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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2010)

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.

    Cover page: In search of a physiological role for amine oxidase,copper containing-3 (AOC3) in adipocytes
    • Article
    • Peer Reviewed
    Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, Volume 21, Issue 6 (2020)
      Cover page: Telemedicine to Decrease Personal Protective Equipment Use and Protect Healthcare WorkersCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2012)
      AOC3 is highly expressed in adipocytes and smooth muscle cells, but its function in these cells is currently unknown. The in vivo substrate(s) of AOC3 is/are also unknown, but could provide an invaluable clue to the enzyme's function. Expression of untagged, soluble human AOC3 in insect cells provides a relatively simple means of obtaining pure enzyme. Characterization of enzyme indicates a 6% titer for the active site 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor and corrected k(cat) values as high as 7 s(-1). Substrate kinetic profiling shows that the enzyme accepts a variety of primary amines with different chemical features, including nonphysiological branched-chain and aliphatic amines, with measured k(cat)/K(m) values between 10(2) and 10(4) M(-1) s(-1). K(m)(O(2)) approximates the partial pressure of oxygen found in the interstitial space. Comparison of the properties of purified murine to human enzyme indicates k(cat)/K(m) values that are within 3 to 4-fold, with the exception of methylamine and aminoacetone that are ca. 10-fold more active with human AOC3. With drug development efforts investigating AOC3 as an anti-inflammatory target, these studies suggest that caution is called for when screening the efficacy of inhibitors designed against human enzymes in non-transgenic mouse models. Differentiated murine 3T3-L1 adipocytes show a uniform distribution of AOC3 on the cell surface and whole cell K(m) values that are reasonably close to values measured using purified enzymes. The latter studies support a relevance of the kinetic parameters measured with isolated AOC3 variants to adipocyte function. From our studies, a number of possible substrates with relatively high k(cat)/K(m) have been discovered, including dopamine and cysteamine, which may implicate a role for adipocyte AOC3 in insulin-signaling and fatty acid metabolism, respectively. Finally, the demonstrated AOC3 turnover of primary amines that are non-native to human tissue suggests possible roles for the adipocyte enzyme in subcutaneous bacterial infiltration and obesity.
        Cover page: Implication for Functions of the Ectopic Adipocyte Copper Amine Oxidase (AOC3) from Purified Enzyme and Cell-Based Kinetic StudiesCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2015)
        To develop tables of normalized glandular dose coefficients D(g)N for a range of anode-filter combinations and tube voltages used in contemporary breast imaging systems. Previously published mono-energetic D(g)N values were used with various spectra to mathematically compute D(g)N coefficients. The tungsten anode spectra from TASMICS were used; molybdenum and rhodium anode-spectra were generated using MCNPX Monte Carlo code. The spectra were filtered with various thicknesses of Al, Rh, Mo or Cu. An initial half value layer (HVL) calculation was made using the anode and filter material. A range of the HVL values was produced with the addition of small thicknesses of polymethyl methacrylate (PMMA) as a surrogate for the breast compression paddle, to produce a range of HVL values at each tube voltage. Using a spectral weighting method, D(g)N coefficients for the generated spectra were calculated for breast glandular densities of 0%, 12.5%, 25%, 37.5%, 50% and 100% for a range of compressed breast thicknesses from 3 to 8 cm. Eleven tables of normalized glandular dose (D(g)N) coefficients were produced for the following anode/filter combinations: W + 50 μm Ag, W + 500 μm Al, W + 700 μm Al, W + 200 μm Cu, W + 300 μm Cu, W + 50 μm Rh, Mo + 400 μm Cu, Mo + 30 μm Mo, Mo + 25 μm Rh, Rh + 400 μm Cu and Rh + 25 μm Rh. Where possible, these results were compared to previously published D(g)N values and were found to be on average less than 2% different than previously reported values.Over 200 pages of D(g)N coefficients were computed for modeled x-ray system spectra that are used in a number of new breast imaging applications. The reported values were found to be in excellent agreement when compared to published values.
          Cover page: Mean glandular dose coefficients (DgN) for x-ray spectra used in contemporary breast imaging systems
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