Micro CT scans for zebrafish on an obesogenic diet (DIO), on caloric restriction (CR) or undergoing catch up growth after 1, 3 or 9 months of CR (CG1, CG3, CG9)

For µCT imaging, adult zebrafish were fixed and decalcified in Bouin's solution at room temperature for 7 days, stored in PBS and imaged using a micro-computed tomography (µCT) device (SkyScan1272, Bruker BioSpin GmbH, Ettlingen, Germany). Zebrafish were placed individually in 1.5ml Eppendorf tubes using and an ultra-focus scan over the whole body was performed in a full-rotation in step-and-shoot mode. 322 projections (1008x672 pixels, 4x4 binning) were acquired per subscan with an x-ray tube voltage of 60 kV, power 0.166 mA, aluminum filter 0.25 mm,exposure time of 363 ms, 6 averages and a object-source distance of 86 mm. All CT images were reconstructed at an isotropic voxel size of 18 µm using a Feldkamp type algorithm (filtered back-projection). Fat-containing regions were appear hypo intense in µCT data and were segmented using Imalytics Preclinical (Gremse-IT GmbH, Aachen, Germany (Gremse et al., 2016; doi:10.7150/thno.13624). The volumetric fat percentage was calculated as the ratio of subcutaneous adipose tissue (SAT) or visceral adipose tissue (VAT) fat volume compared to the entire volume of the body cavity anterior of the anal fin and expressed per skeletal segment.