Abstract

1-01 IL-1R inhibition reduced tumour growth, inflammation, and fibrosis in cachectic tumour bearing Joanna D.C.C. Lima, Estefania Simoes, Stephanie J. Melchor, Marilia Seelaender & Sarah E. Ewald Department of Cell Biology and Development at the Institute of Biomedical Science, University of Sao Paulo, Sao Paulo, Brazil, Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, VA, USA Background: Cancer-associated cachexia (CAC) is a multifactorial disorder characterized by muscle wasting with or without loss of adipose tissue. CAC remains a devastating syndrome affecting 50–80% of cancer patients, and it is responsible for the death of at least 20%. Cachexia is driven by a multiple association of metabolic changes including reduced food intake, enhanced energy expenditure, and inflammation leading to poor prognosis. Inflammatory mediators are derived from tumour cells and host interaction that induces catabolic actions. Commonly, tumour necrosis factor (TNF), IL-6, transforming growth factor-β (TGF-β), IFN-γ, and IL-1 are implicated in mechanisms that include activation of inflammation, fibrosis, proteolysis, autophagy, and lipolysis. Interleukin 1 receptor 1 (IL-1R) recognizes interleukin-1 (IL-1)α and IL-1β, so-called “alarmins” that are released from dead or damaged cells to initiate inflammatory responses and tissue remodelling and trigger behavioural changes like anorexia. Recently, IL-1α blockade was shown to induce weight gain and improve quality of like in metastatic cancer patients. The aim of this study was to investigate the role of IL-1 receptor in the tumour microenvironment of cachectic tumour bearing mice. Methods: C57BL/6 wild type controls or IL-1R / mice (B6.129S7-il1r tm1/mx/J) were obtained from The Jackson Laboratory. Five million LLCs were injected subcutaneously, andmice were euthanized at 21 days post-tumour inoculation. In vivo bioluminescence of LLC-GFP cells was measured using the IVIS® imaging system. The mRNA content was evaluated by real-time PCR (2 ) and protein levels by ELISA assay. Results: Weight loss was significantly higher in WT mice relative to IL-1R / mice after accounting for tumour mass (P = 0.0056). IL-1R deficient mice had significantly smaller tumours than WT mice by weight (P = 0.015) and by LLC-GFP signal intensity (P = 0.020). mRNA levels of IL-1βin the tumour was decreased in IL-1R / compared to WT mice (P = 0.015). Hence, mRNA and protein levels of TNF-α (P = 0.9914 and P =p 0.265, respectively) and IL-6 (P = 0.140 and P = 0.389, respectively) were not significantly different between the groups. There were also no differences between groups in IFN-γ in tumour microenvironment (P = 0.337). Furthermore, gene expression of fibronectin 1 was decreased in the tumour of IL-1R / compared to WT mice (P = 0.014). Expression of collagen 3, collagen 1, and MMP2 were not significantly altered in tumour of IL-1R / mice comparing with WT. Conclusions: The IL-1 axis is necessary for LLC growth and cancer-associated weight loss. The reduction in tumour growth was not due to altered TNF-α, IL-6, and IFN-γ in the tumour environment. The tumour microenvironment showed some evidence of IL-1R dependent fibrotic tissue remodelling. These experiments suggest that inhibiting the IL-1 signalling axis may provide a novel target for tumour progression and cancer cachexia treatment. 1-02 Reservatrol inhibits body weight and skeletal muscle mass losses, decreases pro-myogenic factors, decreases low-degree systemic chronic inflammation, delays the onset of cachexia, and improves cancer-related survival in C57BL/6 mice bearing syngeneic tumour Otávio Cardoso-Filho, Magda Mendes Vieira, Amanda Rodrigues da Silva, Valéria Couto Quintão, Lorrane Katherine Martins Pereira, Maria Isabela Alves Bernardo, Vinicius Dias Rodrigues, Gefter Thiago Batista Corrêa, Erivelton Pereira dos Santos, Amanda Souto Machado, Ludmilla Regina de Souza David & Alfredo Mauricio Batista De-Paula Nucleus of Epidemiologic and Molecular Research Catrumano (Nupemoc), Health Research Laboratory, Post-graduate Programme in Health Sciences, Universidade Estadual de Montes Claros, Unimontes, Montes Claros, Minas Geras, Brazil, Department of Physical Education, Universidade Estadual de Montes Claros, Unimontes, Montes Claros, Minas Gerais, Brazil, Department of Dentistry, Faculdades Independentes do Nordeste, Fainor, Vitória a Conquista, Bahia, Brazil, Department of Dentistry, Universidade Estadual de Montes Claros, Unimontes, Montes Claros, Minas Gerais, Brazil Background: Cancer-related cachexia (CRC) is a paraneoplastic syndrome characterized as progressive, systemic physical consumption state of individual during cancer progression. Transresveratrol (3,4,5-trans-trihydroxystilbene; Resv) is a naturally occurring polyphenol which modulates inflammatory responses that are usually found in a number of low-grade, systemic chronic inflammatory diseases. We investigated the effects of Resv administration on inflammatory plasma ABSTRACTS © 2019 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders Journal of Cachexia, Sarcopenia and Muscle 2019; 10: 1378–1435 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.12513 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. biomarkers, anthropometrical parameters, skeletal muscle (SM) mass, volume and strength, and survival of C57BL/6 mice bearing a syngeneic cutaneous-melanoma model (SCMM). Methods: Murine B16F10 cells were injected into flank of the 58 female C57BL/6 mice in order to establish a SCMM. CRC diagnosis was individually established for each animal using as parameter weight loss ≥5%. Resv was administered in concentrations of 200 and 400 mg/kg body weight using oral gavage in both control and experimental mice. Measurements of water and food consumption, body weight, and tumour size were daily performed. High-sensitive C-reactive protein (CRP) plasma level was measured by an enzyme immunoassay. SM strength, volume, and mass were assessed using a grip strengthmeter, a high-frequency ultrasound, and an analytical balance, respectively. SM samples were collected and posteriorly submitted tomorphometric and gene expression (GAPDH, Myog, IGF1, IGF2, FBXO32, TRIM63, and TRIM55) analysis by using real-time PCR. Control and experimental mice were submitted to cancer-related survival (CRS) analysis. This study was approved by an ethics committee in animal well-being and experimentation (CEEBEA/Unimontes No. 131/2017). Results: Mice treated with Resv significantly reduced plasma concentration of CRP, exhibited a delayed in CRC occurrence, showed gain of body weight, and improved CRS rate. Resv administration increased SM tissue mass, volume, and muscle strength. Moreover, mice treated with Resv showed a higher quantity of SM fibres and higher pro-myogenic factors. The most of these effects promoted by Resv were dosedependent. Conclusions: Resv administration promoted a plethora of anticachectic effects on plasma and SM tissues in C57BL/6 mice bearing SCMM, which might influence CRS improvement in CRS. 1-03 Resveratrol negatively modulates pro-adipogenic gene expression in visceral white adipose tissue of C57BL/6 mice bearing syngeneic cutaneous melanoma Ludmilla Regina de Souza David, Otávio Cardoso-Filho, Andréia de Souza Brito, Bruna Macedo Lima, Gefter Thiago Batista Corrêa, Magda Mendes Vieira, Lorrane Katherine Martins Pereira, Maria Isabela Alves Bernardo, Gabriel Donner Oliveira & Alfredo Mauricio Batista De-Paula Nucleus of Epidemiologic and Molecular Research Catrumano (Nupemoc), Health Research Laboratory, Post-graduate Programme in Health Sciences, Universidade Estadual de Montes Claros, Unimontes, Montes Claros, Minas Geras, Brazil, Department of Dentistry, Faculdades Independentes do Nordeste, Fainor, Vitória da Conquista, Bahia, Brazil, Department of Dentistry, Universidade Estadual de Montes Claros, Unimontes, Montes Claros, Minas Gerais, Brazil Background: During tumour progression, might occur cancer-related cachexia (CRC), a paraneoplastic syndrome characterized as progressive, systemic physical consumption of individual with cancer which modulates inflammatory responses that are usually found in a number of low-grade, systemic chronic inflammatory diseases. We investigated the effects of Resv administration on inflammatory plasma biomarkers, anthropometrical parameters, white adipose tissue (WAT), and survival of C57BL/6 mice bearing a syngeneic cutaneous-melanoma model (SCMM). Methods: Murine B16F10 cells were injected into flank of the 58 female C57BL/6 mice in order to establish a SCMM. CRC diagnosis was individually established for each animal using as parameter weight loss ≥5%. Resv was administered in concentrations of 200 and 400 mg/kg body weight using oral gavage in both control and experimental mice. Measurements of water and food consumption, body weight, and tumour size were daily performed. High-sensitive C-reactive protein (CRP) plasma level was measured by an enzyme immunoassay. SM strength, volume, and mass were assessed using a grip strength meter, a high-frequency ultrasound, and an analytical balance, respectively. Visceral WAT samples were collected and submitted to morphometric and pro-adipogenic gene expression (PPAR-γ and SREBF1) analysis. Control and experimental mice were submitted to cancer-related survival (CRS) analysis. This study was approved by an ethics committee in animal well-being and experimentation (CEEBEA/ Unimontes No. 131/2017). Results: Mice treated with Resv significantly reduced plasma concentration of CRP, exhibited a delayed in CRC occurrenc

Background: During tumour progression, might occur cancer-related cachexia (CRC), a paraneoplastic syndrome characterized as progressive, systemic physical consumption of individual with cancer which modulates inflammatory responses that are usually found in a number of low-grade, systemic chronic inflammatory diseases. We investigated the effects of Resv administration on inflammatory plasma biomarkers, anthropometrical parameters, white adipose tissue (WAT), and survival of C57BL/6 mice bearing a syngeneic cutaneous-melanoma model (SCMM). Methods: Murine B16F10 cells were injected into flank of the 58 female C57BL/6 mice in order to establish a SCMM. CRC diagnosis was individually established for each animal using as parameter weight loss ≥5%. Resv was administered in concentrations of 200 and 400 mg/kg body weight using oral gavage in both control and experimental mice. Measurements of water and food consumption, body weight, and tumour size were daily performed. High-sensitive C-reactive protein (CRP) plasma level was measured by an enzyme immunoassay. SM strength, volume, and mass were assessed using a grip strength meter, a high-frequency ultrasound, and an analytical balance, respectively. Visceral WAT samples were collected and submitted to morphometric and pro-adipogenic gene expression (PPAR-γ and SREBF1) analysis. Control and experimental mice were submitted to cancer-related survival (CRS) analysis. This study was approved by an ethics committee in animal well-being and experimentation (CEEBEA/ Unimontes No. 131/2017). Results: Mice treated with Resv significantly reduced plasma concentration of CRP, exhibited a delayed in CRC occurrence, showed gain of body weight, and improved CRS rate. Resv administration reduced WAT relative weight, adipocyte area and number, and PPAR-γ and SRE expression in WAT. The most of these effects promoted by Resv were dosedependent. Conclusions: Although Resv administration negatively modulated WAT in C57BL/6 mice bearing SCMM, positive effects of Resv on CRC and CRS seem to be caused by its systemic antiinflammatory effects.

