Zacherl, Janelle/R
(2014)
Effects of Trans Fats in Human Macrophages.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The mechanism by which trans fats contribute to atherosclerosis, an important public health issue, is unclear. Trans fats may influence cell membrane stability, inflammatory responses, and signaling. Cellular metabolism of oleate (C18:1Δ9-10 cis), elaidate (C18:Δ9-10 trans), and stearate (C18:0) were compared in adherent peripheral human macrophages, the first responders in atherosclerosis. Metabolism was monitored by acylcarnitine measurement in supernatants by MS/MS, determination of whole cell fatty acid content by GC/MS, and β-oxidation evaluation using radiolabeled fatty acids. Macrophages incubated in elaidate for 44 h accumulated more unsaturated fatty acids, both longer- and shorter-chain, and had reduced C18:0 relative to incubation with oleate or stearate. Cell supernatants exposed to trans fats accumulated both C12:1- and C18:1-carnitines, suggesting inhibited β-oxidation proximal to the trans bond. Next, competitive β-oxidation assays with [9,10-3H]oleate showed that tritium release rates decreased when elaidate replaced unlabeled oleate. Yet, when [1-14C]oleate was compared to [1-14C]elaidate β-oxidation, initial elaidate degradation rates were comparable to oleate, supporting inhibition of double bond isomerization by elaidate. An expression array comparing human macrophages incubated with 30 μM oleate or elaidate showed eight genes associated with zinc homeostasis. Changes in metallothioneins 1X and 2A and SLC39A10 expression were confirmed by qPCR. Parallel qPCR experiments with saturated fatty acids showed elevated metallothionein expression at 44 h, but at 15 h elaidate, stearate, and palmitate have comparable metallothionein expression lower than oleate. Next we investigated these effects on intracellular zinc. Expression changes paralleled intracellular zinc at both time points confirmed quantification in elaidate-, stearate-, and palmitate-treated cells. Elaidate, stearate, and palmitate increased labile zinc at 15 h, but only elaidate-treated remained elevated at 44 h. To determine whether zinc changes corresponded to inflammation, proportional nuclear localization of nuclear factor-κB (NF-κB) was determined. A parallel experiment was conducted with the addition of 5 μM zinc chelator, TPEN. Elaidate, stearate, and palmitate caused the most NF-κB nuclear localization. Addition of TPEN nullified the treatment effect; all conditions, even controls, caused similar effects. These data show the similar initial effects of elaidate, stearate, and palmitate on macrophage zinc homeostasis and NF-κB activation, but the elaidate zinc effect is persistent.
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| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
27 June 2014 |
| Date Type: |
Publication |
| Defense Date: |
8 April 2014 |
| Approval Date: |
27 June 2014 |
| Submission Date: |
11 April 2014 |
| Access Restriction: |
3 year -- Restrict access to University of Pittsburgh for a period of 3 years. |
| Number of Pages: |
122 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Public Health > Human Genetics |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
trans fats, macrophages, lipid metabolism, beta-oxidation |
| Date Deposited: |
27 Jun 2014 20:35 |
| Last Modified: |
01 May 2017 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/21180 |
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