In search of a physiological role for amine oxidase,copper containing-3 (AOC3) in adipocytes

In search of a physiological role for amine oxidase,copper containing-3 (AOC3) in adipocytesThe function of adipocytes was initially thought to be fat storage, thermal insulation, and organprotection. With the discovery of adipokine, cytokine, and chemokine production by adipocytes,they are now considered to have endocrine function with a major role in homeostasis and energybalance. Amine oxidase, copper containing 3 (AOC3) is a major ectoenzyme expressed byadipocytes and catalyzes the oxidative deamination of amines to produce hydrogen peroxide,ammonia, and aldehyde. The physiological function of AOC3 in adipocytes and its endogenoussubstrate(s) are currently unknown. First, a signaling role for the hydrogen peroxide product wasinvestigated. Using an intracellular probe specific for H2O2, it was found that the plasmamembrane of adipocytes is permeable to H2O2 introduced exogenously. However, it was notpossible to detect an increase in intracellular H2O2 even after long incubation times with AOC3substrates. Adipocytes were found to produce extracellular H2O2 at an approximate rate of 0.078uM/min/well upon incubation with AOC3 substrate from whole cell kinetic studies. Due to thisslow rate of H2O2 production, the probe may not be sensitive enough to show an increase inintracellular H2O2 originating from AOC3 turnover. Adipocytes may have an immune function,with possible involvement in chemokine production. Using chemotaxis assays, the migration ofRAW264.7 macrophages was found to be independent of media components released byadipocytes incubated with isoamylamine, suggesting an absence or ineffectively lowconcentration of macrophage-specific chemokine release by adipocytes. However, theseexperiments used unactivated macrophages, which subsequently were reported by others to havechemotactic characteristics that are distinct from activated macrophages; the latter will be thesubject of future studies. In addition to the chemotaxis assay, release of the chemokines byadipocytes was investigated using ELISA. It was found that release of monocytechemoattractant protein-1 (MCP-1) by adipocytes was dependent on both concentration of H2O2exposure and incubation time, with adipocytes also capable of constitutive MCP-1 release.However, the release of MCP-1, as well as other cytokines (including IL-6, KC, and IP-10), wasfound to have little correlation with AOC3 turnover. On the other hand, release of IL-13, a cytokine correlated with populations of alternatively activated macrophages, was shown to bedependent on AOC3 turnover, though the increase was low with no correlation to H2O2. To findadipocyte response(s) to AOC3 turnover, it is important to characterize the enzyme's kineticprofile, since the endogenous substrate(s) of AOC3 is currently unknown. With this goal inmind, the human AOC3 enzyme was expressed and purified using a new expression system andpurification protocol. Using the purified enzyme, a substrate kinetic profile was determined. Itwas found that both cyclic and noncyclic alkylamines, arylamines, and branched chain aminesare all substrates for AOC3 with kcat/Km values ranging from 2.05 - 4.05 x 104 s-1M-1. Substratehydrophobicity did not seem to affect the catalytic efficiency. It is noted that branched chainaliphatic amines are among the best substrates for AOC3, despite the fact that these amines arenot metabolic products in mammals. Finally, the generation of an AOC3 knockout cell line(derived from AOC3 knockout mice) was pursued, providing a potential alternative to the use ofAOC3 inhibitors in cell-based assays. Bone marrow derived mesenchymal stem cells (MSC) aremulti-potent with the ability to differentiate into adipocytes. Using two techniques to enrichmurine MSC, it was possible to reach an adipocyte yield of 1 -2 % of the total cell population.