UC Berkeley

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

The function of adipocytes was initially thought to be fat storage, thermal insulation, and organ

protection. 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 energy

balance. Amine oxidase, copper containing 3 (AOC3) is a major ectoenzyme expressed by

adipocytes and catalyzes the oxidative deamination of amines to produce hydrogen peroxide,

ammonia, and aldehyde. The physiological function of AOC3 in adipocytes and its endogenous

substrate(s) are currently unknown. First, a signaling role for the hydrogen peroxide product was

investigated. Using an intracellular probe specific for H2O2, it was found that the plasma

membrane of adipocytes is permeable to H2O2 introduced exogenously. However, it was not

possible to detect an increase in intracellular H2O2 even after long incubation times with AOC3

substrates. Adipocytes were found to produce extracellular H2O2 at an approximate rate of 0.078

uM/min/well upon incubation with AOC3 substrate from whole cell kinetic studies. Due to this

slow rate of H2O2 production, the probe may not be sensitive enough to show an increase in

intracellular H2O2 originating from AOC3 turnover. Adipocytes may have an immune function,

with possible involvement in chemokine production. Using chemotaxis assays, the migration of

RAW264.7 macrophages was found to be independent of media components released by

adipocytes incubated with isoamylamine, suggesting an absence or ineffectively low

concentration of macrophage-specific chemokine release by adipocytes. However, these

experiments used unactivated macrophages, which subsequently were reported by others to have

chemotactic characteristics that are distinct from activated macrophages; the latter will be the

subject of future studies. In addition to the chemotaxis assay, release of the chemokines by

adipocytes was investigated using ELISA. It was found that release of monocyte

chemoattractant protein-1 (MCP-1) by adipocytes was dependent on both concentration of H2O2

exposure 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), was

found 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 be

dependent on AOC3 turnover, though the increase was low with no correlation to H2O2. To find

adipocyte response(s) to AOC3 turnover, it is important to characterize the enzyme's kinetic

profile, since the endogenous substrate(s) of AOC3 is currently unknown. With this goal in

mind, the human AOC3 enzyme was expressed and purified using a new expression system and

purification protocol. Using the purified enzyme, a substrate kinetic profile was determined. It

was found that both cyclic and noncyclic alkylamines, arylamines, and branched chain amines

are all substrates for AOC3 with kcat/Km values ranging from 2.05 - 4.05 x 104 s-1M-1. Substrate

hydrophobicity did not seem to affect the catalytic efficiency. It is noted that branched chain

aliphatic amines are among the best substrates for AOC3, despite the fact that these amines are

not 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 of

AOC3 inhibitors in cell-based assays. Bone marrow derived mesenchymal stem cells (MSC) are

multi-potent with the ability to differentiate into adipocytes. Using two techniques to enrich

murine MSC, it was possible to reach an adipocyte yield of 1 -2 % of the total cell population.