DOI: 10.15125/bath-00806
title: Dataset for "Divergent immunometabolic changes in adipose tissue and skeletal muscle with ageing in healthy humans"

description: This dataset provides all the raw data collected for a trial investigating the impact of ageing on adipose tissue, skeletal muscle, and systemic inflammatory and metabolic health in healthy, active, and non-obese Younger (20–35 years) and Older (60–85 years) males. This trial was a cross-sectional characterisation investigating the biological effects of ageing, in humans.
description: Recruitment:
One-hundred and nine individuals undertook preliminary screening, from which 24 males aged 20–35 (n = 12 [Young]) and 60–85 (n = 12 [Old]) years participated based on predetermined eligibility criteria (Table 1). Participants were recruited by local advertisement conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent. Participant recruitment was conducted between August 2016 and March 2018.

Design:
participants attended the laboratory for a preliminary assessment, including body composition, resting metabolic rate, and anthropometric measures. Following this initial visit, potential participants had their physical activity levels monitored across seven consecutive days using a multi-sensor physical activity monitor, and completed weighed food and fluid records across three days. Participants meeting eligibility criteria following these initial assessments attended the laboratory on one more occasion (main experimental visit) to undergo fasted blood sampling, provide an adipose tissue and skeletal muscle biopsy, followed by a three-hour meal tolerance test.

For preliminary assessment, participants undertook DEXA and pQCT scanning, and RMR assessments and blood pressure monitoring. After this, participant physical activity was monitored for seven consecutive days using a Sensewear multiaxial monitor and recorded weighed diet diaries during this period for three days (2 week days, one weekend day).

Biochemical measures:
Venous blood samples were collected from an antecubital vein. Samples for serum separation were rested at room temperature for 30 minutes prior to centrifugation at 3,000 x g for 10 minutes at 4oC. Plasma samples were immediately centrifuged upon collection and stored at −80oC until analysis. Peripheral Blood Mononuclear Cells (PBMCs) were isolated by density gradient separation (Ficoll®, Greiner Bio-One; Stonehouse, UK) in Leucosep® tubes for fresh analysis on the day of collection.

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; Hameln Pharmaceuticals; Gloucester, UK). 

Skeletal muscle was obtained from the Vastus Lateralis on the dominant leg under local anaesthesia using the Bergström technique 

A standardised mixed meal test was chosen to produce a physiological response similar to that of a conventional meal, formulated in-house equating to 2 g / kg body mass (BM) carbohydrate, 0.8 g / kg BM fat, and 0.4 g / kg BM protein. The beverage consisted of: Whey protein (MyProtein; Cheshire, UK); Elmlea double cream (Elmlea; Exeter, UK); Maltodextrin (MyProtein; Cheshire, UK); and 1 pint of whole milk as standard across participants, with 5 drops of vanilla flavouring (MyProtein; Cheshire, UK).

Adipose tissue was cultured, ex vivo, in sterile culture plates (Nunc; Roskilde, Denmark), at a final concentration of 50 mg of tissue per millilitre, in endothelial cell basal medium (PromoCell; Heidelberg, Germany) supplemented with 0.1 % fatty acid-free bovine serum albumin (BSA) and 100 U/ mL penicillin and 0.1 mg/ mL streptomycin (Sigma-Aldrich; Gillingham, UK), for 3 hours at 37 oC, 5 % CO2, and 95 ± 5 % relative humidity (MCO-18A1C CO2 incubator; Sanyo, Japan)

Adipose tissue (250 to 500 mg) was digested using 250 U/ mL type-I collagenase (Worthington Biochemical; New Jersey, USA) in PBS containing 2 % BSA (pH 7.4) for 45–60 minutes in a shaking water bath (225 r.p.m) at 37 oC

Isolated adipocytes were incubated at 37 oC and 5 % CO2 in DMEM (supplemented with 10 % FBS), whereupon a fraction was serum-starved in 1 mL of unsupplemented DMEM for 30 minutes and then incubated with or without 100 nM of insulin for 30 minutes. 

A portion of isolated adipocytes was placed onto a glass slide and cover slip for imaging under a light microscope (Olympus; Tokyo, Japan). Adipocyte diameters were measured using ImageJ (National Institutes of Health; Wisconsin, USA) after image file randomisation to mask participant grouping.

Between 35 and 100 mg of skeletal muscle was placed into 5 mL of DMEM (low glucose, with Glutamax™ GIBCO, Fisher Scientific) at 37 oC in a 60 mm Petri dish. Visible signs of blood were washed away by passing samples into successive Petri dishes containing fresh DMEM. Samples were teased apart using sterile tweezers, after-which, samples were reconstituted in 30 mL of PBS and centrifuged at 400 x g for 5 minutes at 4 oC. Cleaned tissue was digested enzymatically with collagenase B (0.5 U/ mL) in DMEM for one hour at 37 oC and 5 % CO2 rotating at ~40 rpm by a MACSmix™ tube rotator (Miltenyi Biotec; Surry, UK).

Total RNA (including microRNAs) was extracted from frozen adipose tissue (approx. 100 mg) or skeletal muscle samples (30 to 50 mg) using miRNeasy Mini Kit (Qiagen; Crawley, UK) according to manufacturer instructions.Following RNA isolation, samples were DNase treated with TURBO DNase (Thermofisher™; Leicestershire, UK) according to manufacturer instructions, followed by phenol-chloroform extraction and ethanol precipitation. The purified RNA pellets were re-suspended in 35 µL of nucleotide-free water each (Thermofisher™; Leicestershire, UK) with 2 μL used for quality control. Thirty microlitres of RNA at a set concentration of 2.1 µg/ 30 µL was sent for RNA-sequencing. RNA-sequencing was performed on RiboZero-treated 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 1.8 and 8.5. Input material was normalised to 200 ng prior to library preparation. Total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal Kit (Epicentre/Illumina, Human [Illumina®; California, US]) following manufacturer’s instructions. 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 for storage. The 10 nM library was 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), generating a raw read count of >38.5 million reads per sample. FastQ sequencing files were uploaded to the Galaxy web platform (usegalaxy.org) for quality control analysis. Raw sequencing files were splice-aligned to the GRCh38/ hg38 reference genome using Hisat2 with a mapping distance <500 kb between reads. Ensembl (Cambridge, UK) was used to annotate sequencing files against the reference genome. Expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) were estimated using Stringtie. Differential expression analysis was undertaken using Cuffdiff v.7 and Deseq2 using Gencode v.29 as the reference database (GRCh38.p12). Functional annotation analysis was performed in the database for annotation, visualisation, and integrated discovery (DAVID) 6.8 (2019 release) and Genesis 1.8.1. 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].

Total proteins from biopsies were recovered from the organic phase of QIAzol-treated tissue samples used for RNA extraction. Organic phases were extracted and processed for western blot analysis. Protein content was determined by BCA protein assay (Thermo Scientific™; Leicestershire, UK).

Multi-parameter flow cytometry on PBMCs and tissue SVFs was undertaken on a FACSAria III.

Baseline blood samples were analysed for plasma glucose, high-density lipoprotein (HDL) cholesterol, total cholesterol, triglycerides, and non-esterified fatty acids (NEFA) using clinical chemistry spectrophotometer (RX Daytona, Randox Laboratories; County Antrim, NI). Quantification of low-density lipoprotein (LDL) cholesterol was achieved using the Friedwald equation. nsulin was measured using Insulin ELISA kits (Mercodia, Mercodia AB; Sweden). All other biomarkers were measured by a commercial electrochemiluminescence technology (Mesoscale Diagnostics).
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