Laboratory analyses

Fasting blood samples were collected in the morning following study inclusion. Cystatin C, fasting plasma glucose (FPG), triglycerides, serum insulin and N-terminal pro–B-type natriuretic peptide (NT-proBNP) were analysed at the Department of Clinical Chemistry, Malmö. The plasma level of cystatin C was determined by an automated particle-based immunoassay, using the Hitachi Modular P analysis system and reagents from DAKO (Dako A/S, Glostrup, Denmark). Plasma triglycerides and FPG were analysed using Cobas c501/Cobas c701 (Roche, Basel, Switzerland). Total serum GIP was analyzed using Millipore’s Human GIP Total ELISA (cat. No. EZHGIP-54 K). Serum concentrations of insulin were analyzed using Cobas 6000/8000 (Roche, Basel, Switzerland). Aliquots (225 µL; REMP, Brooks, Life Sciences, USA) of plasma were stored in − 80 °C in a local biobank in the Region Skåne County Council until proximity extension array analyses (May 2018). Plasma concentrations of Gal-4, Galectin-3 (Gal-3), and Suppression of tumorigenicity 2 (ST2) were determined through a proximity extension array technique, using Proseek Multiplex CVD III 96 × 96 reagents kit (Olink, Uppsala, Sweden). The final proteomic assay read out was given as an arbitrary unit given on a log2 scale meaning that each unit increase corresponds to a doubling in concentration. Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated using the formula: HOMA-IR = (fasting insulin (µU/ml) x fasting glucose (mmol/L)/22.5.