1-04
Tumour-derived Upd3 cytokine coordinates self-growth and host wasting model, we identified two tumour-derived ligands involved in host wasting. However, how yki-gut tumours simultaneously coordinate tumour growth and host wasting is still not fully understood. To search for additional pathways involved in interorgan communication, we developed PathON, a software that comprehensively analyzes canonical Drosophila signalling pathways and their matched ligands. Using PathON, we found that the ligand Upd3 and the targets of JAK/STAT signalling are greatly up-regulated in the tumours and muscle, respectively, of flies bearing yki-gut tumours. We demonstrate that Upd3 is required for both overproliferation of yki-gut tumours and host wasting and that Upd3/JAK/STAT signalling impairs energy balance in both muscle and adipose tissues via suppression of insulin response. Altogether, our results demonstrate that yki-gut tumours produce a single ligand, Upd3, coupling self-growth and host wasting.
Introduction: Cachexia is a syndrome characterized by unintentional weight loss, progressive muscle wasting, and loss of appetite, seen in up to 80% of cancer patients. An antibody (002) that targets the TWEAK receptor (Fn14) has been shown to reverse the symptoms of cachexia in syngeneic tumour-bearing mouse models and extend the lifespan of mice by restoring their body weight. Here, we investigated via positron emission tomography (PET) imaging the glucose changes in tumour-bearing mouse models of cachexia, to explore whether Fn14 plays a role in the metabolic changes occurring during cancer cachexia. Methods: 18 F-FDG PET was performed in non-cachectic MEF H-Ras V12 versus cachectic MEF H-Ras V12 hFn14 tumourbearing Nod SCID gamma mice (NSG) expressing human Fn14. Secondly, 18 F-FDG PET imaging was performed in cachectic C26 tumour-bearing NSG mice treated with anti-Fn14 002 antibody versus vehicle control treated mice. In the C26 model, 002 therapy was commenced before versus after symptoms of cachexia were measurable. Results: 18 F-FDG PET imaging demonstrated increased glucose uptake over time in cachectic versus non-cachectic tumour-bearing mice. This was observed both in the MEF H-Ras V12 hFn14 model as well as in the C26 model. Targeting Fn14 with 002 was able to prevent increased 18 F-FDG uptake in C26 tumours, but more importantly, tumours of cachectic C26 mice with high 18 F-FDG uptake showed reduced 18 F-FDG after 2 days of therapy with 002. Conclusions: Our results demonstrate that cachexia associated with Fn14 signalling is associated with increased tumour glucose metabolism and that 18 F-FDG PET imaging could be used to monitor patient response to mAb 002 cachexia treatments in clinical trials. resistance through activation of AMPK pathway in the skeletal muscle. [1][2][3][4][5][6][7][8][9][10][11][12] β-Hydroxybutyrate replacement therapy does not protect against cachexia in mice with lung adenocarcinoma Seo-Kyoung Hwang, Rahul Grover, Roger Liang, Shakti Ramsamooj, Lewis C. Cantley & Marcus D. Goncalves Weill Cornell, New York, USA Introduction: The cancer-associated cachexia syndrome (CACS) is a systemic metabolic disorder characterized by wasting of body tissues that store nutrients. CACS is particularly prevalent in patients with advanced lung adenocarcinoma where over 80% of people are affected. We have shown that the Kras G12D/+ ;Lkb1 flox/flox (KL) mouse model accurately reproduces the clinical features of lung cancer-induced CACS. In this model, ketogenesis is disrupted in the liver of cachexic mice due to a lack of PPAR-α activity. Treatment with fenofibrate, a PPAR-α agonist, restores ketogenesis and prevents the onset of CACS. We hypothesized that the loss of β-hydroxybutyrate (BHB), the major blood ketone, is necessary for the development of CACS in KL mice, and designed dietary strategies to replace BHB. Methods: In two randomized, controlled, dietary intervention studies, cohorts (N = 21-24) of KL mice were randomized to a normal chow (NC) diet or one of two diets: a ketogenic diet (KD) or a NC diet infused with a 1,3-butanediol acetoacetate diester (KE). Results: The KD effectively increased serum BHB as compared to a NC diet (1.7 ± 0.3 vs. 0.1 ± 0.0, P = 0.0001). The incidence and timing of CACS were similar in both arms of the KD study (6/8 mice on KD and 4/7 mice on NC). There was a trend for the KD to worsen overall survival (7.6w vs. 9.6w, P = 0.07), increase lung mass (420.1 ± 103.7 vs. 669.6 ± 109.5, P = 0.12), and reduce gastrocnemius mass (111.5 ± 3.6 mg vs. 122.5 ± 4.4, P = 0.0649). The KE diet had no effect on any of these parameters. Conclusions: These findings suggest that BHB does not protect against CACS and may actually worsen survival in this model. We conclude that BHB is only a biomarker of a more complex change in hepatic and systemic metabolism and does not play a pathophysiologic role in lung adenocarcinomainduced CACS. [1][2][3][4][5][6][7][8][9][10][11][12][13] Blockade of ACVR2B preserves skeletal and cardiac muscle function in the presence of metastatic colorectal cancer Introduction: Advanced colorectal cancer (CRC), a leading cause of death worldwide, is often accompanied by the development of liver metastases (LM), as well as skeletal muscle (SKM) wasting, i.e. cachexia. Activin receptor type 2B (ACVR2B)-mediated signalling participates in causing SKM wasting in several disease conditions, and its inhibition restores SKM mass and prolongs survival in cancer cachexia. Despite plaguing a majority of CRC patients, cachexia remains understudied and uncured. Moreover, only a single model of LM associated with CRC has been developed for the study of cachexia. We aimed to generate and characterize a new model of CRC and investigate whether systemic blockade of ACVR2B signalling could preserve skeletal and cardiac muscle function in the presence of metastatic cancer. Methods: NSG male mice (8 weeks old) were injected intrasplenically with HCT116 human CRC cells (mHCT116), while sham-operated animals received saline (n = 5-10 per group). Sham and tumour-bearing mice received weekly injections of ACVR2B/Fc, an inhibitor of ACVR2B, and were monitored weekly for skeletal muscle function and body composition. Conscious ultrasound echocardiography was performed the day before euthanasia. Results: mHCT116 hosts presented significant losses in body weight (À7%), SKM mass (quadriceps: À23%) and strength (À21%), along with impaired cardiac function (EF%: À14%, FS%: À14%). Conversely, administration of ACVR2B/Fc completely preserved SKM mass (quadriceps: +31%) and strength (+29%) in mHCT116 hosts. Interestingly, cardiac function was also completely restored in mHCT116 hosts receiving ACVR2B/Fc (EF%: +14%; FS%: +13%). Conclusions: Our model recapitulates the cachectic phenotype of metastatic CRC by displaying reduced SKM mass and strength in mHCT116 hosts. Additionally, we showed that HCT116 LM severely affects cardiac function, supporting the development of cardiac cachexia. ACVR2B antagonism fully preserved SKM mass, strength, and cardiac function, further dictating that activin signalling represents a promising therapeutic target for preservation of skeletal and cardiac muscle size and function in cancer cachexia.
Introduction: Cancer cachexia is a chronic inflammatory syndrome defined by greater than 5% body mass loss with underlying malignancy. It affects more than 50% of advanced cancer patients, contributing to approximately 20% of cancer patient deaths, primarily due to cardiac wasting and failure. The heart undergoes maladaptive remodelling in cancer cachexia, leading to altered function. Molecular mechanisms underlying this cardiopathology are relatively unknown. The aim of this project was to determine potential candidate genes altered in response to elevated cytokines in cardiomyocytes. Methods: Analysis of RNA-sequencing data from cytokine treated HL-1 cardiomyocytes, to emulate cancer cachexia, was performed to identify potential candidate genes altered in response to elevated cytokines. Analysis for differential gene expression was performed using EdgeR and DeSeq2 statistical software and applying two fields, an FDR < 0.1, and a log 2 fold-change deviation of ±0.2 from untreated control levels. Differentially expressed genes that were identified were then validated in cytokine-treated HL-1 cardiomyocytes and colon 26 (C26) carcinoma mouse hearts using qRT-PCR, western blotting, and immunohistochemistry. Results: We have identified alterations in gene expression of key signalling pathways involving Apelin receptor (APLNR) and Oncostatin M receptor (OSMR) in C26 hearts, raising the hypothesis that altered expression of these key signalling molecules may modulate this observed cardiopathology. RNA sequencing results revealed a down-regulation in APLNR gene and protein expression and an increase in OSMR gene and protein expression in both the cytokine-treated HL-1 cardiomyocytes. qRT-PCR, western blotting experiments, and immunohistochemistry showed consistent results in the hearts of C26 mice with cachexia compared to non-tumour bearing controls. Conclusions: The down-regulation of Aplnr, which is implicated in signalling cascades modulating cardiac contractility, and Osmr, which plays a role in signalling cascades involved in the regulation of sarcomeric protein expression, provide new insights into the pathophysiology of cancer induced cardiac cachexia seen in C26 hearts. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] Cardiac cachexia is an independent predictor of survival in dogs with heart failure Deanna L. Ineson, Lisa M. Freeman & John E. Rush Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA, USA Background: Heart disease affects 10-15% of all pet dogs and can result in heart failure, which is often associated with cardiac cachexia (defined as loss of muscle). In humans, cardiac cachexia is associated with shorter survival, but this has not been reported in dogs. Therefore, the aim of this study was to evaluate the effect of cardiac cachexia on survival in dogs with heart failure. Methods: Dogs with heart failure (Stage C or D) due to naturally occurring myxomatous mitral valve disease (MMVD) or dilated cardiomyopathy (DCM) and evaluated between 2015 and 2018 were eligible. Medical records data recorded included body weight, body condition score (BCS), and muscle condition score (MCS). Cachexia was defined as any muscle loss using the validated World Small Animal Veterinary Association scoring system of normal muscle condition or mild, moderate, or severe muscle loss. Results: Median age of the dogs (n = 269) was 11.0 years (range, 1.8-17.1 years); 54% were male. Mean body weight was 7.9 kg (range, 2.1-82.0 kg). Only 12 dogs were underweight (4.5%), 157 were ideal weight (58.6%), and 99 were overweight (36.9%). Cachexia was present in 48.3% of dogs: mild muscle loss: 101/269 (37.6%), moderate muscle loss: 22/269 (8.2%), and severe muscle loss: 7/269 (2.6%). Dogs with cachexia had a median survival time from onset of heart failure of 233 days (range, 0-1200 days) compared to dogs without cachexia (321 days, range 1-1264 days; P = 0.036). On multivariable analysis, cachexia, presence of arrhythmia or azotaemia, and being under-or overweight were independent risk factors for shorter survival time. Conclusions: Cardiac cachexia was present in nearly half of all dogs with heart failure and was associated with a shorter survival time.
Our understanding of the underlying mechanisms that cause cancer cachexia is limited. Several factors and inflammatory mediators released from the tumour have been suggested to contribute to weight loss in cachectic patients. However, inconsistencies between studies are recurrent. Activin A and interleukin 6 (IL-6) are among the best studied factors that seem to be important. Several studies support their individual role in cachexia development. We show that activin A acts in an autocrine manner to promote the synthesis and secretion of IL-6 from cancer cells. By inhibiting activin A signalling, using biological, chemical, or genetic approaches, the production of IL-6 from the cancer cells is reduced by 40-50%. Inhibiting activin signalling also reduces the ability of the cancer cells to accelerate autophagy in non-cancerous cells (up to 43% reduced autophagy flux). In line with the in vitro data, the use of an anti-activin receptor 2 antibody in cachectic tumour-bearing mice reduces serum levels of cancer cellderived IL-6 by 62%, and importantly, it reverses cachexia and counteracts loss of all measured muscle groups. Our data support a functional link between activin A and IL-6 and indicate that interference with activin A-induced IL-6 secretion from the tumour has therapeutic potential for cancerinduced cachexia.

2-02
The mechanical stimulation of myotubes counteracts the effects of tumour-derived factors through IL-4 secretion and the modulation of the activin/follistatin ratio Exercise counteracts cachexia, but it is unclear to which extent the exercise-dependent mechanical stimulation of muscle per se plays a role in exercise beneficial effects. To study the mechanisms underlying mechanical stimulation, we cultured C2C12 myotubes in the absence or in the presence of a cyclic mechanical stretching stimulus (MS) and in the absence or presence of C26 tumour-derived factors (C26-CM), so as to mimic the mechanical stimulation of exercise and cancer cachexia, respectively. We found that C26-CM contains activin and induces activin release by myotubes, further exacerbating its negative effects, consisting in myotube atrophy and in hampering myoblast recruitment and fusion into myotubes. A high level of circulating activin is an adverse prognostic factor in cancer patients, and our in vitro results demonstrate that activin may be a direct player and not just a marker of cachexia. We also found that MS is sufficient to counteract the adverse tumour-mediated effects on muscle cells, in association with an increased follistatin/activin ratio in the cell culture medium, indicating that myotubes actively release follistatin upon stretching. In addition, MS induces IL-4 secretion by muscle cells. Recombinant follistatin counteracts C26 tumour effects on myotubes exclusively by rescuing fusion index, while recombinant IL-4 ameliorates fusion index, as well as the myotube size, both in terms of myotube diameter and number of nuclei per myotube. Our results indicate that tumour cells negatively affect muscle cells by releasing soluble factors and that MS is sufficient to counteract these effects, by affecting the muscle secretome with autocrine/paracrine pathways. Activin and Act-R ligands are becoming increasingly important as triggers of muscle wasting and as pharmacological targets to treat cachexia; however, since follistatin alone is incapable to entirely block the C26-CM effects, the development of novel activintargeted approaches should consider the existence of further significant tumour-secreted factors mediating cachexia.

2-03
Generation of reporter cell lines to identify and characterize cachexia-inducing factors Background: Cancer-induced cachexia resulting in defects in skeletal muscle mass and function is well described, although little is known about the effects of non-bone metastatic cancer on bone, particularly in the absence of metastases. In this study, we investigated the effects of two well-characterized models of cancer cachexia, namely, the mice bearing C26 adenocarcinoma and ES-2 ovarian cancer. Methods and Results: Even though both C26 and ES-2 tumours resulted in comparable body and muscle wasting, the ES-2 hosts showed severe bone loss by microCT analysis, whereas only a modest bone loss was observed in the C26bearing mice. Histomorphometry analysis showed increased osteoclast numbers in femoral trabecular bone from ES-2 hosts, while no significant effects were observed in the C26bearing mice at time of sacrifice. Von Kossa staining of femur sections showed severe reduction of organized osteoblasts as well as osteoid of trabecular bone. Interestingly, both models showed dramatic increase in osteocyte death and empty lacunae, likely due to secreted tumour factors. This was also validated by in vitro experiments showing increased death of MLO-Y4 osteocyte-like cells when exposed to ES-2 or C26 conditioned media. Notably, dramatic depletion of fat vacuoles within the bone marrow, as well as dystrophic mineralization, likely consequence of massive bone marrow cell death, were observed in both models. In addition, the growth plate was also severely affected showing absence of hypertrophic chondrocytes and cartilage degeneration zone in both tumour models. Conclusions: Here, we showed for the first time that bone destruction accompany muscle depletion in two models of non-metastatic cancer cachexia. Overall, this study suggests the importance of monitoring bone quality in cancer patients, even in the absence of bone metastases. Moreover, our ongoing studies will define whether anti-resorptive treatments will preserve bone mass while also improving muscle size and function in cachexia.

2-05
Exploring the contribution of the liver to cancer cachexia development Traditionally, skeletal muscle and adipose tissue have been the focus of research on cancer cachexia. Recent studies suggest that cancer cachexia (CC) is a multi-organ syndrome which involves not only the direct effects of tumour-derived cachexia-inducing factors on target organs but also the crosstalk of these dysfunctional organs. Despite its central role in other metabolic diseases, the liver has received little attention in the context of CC. However, CC is associated with systemic inflammation, and the activation of the hepatic acute phase response (APR) is well-documented in cachectic patients. Serum amyloid A 1 and 2 (SAA 1/2) are classical APR proteins that are mainly produced by the liver and strongly induced in response to inflammatory cytokines. To assess the potential contribution of SAA to tissue wasting in vitro, we treated 3T3-L1 adipocytes and C2C12 myotubes with recombinant SAA1. While lipolysis rates remained unaffected, we found a significant reduction in myotube diameter in response to SAA1 treatment, indicative of an atrophic effect. Next, we addressed the contribution of SAA 1/2 to cachexia development in vivo using a liver-specific knockdown (KD) approach. For this purpose, we injected an AAV-miRNA targeting SAA1/2 or an AAV-control-miRNA into C26 tumour bearing mice. Despite a four-fold reduction in circulating serum levels in the SAA1/2 KD group, SAA1 was still highly up-regulated in tumour bearing mice and no differences were observed in cachexia progression. In order to define novel liver-secreted factors that can potentially impact cachexia development, we integrated hepatocyte specific RNAseq and serum proteome analyses of C26 cachectic animals and observed a high degree of overlap. We are currently functionally characterizing a panel of these hepatocytesecreted proteins in order to investigate their potential adipocyte lipolysis and/or myotube atrophy-mediating properties. These studies might contribute to a better understanding of the pathophysiology of CC and the liver-specific contribution to its development.

2-06
The use of proton pump inhibitors might increase symptoms of cachexia in patients with chronic illness Paulien Vinke 1,2 & Klaske van Norren 1 1 Introduction: Long-term use of proton pump inhibitors (PPIs, e.g. omeprazole), is becoming increasingly common in patients with cachexia-related chronic diseases, as cancer, COPD, and heart failure. Methods: To determine mechanistically, with a literature search, whether the use of PPIs may affect the development of cachexia or the health status of cachectic patients. Results: PPIs increase the pH in the lumen, which changes the microbiota composition and decreases the activity of TRPM6, a major transporter in magnesium metabolism. Decreased TRPM6 activity may lead to lower magnesium absorption from the gut. Low free levels of magnesium, in turn, compromise several processes that are relevant in cachexia. For example, magnesium is necessary for activation of vitamin D. A deficiency in active vitamin D is linked to musculoskeletal and immune function. Hypomagnesaemia can also cause muscle weakness, muscle cramps, and fatigue. Moreover, a PPI-induced change in the microbiome composition may lead to a dysfunctional lipid and energy metabolism. Finally, both microbiota dysfunction and hypomagnesaemia can increase inflammation. Conclusions: Based on a thorough literature search, it can be concluded that PPIs can contribute to hypomagnesaemia, vitamin D deficiency, inflammation, and a change in microbiota. These are properties that all can have an impact on the development of cachexia. Therefore, more research on potential unwanted side-effects of PPI use in diseases related to development of cachexia is necessary. In these studies, it is recommended to screen for PPI-induced nutritional deficiencies and to study the effect of treating those deficiencies.
During extensive literature research, we found that gene and protein expression of muscle mitochondrial dynamics are altered during cancer cachexia in animal models [1]. Therefore, we will measure the oxygen consumption of the rectus abdominis in a subset of the COMUNEX study, an observational study designed to gain insight in changes in muscle gene expression, body composition, muscle function, and muscle metabolism of colon cancer patients. To date, 21 primary colon cancer patients (age 68 ± 7.01) and 3 liver metastasized patients (age 58 ± 15.56) were included. For the most recently included patients of this population (N = 8) oxygen consumption was measured. Tissue of the rectus abdominis was collected in BIOPS buffer during surgery. Oxygen consumption was measured in permeabilized muscle tissue (saponin 50 μg/mL 30 min) using the OROBOROS respirometer. Injection protocol (end concentrations): 5 mM pyruvate, 2 mM malate, 10 mM glutamate, 5 mM ADP + Mg 2+ , 10 μM cytochrome C, 10 mM succinate, 5 mM ADP + Mg 2+ , 5 μM oligomycin, titration with 1 μL steps of 1 mM CCCP, 2.5 μM rotenone, and 5 μM antimycin A. No control patients were included, and therefore, no comparison with 'healthy' patients could be made at this time point of the study. On average, the patients showed a 16% ± 12% increase in oxygen consumption after cytochrome C injection. A typical trace is shown in Figure A. Additionally, Figure B depicts the average of different states and shows that the ET-NS state is lower than the OXPHOS-NS state which is remarkable. We hypothesize that respiration of the rectus abdominis muscle tissue of cancer patients will be overall lower than the respiration of the tissue from controls (N = 8, scheduled coming months). Nonetheless, currently, it is too early to draw any conclusions.

Background:
The majority of patients with pancreatic cancer develop cachexia, which is characterized by progressive muscle loss. However, the mechanisms underlying cancer cachexia development and progression remain elusive. Pancreatic tumour organoids are in vitro 3D organ-like structures that retain specific structures and pathophysiological characteristics of the original tumour. We aimed to establish pancreatic tumour organoids from well-phenotyped cachectic patients to study their cachexia-inducing properties. Methods: Organoids were established from tumour tissue of cachectic (n = 5) and non-cachectic (n = 3) pancreatic cancer patients. A comprehensive pre-operative assessment of cachexia-related parameters was performed (i.e. nutritional status, physical performance, body composition, and inflammation). Tumour-and cachexia-related characteristics of the tumour organoids were analysed using histological stainings, sequencing, and RT-qPCR. C2C12 myoblasts were exposed to organoid conditioned medium (CM) and mRNA expression of myogenic markers and myosin heavy chain (MyHC) isoforms was analysed by RT-qPCR. Results: Organoids presented typical features of malignancy similar to the primary tumour (i.e. nuclear enlargement, nucleoli, mitosis, apoptosis, and mutated KRAS and/or TP53). mRNA expression of known cachexia-related factors was highly variable among the organoid lines. Strikingly, IL-8 (1.4-fold, P = 0.01), LIF (1.9-fold, P = 0.0043), GDF15 (2.3-fold, P < 0.001), and MIF (3.0-fold, P < 0.001) expressions were significantly increased in organoids from cachectic versus non-cachectic patients. Unexpectedly, exposure of C2C12 myoblasts to organoid CM accelerated myogenic differentiation, as evident from reduced expression of PAX7 (2.0-fold) and CCND1 (1.7-fold), and increased expression of MYOG (1.6-fold), MYMK (2.8-fold), and MCK (3.9-fold) early after initiation of differentiation. Subsequent myotube formation was accompanied by increased expression of MyHC-2b (8.7-fold) and decreased expression of MyHC1 (1.3-fold), consistent with the shift from slow-to fast-twitch muscle fibres often observed in cachectic patients. Conclusions: Tumour organoid-derived factors from cachectic patients with pancreatic cancer affect both differentiation and maturation of C2C12 myoblasts. Organoids represent a promising model to uncover mechanisms underlying cancerinduced cachexia.

