Experimental design:
Twenty healthy, young (20–45 years old) males undertook 60 days of complete bed rest (24h-a-day) preceded by a 14-day ambulatory control period. The study was conducted in accordance with ESA Standardisation of Bed Rest Study Conditions guidelines (Heer et al. 2009) at the Médecine et de Physiologie Spatiales (MEDES) clinical institute in Toulouse, France. The study was sponsored by the ESA and French National Space Agency (CNES) and was conducted in two campaigns (n = 10 per campaign) from January–April 2017 and September–December 2017. The protocol was reviewed and approved by the local ethics committee (CPP Sud-Ouest et Outre-Mer I, France, RCB: 2016-A00401-50) and was registered on ClinicalTrials.gov (NCT03594799). All procedures conformed to the Declaration of Helsinki. Participants were recruited by online advertisements and press announcements. 

Within each campaign, half of participants (n = 5 in each campaign) consumed an antioxidant/ anti-inflammatory nutritional cocktail during the bed rest period (Cocktail Group), and the other half were a Control Group. Group allocation was blinded from all researchers until the completion of each bed rest campaign. Six pills were consumed each day, two per meal. No placebo was administered to the Control group due to the inability to mask the fish oil odour of ω-3 supplementation. Our primary focus was on the impact of bed rest per se on adipose tissue and there was no evidence of a supplement effect (see results). 

Body composition:
Body composition, Fat Mass Index (FMI) and central fat mass (fat mass between L1 and L4 vertebrae)  was determined using dual-energy X-ray absorptiometry (DEXA; Discovery, Hologic; Bedford, UK) two days prior to the start of bed rest and following 58 days of bed rest. 

Physical activity baseline standardisation:
During the 14-day pre-bed rest period in the clinical facility, participants undertook approximately 8,000 steps/ d measured with a wrist-mounted pedometer (Polar Loop; Polar; France). Participants also undertook bouts of supervised structured exercise during this standardisation period on a treadmill and cycling ergometer,.

Dietary control:
Diet was strictly controlled throughout the study period, participants were confined to the MEDES facility according to guidelines detailed in Heer, Liphardt, Frings-Meuthen (2009). Caloric intake was based on basal metabolic rate (BMR) measured by indirect calorimetry (22). During the pre-bed rest period 140 % of BMR was consumed, which was reduced to 110 % BMR during the bed rest period in order to keep fat mass stable. An additional 1000 IU of 25 (OH) vitamin D was supplemented daily by oral administration as specified by Heer, Liphardt, Frings-Meuthen (2009). 

Blood sampling:
Fasted venous blood samples were collected from an antecubital vein at 07:00 six days prior to the start of bed rest and following 56 days of bed rest. Plasma samples were immediately centrifuged and stored at −80oC. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient separation (Ficoll®, Greiner Bio-One; Stonehouse, UK) in Leucosep® tubes (Greiner Bio One Inc.; Kremsmünster, Austria) for analysis on the day of collection.

Adipose tissue sampling:
Pre- and post-bed rest subcutaneous adipose tissue samples were obtained from ~5 cm lateral to the umbilicus with a 14G needle using the needle aspiration method under local anaesthesia (1 % Lidocaine hydrochloride containing 0.005 mg/ mL adrenaline; Xylocaine; Dublin, Ireland). Adipose tissue was taken following blood draws 6 days prior to the start of bed rest and following 56 days of bed rest. Adipose samples were cleared of visible connective tissue, blood, and vasculature prior to washing the remaining tissue with 0.9 % NaCl solution (B.Braun; Sheffield, UK) over a single-use sterile 100 µm gauze to minimise blood contamination. Samples were placed into unsupplemented endothelial cell basal medium (PromoCell; Heidelberg, Germany) at room temperature for transfer to another laboratory for processing. Snap frozen tissues were stored at −80 oC until analysis.

Ex vivo adipose tissue measures:
Between 20 and 25 mg of adipose tissue was cultured ex vivo at a final concentration of 50 mg of tissue per millilitre for 3 hours .

