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Background Constitutional thinness (CT) is a natural state of underweight (13–17.5 kg/m2) without the presence of any eating disorders and abnormal hormonal profile, and with preserved menses in women. We previously conducted a four-week fat overfeeding study showing weight gain resistance in CT women and one of our main results was the identification of an energy gap: a positive energy balance (higher energy intake than energy expenditure). Objective This new overfeeding study is designed to confirm the energy gap and propose mechanistic hypothesis. Design A 2-week overfeeding (daily consumption of one bottle of Renutryl® Booster (600 kcal, 30 g protein, 72 g carbohydrate, 21 g fat) on top of the dietary intake) is performed to compare 15 women and men in each CT group (Body Mass Index [BMI] Results and conclusions Data inter-linking will be able to be established with results of this study. The findings could possibly open to therapeutic approaches to help CT patients to gain weight as well as provide a better understanding of energy regulation with regard to treat obesity (resistance to weight loss), a mirror image of CT (resistance to weight gain).

Objectives: Salivary α- amylase (AMY1) is responsible for the breakdown of starch into oligosaccharides, tri and di-saccharides giving a start to the starch digestion in the oral cavity on food consumption. Several studies recently reported contradicting results regarding the link between AMY1 copy numbers (CNs) and obesity and type 2 diabetes. Objective: We investigated whether CN in the AMY1 gene was associated with differential anthropometrics and glycaemic outcomes in obese individuals who underwent a dietary plan varying in macronutrient intake, as a part of weight loss and weight maintenance program. We also investigated whether there existed an interaction between nutrient intakes and AMY1 CNs and if AMY1 CNs have influence on body weight, body composition and glycemic trajectories during dietary interventions.Methods: Using the Paralogue Ratio Test, we accurately measured the AMY1 CNs in 761 obese individuals from the Diogenes study. Subjects underwent first an 8-week low-caloric diet (LCD, at 800 kcal/d) and those achieving >8% weight loss were then randomized to a 6-month weight maintenance dietary (WMD) intervention. The association between AMY1 CNs and weight- and glycemic- parameters was tested at baseline and following each intervention phase (LCD, WMD) with the use of linear mixed effect models adjusting for gender, age, center and total energy intake.Results: At baseline, a modest association between AMY1 CN and BMI (P = 0.04) was observed. AMY1 CN was not associated with baseline glycemic variables. Additionally, AMY1 CN was not associated with anthropometric or glycemic-outcomes following either LCD or WMD. Interaction analyses between AMY1 CN and nutrient intake did not reveal significant association with any clinical parameters (at baseline and following LCD or WMD) or when testing gene x WMD interactions during the WMD phase.Conclusions: In the absence of association with weight trajectories or glycemic improvements, the AMY1 CN cannot be considered as an important biomarker for response to a clinical weight loss and weight maintenance programs in overweight/obese subjects.Funding Sources: European Commission, Food Quality and Safety Priority of the Sixth Framework Program (FP6-2005-513946), Nestlé Institute of Health Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction et but de l’etude Les microARNs (miRs) sont des petits ARNs endogenes qui regulent negativement l’expression des genes de facon post-transcriptionnelle. Ils auraient un role important dans la regulation de certaines voies metaboliques dans le tissu adipeux (TA). Les miRs sont particulierement stables et representent de potentiels biomarqueurs d’interet. Notamment, dans le domaine de la prise en charge de l’obesite, ils pourraient etre des marqueurs de la reponse a une intervention dietetique de restriction calorique. Le but de ce travail est d’analyser les changements du miRnome du TA de patients obeses ou en surpoids soumis a une restriction calorique, en comparant les niveaux d’expressions en basal a ceux a la fin de l’intervention. Materiel et methodes Un essai clinique randomise consistant en 8 semaines de restriction calorique a recrute 548 sujets, qui ont beneficie de multiples investigations cliniques : enquetes dietetiques, parametres anthropometriques et sanguins. Des biopsies de TA sous-cutane a l’aiguille ont ete realisees en basal et a 8 semaines. Le profil d’expression des miRs a ete determine par des puces a microARNS et valide par RTqPCR. Des modeles mixtes ont ete construits, avec la difference d’expression des miRs comme variable dependante, et ajustes sur l’âge, le sexe, le pourcentage de perte de poids et la difference d’apport energetique, le centre etant considere comme un effet aleatoire. Les calculs ont ete faits grâce au logiciel R (version 3.2.2), implemente des packages lme4 (version 1.1-11) et boot (version 1.3-17). Resultats et analyse statistique Cent cinquante huit sujets ayant a la fois des donnees disponibles d’expression des miRs et des recueils dietetiques ont ete inclus. Neuf miRs etaient differentiellement regules (p > 0,05). Tous sauf un avaient un b negatif, ce qui signifie que plus la restriction calorique est forte, plus l’expression du miR diminue. Les miRs les plus significativement regules ont ete valides par RTqPCR sur un groupe de validation de 142 sujets, selon les memes methodes. Un seul miR s’est avere proche de la significativite, miR-22-3p, (p = 0,086). Un b negatif (−0,03) indique que la diminution du niveau d’expression du miR-22-3p dans le TA est associe a une restriction calorique plus forte. Conclusion Une restriction calorique forte chez des individus obeses est associee negativement au niveau d’expression du miR-22-3p dans le TA sous-cutane. Des etudes futures determineront si ce miR est detectable dans le plasma sanguin, potentiellement produit par le TA, constituant ainsi un biomarqueur de la restriction calorique.

