Leptin

Serum insulin, leptin, and adiponectin levels were measured with ELISAs for mouse insulin (ALPCO Diagnostics, Salem, NH, USA), adiponectin ELISA Kit (R&D Systems, Oxon, UK), and leptin (AdipoGen, Seoul, Korea) according to the manufacturer's instructions.

Serum concentrations of all adipokines under investigation (adiponectin, chemerin, FGF-19/21/23, progranulin, AFABP, vaspin, IL-6, leptin, PENK, irisin, AGF) were determined with a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (AGF, irisin, vaspin and progranulin: AdipoGen Inc, Seoul, Korea; FGF-19, FGF-21, adiponectin, chemerin and AFABP: BioVendor Inc, Brno, Czech Republic; IL-6: R&D Systems, Minneapolis, MN USA; leptin: Mediagnost, Reutlingen, Germany; FGF-23: Immutopics, San Clemente, CA, USA). PENK and pro-NT was centrally quantified in a single lab by sphingotec (sphingotec GmbH, Hennigsdorf, Germany) using a chemiluminometric sandwich immunoassay [18 (link)]. Serum IGF-1 concentrations were measured by commercially available automated two-site chemiluminescent immunometric assays (Immulite 2000, Siemens Healthcare Diagnostics GmbH, Bad Nauheim, Germany). Whereas all adipokines were measured in the Sorbs cohort, only a subset of them (chemerin, vaspin, pro-NT, PENK) was recorded in the LIFE-Adult cohort. Therefore, the more comprehensively phenotyped Sorbs cohort was taken for the primary analyses, whereas the replication analyses of four adipokines were performed in the larger LIFE-Adult cohort.

The effects of fatty acids (FA), adipokines and hypoxia on MDM reagibility were further analysed.
Therefore the saturated FA palmitate (P9767, MilliporeSigma, St. Louis, Missouri, USA) and its unsaturated analogue palmitoleic acid (P9417, MilliporeSigma, St. Louis, Missouri, USA) were solubilized in 70% ethanol as stock solutions of 50mM. BSA (A3294, MilliporeSigma, St. Louis, Missouri, USA) was dissolved 5% in cell assay medium RPMI-1640. Stock solutions of fatty acids were added to the BSA medium to achieve a molar ratio of FA–BSA of 4:1. The pH was adjusted to 7.4 with 0.25 M NaOH and the solution was filtered using a 0.2 µm low protein-binding filter (Filtropur S 0.2, Sarstedt, Nümbrecht, Germany). BSA medium alone was used in control solutions.
The four adipokines Leptin, Chemerin (both R&D Systems, Minneapolis, Minnesota, USA), FABP4 and Progranulin (both Adipogen Corporation, San Diego, CA, USA) have been diluted in TRIS (Leptin, FABP4) and PBS (Chemerin and Progranulin) respectively. Experiments were done using named adipokines as costimulatory substance during stimulation period or during maturation of monocytes towards macrophages.
In a separate experiment hypoxic conditions were maintained by transfer of cell culture plates into bags, aspiration of room air and insufflation of hypoxic air (5% CO₂, 1% O₂, 94% N₂).