J Cachexia Sarcopenia Muscle (2012) 3:37–43 Table 1 Effects of sActRIIB treatment on body weights and food intake in mice bearing the Lewis lung carcinoma Initial body weight (g) Final body weight (g) Body weight increase (%) Carcass Total food intake C 19.4±0.3 19.8±0.3 77±2 13.5±0.2 T 19.3±0.2 17.7±0.2 −8.20% 57±0.7 11.8 ±0.2 T+A 19.2±0.4 21.0±0.6 9.30% 70±2.1 13.0±0.4 ANOVA A ns 0.0032 0.0000 0.0008 39 B ns 0.0000 0.0000 0.0053 Results are mean±S.E.M. Food intake is expressed in g/100 g of initial body weight and refers to the ingestion during the period of the experiment prior to sacrifice which took place 14 days after tumor inoculation. Final body weight excludes the tumor weight. Carcass (body without organs or tumor) is expressed as g/100 g of initial body weight (IBW). Statistical significance of the results by two-way analysis of variance (ANOVA) C control mice (n=6), T tumor-bearing mice (n=8), T+A tumor-bearing mice treated with sActRIIB (n=8), ns nonsignificant differences, A (tumor effect), B (treatment effect) CCA TCA GGA GAA GTG GAT CTA TGT T 3′; REVERSE 5′ GCT TCC CCC AAA GTG CAG TA 3′); m-calpain (FORWARD 5′ TTG AGC TGC AGA CCA TC 3′; REVERSE 5′ GCA GCT TGA AAC CTG CTT CT 3′), cathepsin B (FORWARD 5′ CTG CTG AGG ACC TGC TTA C 3′; REVERSE 5′ CAC AGG GAG GGA TGG TGT A3′) and p0 (FORWARD 5′ GAG GTC CTC CTT GGT GAA CA 3′; REVERSE 5′ CCT CAT TGT GGG AGC AGA CA 3′). To avoid the detection of possible contamination by genomic DNA, primers were designed in different exons. The real-time polymerase chain reaction (PCR) was performed using a commercial kit (LightCycler TM FastStart DNA MasterPLUS SYBR Green I, Roche, Barcelona, Spain). The relative amount of all mRNA was calculated using comparative CT method. Acidic ribosomal phosphoprotein P0 mRNA was used as the invariant control for all studies. 2.4 Total physical activity Total physical activity (IR ACTIMETER System and ACTITRAK software from Panlab, Barcelona) was determined during the last 24 h before the sacrifice of the animals in a two subgroups of four TB mice (non-treated) and four TB mice (treated with the sActRIIB) using activity sensors that translate individual changes in the infrared pattern caused by movements of the animals into arbitrary activity counts. For the measurements, animals remained in their home cage. A frame containing an infrared beam system was placed on the outside of the cage; this minimized stress to the animals. 2.5 Grip force assessment Skeletal muscular strength in mice was quantified by the grip strength test [30, 31]. The grip strength device (Panlab- Harvard Apparatus, Spain) comprised a triangular pull bar connected to an isometric force transducer (dynamometer). Basically, the grip strength meter was positioned horizon- tally and the mice are held by the tail and lowered towards the device. The animals are allowed to grasp the triangular pull bar and were then pulled backwards in the horizontal plane. The force applied to the bar just before it lost grip was recorded as the peak tension. At least three measure- ments were taken per mouse on both baseline and test days, and the results were averaged for analysis. This force was measured in grams/gram initial body weight. 2.6 Statistical analysis Statistical analysis of the data was performed by means of one-way and two-way analysis of variance. 3 Results and discussion The mouse Lewis lung carcinoma is a suitable model system to study the mechanisms involved in the establishment of Table 2 Effects of sActRIIB treatment on muscle weights in mice bearing the Lewis lung carcinoma C T T+A ANOVA Gastrocnemius Tibialis EDL Soleus 581±11 174±5 84±3 54±5 446±9 130±4 25±3 23±2 587±15 177±7 31±3 28±2 Results are mean±S.E.M. Muscle weights are expressed as mg/100 g of initial body weight. Statistical significance of the results by two- way analysis of variance (ANOVA) EDL extensor digitorum longus, C control mice (n=6), T tumor-bearing mice (n=8), T+A tumor-bearing mice treated with sActRIIB (n=8), ns nonsignificant differences, A (tumor effect), B (treatment effect) A 0.0000 0.0000 0.0000 0.0000 B 0.0000 0.0000 ns ns