 31, 43]. Oxidative stress-mediated up-regulation of the amyloidogenic pathway has been reported in human cerebral vascular smooth muscle cells [44]. Enzymes participating in the cellular defense against oxidative stress are accumulated in IBM muscle fibers suggesting that evidence for free radical mediated IBM pathogenesis [9, 45, 46]. Insulin amyloid produced only a minimum amount of superoxide anion. The results indicate that AE25-35 fibril is able to trigger the free-radical production even in the presence of less toxic synthetic amyloid. Our data also suggests that oxidative stress is involved in amyloid fibrils produced toxicity in myoblasts. Previous reports have shown that dysregulation of IBM specific protein including amyloid precursor protein, apolipoprotein A-1 and transthyretin, which are associated with amyloidosis. Furthermore, superoxide dismutase, enolase and various molecular chaperones were also dysregulated in IBM, indicating perturbation in detoxification, energy metabolism, and protein folding processes [47]. Neuronal damage may occur partly due to oxidative processes initiated through amyloid-derived free radicals species [48]. Amyloids are likely to be involved in the overall regulation/ disruption of cellular functions [49]. Besides their obligate role in the fight against ROS, antioxidants may also function as regulators of growth factor receptors, cellsignaling reactions, or activators or in-activators of enzymes and proteins involved in transcription and translation [50]. Glutathione is an antioxidant and its intracellular level is a sensitive indicator oxidative stress. The expression of oxidative stress handling genes, including those encoding GPx, GSH reductase, Zn-SOD was reported to increase in the Alzheimer’s disease [51]. Indeed, decrease of antioxidant systems in Alzheimer’s disease, such as GPx, GSH reductase and catalase activities

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was observed [43]. Therefore, measurements of an antioxidant system are important to compare the alterations in myoblast induced by AE25-35 fibril, insulin fibril with those found in IBM and AD patients. Millet et al (2005) have showed apoptotic and differentiating activity of amyloid over neural progenitor cells [52]. However beta amyloid fibril treatment significantly slows cell differentiation due to augmented cell death. Insulin associated muscle cell differentiation has been reported earlier [53]. The present result is consistent with earlier reports. AE inhibition of myoblast differentiation is reversed by the exogenous addition of native insulin and one fold increase in CK activity with insulin fibril, shows that amyloid treatment does not permanently block the expression of muscle differentiation marker. CK is a marker of various physiologic abnormalities, such as the AD and it has been shown that decreased CK activity in soluble fractions of AD brain [54, 55]. Decreased CK activity in Abeta fibril treated myoblast suggesting that existence of AD-like pathology in IBM muscle. Taken together, the myoblast differentiation is significantly inhibited by AE25-35 fibril treatment. IBM is a debilitating disorder, its diagnostic method and molecular basis of pathogenesis remains poorly defined. It has been previously reported that cytotoxicity of over-expressed betaamyloid in cultured skeletal muscle fibers [56]. Increased amount of deposition of multiple subfragments of beta amyloid precursor protein as amyloid aggregates were observed in muscle fibers from IBM biopsies [57]. Similar abeta-containing amyloid deposits were observed in vessel walls in AD brain smooth muscle cells (SMCs), endothelial cells (ECs) and their basement membranes [58, 59]. In conclusion, we have shown that AE25-35 and insulin fibrils bind to myoblast membrane. The present data support the hypothesis that oxidative stress mediated amyloid cytotoxicity in skeletal myoblast through reducing efficiency of antioxidant system. Thus, oxidative stress

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coupled cytotoxicity and impaired cell differentiation are the possible amyloid induced pathologies observed in myoblasts associated with IBM.

Acknowledgement: We are thankful to Director, CLRI, for the support and encouragement to this work. The author J.M thanks Council of Scientific and Industrial Research, INDIA for awarding Senior Research Fellowship. We thank Dr. Ravindra Kodali for his critical comments on the manuscript.

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Figure captions: Fig: 1. Binding curves showing the effect of Insulin fibril and AE25-35 fibril binding to rat skeletal myoblast membrane. Myoblast membrane incubated with (a) insulin fibril (b) insulin fibril and cold native insulin (c) AE25-35 fibril, (d) AE25-35 fibril and cold native insulin. The binding data were analyzed with the aid of curve fitting software (Graph pad Prism version 1.0). Values are mean ± SEM (n=3). Fig: 2. Mean percentage survival of myoblast exposed to AE25-35 fibril and insulin fibril intermediates measured from MTT reduction assay. Cells were treated with 10μM of AE25-35 fibril (a), AE25-35 fibril and insulin fibril (b), AE25-35 fibril and native insulin (c), insulin fibril intermediates formed during heat treatment at 1h (d), 2h (e), 3h (f), 4h (g) and 5h (h) time intervals and denatured insulin (i) respectively. Data are r SD from 4 dishes. *P ” 0.05; **P ” 0.01. Fig: 3A. ROS levels were measured by DCF fluorescence assay. Myoblasts incubated in the absence (control) or presence of 10 PM of each amyloid fibril. Data are given as difference in fluorescence measured at 0 min and 30 min of incubation and are the mean of at least four independent experiments. *P ” 0.05. Fig: 3B. Fluorescence photomicrographs of myoblast control (a), exposed to 10 PM of Insulin fibril (b) and 10 PM AE25-35 fibril (c). Photographs were captured 30 min from incubation. Fig: 4. Effect of amyloid treatment on myoblast on cellular GSH. Cells were treated with 10 PM of each peptide. *P ” 0.05. Fig: 5. Effect of amyloid treatment on myoblast on cellular GPx. Cells were treated with 10 PM of each peptide. *P ” 0.05. 20

Fig: 6. Effect of amyloids on Creatine kinase activity in myoblast. Time dependent change in Creatine kinase activity in control myoblast (i), during incubation of 50 nM native insulin (Ŷ), 100 nM AE25-35 fibril (Ÿ), 100nM insulin fibril (Ɣ), co-treatment of 100 nM of each insulin fibril and AE25-35 fibril ('), co-treatment of 100 nM fAE25-35 and 50 nM native insulin (ż) respectively

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