3-02
Early changes in muscle and adipose tissue in stage IV non-small cell lung cancer treated with immune-checkpoint inhibitors Introduction: Early loss of skeletal muscle (SM) during chemotherapy has shown to be poor prognostic for overall survival (OS) in stage IV non-small cell lung cancer (NSCLC) [1]. Treatment options have expanded with immune checkpoint inhibitors (ICI). Data on changes of SM and adipose tissue (AT) during treatment with ICI in stage IV NSCLC are lacking. Methods: This was a single centre study. All patients were treated with ICI between June 2015 and December 2018. SM, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) were characterized at the first lumbar level on CT images in stage IV NSCLC. CT scans of 130 patients were obtained before the start of ICI treatment (baseline) and after 6 and 12 weeks. The contribution of changes in different body compartments to OS was assessed. Results: Between baseline and 12 weeks, a significant loss was observed of SM cross-sectional area (CSA) (À2.4 ± 9.8%, P = 0.003), VAT CSA (À9.5 ± 30%, P = 0.001), and SAT CSA (À10 ± 26.3%, P = 0.001) but not in muscle attenuation. Mean weight change was À0.2% (±4.9, NS). Median OS was significantly shorter among patients losing VAT (14 vs. 25 months; HR 2.19, 95% CI 1.26-3.81, P < 0.05) and SAT (11 vs. 20 months; HR 2.91, 95% CI 1.53-5.55, P < 0.05). In the subgroup of 90 patients without progression of disease within 12 weeks, multivariate analysis showed that loss of VAT and SAT CSA remained significantly associated with poor OS, independent from age, gender, smoking-status, WHO-PS, and pre-existent weight loss. Conclusions: Loss of adipose tissue in stage IV NSCLC patients treated with immune checkpoint inhibitors is associated with poor prognosis. The research was a mixed-methods study conducted at a cancer centre in the UK. A questionnaire to assess eating problems, which included the Patient-Generated Subjective Global Assessment (PG-SGA), was administered to patients with stage I-III colorectal cancer in treatment (n = 92). Telephone interviews were then conducted with a maximum variation sample of these patients (n = 20), to explore factors influencing nutritional self-care. The survey data were analysed using descriptive statistics and the interview data using framework analysis. New insights into patients' nutritional self-care during treatment were then generated by synthesis of results.

Results:
We categorized knowledge of eating-related distress in patients and family members as (i) nutrition impact symptoms, (ii) eating-related distress and perceived need for nutritional support in patients, (iii) eating-related distress and perceived need for nutritional support in family members, (iv) conflicts over food between patients and family members, (v) role of palliative and supportive care, (vi) role of nutritional care, and (vii) integration of palliative, supportive, and nutritional care.

Conclusions:
We found limited information about eatingrelated distress among patients and family members. There are no well-developed educational tools or psychosocial interventions to alleviate their distress. There is a lack of validated tools for the clinical assessment of eating-related distress and its response to interventions. More robust evidence regarding nutrition impact symptoms and their association with distress is needed. To inform and support patients and family members to cope effectively, health care professionals must appreciate the complexities of eating-related distress. We propose that this will be facilitated by close coordination of palliative, supportive, and nutritional care. Introduction: Despite decades of research, pancreatic adenocarcinoma remains a devastating disease. While the difficulty of detection and aggressive biology of this tumour are significant contributors to the high mortality rates of this disease, body weight loss, and particularly the loss of skeletal muscle mass, also contributes to poor survival of patients with pancreatic cancer. Methods: Because pancreatic cancer incidence and average body weight losses are similar between men and women, little attention has been paid to how cachexia may differ between the sexes. We sought to investigate sex differences in the clinical and biological data contained in The Ohio State University Comprehensive Cancer Center Pancreatic Cancer Cachexia Biobank. Consistent with an international consensus, patients were classified as cachectic if they had lost greater than 5% of their pre-illness body weight. Results: Consistent with previous reports, we found cachectic patients (n = 59) had higher pre-illness body mass indices (BMI) than weight-stable cancer patients (n = 30). However, when men and women were assessed separately, only cachectic women tended to have higher pre-illness BMIs than their weight-stable counterparts. Importantly, cachectic men and women had similar changes in BMI. To further characterize this potential sex difference, CT scans were analysed for a subset of patients. While there were no differences between cachectic and weight stable men or women in estimated fat mass, we did identify an interaction effect in fat free mass, suggesting a sex-dependent effect of cachexia in pancreatic cancer patients. RNA sequencing data from muscle support this dissimilarity between men and women, as clear gene expression clustering is present in male patients, yet absent in female patients. Conclusions: While there are caveats to this work, including the limited power and cross-sectional nature of the study, these data provide strong rationale for consideration of sex as a biological variable in future cancer cachexia studies.

3-09
Inflammatory pathways involved in the gut-brain axis in cachexia Background: Cancer-related cachexia is characterized by skeletal muscle wasting and systemic inflammation. The gut and hypothalamus have been indicated to play a key role. However, the mechanistic details remain largely unexplored. To determine the mechanisms behind the cross talk between the tumour, the gut, and the hypothalamus that lead to muscle wasting in cachexia.

Methods:
To imitate the influence of decreased integrity of the gut, sub-inflammatory levels (31.6 ng/mL) of the gutderived bacterial compound lipopolysaccharides (LPS) were added to hypothalamic (HypoE-N46) cells. These cells were incubated with the secretome of cachexia inducing tumour cells from murine colon (C26). Subsequently, hypothalamic cells were exposed for 24 h to LPS in combination with tumour-secretome or a mimic of this secretome (110 pg/mL IL6, 30 000 pg/mL MCP-1, 100 pg/mL LIF, 6500 pg/mL PGE2). Effects on inflammation were assessed by measuring Il-6, MCP-1, and LIF secretion using a commercial ELISA.

Results:
The IL6-release of hypothalamic cells reached 1100 pg/mL when incubated with the combination of the C26 tumour-secretome and 31.6 ng/mL of LPS. This was several magnitudes higher than the release of hypothalamic cells incubated with C26 tumour-secretome or LPS separately (50 and 365 pg/mL, respectively, P < 0.05). A similar finding was observed with the C26 tumour-secretome mimic (separately: 26 pg/mL, combination LPS and tumour-secretome mimicking medium: 940 pg/mL, P < 0.05). The MCP-1 and LIF release of the hypothalamic cells showed no or less additive effects of the tumour-secretome and LPS, indicating that for secretion of these inflammatory mediators, different pathways might be involved.

Conclusions:
These data indicate that the inflammatory hypothalamic response to a tumour can be significantly more pronounced in the presence of low levels of gut-derived bacterial compounds like LPS. This increased activation seems to be associated with the inflammatory mediators secreted by the tumour. Introduction: Recent studies showed that physical activity increased survival in cancer patient and animal models of cancer cachexia. The underlying mechanisms, however, are still largely unknown. Methods: To identify signalling pathways involved in exercise-dependent maintenance of muscle mass and function in cachexia, we investigated the role of serum response factor (SRF)-a transcription factor playing a pivotal a role in muscular growth, differentiation and regeneration-in C26-bearing mice in the absence or presence of voluntary exercise (wheel running). Results: SRF levels are decreased at protein level in cachexia. Consistently, a decrease in the expression of SRF target genes such as MyoD and SK-actin occurs in C26-bearing mice, suggesting a decrease of SRF transcriptional activity. These tumour effects were counteracted by wheel running and associated to the rescue of muscle mass and function. However, a minimum amount of exercise (2 km/day) is necessary to keep SRF levels elevated in cachexia over a threshold which is necessary to exert beneficial effects. SRF levels inversely correlate with wasting in mice, suggesting that SRF play a role in maintaining body mass (mostly accounted for by muscle mass). We also observe the recruitment of nuclei within the muscle fibres in response to exercise, which could contribute to muscle homeostasis and is consistent with the previously observed opposite effects of tumour and exercise on MyoD and Pax7 expression.

Conclusions:
Our results suggest that physical activity rescues SRF expression as well as its transcriptional activity, highlighting the importance of genetic activation induced by skeletal muscle activity for muscle rescue and homeostasis. These effects could be extended to the fibre microenvironment, including myogenic stem cell activity.

3-11
Toward the identification of receptor for advanced glycation end-products (RAGE) as a muscle biomarker of cancer cachexia Introduction: Cachexia is a debilitating syndrome affecting more than 50% of patients with advanced cancer. Its major clinical feature is skeletal muscle atrophy leading to pronounced weight loss, reduced quality of life, and poor prognosis. The identification of valuable biomarkers of early cachexia is of great importance to identify the patients at risk of cachexia and to treat patients in the reversible phase of the disease (Porporato et al., Oncogenesis 2016, Loumaye-Thissen, Clin Biochem 2017). RAGE (receptor for advanced glycation end-products) signalling concurs to skeletal muscle development and homeostasis (Riuzzi et al., JCSM 2018); however, in cancer conditions, RAGE hyperstimulated by high levels of its ligands leads to muscle wasting, sustains inflammation, and reduces survival of mice (Chiappalipi et al., submitted). Here, we investigated whether RAGE might represent a biomarker of cachexia. Methods: We analysed RAGE expression in muscle tissue of different tumour-bearing mice in the absence or presence of endurance exercise and correlated it with myofiber CSA and hallmarks of atrophy (body and muscle weights, protein degradation, and activation of the proteolytic systems). We performed RAGE expression analysis in muscle biopsies of cancer patients. Results: We found that (i) Lewis lung carcinoma (LLC)-bearing C57Bl/6 mice and colon adenocarcinoma (C26-ADK)-bearing BALB/c mice express RAGE in myofibres in coincidence with reduced body and muscle weight and induction of proteolysis; (ii) an inverse relationship exists between RAGE expression in muscles, tumour masses, and the beneficial effects of endurance exercise in LLC-bearing mice; (iii) RAGE expression increases in muscles during cachexia progression; (iv) LLC or melanoma A375 cells injected in athymic-nude mice are not able to induce neither cachexia nor RAGE expression in muscle; and (v) muscles of cachectic patients express higher amounts of RAGE than non-cachectic subjects. Conclusions: RAGE might represent a muscle biomarker of the cachectic stage.

3-12
Musclin, a myokine induced by aerobic exercise, retards muscle atrophy during cancer cachexia Physical activity improves the prognosis of cancer patients, partly by contrasting the associated muscle wasting (cachexia), through still unknown mechanisms. Since aerobic exercise seems to be the most effective to preserve muscles during cancer, we asked whether it promotes secretion of proteins by muscles (i.e. myokines) that may contrast cachexia. Media conditioned by PGC1-α-expressing myotubes, reproducing some metabolic adaptations of aerobic exercise, as increased mitochondrial biogenesis and oxidative phosphorylation, restrained caFoxO3-induced proteolysis. Microarray analysis identified AREG, NppB, musclin, and FGF18 as myokines highly induced by PGC1-α. Notably, only musclin tended to be low in muscle of mice with a rare human renal carcinoma, and it was reduced in plasma and in muscles of C26-bearing mice and in atrophying myotubes, where PGC1α expression is impaired. So we electroporated tibialis anterior of C26-bearing mice with musclin or (its receptor) Npr3-encoding plasmids and found preserved fibre area, as a result of restrained proteolysis. Running protected C26bearing mice from cachexia, unchanging tumour growth, and rescued the C26-induced down-regulation of musclin in muscles and plasma. Musclin expression did not change in overloaded plantaris of mice, recapitulating partly muscle adaptations to anaerobic exercise. Furthermore, the expression of musclin, but not its receptor Npr3, was reduced in sarcopenic muscles of aged mice. Musclin might therefore be beneficial to cancer and perhaps also to sarcopenic old patients who cannot exercise for various reasons and may be added to the mechanisms by which aerobic exercise alleviates muscle wasting.

3-13
Mechanisms of tumour metabolic reprogramming by Fn14 as a driver of cancer cachexia Introduction: Cachexia is a multifactorial metabolic syndrome characterized by weight loss, distal muscle, and fat wasting and is seen in~50% of cancer patients. The molecular mechanisms of cachexia are poorly understood with no FDA approved treatments available. We have developed an antibody that targets the TWEAK receptor Fn14, on the tumour surface, which is able to abrogate symptoms of cachexia in syngeneic mouse models (Johnston et al., 2015, Cell). This anti-Fn14 antibody (mAb 002) prolongs life by blocking body weight loss associated with cancer cachexia. We have shown that 18 F-FDG PET imaging links Fn14 to glucose metabolic pathways in tumours that induce cachexia. Here, we aim to further understand the role of Fn14 in tumour metabolism to drive cancer cachexia as well as the therapeutic effect of mAb 002. Methods: Tumours from either vehicle or mAb 002 treated cachectic mice were subjected to large-scale transcriptomic and proteomic profiling followed by gene ontology and pathway enrichment analysis. Metabolic assays were used to measure serum and tumour lactate levels. Digital droplet PCR was employed to assess metabolic response to cachexia in muscle and white/brown adipose tissue. Results: 18 F-FDG PET imaging demonstrated increased glucose uptake in cachectic versus non-cachectic tumourbearing mice, suggesting a higher glycolytic activity in tumours of cachectic mice. This occurs alongside an upregulation of pentose phosphate and nucleotide salvage pathways and a concomitant decrease in oxidative phosphorylation due to down-regulation of mitochondrial ribosome subunits. Treatment of cachectic mice with mAb 002 prevents increased 18 F-FDG uptake, reduces tumour and plasma lactate levels, and blocks weight loss. Conclusions: These results suggest that cachexia is driven by specific reprogramming of tumour metabolism, with downstream signalling events responsible for distal muscle and fat loss. This increase in glycolytic activity, body weight loss, and tumour growth can be reversed with anti-Fn14 antibody treatment.

3-14
Small-molecule inhibition of MuRF1 attenuates skeletal muscle atrophy and dysfunction in tumour cachexia Background: Skeletal muscle atrophy is the most prominent phenotypic feature of cancer cachexia contributing to a reduced life expectancy and affecting quality of life. During tumour progression, the activation of catabolic processes, including the activation of muscle RING finger 1 (MuRF1), is well established. The Aim of the present study was to test the effect of a recently identified MuRF1 inhibitor, MCEMBL#205, on the development of muscle atrophy/dysfunction and molecular alterations. Methods: Male C57BL/6N mice (n = 30) were included into the study. At an age of 6 to 8 months, the animals were randomized into the following groups (n = 10 in each group): (i) control group (Con), normal chow, no tumour; (ii) tumour group (Tu): inoculation with tumour cells and normal chow; (iii) tumour group on MCEMBL#205 (Tu-205): inoculation with tumour cells and chow with MCEMBL#205. The chow containing Embl205 was started 3 days after inoculation with tumour cells (5*10 5 cells per animal in 0.1 mL) into the right thigh. After 3 weeks, muscle force was determined, and tissue was harvested for molecular analysis. Results: Functional analysis revealed impairment of muscle strength, which was attenuated by MCEMBL#205. This goes along with muscle mass. Molecular analysis showed increased expression of MuRF1, markers for oxidative stress (Nox2, UCP3, ubiquitin, and nitrotyrosine) and autophagy (LC3). In addition, actin content was decreased. These molecular effects were reversed by MCEMBL#205. Conclusions: Tumour development is associated with functional impairment and muscle atrophy related to increased ROS and autophagy. MCEMBL#205 might be a future pharmacological treatment to prevent functional decline in tumour cachexia patients.
Taken together, these results suggest that a combination of bisoprolol and oxypurinol allows the reduction of the respective dose to 75% of mono-therapy in experimental cancer cachexia.