s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S77 most strongly associated with activated macrophages in the capsule of the tibiofemoral (TF) compartments (p1⁄40.0063) and the whole joint synovium (p1⁄40.0017). sCD163 was also associated with severity of radiographic OA, specifically joint space narrowing (p1⁄40.0554), but not osteophyte severity. The use of the markers in combination improved the prediction of activated macrophages in the capsule of the PF knee compartment (p1⁄40.013) and joint space narrowing (p1⁄40.0099) independent of osteophyte severity. Conclusions: These findings suggest that cell-surface markers shed from activated macrophages may be used as biomarkers to identify a subtype of OA patients with an active inflammatory disease state. The combined analysis of both CD14 and CD163 indicate synergism between the markers for identifying the presence of activated macrophages in the capsule of the PF knee and joint space narrowing. Furthermore, these results contribute to a growing literature identifying macrophages and macrophage-activation pathways as potential targets for new OA therapies. Acknowledgements: Samples were obtained with support of a grant from Lilly USA, Inc; support for the assays was provided by laboratory research funds of the PI (VBK), and support for personnel was provide by NIH 5T32AI007217-30 (HND) and P01 AR050245. 127 CHONDROCYTE HYPERTROPHY, MEASURED BY THE SECRETION OF COLLAGEN TYPE X, IS A HALLMARK OF PATHOLOGICAL CHANGES IN OSTEOARTHRITIS Y. He y, N. Urik Brndt-Hansena y, J. Wang z, D. Su z, Q. Zheng z, O. Simonsen x, K. Petersen k, M. Karsdal y, A.-C. Bay-Jensena y. yNordic BioSci., Herlev, Denmark; zNordic BioSci. China, Beijing, China; xDept. Orthopedic Surg, Aalborg Univ. Hosp., Aalborg, Denmark; kCtr. for Sensory-Motor Interaction, Dept. of Hlth.Sci. and Technology, Aalborg Univ., Aalborg, Denmark Purpose: Collagen type X (Col X) is a short chain and network-forming collagen, which is specifically synthesized by hypertrophic chondrocyte in the growth plate during endochondral ossification, the calcified cartilage in normal adult or the deep zone of osteoarthritic (OA) cartilage; thus Col Xmay play an important role in cartilage calcification. The aim of the study was to measure serum levels Col X in OA patients as well characterize the presence of the ColX in OA cartilage section. Methods:Amonoclonal antibody (mAb)was raised against the 10aa at Cterminus of NC1 domain of Col X. ThismAbwas characterized bywestern blot using U2-OS osteosarcoma cell as positive control, and by immunohistochemistry on 5 human OA knee cartilages sections. A solid-phase competitive ELISAwas developed applying the mAb and tested in a small cohort including: 47 OA patients undergoing total knee replacement surgery (mean age SD: 64 15), and 13 controls with knee problems having arthroscopy (mean age SD: 34 21), all women. Mann-Whitney test was applied to compare the serum Col X levels in TKR and control group. Pearson correlationwas done on log transformed data. Results: The mAb, NB509-11G8, was specific for the immunogen; it did not recognize truncated or nonsense peptides when tested in the ELISA. The mAb detected the monomeric Col X (59KDa) and dimeric Col X (120KDa) in the U2-OS cell lysate. Moreover, these bands were blocked by the specific peptide. NB509-11G8 stained the extracellular matrix of chondrocytes in the deep zone and calcified cartilage of human OA samples but not the middle or upper zone (fig. A). The competitive ELISA had a measuring range of 0.02-5 ng/ml, with intraand interassay CVs of 4.19% and 10.62%. The mean serum Col X level was significantly higher in the TKR than the control group (P1⁄40.017, fig. B). Serum Col X level did not correlate with age (R21⁄40.055), but weak Ă relationwith BMI (R21⁄40.17). The AUC for serum Col X for discriminating TKR from controls was 0.72 (P1⁄40.017). Conclusions: Secretion of Col X as a marker of cartilage hypertrophy may serve as an early biomarker for OA burden of disease. 