Adipose tissue digestion:
Between 200 and 500 mg of adipose tissue was digested using collagenase as previously described . Isolated adipocytes were recovered by flotation and the SVF cells were recovered following centrifugation at 300 x g for 5 minutes.

Adipose tissue RNA isolation:
Total RNA (including microRNAs) was extracted using miRNeasy Mini Kit (Qiagen; Crawley, UK) according to manufacturer instructions. Following RNA isolation, samples were DNase-treated and purified as previously described . Thirty microlitres of RNA at a set concentration of 2.1 µg/ 30 µL were used for transcriptomic analysis.

Quantitative polymerase chain reaction (qPCR):
Quantitative polymerase chain reaction analysis was performed on DNase-treated RNA from adipose tissue on a StepOne™ analyser (Applied Biosystems; Warrington, UK) using pre-designed TaqMan Assays from Applied Biosystems (Invitrogen; California, US) for the measurement of PDK4 (hs00176875_m1), SREBP1c (hs01088691_m1), AKT2 (hs01086099_m1), INSR (hs00961557_m1), GLUT4 (hs00168966_m1), IRS2 (hs00275843_s1), AMPK1/2 (hs01562315_m1 and hs00178903_m1), AS160 (hs00952765_m1), FAS (hs00188012_m1), HK2 (hs00606086_m1), IRS1 (hs00178563_m1), and PPARG (hs01115513_m1). Data were normalised to an internal calibrator (peptidylprolyl isomerase A [PPIA] ; hc04194521_s1), using the ΔΔ comparative threshold (Ct) method .

Transcriptomic and bioinformatics analyses:
RNA-sequencing was performed on polyA-enriched total RNA, on a HiSeq4000 (Illumina, Inc.; California, US) by the Oxford Genomics Centre (Wellcome Trust; Oxford, UK). In brief, total RNA was quantified using RiboGreen (Invitrogen; California, US) on the FLUOstar OPTIMA plate reader (BMG Labtech GmbH; Aylesbury, UK) and the size profile and integrity analysed on the 2200 or 4200 TapeStation (Agilent, RNA ScreenTape [Agilent Technologies; California, US]). RIN estimates for all samples were between 4 and 8.4. Input material was normalised to 200 ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA kit (New England Biolabs Inc.; Massachusetts, US) following manufacturer’s instructions. Libraries were amplified (11 cycles) on a Tetrad (Bio-Rad Laboratories; California, US) using in-house unique dual indexing primers. Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to ~10 nM, denatured and further diluted prior to loading on the sequencer. Paired end sequencing was performed using a HiSeq4000 75bp platform (Illumina, HiSeq 3000/4000 PE Cluster Kit and 150 cycle SBS Kit),

FastQ sequencing files were processed as previously described , using the Galaxy web platform (usegalaxy.org). GRCh38/hg38 was used as the reference genome. P-values were adjusted for transcriptome-wide false discovery rate (FDR), with an adjusted significance threshold of q < 0.05. Functional annotation was performed in the database for annotation, visualisation, and integrated discovery (DAVID) 6.8 (2019 release) and Genesis 1.8.1 (35). Pathway analysis was performed using Kyoto encyclopaedia of genes and genomes (KEGG) and gene ontology (GO)-terms, using a modified Fisher exact test (EASE [expression analysis systematic explorer]) with a significance threshold of p ≤ 0.01.

Immunoblotting:
Organic phases were extracted from QIAzol-treated tissue samples and processed for immunoblot analysis as previously described . Protein content was determined by BCA protein assay (ThermoFisher Scientific™; Leicestershire, UK). Isolated primary adipocytes were thawed on ice and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.4], 150 mM NaCl; 0.5 % sodium deoxycholate; 0.1 % SDS; 0.1 % NP-40), supplemented with HALT™ protease inhibitor cocktail (ThermoFisher™; Leicestershire, UK) and PhosSTOP EASYpack phosphatase inhibitor (Roche AG; Basel, Switzerland). 

Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane for immunoblot analysis using the following antibodies: Akt2 (Cell Signalling Technology; London, UK [1 in 500 dilution]); Akt substrate of 160 kilo-Daltons (kDa) (AS160) (Millipore; Massachusettes, US [1 in 500 dilution]); glyceraldehyde 3-phosphate dehydrogenase (GAPDH [1 in 2000 dilution]) (Proteintech; Manchester, UK); glucose transporter 4 (GLUT4)(1 in 5000 dilution); and Insulin receptor β-chain (InsRβ) (Santa Cruz Biotechnology; Texas, US [1 in 500 dilution]). Images were acquired with EPI Chemi II darkroom (UVP) and bands were quantified using ImageStudio Lite (LI-COR Biosciences®; Nebraska, US).

Flow cytometry:
Cells from the SVF were divided into two tubes, and additionally 250,000 PBMCs were place into two additional tubes. For each, one tube contained a T cell panel and the other a monocyte/ macrophage panel. The T cell panels contained the following fluorophore-conjugated antibodies: cluster of differentiation (CD)3–V450 clone UCHT1, CD4–APC clone RPA–T4, CD8–PerCP Sk1, CD45RA–FITC clone HI100, CD27–PE clone MT271, CD45–BV510 clone HI30, and human leukocyte antigen–DR isotype (HLA–DR)–APC-Cy7 clone L243. The monocyte/ macrophage tubes comprised of the following fluorophore-conjugated antibodies; CD14–PE Vio770 clone TUK4, CD16–FITC clone 3G8, CD206–APC clone 19.2, HLA-DR–APC Cy7 clone L243, and CD45–BV510 clone HI30. Isotype control antibodies for CD206 (APC–IgG1, κ clone MOPC-21) and HLA-DR (APC Cy7 IgG2a, κ clone G155-178) were used with PBMCs and informed the gating strategy for both PBMCs and SVF. Endothelial/ progenitor cells were identified in the SVF using the following fluorophore-conjugated antibodies; CD31–V450 clone WM59, CD34–PerCP clone 8G12, and MSCA-1–PE clone W8B2. Gating strategies are detailed in Supplementary Figure 3–5. Absolute cell counts were obtained using counting beads (100 µL of Perfect-Count Microspheres; Cytognos, Spain), according to manufacturer instructions. Flow cytometry was performed on a FACS Canto II (Becton Dickenson; Oxford, UK) and results analysed using FlowJo v.10. 

Biochemical analysis of plasma, serum, and ex vivo adipose tissue supernatants:
Fasted plasma insulin was measured by ELISA (Mercodia, Mercodia AB; Sweden). A further 32 biomarkers were measured in fasted plasma and adipose cell culture supernatant using multiplex assays (R-plex, U-plex and V-plex kits on a QuickPlex SQ120; Mesoscale Diagnostics, LLC; Maryland, US). Biomarkers included MCP-1, MIP-1α, MIP-1β, RANTES, MIP-3α, IP-10, GM-CSF, IFN-γ, TNF-α, IL-1β, IL-4, IL-6, IL-10, IL-13, IL-15, IL-17A, IL-17B, IL-17C, IL17-D, ICAM-1, VCAM-1, SAA, VEGF-A, VEGF-D, granzyme-A, FGF-21, leptin, adiponectin, resistin, adipsin, and osteopontin. Ex vivo Leptin release by adipose tissue was assessed using ELISA kit (Quantikine, Bio-Techne Ltd; France). Blood glucose was measured using fresh whole-blood samples at the MEDES clinic using an automated analyser (Architect C8000; Abbott, CA) four days prior to the start of bed rest and following 49 days of bed rest.

Statistical analysis:
The influence of the nutritional countermeasure on the effect of bed rest was analysed by two-way repeated measures ANOVA. Pre- to post-bed rest comparisons for the whole cohort (groups collapsed) were assessed using paired samples t-tests where data were normally distributed, and Wilcoxon signed rank tests where not normally distributed (Shapiro Wilks: p > 0.05). Linear regression analysis was performed using Pearson’s r. Descriptive data are presented as mean ± standard deviation (SD), unless otherwise stated. Statistical analysis was performed using GraphPad Prism v.8.0.0 for Windows (GraphPad Software; California, US) and SPSS v.22 (IBM Corp.; New York, US). Significance was set at p ≤ 0.05.