3-19
The is no effective treatment of cancer cachexia. Erythropoietin is a hormone that plays tissue protective role in different tissues. Based on the structure of erythropoietin, small peptides have been synthesized which are not erythropoietic but activate the tissue protective signalling pathways. In this study, we investigated the influence of the tissue protective peptide ARA 284 on cancer cachexia in rats. We show that administration of this compound (1.7 μg/kg/day) counteracted the loss of total body weight (12.46 ± 4.82% ARA 284 vs. 26.85 ± 0.88% placebo, P = 0.0058), fat mass (P = 0.027), and lean mass (P = 0.012), as well as significantly improved the spontaneous activity of the ARA 284 treated animals. Further, gastrocnemius muscle mass was shown to be increased (13.2% ARA 284 vs. placebo, P = 0.015) in association with a higher level of activated Akt (P = 0.0038), and a decrease in activated p38 MAPK, GSK-3β, and myostatin (all P < 0.0001). These results suggest that an induction of anabolic pathways underlies the increase of muscle tissue. At the same time, we observed the significant increase in the survival of animals (P < 0.05). Taken together, these results suggest that ARA 284 can be considered as a prospective drug to improve conditions of patients with cancer cachexia.    2). Furthermore, miRNAs can be secreted in the blood, free or clotted in extracellular vesicles (microvesicles and exosomes), and distributed throughout the whole organism. Evidences demonstrate that these miRNA-containing vesicles can be up-taken by cells and contribute to signalling (3). Similar to muscle, circulating miRNAs are found modulated in many pathologies, therefore being good potential candidates for diagnosis and treatment.

Methods:
In the attempt to characterize the miRNA pattern in a setting of cancer cachexia, we isolated total RNA from gastrocnemius muscle and plasma-derived microvesicles of both healthy and cachectic mice bearing the transplantable C26 colon carcinoma. As for plasma, samples of two mice were pooled, and consecutive centrifugations were applied for microvesicle isolation. Next-generation sequencing (Illumina) was used to sequence whole miRNA transcriptome. We also extended the alignment analysis of the remaining unmapped sequences to other candidate small non-coding RNAs.
Results: About 30 out of the 304 detected miRNAs were differentially regulated in the skeletal muscle of C26-bearing mice. By contrast, miRNAs extracted from plasma-derived microvesicles appear poorly modulated in the C26 hosts; only miR-181a-5p, miR-375-3p, and miR-455-5p were found dysregulated from a total of 118. Finally, the analysis of the remaining unmapped sequences revealed that muscle from mice bearing the C26 had eight up-regulated snoRNAs, while a general down-regulation of mt-tRNAs was observed (12 out of the 21 detected). Conclusions: These results bring new insight into the modulation of muscle and circulating miRNA expression during cancer cachexia, as well as new data concerning the involvement of other types of small non-coding RNAs.

Introduction:
The loss of skeletal muscle mass is recognized as a complication of several chronic diseases and is associated with increased mortality and a decreased quality of life. Relevant and reliable animal models in which muscle wasting can be monitored longitudinally are instrumental to investigate and develop new therapies. However, the non-invasive follow-up of time-dependent changes in muscle mass is time consuming and challenging due to technical requirements. In this work, we developed a fully automatic deep learning algorithm for segmentation of micro cone beam CT (μCBCT) images of the lower limb muscle complex in mice and subsequent muscle mass calculation. Methods: Whole body μCBCT images were obtained from anaesthetized mice using a X-RAD 225Cx image-guided biological irradiator system, and the lower hind leg musculature was manually segmented using SmART-ATP software. A deep learning algorithm was constructed (two-step 3D U-Net model) and trained on manually segmented data from 32 mice. Muscle wet mass measurements were obtained of 47 mice and served as a dataset for model validation and the reverse model validation.

Results:
The automatic algorithm performance was approximately 150 times faster than manual segmentation. Reverse validation of the algorithm showed high quantitative metrics, i.e. a Dice Similarity Coefficient (DSC) of 0.93 and a submillimeter precision in Hausdorff distance and center of mass displacement, substantiating the robustness and accuracy of the model. A high correlation (R 2 = 0.92) was obtained between the CT-derived muscle mass measurements and the muscle wet masses. Longitudinal follow-up revealed time-dependent changes in muscle mass that separated control from lung tumour bearing mice, which was confirmed as cachexia.
Conclusions: This deep learning model for automated assessment of the lower limb muscle complex provides highly accurate non-invasive longitudinal evaluation of skeletal muscle mass. Furthermore, it facilitates the workflow and increases the amount of data derived from mouse studies, while reducing the animal numbers.

5-01
Longitudinal characterization of muscle alterations in a rodent model of NAFLD Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. A substantial body of literature supports that a low muscle mass, low strength, or a higher muscle fatty infiltration are associated with NAFLD presence and severity. Here, the muscle compartment was evaluated longitudinally in a widely used NAFLD rodent model.

Conclusions:
In CSAA-HFD mice, mild myosteatosis develops together with mild liver steatosis and metabolic syndrome.
In CDAA-HFD, loss of muscle mass occurs together with severe steatohepatitis, but without myosteatosis or metabolic syndrome. Further studies are needed to decipher the respective contribution of dietary choline deficiency, metabolic alterations, and liver disease to changes in the muscle compartment. ) measured by micro CT-scan at W0, W4, W6, and W8, (C) quadriceps weight (mg) at W4 and W8. Student's t-test, two-way ANOVA, mean ± SEM. ***

5-02
Micro-CT imaging is an effective tool to detect muscle alterations non-invasively Background: Cross-sectional clinical studies support that muscle alterations (i.e. low muscle mass, low strength, or a high muscle fatty infiltration) are associated to poor outcomes in almost all chronic diseases. To better understand how the muscle compartment influences disease progression, longitudinal studies in disease models are needed. To develop and validate a micro-CT based methodology to measure muscle mass and fatty infiltration non-invasively in small rodents. Methods: Anaesthetized lean and obese mice, exhibiting a wide range of muscle alterations (i.e. low muscle mass and myosteatosis), were imaged using a Skyscan 1278 (Bruker, Belgium) at 50 μm voxel resolution. Whole body fat, lean (muscles and organs), and bone volumes were measured (Figure 1A) and transformed in grams to estimate total body weight. The surface (mm 2 ) and density (in HU) of dorsal muscles (i.e. erector spinae/quadratus lumborum or E/Q, and psoas), were semi-automatically measured at 4th and 5th lumbar levels ( Figure 1B). A virtual liver biopsy was performed by placing a 3D cylindrical region of interest (±1.3 cm 3 ) in the liver to measure the mean density (HU). Dorsal muscles and liver densities ( Figure 1B) were respectively normalized to spleen density and expressed as a ratio. After scanning, mice were sacrificed, and muscles (i.e. E/Q, gastrocnemius, and quadriceps) and liver were harvested and weighted, and lipids were extracted for quantification. Correlations were tested with Pearson's coefficient. Results: The procedure was feasible in 100% of tested mice, repeatable and well tolerated. Scanning time was 2.5 min. We found an excellent agreement between micro-CTestimated and measured body weight (Figure 2A, r = 0.99, n = 69, P < 0.001). Dorsal muscle area was strongly correlated with both gastrocnemius ( Figure 2B) and gastrocnemius + quadriceps weight (r = 0.85 and 0.83, respectively, n = 18, P < 0.001). Dorsal muscles density was strongly negatively correlated with dorsal muscles lipid content ( Figure 2C, r = À0.83, n = 16, P < 0.0001). Liver density was strongly correlated with liver lipid content (r = À0.91, n = 69, P < 0.001). Conclusions: Micro-CT-derived measurements accurately capture lean mass, muscle mass, and muscle and liver lipid content. This innocuous and high-throughput methodology is readily applicable for longitudinal evaluation in pre-clinical studies.

5-03
Non-invasive detection of myosteatosis predicts NASH in the context of metabolic syndrome and obesity Background: A growing body of evidence links muscle alterations (i.e. loss of mass, strength, and myosteatosis) to the presence and severity of non-alcoholic fatty liver disease (NAFLD). Here, we evaluated the muscle compartment with gold standard techniques, both longitudinally in a pre-clinical NAFLD model and cross-sectionally in a clinical cohort of morbidly obese patients. Methods: For over 34 weeks (34W), we followed WT mice fed a standard chow as controls (Ctl), WT mice fed a high fat (HF) diet (60% fat) as a model of fatty liver (NAFL;WT HF) and foz/foz mice fed a HF diet as a model of progressive NASH (FOZ HF). We monitored muscle mass and density (i.e. myosteatosis) by micro-CT and analysed muscles and liver histology at monthly intervals. We evaluated muscle mass and myosteatosis at fourth lumbar level with CT in a cohort of 185 morbidly obese patients with biopsy-based liver histology (62.8%, 19.9%, and 17.3% had NASH, NAFL, and normal liver histology, respectively). Results: Ctl had normal liver histology, WT HF developed obesity and isolated mild steatosis, and FOZ HF exhibited obesity and histologically proven NASH from 12W on. In FOZ HF at 34W, muscle size, strength, and density were significantly lower than in WT HF. Strikingly, muscle density was already significantly lower in FOZ HF after 4W of HF (0.79 ± 0.02) than in Ctl (0.91 ± 0.02) and further dropped in FOZ HF to reach a minimum at 12W (0.37 ± 0.05). Muscle density strongly negatively correlated with the NAFLD activity score (r = À0.87, P < 0.001, n = 67) and robustly discriminate NASH from NAFL (AUROC = 0.96, P < 0.0001, n = 67) ( Figure  1A). In the cohort of 185 obese patients, muscle mass was unexpectedly significantly higher in patients with NASH than in those with NAFL or normal liver histology. By contrast, muscle density distinguishes NASH from NAFL with a high diagnostic power ( Figure 1B, AUROC = 0.74, P < 0.0001) and independently from multiple confounders in these patients.

Conclusions:
Myosteatosis strongly relates to NAFLD severity. We propose to further exploit this parameter to discern NASH from NAFL in NAFLD and monitor disease status.

Michaiah Halley & Chitra Iyer
The Ohio State University, Columbus, Ohio, USA Background: Sarcopenia is an important contributor to loss of physical function in older adults. Neurodegeneration has increasingly been recognized as a potential factor in the pathogenesis of sarcopenia. Prior ex vivo recordings of neuromuscular junction (NMJ) transmission have consistently demonstrated accentuated transmission rather than failure, but ex vivo recordings do not directly assess muscle fibre action potential generation. Single fibre electromyography (SFEMG) is a sensitive assessment of NMJ transmission routinely used in the clinic. Here, we investigated the following questions in aging C57BL/6J mice using the SFEMG technique: (i) Do aged mice show features of NMJ failure? (ii) At what ages does NMJ failure become evident? and (iii) Can therapeutic agents used in genetic and acquired NMJ disorders (myasthenic disorders) have a positive effect on NMJ transmission in aged mice? Methods: Motor unit number estimation (MUNE), SFEMG, and contractility were obtained from the gastrocnemius muscle of mice aged 12, 20, 24, 27, and 29 months (n = 9-11 per age, balanced for sex). An additional cohort of aged mice (n = 7-8 per group) were treated with salbutamol 8 mg/kg daily or vehicle by intraperitoneal injection for 3 days and were compared using SFEMG, grip strength, and rotarod. Results: MUNE and muscle contractility were significantly reduced at 20, 24, 27, and 29 compared to 12 months. In contrast, SFEMG jitter (variability of NMJ transmission) and blocking (failed NMJ transmission) were not significantly changed until 27 and 29 months. Salbutamol significantly improved SFEMG jitter (P < 0.0001) and blocking (P = 0.003), and rotarod (P = 0.046), but not grip (P = 0.074). Conclusions: NMJ transmission failure occurred late in the lifespan of C57BL/6J mice. Salbutamol improved NMJ transmission and muscle performance in aged mice. Work is ongoing to investigate other potential therapeutics and exercise on NMJ failure. Future studies are needed to understand if similar NMJ defects are present in sarcopenic older adults.

5-06
Anthropometric muscle mass measurements, mid-arm muscle, and calf-circumference as a mortality predictor in obese and non-obese older adults: Cohort Elderly Project/Goiania Introduction: Sarcopenia is associated with mortality however reaming unclear whether anthropometric measure of muscle mass such mid-arm muscle circumference (MAMC) and calf circumference (CC) may be mortality predictors in older adults. We examined the impact of MAMC and CC in mortality in obese elderly and non-obese older adults. Methods: This is a longitudinal cohort study which data were extracted from the 'Elderly Project/Goiania'. MAMC was calculated using the standard formula: MAMC = mid-arm circumference À (0.314 × triceps skinfold thickness). We divided the MAMC into tertiles, and the lowest tertile was set as the reference group. Cut-off points to define low CC were <34 cm for men and <33 cm for women. We used specific older adults body mass index (BMI) classification which obesity status was BMI >27.0 kg/m 2 . Data mortality was collected from the Brazilian Mortality Information System of the Health Ministry. We used Cox proportional hazards regression to estimate the hazard ratio (HR) of these anthropometric indices and mortality. Results: Of the 416 participants, 66.1% were women, mean age 70.1 ± 7.1 years. During a mean 8.5-year follow-up period, we observed 144 all-causes deaths (34.62% of the total cohort). Mean MAMC was 24.4 ± 3.12, and mean CC was 34.3 ± 3.4. Obesity prevalence was 49.3% (95% CI: 44.45-54.10). Men and women differed in their MAMC (25.8 ± 2.77 vs. 23.6 ± 3.0; P < 0.01, respectively) but not in CC measure (P = 0.299). The prevalence of low MAMC was higher among women than men (43.3%, 95% CI: 37.38-49.160 vs. 14.2%, 95% CI: 8.35-20.01; P < 0.01). Cox proportional regression analyses for all-cause mortality in obese older adults with low MAMC was HR = 1.89, 95% CI: 1.01-3.55; P = 0.016, but not associated in non-obese (P = 0.127). All-cause mortality is associated with low CC in non-obese (HR = 2.22, 95% CI: 1.33-3.69; P = 0.001) but not in obese older adults (P = 0.055). Conclusions: These findings indicate that muscle mass by anthropometric measures has different impact in all-cause mortality according to nutritional status. Low MAMC in obese and low CC in non-obese are strong predictors of all-cause mortality risk among non-institutionalized older adults.

5-07
Comparison between sarcopenia and frailty as predictors of post-operative complications: a cohort study Background: Although sometimes used interchangeably, sarcopenia and frailty represent different concepts. Their correlation and whether one is a better predictor of postoperative adverse outcomes have not been well studied. To evaluate the correlation between psoas muscle area (PMA), used as a surrogate for sarcopenia, and the Clinical Frailty Scale (CFS) in a cohort of non-cardiac surgery patients. Our secondary objective is to evaluate the association between low PMA and the occurrence of post-operative complications. Methods: We conducted a retrospective observational cohort study. Patients 65 years and older undergoing elective non-cardiac surgery in a tertiary academic centre were included. We collected post-operative complications as defined by the National Surgical Quality Improvement Program. Right and left psoas areas were measured on CT scan at the level of the fourth lumbar vertebra body using the CORESLICER software. The total psoas area was calculated by adding left and right psoas areas and normalized for height. Patients were divided into tertiles according to PMA, the lowest tertile representing the low PMA group. Correlation between CFS and PMA was analysed using the Spearman correlation, and we performed a negative binomial regression analysis to assess the association between CFS and PMA and postoperative complications. Results: A total of 78 patients were included. The median value of corrected PMA was 7.15 [interquantile range (IQR): 2.23]. The median age was 72 years old (IQR: 7) and median Charlson Co-morbidity Index was 6 (IQR: 3). The correlation coefficient between PMA and CSF was À0.256 (P = 0.031). In multivariate analysis, low PMA was significantly associated with increasing number of complications [incidence rate ratio (IRR): 3.53, 95% confidence interval (CI): 1.23-10.13, P = 0.019] while CFS was not (IRR: 0.22, 95% CI: 0.02-2.25, P = 0.199). Conclusions: In our cohort, correlation between PMA and CFS was weak, and PMA was a better predictor of postoperative complications than CFS.