128 CTX-II IS A MARKER OF CARTILAGE DEGRADATION BUT NOT OF BONE TURNOVER J. Catterall y, S. Dewitt Parr z, K. Fagerlund x, B. Caterson z. yDuke Univ., Durham, NC, USA; zCardiff Univ., Cardiff, United Kingdom; x Pharmatest Services Ltd, Turku, Finland Purpose: The CTx-II biomarker has been widely investigated and used to determine the status of cartilage turnover in osteoarthritis and rheumatoid arthritis. Recent publications have discussed the possibility that CTx-II release may partially reflect bone turnover rather than being a specific marker of cartilage turnover. As CTx-II has been used so widely, it is clear that the specificity of CTx-II for cartilage needed to be re-evaluated. To this end, we decided to investigate the specificity of CTx-II for cartilage in comparison to the gold standard CTx-I bone degradation marker. We investigated the specificity of the ELISAs for their respective epitopes, release of CTx-I and CTx-II from bone and cartilage explant cultures, and levels of CTx-II and CTx-I in a small acute injury and osteoarthritis (OA) cohort. Methods: ELISA specificity for CTx-I and CTx-II was determined by depletion of the epitopes from urine and by affinity purification of the epitopes. Release of CTx-I and CTx-II from bone and cartilage was investigated by digesting both cartilage and bone with either cathepsin K or matrix metalloproteinases, and release of CTx-I and CTx-II determined by ELISA. We also investigated the release of CTx-I and CTx-II in explant culture from both bone by osteoclasts mediated bone degradation and from pro-inflammatory cytokine-stimulated cartilage explant cultures. Finally, we investigated the changes in CTx-I and CTxII in patient cohorts. Results: In vitro, cathepsin K digestion of bone released CTx-I alone while digestion of bone with activated MMPs was unable to release either CTx-I or CTx-II from bone. Only digestion of cartilage by activated MMPs released CTx-II, while digestion of cartilage by either MMPs or cathepsin K was unable to release detectable CTx-I. Osteoclast digestion of bone slices released only CTx-I and no detectable CTx-II, while the reverse was true in chondrocyte-mediated cartilage degradation, with high CTx-II release and no detectable CTx-I. In our OA patient cohorts we observed an increase in CTx-II levels when compared to healthy controls but no change in CTx-I, while acute injury patients demonstrated a reduction in CTx-I levels after injury and no change in CTx-II. Conclusions: Our data strongly suggest that CTx-I reflects bone degradation and CTx-II reflects cartilage degradation, and that in acute injury and in osteoarthritis, bone turnover and cartilage degradation/ repair processes differ. As CTx-II reflects cartilage degradation, it is a valid biomarker of cartilage breakdown and not a biomarker of bone turnover, although in some cases it may reflect the connected pathology of bone and cartilage in joint diseases. 129 CTX-II LEVELS ARE ASSOCIATED WITH PERIPHERAL BONE DENSITY, SUGGESTING CTX-II EPITOPE RELEASE FROM BONE: DATA FROM COHORT HIP AND COHORT KNEE (CHECK) W.E. van Spil, K.L. Vincken, W.F. Lems, F.P. Lafeber. Univ. Med. Ctr. Utrecht, Utrecht, The Netherlands Purpose: Recent data have suggested that CTX-II (C-terminal telopeptide of collagen type II) may not only originate from articular cartilage but also from bone changes. Therefore, the purpose of this study was to look for cross-sectional associations between urinary CTX-II levels and bone density distant from the subchondral bone area. Methods: This study was performed in 366 subjects with early-stage symptomatic knee and/or hip osteoarthritis from Cohort Hip and Cohort Knee (CHECK). In urine (u-) and serum (s-) samples from these subjects, uCTX-II, the cartilage markers sCOMP, sPIIANP, and sCS846 (cartilage oligomeric matrix protein, N-terminal propeptide of procollagen type IIA, and chondroitin sulphate 846), and the bone markers uNTX-I, uCTX-I, sPINP, and sOC (N and C-terminal telopeptides of collagen type I, N-terminal propeptide of procollagen type I, and osteocalcin) were assessed by enzyme-linked immunosorbent assay or radioactive immunoassay. Bone density was determined at the tibia and