AGMO: Impact on adipocyte differentiation

Tetrahydrobiopterin is a vitamin-like cofactor with well-defined roles in the reactions catalysed by the aromatic amino acid hydroxylases and nitric oxide synthases. We recently identified the fifth enzyme dependent of this cofactor, alkylglycerol monooxygenase, and discovered a new and unanticipated impact of both tetrahydrobiopterin and alkylglycerol monooxygenase on lipid metabolism and cellular lipid signalling. This proposal will explore, for the first time, entirely novel roles for alkylglycerol monooxygenase in signalling and cell differentiation, where recent findings in fat cell maturation indicate that alkylglycerol monooxygenase is expected to play a central role. We will tackle this central aim by spanning a bridge from cell lines to primary cells and by combining established methods with ground- breaking high resolution lipidomics and novel, unique mouse models for alkylglycerol monooxygenase. Pivotal roles for alkylglycerol monooxygenase in lipid metabolism would identify an entirely new therapeutic target with great future potential for treatment of obesity and type 2 diabetes, two of the worlds most threatening health issues. AGMO IN ADIPOCYTE DIFFERENTIATION KATRIN WATSCHINGER1

Alkylglycerol monooxygenase (AGMO) is an extremely hydrophobic membrane protein, which is responsible for cleaving alkylglycerols in the final part of etherlipid degradation. The genetic information of AGMO is available only since 2010. Since this time, we have been investigating the biochemical properties and the physiological function of this enzyme. In this project, we assessed the role of AGMO in 3T3-L1 adipocyte differentiation, established a method to genotype the AGMO knockout mouse produced in our laboratory and performed a first phenotypic characterisation of these animals. We could successfully show that AGMO is expressed and active in 3T3-L1 fibroblasts and the enzymatic activity increases during their conversion to mature adipocytes. Knockdown of AGMO did not interfere with the ability of these cells to undergo adipogenesis and form mature lipid droplet. However, in an untargeted lipidomics approach, where we compared control and AGMO knockdown cells prior and at the end of the differentiation process we found that a special subclass of ether lipids, the plasmalogens, were preferentially accumulated upon Agmo knockdown, and that specific lipids presented with longer and more polyunsaturated acyl side chains. We also had significant advances in our understanding on the genetic make-up of the AGMO knockout mouse, which had been previously established in our laboratory. It turned out that the assumed embryonic lethality deduced from presence of a wild type band in all genotyping PCR reactions was not the underlying reason for these bands. By a innovative method to sequence very long DNA stretches, the nanopore sequencing, we could show that indeed during AGMO knockout stem cell production an integration of a piece of wild type AGMO gene into the AGMO locus carrying the knockout cassette had occurred. This led to constant presence of wild type PCR bands. To circumvent this problem, we established an alternative, quantitative PCR genotyping protocol, which does not rely on the presence or absence of wild type signal but instead counts the presence of transgenic alleles allowing to correctly assigning the genotype to the animals. With this major breakthrough, we were then able to characterise the knockout animals, which are not presenting a phenotype so far under unchallenged conditions.