5-08
Sarcopenia as a mortality predictor in community-dwelling older adults: a comparison of the diagnostic criteria of the European Working Group on Sarcopenia in Older People Introduction: The definition of sarcopenia remains a matter of discussion, and there is no globally accepted consensus for its diagnosis. The aim of this study was to assess the effect of sarcopenia diagnostic components on mortality, as well as to compare the associations between sarcopenia diagnosed via the 2010 and 2018 Consensuses of the European Working Group on Sarcopenia in Older People (EWGSOP) and mortality. Methods: Prospective cohort study involving noninstitutionalized older adults aged ≥60 years. For the diagnosis of sarcopenia, the definitions proposed by the 2010 (EWGSOP) and 2018 (EWGSOP2) Consensuses were used. The diagnostic components corresponded to muscle mass, muscular strength, and physical performance. The associations of sarcopenia and its components with mortality were investigated using Cox proportional hazard regression models. Results: The sample consisted of 1291 older adults. After an average of 2.6 years of follow-up, 88 (6.8%) participants had died. The diagnosis of severe sarcopenia by both consensuses was associated with an increased risk of mortality. Severe sarcopenia was associated with an increased risk of death compared to that in people without sarcopenia when using EWGSOP [hazard ratio (HR) 3.15, 95% confidence interval (CI): 1.44-6.90] and EWGSOP2 (HR 4.11, 95% CI: 1.88-9.00). Older adults with decreased gait speed had a 76% higher risk of dying (P = 0.033). There was no statistically significant association between the other sarcopenia components and mortality risk. Conclusions: Older adults with severe sarcopenia and those with changes in physical performance had an increased risk of death in the short term. Introduction: SarcDay is a project which intend to increase the awareness of the sarcopenia risk and its consequences in hospitalized elderly patients. In a 1-day audit, all the elderly patients hospitalized in the last 72 h will have the sarcopenia risk assessed using SARC-Calf and their outcome after 30 days. The objective of this study is to verify the association of SARC-Calf and mortality. Methodology: Multicentre observational study, with assessment of elderly patients (≥60 years) hospitalized in the last 72 h. Incapacity of answering the questions or calf circumference (CC) measurement was considered exclusion criteria. SARCF questions and low CC (<33 cm for male and <32 cm for female) were used to create SARC-Calf 1 (score ≥ 11 = sarcopenia risk). Other information, such as diagnostic and previous hospitalizations, were also collected. Outcome (mortality, still hospitalized, or hospital discharging) was verified after 30 days. Results: A total of 162 patients was analysed (72.9 ± 10.2 years; 50.6% female). Among these patients, 18% had a previous hospitalization in the last 30 days, with a median length of stay of 7 days [interquartile range (IQR): 1-60] and 13.6% was bedridden at home. After 30 days, 133 patients were discharged (82%) from the hospital, and 17 patients (10.5%) died. Low CC and sarcopenia risk were found in 44% and 40% of the sample, respectively. Among the patients who died, 77% had SARC-Calf ≥11. The mortality in patients with sarcopenia risk was five times higher than in patients without risk (20.3% vs. 4.1%; RR: 4.98, 95% CI: 1.70-14.59). Conclusions: SarcDay showed that sarcopenia risk is already present in the first 72 h of hospitalization in elderly patients, and this risk is associated with a higher mortality. SARC-Calf is a simple tool to identify the higher risk patients who will need an early intervention during the hospitalization.

5-10
Impacts of cardiovascular disease risk on different status of sarcopenia in the community-dwelling old adults Results: A total of 709 elderly participants included (305 men). In old men with dynapaenia (n = 47) had 17.70 ± 5.08% CHDRS. In old women with sarcopenia (n = 74) had 7.74 ± 6.06% CHDRS. Participants with pre-sarcopenia had the lowest CHRDS in both gender (15.41 ± 5.35% in men and 5.25 ± 3.70% in women). The CHDRS in the participants with dynapaenia was significant higher than sarcopenia in men [odds ratio (OR) = 2.52, 95% confidence interval (CI) = 1.04-6.09]. The CHDRS in the participants with sarcopenia was significant higher than dynapaenia in women (OR = 2.84, 95% CI = 1.11-7.25). Conclusions: Old men with dynapaenia had higher risk of 10year coronary heart disease; nevertheless, this higher risk existed in old women with sarcopenia. Old adults with presarcopenia had the lowest coronary heart disease risk in both gender.

5-11
Sarcopenic obesity and its association with frailty and protein-energy wasting in haemodialysis patients: preliminary data from a single centre in Japan In the multivariate analysis, the sarcopenic obesity group had a significantly higher risk of frailty than the normal group in the multivariate analysis after adjusting for age and gender (OR 4.518, 95% CI 1.218-16.752, P = 0.024). However, sarcopenic obesity was not associated with a higher likelihood of PEW, and sarcopenia imposed a significantly higher risk of PEW (OR 4.272, 95% CI 1.157-15.778, P = 0.029) than that in the normal group after adjusting for confounding factors. Conclusions: Sarcopenic obesity was closely associated with frailty but not with PEW compared with the normal condition in haemodialysis patients.

Differences of clinical and vascular parameters between dippers and non-dippers in patients with maintenance haemodialysis
Background: Cardiovascular disease is the leading cause of death in ESRD patients. The cardiovascular complications of non-dippers are known to be greater than the dippers in the stratification of hypertensive patients. Oxidative stress plays key roles in developing cardiovascular disease and non-dippers related with higher oxidative stress markers and lower antioxidant levels. The aim of this study was to compare clinical parameters and oxidative stress between dippers and non-dippers in patients with maintenance haemodialysis.
Methods: A total of 55 patients who were on maintenance haemodialysis were enrolled, and 49 patients were analysed. All the participants were performed 24 h ambulatory blood pressure (ABP) monitoring to divide the patients into two groups: dippers and non-dippers. Non-dippers were defined by a nocturnal reduction in daytime systolic blood pressure of less than 10%. Bioimpedance analysis (BIA) and oxidative stress marker (myeloperoxidase, MPO) were performed with routine laboratory tests for baseline study. Results: Dippers and non-dippers were 11 and 38 patients, respectively. Age, body mass index, proportion of abnormal results in ABI, IMT, and all the laboratory tests were not different between the two groups. The mean systolic BPs in daytime were not different between dippers and non-dippers (131 vs. 136 mmHg, P = 0.320). However, the nocturnal mean systolic BPs were significantly different between the two groups (112 vs. 137 mmHg, P < 0.001). The fat free mass, skeletal muscle mass, appendicular muscle mass index, body mass index, and body cell mass were not different between dippers and non-dippers. The level of MPO was significantly low in non-dippers than in dippers (2.52 vs. 1.49 ng/mL, P = 0.018).

Conclusions:
This study showed that skeletal muscle mass and appendicular muscle mass index were not associated with the non-dipping BP pattern, and MPO was significantly associated with non-dipping BP pattern in haemodialysis patients.

5-13
Low muscle mass in patients receiving haemodialysis: correlation with noncoronary vascular calcification and incidence of repeat vascular intervention Introduction: According to recent studies, vascular calcification was negatively correlated with sarcopenia. We aimed to investigate the correlation between sarcopenia and quantified vascular calcification score (VCS) of the arm including vascular access and whether low muscle mass (LMM) is associated with incidence of repeat vascular intervention. Methods: Non-contrast arm CT scan including vascular access was taken. Later, VCS was measured by using Agatston method. Skeletal muscle mass was estimated using bioelectrical impedance in supine position. LMM was defined as patients with skeletal muscle mass at both legs normalized to height-squared less than the median. Vascular calcification (VC) group was assigned to patients with a VCS of 500 or higher. Statistical differences between the two groups were determined using the Mann-Whitney U-test for continuous variables and the χ 2 test for categorical variables. Univariate and multivariate logistic regression analyses were used to determine the association between LMM and VC.

Conclusions:
We quantified the VC and found for the first time that it is associated with LMM. LMM may be suggested as a potential predictor of VC. LMM increased a risk of repeat vascular intervention.

5-14
Phase angle (PA) and Barthel score as prognostic tools for sepsis and mortality in critically ill older patients Introduction: Loss of lean body mass and functionality are characteristics of aging and disease severity and maybe associated with prognosis. The objective of this study was to verify PA at 1st day (PA 1 ) correlation with other prognostic scores, including Barthel score, and their association with sepsis and mortality in 28 (D 28 ) and 60 (D 60 ) days after ICU admission in critically older patients. Methods: Patients, older than ≥60 years, under mechanical ventilation ≥48 h, length of stay ≥3 days, haemodynamically stable were studied. PA 1 was measured by a single frequency bioelectrical impedance (BIA) after haemodynamic stability. Severity (APACHE II, SOFA, and SAPS III) and functional scales (Barthel Index) were assessed in the first 24 h. Mortality at 28 (D28M) and 60 days (D60M) were also assessed. Results: Seventy-three older patients were enrolled (mean age 80 ± 8.8 years, 53% female). Sepsis was present in 36 patients (49.3%). The mortality incidence was 37% and 41% at D 28 and D 60 , respectively. There was only a significant correlation between PA 1 and Barthel score (0.43, P < 0.001). PA 1 was significantly lower in septic patients than non-septic (3.36 ± 0.78 vs. 3.74 ± 0.90, respectively) and in patients who died at D 60 (3.26 ± 0.85 vs. 3.73 ± 0.81, respectively), but there is no significant difference in PA 1 between survivors or not at D 28 . From the other prognostic scores, only Barthel scores are significantly different between survivors and nonsurvivors at D 60 [median 100 and interquartile range (IQR): 60-100 and median 60 and IQR: 45-100, respectively). Conclusions: PA 1 showed a significant correlation only with Barthel score. PA 1 could differentiate septic from non-septic patients. From all the prognostic scores, only PA 1 and Barthel score could differentiate survivors from non-survivors at D 60 , but none of them could be used as a prognostic tool to identify mortality at D 28 .

5-15
Low phase angle is associated with cirrhosis and low muscle mass in chronic hepatitis C patients . The diagnosis and staging of liver disease were based on clinical, biochemical, histological, and radiological criteria. The PhA values were classified into percentiles according to the age/sex and the 5th percentile was adopted as cut-off point. Low muscle mass was defined as <15th percentile for mid-upper-arm muscle area (MAMA). Data were analysed in logistic regression models. Results: Low PhA and reduced MAMA were identified in 52 (23.4%) and 55 (24.8%) patients, respectively. The aspartate aminotransferase to platelet ratio index (APRI) in cirrhotic and non-cirrhotic patients was 3.4 ± 2.8 and 0.8 ± 0.7, P ≤ 0.001, respectively. In the multivariate analysis, adjusted for age, body mass index, and gender, low PhA was significantly and independently associated with cirrhosis (OR = 3.74; 95% CI = 1.68-8.31; P = 0.001) and low MAMA (OR = 5.66; 95% CI = 2.56-12.68; P ≤ 0.001).
Conclusions: Low PhA is associated with negative conditions such as cirrhosis and low muscle mass. Reduced PhA is associated with poor clinical and nutritional prognosis in CHC patients.

5-16
Predictive accuracy of muscle wasting is improved by combination of anthropometric indicators with nutritional status score in patients with heart failure Background: We aimed to investigate whether the predictive accuracy of muscle wasting (MW) in CHF is improved by adding an index of nutritional status to anthropometric indicators.

5-17
Impact of the obesity degree in the densitometric diagnosis of sarcopenia Introduction: Skeletal muscle metabolism changes may occur in obesity and may lead to altered body composition with higher fat mass and substantial impairment of muscle mass and quality. The degree of obesity may influence on the prevalence of muscle mass loss. The study purpose was to evaluate the presence of pre-sarcopenia and sarcopenia in individuals with different degrees of obesity and to correlate the diagnosis with anthropometric data and characteristics of obesity.

Methods:
The study included obese patients (BMI ≥ 30 kg/m 2 ), ≥18 years old, excluding those in use of medications or co-morbidities that affect lean mass. Patients answered questionnaires to assess demographic data, physical activity level, and health status. Anthropometric data collection, total body densitometry examination by dual X-ray emission, and handgrip strength were performed. Sarcopenia was diagnosed by dynamometry and FNIH (Foundation for the National Institutes of Health) criteria for densitometry, and classified as normal (two normal criteria), pre-sarcopenia (altered FNIH only), and sarcopenia (altered dynamometry plus FNIH). Patients were classified in degrees of obesity, as obesity I (GI), II (GII), and III (GIII), and compared to a control group (CG).

Results:
The study selected 121 patients, 92.3% women, mean age 46.8 ± 14 years. The mean BMI was 37.1 ± 5.0 kg/m 2 being GI (36.6%), GII (38.3%), and GIII (24%). Presarcopenia was observed in 33 (27.2%) obese patients (35 women) compared to 4 (3.5%) individuals in CG, P < 0.001. There was a positive association between pre-sarcopenia and degree of obesity (P < 0.001). Sarcopenia was present in 6 (4.9%) obese patients and none of CG, and it was correlated with the higher percentage of android fat (P = 0.04), with no correlation between BMI and age. Conclusions: Sarcopenia was prevalent in all degrees of obesity, and there is still no well-established criterion for the diagnosis of obesity sarcopenia in the literature.

5-18
Body composition analysis in obese patients using bioelectrical impedance Twenty-five patients with benign diseases and 17 patients without tumour or inflammatory diseases served as controls. IL-6 was significantly higher in cancer patients versus control patients (P = 0.043). CRC patients revealed higher IL-6 compared to other tumour entities and controls. Furthermore, IL-6 was significantly increased in CRC patients with sarcopenia compared to those without (P = 0.022). There was a significant negative correlation between plasma IL-6 and SMAI in CRC male patients (R = À0.437; P = 0.023). However, no significant differences could be found in IL-6 plasma levels between cachectic and non-cachectic patients.

Conclusions:
In CRC patients, the proinflammatory cytokine IL-6 may be a suitable marker for sarcopenia, but not for cachexia in general. To link cachexia and sarcopenia to the inflammatory state under tumour burden on a molecular level, further investigations are needed.

5-21
Comparison of EWGSOP2 and EWGSOP1 for their impact on the capability of sarcopenia to predict prognosis after radical gastrectomy: analysis from a large-scale prospective study Conclusions: Sarcopenia at uniform diagnosis standard was an independent risk factor for survival in patients undergoing radical gastrectomy for gastric cancer. The EWSGOP2 improved the capability of sarcopenia to predict prognosis.

5-22
The impact of computed tomography-defined sarcopenia on survival in adult patients undergoing radiotherapy ± other treatment modality of curative intent for head and neck cancer: a systematic review and meta-analysis

University of Eastern Finland, 2 Bar-Ilan University, Israel
Introduction: Diseases of muscles, specifically sarcopenia, are of clinical importance as sarcopenia is a common condition of old age. Widening the scope of knowledge in the field of muscle mass/strength genetics is important in the sense that it allows us to identify new genetic markers for musculoskeletal diseases or identify patients with an increased risk to develop a specific condition. It might also allow us to identify drugs that affect muscle in ways unknown before and therefore to reposition drugs to other uses, in accordance to their newly found target. Method: The aim of this project was to show that a mutation in a specific locus is associated with muscle health phenotypes. We identified by statistical, bioinformatic tools loci responsible for regulating relevant genes for muscle health, which can then be a target for downstream lab experimentation validation. SNPs associated with various disease traits for the muscles and specific loci were chosen according to their muscle phenotype association P-value, as traditionally done in the GWASs. We developed and applied a combination of expression quantitative trait loci study (eQTLs) and GWAS summary information, to prioritize causative SNP and point out the unique genes associated in the tissues of interest (muscle).

Results and Conclusions:
We found NUDT3 and KLF5 for lean mass & HLA-DQB1-AS1 for handgrip skeletal muscles traits candidate target genes to target for these phenotypes. SNPs associated with these genes for regulation can then be seen in perspective of TADs and can be targeted for knock out in either C2C12 mouse myoblast cells, adipocytes, or any other relevant cell.

6-02
Spsb1 is involved in inflammation-induced muscle atrophy Introduction: Critically ill intensive care unit (ICU) patients often develop a significant loss of muscle weight leading to muscle weakness during their ICU stay (ICU acquired weakness, ICUAW). Sepsis is one of the major causes of ICUAW. However, exact mechanisms underlying muscle atrophy in ICUAW are not well defined. By RNA sequencing, we observed the up-regulation of Spsb1 in the tibialis anterior muscle of septic mice undergoing cecal ligation and puncture surgery. The SPRY domain-and SOCS box-containing protein 1 (Spsb1) was shown to inhibit TGF-β signalling by targeting TGF-β receptor type-2 (TβRII), but its relevance in muscle atrophy is unknown. Our objectives were to investigate the role of Spsb1 in inflammation-induced muscle atrophy and identify the downstream targets and signalling pathways that contribute to muscle atrophy in cultivated myocytes.

