P070 The influence of Crohn’s disease-derived blood plasma upon anabolic signaling responses to leucine stimulation in skeletal muscle cells in vitro

Abstract

Abstract Background Many Crohn’s Disease (CD) patients have low muscle mass and function (sarcopenia), yet the precise drivers of this pathophysiology are unclear. Positive muscle protein balance is driven by essential amino acids (EAA) that stimulate muscle protein synthesis (MPS), with the EAA leucine (L) playing a key role. However, if circulating factors in CD impact muscle anabolic responses to feeding remain unclear. Our aims were to use plasma from healthy human volunteers (HV) and active CD patients to investigate the impact on anabolic signaling and responses to feeding. Methods HV were 32 ± 4 y with a BMI 24 ± 1 and CD 26 ± 3 y with a BMI 23 ± 1 disease duration 5 ± 1 y faecal calprotectin 720 ± 12 ug/g. C2C12 myotubes were treated with 4 conditions of media conditioned with 5% plasma from HV or CD, in addition to being treated ± 5mM L for 4 hrs. Protein content was quantified via nanodrop and muscle anabolic signaling measured via immunoblotting for p-mTORC1 (Ser2448), p-4E-BP1 (Thr37/46) and p-P70S6K1 (Thr389). Results Immunoblotting revealed no difference in mTORC1 phosphorylation between HV and CD (HV:18.9 ± 2.7 AU, CD: 19.9 ± 3.2 AU), with no change in response to L treatment (HV:18.0 ± 2.4 AU, CD: 16.6 ± 2.3 AU). There was no difference in P70S6K1 phosphorylation between HV and CD (HV: 33.7 ± 3.2 AU, CD: 28.0 ± 4.7 AU) with both groups showing significant increases in phosphorylation with L treatment (HV: 56.4 ± 4.2 AU (P<0.05), CD: 54.5 ± 9.2 AU (P<0.001)). There was no difference in 4EBP1 phosphorylation between HV and CD (HV: 49.8 ± 6.4 AU, CD: 64.6 ± 7.5 AU) with only HV showing a significant increase in phosphorylation with L (HV: 105.5 ± 8.7 AU (P<0.0001), CD: 47.8 ± 5.3 AU). There was difference in total protein concentration between HV and CD, or with L treatment. Conclusion We found CD derived blood plasma did not impact the leucine stimulated increase in P70S6K1 phosphorylation but did result in blunted phosphorylation of 4EBP1. These disparities in anabolic signaling indicate that systemic factors in CD may impact muscle anabolic responses to feeding. Yet, the relationship between anabolic markers and rates of MPS remains complex.