Our previous work at a cellular and molecular level suggest that the glycopeptide wnt5a is a novel cytokine being secreted by macrophages in response to metabolic abnormalities. This cellular data is supported by recent clinical studies carried out by our group, revealing that wnt5a is elevated in the serum of human subjects with (1) obesity, (2) rheumatoid arthritis, (3) psoriasis and (4) sepsis, which are all diseases combining inflammation with metabolic abnormalities, e.g. insulin resistance. Furthermore, our own molecular data suggests that the wnt5a expression in macrophages is inhibited by the GPR120 receptor, which is known to be stimulated by Ω-3 polyunsaturated fatty acids (FA). Since GPR120, like wnt5a, has recently been shown to be involved in the pathogenesis of obesity and diabetes, the GPR120/wnt5a system might serve as a future target for the development of preventive (nutrition) and therapeutic (pharmacology) strategies for both, chronic metabolic and chronic inflammatory diseases. Interestingly, recent population based genetic studies revealed that the R270H polymorphism of the GPR120 gene resulting in reduced receptor function with a minor allele frequency of 3% is associated with obesity and type 2 diabetes. However, until now no data exists on the effect of that polymorphism on wnt5a physiology in human subjects. Within the last three years, and funded by the BMBF, our group has built up the Food Chain Plus (FoCus) cohort, comprising n=1500 cross sectional controls and n=500 severe obese human subjects. The subjects recruited received a detailed phenotyping programm, including food frequency questionnaires (e.g. on intake of Ω-3 FA) and a biobanking of DNA, serum, urine and stool was performed. In the serum samples of those subjects, besides glucose, insulin, IL-6, etc., the concentrations of wnt5a have also been determined by ELISA.
In the present proposal in workpage (WP-)1, we will genotype the GPR120 polymorphism R270H in the FoCus cohort and relate the serum wnt5a levels to the genotype. These experiments will prove the concept in humans in vivo that GPR120 signaling is important for regulating wnt5a. In WP2 and WP3, this data at a genetic level will be completed by experiments at a molecular and cellular level using adipose tissue biopsies obtained during elective surgery.
WP1 GPR120 R270H genotyping of the FoCus cohort (in vivo experiments): In a first set of experiments, genotyping the common R270H polymorphism in the FoCus cohort, a nutritional intervention cohort comprising n=1500 cross-sectional controls and n=500 human subjects with severe obesity, is assumed. Importantly, detailed data on the nutritional intake of different FA (including ω3-FA) exist for each proband in addition to wnt5a serum levels, which have been analyzed by ELISA. Thus, after genotyping the GPR120 R270H polymorphism it will be possible to relate wnt5a serum levels to the genotype, which will clarify whether reduced GPR120 receptor activity might be associated with higher wnt5a levels. This would suggest that, in humans, in vivo wnt5a is indeed regulated by GPR120 receptor signaling.
WP2: Molecular and cellular analysis of the GPR120/wnt5a system using human adipose tissue biopsies (ex vivo experiments): Interestingly, it has been published that obese humans express less GPR120 in adipose tissue macrophages (ATM)13 which might explain their higher inflammatory activity and the limited anti-inflammatory effect of an increased nutritional ω3-FA supply. In the present project, it will be examined (1) if wnt5a expression levels in ATM (isolated from adipose tissue biopsies by collagenase digestion and MACS) are inversely related to GPR120 expression levels, (2) if GPR120 and wnt5a are differentially expressed in isolated pro-inflammatory M1 ATM and anti-inflammatory M2 ATM (which are both known to be present in adipose tissue of obese human subjects13) and (3) to what extent ω3-FA or the small molecule GPR120 agonist cpdA can alter wnt5a expression levels ex vivo in isolated M1 vs. M2 ATM.
WP3: Effect of ω3-FA and small molecule GPR120 agonists on activity of GPR120 wt and GPR120 R270H receptors (in vitro experiments): This WP aims to identify if small molecule GPR120 agonists, which are much more potent compared to ω3-FA, will be able to overcome the reduced activity of the GPR120 R270H receptor in response to natural ligands. Therefore, the cDNA of the GPR120 receptor will be cloned into a pcDNA3.1 expression plasmid and an in vitro mutagenesis (wt → R270H) will be performed. Subsequently, wt or mutant receptors will be overexpressed in THP1 macrophages followed by stimulation by ω3-FA versus small molecule agonists. Finally, mRNA will be isolated and the wnt5a expression will be analyzed by real-time RT-PCR and a comparison carried out between the groups.