Methods:
The effects of Spsb1 on undifferentiated and differentiated cultured C2C12 skeletal muscle cells were studied by analysing gene expression, protein content, and the atrophy phenotype. Co-immunoprecipitation assays were performed to identify downstream targets of Spsb1. Results: Spsb1 over-expression significantly impaired proliferation, differentiation, and fusion of C2C12 myoblasts, which resulted in the formation of atrophied myotubes. Myogenin, a critical transcription factor regulating myogenic differentiation, was down-regulated in Spsb1 over-expressed cells. The transcripts of Mymk and Mymx, which encode key factors that govern the myoblast fusion, were severely decreased by Spsb1 overexpression. A search for the molecular mechanisms revealed that the expression of TβRII was up-regulated during differentiation and Spsb1 targeted TβRII for degradation. Conclusions: Spsb1 is involved in myoblast fusion and differentiation which contributes to myotube atrophy.
Introduction: Sarcopenia is a common disease in old age and can lead to falls and fragility fractures. It is unclear to what extent the muscle cell metabolism is affected in patients with sarcopenia with proximal femur fractures. Methods: Muscle biopsies of the vastus lateralis muscle were taken from 38 patients (mean age: 81 years, 26 women) who underwent operative treatment by osteosynthesis or endoprosthesis implantation due to a proximal femur fracture. The myoblasts from primary cell cultures of the extracted biopsies were analysed for mitochondrial respiration and glycolysis using a Seahorse XFp Analyzer. The patients were examined by isometric handgrip strength measurement and bioelectrical impedance analysis for determination of the muscle mass index. According to the criteria of EWGSOP 2, a z-score was calculated as a measure of the degree of sarcopenia. Data were analysed, using regression analysis, considered significant with a P-value <0.05. Results: There was a significant association between the degree of sarcopenia and the glycolytic capacity (β = À0.386, P = 0.020) and the glycolytic reserve (β = À0.497, P = 0.002). This relationship persisted after adjusting for the age of the patients. The effect was more pronounced for women than for men. Conclusions: Sarcopenia seems to be associated with a limitation of metabolic-energetic glycolysis. A gender-related effect was shown but requires further investigation due to the low number of men. Confirmation of our results could open a new door for therapeutic interventions of sarcopenia.

6-05
A subset of canonical Wnt and Hippo pathway transcriptional regulators ensure physiological synaptic gene transcription at the neuromuscular junction

Said Hashemolhosseini, Danyil Huraskin & Nane Eiber
Friedrich-Alexander-Universität Erlangen-Nürnberg, Institut für Biochemie, Erlangen, Germany We inquired the neuromuscular role of canonical Wnt/βcatenin and Hippo signalling pathways and their nuclear effectors β-catenin, YAP, TAZ, and members of the TCF, TEAD, and Groucho/TLE families. Denervation caused a muscular abrogation of canonical Wnt/β-catenin and a gain in YAP/TAZ/TEAD signalling activity. Transcriptional profile changes in the myogenic lineage and in response to agrin pointed at potential importance of Tle3, Tle4, Tead1, and Tead4 at the post-synapse. Knockouts of these genes via CRISPR/Cas9 gene editing led to reduced agrin-dependent AChR clustering and diminished synaptic gene transcription in differentiated primary muscle cells. In silico analysis of previously reported TEAD1/4 genomic occupation sites revealed evolutionary conserved areas with potential TEAD binding motifs in important synaptic genes, which relevance was functionally confirmed by luciferase assays. Overall, our data point to a role of TLE3, TLE4, TEAD1, and TEAD4 in acetylcholine receptor clustering and regulating expression of synaptic genes at the neuromuscular junction.

6-06
Muscle wasting in cancer involves suppression of ribosomal production and increased expression of the ribophagy receptor NUFIP1 Introduction: Muscle wasting/cachexia is a major contributor of mortality and morbidity in cancer and is frequently accompanied by functional and metabolic deficits. Muscle mass is determined by the balance between protein synthesis and degradation, yet while previous studies revealed that protein degradation plays an important role in muscle wasting, the contribution of anabolic deficits (i.e. lower ribosomal content) in muscle wasting in cancer remains understudied. Ribosomal content is regulated by transcription of the ribosomal (r)DNA genes and by degradation of ribosomes via ribophagy. We hypothesized that muscle loss in a previously described model of ovarian cancer cachexia is associated with reduced ribosomal mass. Methods: Nod SCID gamma mice were injected with 1 × 10 7 ES-2 human high-grade serous ovarian cancer cells for 14 days. Translational capacity was estimated by quantitating rRNA content, and the mechanisms of ribosomal production and degradation by measuring rDNA gene transcription rates, and expression of the ribophagy receptor Nuclear FMR1 Interacting Protein 1 (NUFIP1), respectively. Results: Gastrocnemius mass was 24% (P < 0.0001) lower than control. Muscle wasting was associated with a~50% (P = 0.0003) reduction in ribosomal capacity and a significant suppression in rDNA gene transcription rates (35%, P = 0.0086). This anabolic deficit also involved a four-fold increased expression (P < 0.0001) of the ribophagy receptor NUFIP1.
Conclusions: In this model of ovarian cancer, a reduction in ribosomal mass may drive muscle wasting and is associated with deficits in ribosomal production. Additionally, the elevation in NUFIP1 expression suggests that during wasting conditions, degradation of muscle ribosomes may also contribute to a lower translational capacity and thereby exacerbate muscle wasting. Overall, these data suggest that in cancer cachexia, the ability of the muscle to synthesize protein is diminished in part due to reduced ribosome production and increased ribosome degradation.

6-07
Skeletal muscle mTORC1 regulates neuromuscular junction stability Background: Skeletal muscle is a plastic tissue which can adapt to different stimuli. It is well established that mammalian target of rapamycin complex 1 (mTORC1) signalling is a key modulator in mediating increases in skeletal muscle mass and function. However, the role of mTORC1 signalling in adult skeletal muscle homeostasis is still not well defined. Methods: Inducible, muscle-specific Raptor and mTOR k.o. mice were generated. Muscles at 1 and 7 months after deletion were analysed to assess muscle histology and muscle force.

Results:
We found no change in muscle size or contractile properties 1 month after deletion. Prolonging deletion of raptor to 7 months, however, leads to a very marked phenotype characterized by weakness, muscle regeneration, mitochondrial dysfunction, and autophagy impairment. Unexpectedly, reduced mTOR signalling in muscle fibres is accompanied by the appearance of markers of fibre denervation, like the increased expression of the neural cell adhesion molecule (NCAM). Both muscle-specific deletion of mTOR or raptor, or the use of rapamycin, was sufficient to induce 3-8% of NCAM-positive fibres (P < 0.01), muscle fibrillation, and neuromuscular junction (NMJ) fragmentation in 24% of examined fibres (P < 0.001). Mechanistically, reactivation of autophagy with the small peptide Tat-beclin is sufficient to prevent mitochondrial dysfunction and the appearance of NCAM-positive fibres in raptor k.o. muscles. Conclusions: Our study shows that mTOR signalling in skeletal muscle fibres is critical for maintaining proper fibre innervation, preserving the NMJ structure in both the muscle fiber and the motor neuron. In addition, considering the beneficial effects of exercise in most pathologies affecting the NMJ, our findings suggest that part of these beneficial effects of exercise are through the well-established activation of mTORC1 in skeletal muscle during and after exercise.
Background: Transforming growth factor β (TGF-β) ligands, TGF-βs, myostatin, GDF11, and activins are major negative regulators of skeletal muscle mass. Dysregulation of TGF-β proteins and their associated signalling components is increasingly being implicated in muscle wasting associated with chronic diseases such as myopathies, cancer, heart failure, and aging. Myofibre atrophy results from a combination of increased protein degradation, predominantly involving the ubiquitin-proteasome pathway, and decreased protein synthesis. However, underlying molecular mechanisms involved in TGFβ-mediated myofibre atrophy are not fully elucidated as well as impact of chronic TGF-β dysregulation on muscle physiology (i.e. muscle energy metabolism, calcium homeostasis, regenerative capacities, and neurotransmission). Studies conducted so far mostly relied on loss-of-function approaches and ligand gain-of-function, resulting in complex intricated effects not restricted to muscle cells. Methods: A conditional mice model was generated to specifically activate TGF-β signalling in adult myofibres through inducible expression of a constitutively active TGF-βI receptor, TGFβ-RI-CA (RCA). Consequences of dysregulated TGF-β signalling on muscle pathophysiology were investigated in RCA muscles to explore the mechanisms involved. Transcriptomic analysis was performed to identify new molecular signatures of TGF-β-mediated muscle wasting. Results: Conditional activation of TGF-β signalling in adult myofibres rapidly resulted in significant muscle atrophy, reduced muscle force, skeletal muscle metabolic modifications, and disturbed calcium homeostasis. A progressive fibre type switch toward more oxidative fibres was observed in both glycolytic and oxidative muscles. Mechanistically, constitutively active TGF-βI receptor promoted activation of Smad2/3 signalling in myofibres leading to target genes expression and an imbalance between muscle protein synthesis and protein degradation. Crosstalk on AKT/mTOR signalling pathway was observed. Identified molecular signatures were investigated.
Conclusions: This study validated a new unbiased musclespecific mice model to investigate impact of chronic activation of TGF-β signalling on skeletal muscle physiology and uncover new potential therapeutic targets for muscle-wasting disorders.

6-09
Relationship between the severity spectrum of sarcopenia and osteoporosis in elderly men and women in Singapore Introduction: Sarcopenia may increase the risk of osteoporosis in older individuals, but evidence is equivocal in Asia. We examined the association between the severity spectrum of sarcopenia and osteoporosis in elderly Singaporeans.

Methods: We included individuals from The PopulatION
HEalth ProfilE in EldeRly Singaporeans study (PIONEER), a nationally representative, population-based study of Singaporean Chinese, Malays, and Indians aged ≥60 years. Participants underwent body composition and bone density (dual energy X-ray absorptiometry), grip strength (hand dynamometer), and habitual 4 m-walking speed assessments. Presarcopenia was defined as low appendicular lean mass (LALM; men <7 kg/m 2 , women <5.4 kg/m 2 ); sarcopenia as LALM and low muscle strength (LMS; men <26 kg, women <18 kg) or slow walking speed (SWS; ≤0.8 m/s); and severe sarcopenia as LALM, LMS, and SWS. Osteoporosis was defined as a low bone mineral density (BMD; T-score, ≤ À 2.5) at either the hip, femoral neck, or lumbar spine sites. Modified Poisson regression models were used to determine the cross-sectional sarcopenia-osteoporosis relationship. Conclusions: Sarcopenia is highly prevalent in Singapore, and its severity spectrum is independently associated with increased risk of osteoporosis. Strategies to prevent and delay the progression of sarcopenia are warranted.

Introduction:
The aim of this study was to investigate changes in knee-extension strength and physical function in older adults during and after acute hospital admission. Methods: A repeated measures cohort study, using a convenience sample of participants were recruited during the first 24 h of their admission. Measurements of knee-extension strength and functional mobility were taken at recruitment, on day 7 of admission (or at discharge if earlier) and again at follow-up 4-6 weeks post-discharge. Self-reported general functional ability was scored for 2 weeks before admission and at follow-up. During the first 7 days of admission, daily measurements of muscle strength were taken. Results: We recruited 70 participants, of which 65 (28 women) had at least one repeated measure in hospital. Median age was 84 years. Whilst in hospital, participants were active for less than 4% of the day. Knee-extension strength reduced during hospitalization by approximately 11% (P < 0.001), but there was no change post-hospitalization (P = 0.458). There was a reduction in general functional ability between 2 weeks before admission and follow-up (P = <0.001). Functional mobility improved during hospitalization (P < 0.001), but there was no change posthospitalization (P = 0.508). A repeated-measures mixed model including 292 in-hospital observations from 62 participants showed that greater loss in knee-extension strength during hospitalization was associated with increased sedentary time on days 2 to 7 of the study, and with increased frailty, higher baseline strength, and lower baseline inflammatory levels.
Conclusions: Knee-extension strength deteriorated during hospitalization and showed no recovery at follow-up. Although functional mobility improved during hospitalization, it did not continue to improve between discharge and follow-up, despite general functional ability not having recovered to pre-hospital levels. Greater loss of knee-extension strength was associated with increased sedentary time, frailty, lower admission inflammatory levels, and higher baseline strength.
Background: The aim of this study was to determine factors that underlie association of chronic heart failure (CHF) with osteoporosis. Background: Therapeutic glucocorticoids (GCs) are used to treat chronic inflammatory disease. Despite their antiinflammatory efficacy, chronic exposure to GCs elicits undesirable side effects, including muscle atrophy. 11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) activates GCs within muscle, is induced by inflammation, and has previously shown to drive GC-induced muscle wasting. We examined the role of 11β-HSD1 in mediating muscle wasting in chronic inflammatory disease when treated with therapeutic GCs. Methods: Muscle biopsies were taken from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Cortisol production in the muscle was measured using thin-layer chromatography (TLC), and catabolic and inflammatory gene expression were assessed. Global 11β-HSD1 knock out (KO) animals were crossed onto the TNF-tg murine model of polyarthritis and received vehicle or corticosterone (100 μg/mL) over 3 weeks in drinking water. Muscles were histologically assessed, and anabolic, catabolic and inflammatory gene, and protein expression were examined by RT-qPCR and western blot. WT and 11β-HSD1/KO murine muscle cultures were exposed to TNF-α, dehydrocorticosterone, or both. Catabolic and inflammatory gene expression was measured. Results: Cortisol activation in muscle was increased in RA patients than OA patients, and correlated with serum CRP levels. Local inflammation (IL-6 mRNA) was increased in RA compared to OA, correlated with 11β-HSD1 and was accompanied by elevated Mstn and FoxO1 expression. The myopathy previously described in TNF-tg and TNF-tg 11β-HSD1KO mice (1,2), was aggravated by therapeutic GCs in TNF-tg mice based on reduced muscle weights and fibre size, while their TNF-tg 11β-HSD1KO counterparts were protected from muscle wasting. This was accompanied by an attenuated GC-induced elevation of FoxO1 and Trim63 mRNA abundance, and alterations in FoxO1 and ribosomal S6 phosphorylation. These data were in part recapitulated in primary muscle cultures. Together, these data suggest that 11β-HSD1 inhibition may protect against therapeutic GC-induced muscle wasting in chronic inflammatory disease.

6-14
Association between vitamin D level and muscle strength in patients undergoing haemodialysis Introduction: Considering conflicting results or heterogeneity in study design, further investigations are needed to identify the definite association between vitamin D level and muscle health. Our study aimed to address these issues and to evaluate the association between vitamin D level and muscle mass indices, strength, or physical performance through comprehensive measurements in patients undergoing haemodialysis.
Methods: This study was performed in a tertiary medical centre. We included patients undergoing haemodialysis with age ≥ 20 years. A total of 84 patients were enrolled. The patients were divided into tertiles based on the 25-hydroxy (25-OH) vitamin D level as follows: lowest tertile (Lowest T, n = 28), middle tertile (Middle T, n = 28), and highest tertile (Highest T, n = 28). We evaluated the association between the tertiles and clinical outcomes including nutritional status, muscle mass, muscle function, handgrip strength (HGS), physical performance, and health-related quality of life scales (HRQoL).
Results: There were no significant differences in the muscle mass indices and nutritional markers according to tertiles of 25-OH vitamin D level. However, 25-OH vitamin D level as a continuous variable or the tertile of 25-OH vitamin D level as a categorical variable was positively associated with HGS. Logistic and linear regression analyses showed a consistent superiority of the Highest T in HGS compared with the Lowest or Middle T. Although the statistical significance was weak, the scores of various physical performance tests and the HRQoL scales were the highest in the Highest T, among the three tertiles.

Conclusions:
The present study demonstrated that serum vitamin D level is associated with HGS in patients undergoing haemodialysis regardless of muscle mass indices or nutritional status.

6-15
Clinical significance of volume status in body composition and physical activity measurements in haemodialysis patients Methods: A total of 84 patients were enrolled. The participants were divided into tertiles on the basis of oedema index: low, middle, and high tertiles. Serum albumin and hs-CRP levels were measured. Thigh muscle area index (TMA/Ht 2 , cm 2 /m 2 ) was measured using computed tomography. Extracellular and total body water, and phase angle were obtained from the bioimpedance analysis. The oedema index was defined as the ratio of extracellular water to total body water. Subjective global assessment (SGA), handgrip strength (HGS), and gait speed (GS), Short Physical Performance Battery (SPPB), sit-to-stand for 30 s (STS30), timed up and go (TUG), sit-to-stand test performed 5 times (STS5), and 6-min walk (6-MW) tests were also evaluated.

Results:
The levels of oedema index in low, middle, or high tertile was 0.338 ± 0.010, 0.356 ± 0.003, and 0.370 ± 0.007, respectively. On univariate analysis, SGA score and phase angle in high tertile were lowest among the three groups. On multivariate analysis, TMA/Ht 2 and phase angle in high tertile were lowest among the three groups. Inverse correlations were observed between oedema index and TMA/Ht 2 , SGA score, phase angle, HGS, GS, SPPB, STS30, or 6 MW. Positive correlations were observed between oedema index and STS5 or TUG. AUROC, sensitivity, and specificity for predicting low GS were 0.641%, 34.5%, and 89.7%, respectively (P = 0.025). Those for predicting low SPPB were 0.791%, 68.0%, and 79.7%, respectively (P < 0.001). Results from correlation and subgroup analyses showed similar trends.

Conclusions:
The present study demonstrates that high volume status is associated with decrease in muscle mass and physical performance regardless of inflammatory or nutritional status.

6-16
Association of growth differentiation factor 15, a novel cachexia marker, with body anthropometry in a prospective haemodialysis cohort There was also a trend between higher GDF15 levels and lower MAC, waist circumference, and biceps skinfold, although associations did not achieve statistical significance. Conclusions: In haemodialysis patients, higher GDF15 levels were associated with lower levels of skeletal muscle, fat, and total body mass. Further studies are needed to determine whether reduction of GDF15 levels improves body composition and ameliorates protein-energy wasting in this population.

6-17
Thyroid status and body composition in a prospective haemodialysis cohort Introduction: Thyroid dysfunction is a highly prevalent metabolic disorder in haemodialysis (HD) patients. In the general population, hypothyroidism is associated with increased body weight due to reductions in energy expenditure. However, little is known about the impact of thyroid status on body composition in end-stage renal disease patients. We thus sought to examine the relationship between serum thyrotropin (TSH) levels, the most sensitive and specific single biochemical metric of thyroid function, with body composition measured by dual-energy X-ray absorptiometry (DXA) scan in HD patients. Methods: Among 103 patients from the Anti-Inflammatory and Anti-Oxidative Nutrition in Hypoalbuminemic Dialysis Patients (AIONID) trial, we examined cross-sectional associations of TSH [categorized as the highest quartile (Q4) versus lowest three quartiles (Q1-3)] with DXA-ascertained body composition parameters (i.e. fat, bone, and lean mass). Using logistic regression models, we estimated associations between TSH with higher levels of fat, muscle, and bone [defined as the highest three quartiles (Q2-4) versus lowest quartile (Q1)] in analyses adjusted for case-mix and expanded case-mix covariates. Results: In case-mix analyses, the highest quartile of TSH levels was associated with higher levels of whole-body total fat mass, subtotal fat mass, whole-body total fat mass index, and whole-body total mineral content (ref: 3), and 5.78 (1.29-25.9), respectively. While there was a trend toward higher TSH levels and higher levels of lean mass parameters, associations did not achieve statistical significance. A similar pattern of findings was observed in expanded case-mix analyses. Conclusions: In HD patients, higher TSH levels were associated with increased fat mass and bone mineral content. Further studies are needed to determine whether thyroidrelated alterations in body composition impact the health and survival of HD patients and whether reduction of TSH with treatment influences muscle and bone parameters in this population.
Introduction: Clear cell renal cell cancer (ccRCC) patients who have low skeletal muscle index (SMI) at the time of nephrectomy experience worse cancer-specific and overall survival than patients with high SMI, but potential mechanisms underlying this association are unknown. ccRCC is a heterogeneous disease that can be classified into two molecular subtypes (ccA and ccB) based on gene expression patterns. ccB tumour subtype is considered more aggressive and has a significantly worse prognosis than ccA subtype. We examined how SMI at the time of nephrectomy was associated with molecular tumour subtype while considering age, sex, and stage at diagnosis. Methods: The study cohort consisted of 76 incident ccRCC patients treated by nephrectomy whose tumours were transcriptomically profiled by the Cancer Genome Atlas. The ClearCode34 gene expression classifier categorized patients into either ccA or ccB molecular subtypes. Computerized tomography scans without contrast performed within 60 days of surgery were reviewed using Slice-O-Matic software to determine SMI (skeletal muscle cross-sectional area divided by height in meters squared). SMI was classified into high versus low using the gender-specific cut points of <55 cm 2 /m 2 for men and <39 cm 2 /m 2 for women. Odds ratios and 95% confidence intervals (OR 95% CI) describe associations between SMI and molecular subtype in logistic regression models that adjust for age, sex, BMI, and stage. A subgroup analysis restricted to patients with early stage disease (n = 47) was performed to address the possibility of muscle wasting due to advanced stage disease. Statistical significance was regarded as P-value < 0.05. Results: Patients were predominantly male (77%) and had early stage disease (62%). Median age was 59 years (IQR: 51-68). Overall, 40% of patients were classified as having low SMI and 42% of tumours were ccB subtype. Patients with low SMI were significantly more likely to have the aggressive ccB tumour subtype (66%) than patients with high SMI (26%); adjusted OR 9.1 (95% CI: 2.1-40.1). This pattern persisted even when analyses were restricted to patients with early stage disease (P-value = 0.01). Conclusions: While preliminary, our findings suggest that patients with low SMI harbour more aggressive ccB tumours and lend biologic support to the observation that low SMI is associated with poor prognosis. Given the cross-sectional nature of this analysis, it is not clear whether low SMI is a cause or consequence of tumour aggressiveness, or whether addressing low SMI could improve clinical outcomes among ccRCC patients.

6-19
Systemic low-grade inflammation and its association with muscle mass throughout the age-span-Copenhagen Sarcopenia Study Introduction: Chronic low-grade inflammation has been associated with sarcopenia [1,2]. However, results regarding the role of systemic low-grade inflammation on muscle mass are inconsistent, and lack of unequivocal evidence as to whether systemic levels of inflammatory markers could represent biomarkers of sarcopenia. Thus, the aim of this study was to investigate the association between circulating inflammatory biomarkers and appendicular lean mass (ALM/h 2 ) throughout the age-span. Methods: 1177 healthy men and women (range: 22-93 years) were included in the present study. ALM/h 2 (kg/m 2 ) was assessed by DEXA (iDXA, GE Lunar). Blood samples were analysed for IL-1β, IL-4, IL-6, IL-13, IFN-γ, and TNF-α using multiplex bead-based immunoassays (Bio-Rad) and for hsCRP using latex particle-enhanced immunoturbidimetric assays following the manufacturer's instructions (Roche Diagnostics). Biomarkers were log-transformed, and analyses were stratified by gender and adjusted for age, visceral fat, and assay plate ID.

Results:
There was an inverse association between hsCRP and ALM/h 2 in young men (22-39 years), but no other associations were found in subjects under 80 years. In subjects over 80 years, IL-13, IL-4, IFN-γ, and TNF-α were inversely associated with ALM/h 2 in men. Women over 60 years with high (>0.71 pg/mL) or intermediate (0.28-0.71 pg/mL) levels of IL-13 had more than two-fold greater odds of having lower ALM/h 2 compared to women with low levels (<0.28 pg/mL).

Conclusions:
The present study demonstrated an association of low-grade inflammation with aging in apparently healthy men and women. Except for an inverse association between hsCRP and ALM/h 2 in young men, there was no association between ALM/h 2 and inflammatory biomarkers in healthy subjects under the age of 80 years. Of note, an inverse association of both proinflammatory and anti-inflammatory markers with ALM/h 2 was observed in men over 80 years. In contrast, increased levels of IL-13 were associated with having lower ALM/h 2 in women over 60 years.
supplementation of VitD and leucine-enriched whey protein affected CLIP in subjects enrolled in the PROVIDE-study, as a secondary analysis. Methods: Sarcopenic adults (low skeletal muscle mass) aged ≥65 years with mobility limitations (Short Physical Performance Battery 4-9) and a body mass index of 20-30 kg/m 2 were randomly allocated to two daily servings of active (n = 137, including 20 g of whey protein, 3 g of leucine and 800 IU VitD) or isocaloric control product (n = 151) for a doubleblind period of 13 weeks. At baseline and after 13 weeks, circulating interleukin (IL)-8, IL-1 receptor antagonist (RA), soluble tumour-necrosis-factor receptor (sTNFR)1, IL-6, highsensitivity C-reactive protein, pre-albumin, and 25hydroxyvitamin (OH)D were measured. Data analysis included repeated measures analysis of covariance (corrected for dietary VitD-intake) and linear regression. Results: IL-6 and IL-1RA serum levels showed overall increases after 13 weeks (P = 0.006 and P < 0.001, respectively). For IL-6 a significant time*treatment interaction (P = 0.046) was observed, with no significant change over time in the active group (P = 0.155) compared to control (significant increase P = 0.012). IL-8 showed an overall significant decrease (P = 0.03). The change in pre-albumin was a significant predictor for changes in IL-6 after 13 weeks. Conclusions: We conclude that 13 weeks of nutritional supplementation with VitD and leucine-enriched whey protein may attenuate the progression of CLIP in older sarcopenic persons with mobility limitations.

7-02
Intramuscular fat is predictive of hypoglycaemia incidence: results from the MENU study Background: To analyse body composition among noncritically ill patients with high risk of malnutrition, with and without hypoglycaemia. Methods: Included were patients enrolled in the MENU study that underwent CT scanning during their hospitalization. The NRS2002 was used for nutritional screening. Body composition was analysed at the level of L3 using Slice-O-matic software (TomoVision, Montreal, Canada). Patients were categorized as hypoglycaemic if they had at least one documented hypoglycaemic event during the hospitalization period (glucose ≤70 mg/dL). Regression analysis was used to examine the association of body composition with incident hypoglycaemia. Results: Included were 155 patients (mean age 69.7 ± 15.7, 51.6% were men, 52.9% had diabetes mellitus). Rate of positive NRS2002 was 57.8%, and 26 patients (16.7%) had at least one documented hypoglycaemic event. Patients at risk of malnutrition had lower muscle mass (44.9 ± 12.5 vs. 49.3 ± 13.6 cm 2 /m 2 , P = 0.045) and lower subcutaneous fat (59.4 ± 40.7 vs. 86.8 ± 49.7 cm 2 /m 2 , P < 0.001) and visceral fat (64.3 ± 40.2 vs. 93.4 ± 58.1 cm 2 /m 2 , P = 0.001). Regression analysis showed that the NRS2002 (OR 4.986, 95% CI 1.052-23.632, P = 0.043) and insulin treatment (OR 7.769, 95% CI 1.529-39.461, P = 0.013) were predictive of hypoglycaemia in this patient population. Furthermore, intramuscular fat was also indicative of hypoglycaemia incidence (OR 1.091, 95% CI 1.002-1.187, P = 0.044). Muscle mass, subcutaneous, and visceral fat mass, as well as sex, albumin, and diabetes mellitus status did not affect incident hypoglycaemia. Conclusions: Our data suggest that intramuscular fat is predictive of hypoglycaemia incidence among patients admitted to internal medicine units, irrespective of malnutrition risk.
Introduction: Evidences showed that exposure to diesel exhaust particles is associated with cardiopulmonary, vascular, and oncologic diseases. Recent data from large cohorts of subjects have highlighted associations between exposure to ambient air particulate matter (PM) 2.5 pollution and increased cardiovascular morbidity and mortality, as well as increased risk for obesity and diabetes. In this light, we aimed to identify association(s) between nutritional, metabolic, and hormonal derangements and exposure to environmental pollution in an Italian population of traffic policemen professionally exposed to high levels of air PM. Methods: We performed a cross-sectional study considering adult male municipal policemen of the city of Naples, Italy, professionally exposed to airborne nanoparticles in an urban area at high traffic density (Exposed group) compared to nonexposed municipal policemen (at least 1 year indoor working) (Non-exposed group) matched by age and body mass index (BMI). Clinical and laboratory assessments were performed, including serum levels of leptin, adiponectin, and ghrelin by ELISA. Results: A total of 199 participants were consecutively enrolled, 100 adult males in the Exposed-group and 99 adult males in the Non-exposed group. No differences were observed between the two groups in terms of body weight, BMI, and lipid profile, whereas plasma glucose levels and HOMA-IR were higher in the Non-exposed group compared to the Exposed one (P = 0.009 and P = 0.03, respectively). Metabolic syndrome was documented in 32% of the Exposed group and in the 52.5% of the Non-exposed group (P = 0.008); no differences were seen between the two groups in terms of adiponectin, ghrelin, and leptin serum levels. In the Exposed group, we found a negative correlation between BMI and serum adiponectin levels (P = 0.04) and a positive correlation between BMI and serum leptin concentrations (P < 0.0001), whereas in the Non-exposed group, we found only a positive correlation between BMI and serum leptin (P < 0.0001). Subjects in the Exposed-group with metabolic syndrome showed lower serum adiponectin levels and higher serum leptin levels with respect to those without metabolic syndrome (P < 0.0001 and P = 0.002, respectively), while in subjects with metabolic syndrome within Non-exposed group, we found higher serum leptin levels when compared to those without metabolic syndrome (P = 0.58 and P = 0.01; respectively). Stratifying participants for the presence of metabolic syndrome, when comparing the two groups, we found lower serum adiponectin levels in the Exposed group with respect to the Non-exposed (P = 0.007). Finally, when comparing the two groups, after stratifying participants for HOMA-IR >2.5, we found lower adiponectin serum levels in the Exposed group with respect to the Non-exposed (P = 0.038). Conclusions: Our results seem to suggest that exposure to air PM pollution is associated with the presence of markers of insulin resistance and metabolic syndrome, likely due to low adiponectin circulating levels, determining impaired protective function against systemic inflammation.

Nguyen & Connie M. Rhee
University of California Irvine, Orange, CA, USA Introduction: Growth differentiation factor 15 (GDF15) is a protein in the transforming growth factor-β family that acts directly upon the hypothalamus to reduce food intake and energy expenditure. In animal models and clinical studies of cancer-associated cachexia, higher GDF15 levels have been associated with satiety, weight loss, and death. While weight loss and protein-energy wasting (PEW) are also known to be strong predictors of mortality in haemodialysis patients, little is known about the relationship between GDF15 levels and nutritional status in this population.
Methods: Among the 205 haemodialysis patients from the multicentre prospective 'Malnutrition, Diet, and Racial Disparities in Chronic Kidney Disease' (MADRAD) study, we examined cross-sectional associations of serum GDF15 levels measured over October 2011 to April 2012 with results from their Malnutrition Inflammation Score (MIS) instruments, a validated and quantitative nutritional assessment tool used to estimate PEW in kidney disease patients (i.e. higher levels indicate worse levels of PEW). Associations of GDF15 levels categorized as tertiles with higher MIS levels (defined as >median of observed values; reference: ≤median) were estimated using case-mix adjusted logistic regression models. In secondary analyses, we examined GDF15 defined as (i) >median versus ≤median and (ii) categorized as quartiles. Results: In case-mix analyses, patients with incrementally higher GDF15 levels had greater likelihood of higher MIS levels (ref: lowest GDF15 tertile): ORs (95% CIs): 2.70 (1.21-6.03) and 2.87 (1.22-6.74) for the first and second GDF15 tertiles, respectively. In secondary analyses, higher GDF15 levels defined as >median and the highest GDF15 quartile were also associated with higher MIS levels: 2.27 (1.14-4.55) and 3.89 (1.40-10.8), respectively. Conclusions: In a prospective haemodialysis cohort, higher GDF15 levels were associated with poorer nutritional status defined by MIS level. Further studies are needed to determine whether GDF15 may be a novel target for PEW in the haemodialysis population.

Introduction:
In the general population, epidemiologic data suggest that higher dietary fibre intake is associated with lower mortality, presumably due to favourable effects on nutritional and metabolic health, including decreased glucose absorption, lowering of cholesterol, gut microbiome-induced production of immunomodulatory and anti-inflammatory short fatty chain acids, reductions in blood pressure, and trapping of carcinogens. Haemodialysis patients are subject to a number of dietary restrictions (i.e. dietary potassium restriction), which may subsequently limit their fibre intake. We thus sought to examine the relationship between dietary fibre intake and mortality in a prospective cohort of haemodialysis patients. Methods: Among the 415 haemodialysis patients from the multicentre prospective 'Malnutrition, Diet, and Racial Disparities in Chronic Kidney Disease' study, information regarding dietary fibre intake was obtained using the Block Food Frequency Questionnaire administered over October 2011-March 2015, with follow-up through 2019. Associations of baseline dietary fibre intake categorized as tertiles with allcause death risk were examined using unadjusted, case-mix, expanded case-mix, expanded case-mix + laboratory, and expanded case-mix + laboratory + nutrition adjusted Cox models. Results: The mean ± SD age of the cohort was 56 ± 15 years, among whom 45% were female, and 36% and 48% were African-American and Hispanic, respectively. Across all levels of adjustment, patients with the lowest tertile of dietary fibre intake had higher mortality risk ( Introduction: Frailty is defined as a syndrome of physiological decline in late life. In patients with cirrhosis the dysregulation of catabolic and anabolic muscular pathways cause, an increased risk to anticipate sarcopenia and frailty. We aimed to evaluate 3 g/die β-hydroxy-β-methylbutyrate (HMB) supplementation for 12 weeks (T1), on muscular mass and performance in patients with cirrhosis Methods: For each patient, at the beginning of the study (T0), clinical history and blood parameters were collected; cognitive tests (MMSE, PHES, and ANT) and physical test (6MWT, FCS, and HGT) anthropometric measurements using 'quadriceps femoris ultrasound' and bioimpedentiometry and Liver Frailty Index (LFI) were also performed. Patients were randomized into a treatment group (3 g per die HMB supplementation for 12 weeks) and a control group. (3 g per die sorbitol supplementation for 12 weeks). Both groups received nutritional counselling and indications on physical activity to carry out during treatment period. At the end of the 12 weeks (T1), all the data collected at T0 were repeated and compared in each patient. Results: At present, 22 cirrhotic patients were enrolled, and preliminary results are shown. Patients receiving HMB showed a statistically significant improvement in muscular performance at FCS ranging from 14.5 s (±5.4 DS) to 11.6 s (±2.7 DS), at 6MWT rising from 346.8 m (±66 DS) to 416 m (±57 DS) and LFI, decreasing from 4.0 (±0.4 DS) to 3.7 (±0.4 DS) with P value = 0.008. No significant variation, between T0 and T1, were reported in patients assigned to the control group. Conclusions: The preliminary results of this controlled randomized study suggests efficacy of HMB supplementation in enhancing muscular performance and, consequently, reducing frailty in cirrhotic patients.

7-07
Nutritional status affects the risk of contrast induced nephropathy after percutaneous coronary intervention Introduction: Contrast induced nephropathy (CIN) is the major common cause of hospital acquired acute kidney injury and associated with longer hospital stay, increased morbidity, and mortality. Various risk factors for CIN after coronary artery angiography are known; however, the risk of malnutrition is not validated well. The aim of this study is to evaluate the effect of nutrition for CIN after coronary intervention. Method: A total of 1913 subjects who got percutaneous coronary intervention (PCI) in Pusan National University hospital from January 2014 to November 2018 were included in this study. The subjects who already got haemodialysis, had eGFR under 15 mL/min/1.73 m 2 , or had no baseline creatinine were excluded. CIN is defined as an elevation of serum creatinine (Scr) of more than 50% or ≥0.3 mg/dL from baseline within 48 h. Patients with a serum albumin less than 3.5 g/dL and/or a TLC less than 1500 cells per mm 3 were classified as having protein energy malnutrition (PEM). Results: 95 (4.9%) developed CIN after PCI. The subjects with CIN were older, had higher proportion of female, diabetes, and hypertension. The level of haemoglobin, total lymphocyte count, albumin, estimated glomerular filtration rate was lower in CIN group, whereas the level of creatinine and C-reactive protein was higher in CIN group. In multivariate logistic regression analysis for CIN, body mass index, heart rate, albumin, estimated glomerular filtration rate, proteinuria, and contrast volume were significant related. When we combine serum albumin level and TLC for PEM, PEM was still significant risk factors in multivariate logistic regression analysis (HR 3.947, 95% CI 1.522, 10.235, P = 0.005). Conclusions: PEM raised the risk of CIN after coronary intervention. Clinicians should be aware of nutritional status and increased risk of CIN.

7-08
Assessment of sarcopenia and malnutrition in patients with head and neck cancer undergoing treatment of curative intent-implications for practice Teresa Brown 1,2 , Merrilyn Banks 2 , Louise Campbell 2 , Brett Hughes 1,2 , Elizabeth Ahern 2 , Charles Lin 1,2 , Liz Kenny 1,2 & Judy Bauer 1 1 The University of Queensland, Australia, 2 Royal Brisbane and Women's Hospital, Brisbane, Australia Background: Malnutrition and sarcopenia are poor prognostic factors for survival in patients with head and neck cancer (HNC). Assessments of malnutrition and sarcopenia are important factors to consider in the management of these patients. To determine the prevalence of CT-defined sarcopenia and malnutrition in patients with HNC undergoing radiotherapy ± other treatment modality of curative intent. Methods: Nutritional status and body composition of patients with HNC undergoing radiotherapy of curative intent ± other treatment modality participating in a RCT were included. Outcomes were measured at baseline and 3 months post-treatment. Malnutrition was assessed using the Patient Introduction: Age-related abdominal fat mass accumulation and muscle weakness have been associated with several adverse health effects, including non-alcoholic steatohepatitis (NASH). Lifestyle recommendations remain the primary treatment for NASH patients, although the specific effects of exercise and dietary changes on NASH and underlying molecular pathways involved remain elusive. We therefore investigated the effects of exercise, dietary change, and the combination thereof on already manifest NASH in mice with sarcopenic obesity. Methods: Ldlr À/À .Leiden mice received a high fat diet for 30 weeks to induce NASH. After 30 week mice were left untreated (control group) or received a running wheel, were switched to an isocaloric healthy chow diet, or the combination thereof, for another 20 weeks. Effects on body composition, grip strength, plasma and liver biochemical variables, and liver histology were assessed. Results: Both exercise and dietary change significantly reduced body weight and fat mass, improved grip strength, and the combination treatment had additive effects. While exercise significantly reduced micro-vesicular steatosis, dietary change significantly reduced macro-vesicular steatosis. The combination treatment revealed additive effects for both macro-vesicular and micro-vesicular steatosis. Hepatic inflammation was almost fully reduced by each monotreatment. Hepatic fibrosis was significantly reduced by dietary switch, tended to be reduced by exercise, and combined treatment had no additive resolving effect. Conclusions: Both exercise and dietary lifestyle interventions showed beneficial effects in mice with clear additive effects of the combination treatment for body weight, fat mass, grip strength and steatosis, and non-additive effects on hepatic inflammation and fibrosis. This suggests that most effects of exercise and dietary switch are mechanistically complementary and improve sarcopenic obesity, while anti-inflammatory and anti-fibrotic effect may involve similar pathways that do not add up.

8-02
Exercise pre-conditioning diminishes solute carrier protein expression and doxorubicin accumulation in the diaphragm Introduction: Doxorubicin (DOX) is an anthracycline antibiotic used in cancer treatment. Unfortunately, the clinical use of this highly efficacious anticancer drug is limited due to the development of respiratory and diaphragm muscle dysfunction in patients. DOX-induced ventilatory impairment is a debilitating condition that promotes the onset of dyspnoea, fatigue,and exercise intolerance. While the mechanisms responsible for DOX-induced respiratory insufficiency are unclear, previous work demonstrates that the incidence of ventilatory dysfunction greatly correlates to the concentration of DOX taken up by the diaphragm. In this regard, we recently demonstrated that endurance exercise performed prior to DOX administration is sufficient to reduce the accumulation of DOX within the diaphragm and prevent ventilatory dysfunction. While the mechanisms for the exercise-induced reduction in diaphragm DOX levels are unknown, we hypothesize that endurance exercise alters the expression of solute carrier proteins (SLCs) required for the influx of DOX into the diaphragm. Methods: To determine if exercise training can alter SLC protein expression in the diaphragm, animals underwent 2 Introduction: Exercise and enhancing nutrient intakes are expected for improvement in muscle wasting, and insulin-like growth factor-1 (IGF-1) is known to be involved in muscular hypertrophy. We investigated the effects of 5-day of endurance exercise programme on protein metabolism including changes in plasma IGF-1 levels under dietary conditions of positive energy balance. Methods: Seven healthy male volunteers (age, 21 ± 1 years; body mass index, 21.7 ± 2.2 kg/m 2 ; means ± SD) participated in a 5-day body weight gain programme preceded by a 5-day period which was set for stabilization of nutritional status. During the stabilization period, subjects were fed a diet based on estimated energy requirement, recommended dietary allowance, or adequate intake published in the Dietary Reference Intakes for Japanese. During body weight gain programme, energy intake was increased by 30% of the level during the stabilization period, and all foods were increased in proportion to the energy intake. In addition, the subjects exercised on a bicycle ergometer to consume an additional energy correspond to 20% of the level during the stabilization period. Thus, energy balance was positively set by 10% of the energy intake level during the stabilization period. Results: Body weight significantly (P < 0.05) increased during body weight gain programme (before vs. after: 63.46 ± 7.85 vs. 63.78 ± 7.75 kg). Basal metabolic expenditure also significantly increased after the programme. Serum urea nitrogen (UN) levels tended to increase (P = 0.063), and the ratio of urinary UN excretion levels to energy intake levels was significantly lower in weight gain programme than in stabilization period. On the other hand, plasma IGF-1 levels were significantly decreased, and testosterone levels were unchanged.

Conclusions:
At the beginning of the exercise period, a decrease in fasting plasma IGF-1 levels early in the morning is induced even under the dietary conditions of positive energy balance.

8-05
Determinants of worsening in the chair-stand-test in older men-the prospective STRAMBO study Introduction: The five-time chair-stand-test (CST) is a measure of lower body strength in the elderly. Our aim was to assess the risk factors of incident worsening in CST in older men. Methods: Eight hundred and one men aged 60-87 and able to perform CST were followed up prospectively for 8 years. During this period, 641 men had at least one follow-up test. The worsening in CST was defined by an increase in the time necessary to perform CST by >1 s/year or an incident incapacity to perform CST. Results: The worsening was found in 147 men and increased with age (OR = 1.39 per 5 years, 95% CI: 1.18-1.63, P < 0.001). Compared with men having BMI 21-25 kg/m 2 , worsening was more frequent in 9 men with BMI < 21 kg/m 2 (OR = 6.22, 95% CI: 1.36-28.47, P < 0.05) and in 43 men with BMI > 33 kg/m 2 (OR = 2.59, 95% CI: 1.06-6.30, P < 0.05). Longer time to perform a 10-step tandem walk (>16 s, upper quartile) was associated with higher risk of worsening (OR = 2.29 vs. three lower quartiles combined, 95% CI: 1.48-3.54, P < 0.001). The risk of worsening was higher in men selfreporting fragility fracture of the lower limbs (OR = 3.77, 95% CI: 1.37-10.33, P = 0.01) and of upper limbs (OR = 3.33, 95% CI: 1.36-8.17, P < 0.01). It increased with the number of limb fractures (trend P < 0.001) being the highest in men with ≥2 fractures (OR = 16.08 vs. no fracture, 95% CI: 2.42-180.94, P < 0.05). The risk of worsening was higher in men with parathyroid hormone (PTH) level > 72 pg/mL (>mean + 3SD in young men) versus men with PTH < 36 pg/mL (<mean in young men): OR = 2.33, 95% CI: 1.13-4.79, P < 0.05. The worsening in CST was not related to the lifestyle, co-morbidities, or testosterone deficit. Conclusions: In older men, worsening in CST is predicted by higher age, extreme BMI, poor dynamic balance, prior fragility fracture of the limbs, and high PTH concentration.

8-06
Three months of strength training changes the gene expression of inflammation-related genes in PBMC of older women Introduction: Exercise can counter chronic low-grade inflammatory profile (CLIP). The involvement of peripheral blood mononuclear cells (PBMC) remains unclear. We investigate changes of inflammation-related gene-expression in PBMC by strength training at different modalities. Methods: Fourteen women aged ≥65 years were randomized into 3 months of either 3×/week intensive strength training (IST: 3 × 10 reps at 80% 1RM), strength endurance training (SET: 2 × 30 reps at 40% 1RM), or control (CON: 3 × 30 s stretching). RNA was extracted from isolated PBMC from blood samples taken before and after 3 months training. Targeted RNA sequencing including 407 inflammation-related genes was performed, and differentially expressed genes were identified. Fold changes ≤0.5 or ≥1.5 were considered as significant. Pathway analysis was performed using IPA, zscores ≥2 or ≤ À2 were considered as significantly enriched. Results: Eighty-five genes, mostly pro-inflammatory (n = 56), showed significant exercise-induced changes in expression. IST and SET altered only 9 genes in similar direction (e.g. MXRA5 FC IST = 23.54 and FC SET = 6.78) whereas 26 genes were altered in opposite direction (e.g. IL1A FC IST = 0.15 and FC SET = 2.27). Compared to CON, IST induced changes in expression of 5 genes in the same direction, and for 15 genes in the SET group (e.g. ILTRAPL2 FC IST = 20.52, FC SET = 6.94 and FC CON = 5.54). Likewise, 13 and 7 genes were oppositely expressed for respectively IST and SET compared to CON (e.g. MXRA5 FC CON = 0.29). For IST and SET, proinflammatory pathways were inhibited such as dendritic cell maturation pathway (IST z-score = À2.94) and Sirtuin pathway (SET z-score = À2.52). None of the enriched pathways overlapped between IST and SET. LXR/RXR and TREM1 pathways were enriched oppositely in both groups (respectively IST z-score = 2.04 and z-score = À2.04, SET z-score = À2.04 and z-score = 2.41). Conclusions: Three months strength training at high and at moderate external load can both induce changes in CLIPrelated gene expression in PBMC, but by affecting different genes and related pathways. and treatment of heart failure. Each patient underwent a dual energy X-ray absorptiometry (DEXA) scan to measure appendicular skeletal mass index (ASMI) as an indication of MM and a five-times sit-to-stand test (FTSS) as an index of MF. In the FTSS, the time a patient took to consecutively stand up 5 times from a seated position on a chair was recorded. Low ASMI and low MF were defined as follows: low ASMI, <7.0 kg/m 2 in men and < 5.4 kg/m 2 in women; low MF, ≥11 s in men and ≥ 12 s in women. Functional dependence (FD) was defined as a Barthel Index score of 85 points or lower. Results: Fifty-four patients (26%) were diagnosed as having functional dependence. ASMI was significantly lower (5.25 vs. 5.90 kg/m 2 , P < 0.01), and FTSS time was significantly longer (15.0 vs. 10.6 s, P < 0.01) in patients with (FD) than in those without FD. In multivariate logistic regression analysis with adjustments for potential confounders, low MM, and low MF were independent predictors of FD. An increase in adjusted odds ratio for predicting the presence of FD was observed across subgroups in the following order: patients with low MM (5.42), those with low MF (9.48), and those with low MM and low MF (18.58). There was no significant association between ASMI and FTSS time (r = À0.11, P = 0.11). Conclusions: Reduction in MM and decline in MF independently and incrementally predict the presence of FD in elderly heart failure patients.

8-10
Anaerobic threshold-based exercise training programme stimulates genes that control skeletal muscle differentiation and function in heart failure patients Renata  Background: Heart failure is associated with skeletal muscle wasting and limited ability to exercise leading to worsening of heart failure-induced metabolic disorders. The aim of this study was to evaluate the signalling response to personalized exercise therapy in heart failure patients in order to determine the molecular mechanisms behind therapeutic effects and to provide the background for the search of new therapeutic targets for treatment of muscle wasting. Methods: 234 patients (176 male, 61 female) with HF NYHA III class were enrolled. The individual training programme was determined at 90% of anaerobic threshold; all patients underwent cardiopulmonary exercise testing (VO2 peak), echocardiography (LVEF), quality of life, and exercise tolerance assessment before and after 24 weeks of training. The muscle biopsies were taken from subgroup of eight patients before and after of 12 weeks of training: histology analysis was used to evaluate changes in muscle morphology; RNA-Seq analysis was employed to uncover gene expression changes that may contribute to beneficial effects of exercise training programmes in HF patients. Results: 95% of patients responded positively to physical rehabilitation programme and 30% of those showed the improvement of all indicators used in the study: LVEF, VO2 peak, exercise tolerance, and quality of life. RNA-Seq analysis revealed training-induced coordinated up-regulation of expression of signalling pathways and genes that involved in control of skeletal muscle differentiation and function including: significant up-regulation of RYR1, a calcium release channel; FLNC, the muscle-specific filamin critical for maintaining the structural integrity of the muscle fibers; CAVIN4, that facilitate myofibrillar organization; GSN, calcium-regulated, actin-modulating protein that promote the assembly of monomers into filaments; NACA, involved in regulation of post-natal skeletal muscle growth and regeneration, as well as PLEC, involved in regulation of filaments network; CYR61 that promotes angiogenesis and regeneration. Conclusions: Our data reveal the specific genes that may be considered as the potential contributors to the therapeutic response to personalized exercise therapy in heart failure patients, as well as prognostic markers and therapeutic targets.