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. 2015:4:14-22.
doi: 10.1016/j.redox.2014.11.009. Epub 2014 Nov 28.

Uncoupling protein-2 attenuates palmitoleate protection against the cytotoxic production of mitochondrial reactive oxygen species in INS-1E insulinoma cells

Jonathan Barlow  1 , Verena Hirschberg Jensen  1 , Charles Affourtit  2
Affiliations

Uncoupling protein-2 attenuates palmitoleate protection against the cytotoxic production of mitochondrial reactive oxygen species in INS-1E insulinoma cells

Jonathan Barlow et al. Redox Biol. 2015.

Abstract

High glucose and fatty acid levels impair pancreatic beta cell function. We have recently shown that palmitate-induced loss of INS-1E insulinoma cells is related to increased reactive oxygen species (ROS) production as both toxic effects are prevented by palmitoleate. Here we show that palmitate-induced ROS are mostly mitochondrial: oxidation of MitoSOX, a mitochondria-targeted superoxide probe, is increased by palmitate, whilst oxidation of the equivalent non-targeted probe is unaffected. Moreover, mitochondrial respiratory inhibition with antimycin A stimulates palmitate-induced MitoSOX oxidation. We also show that palmitate does not change the level of mitochondrial uncoupling protein-2 (UCP2) and that UCP2 knockdown does not affect palmitate-induced MitoSOX oxidation. Palmitoleate does not influence MitoSOX oxidation in INS-1E cells ±UCP2 and largely prevents the palmitate-induced effects. Importantly, UCP2 knockdown amplifies the preventive effect of palmitoleate on palmitate-induced ROS. Consistently, viability effects of palmitate and palmitoleate are similar between cells ±UCP2, but UCP2 knockdown significantly augments the palmitoleate protection against palmitate-induced cell loss at high glucose. We conclude that UCP2 neither mediates palmitate-induced mitochondrial ROS generation and the associated cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity.

Keywords: Cytoprotection; Glucolipotoxicity; INS-1E insulinoma cells; Mitochondrial dysfunction; Non-esterified fatty acids; Obesity; Pancreatic beta cells; Reactive oxygen species; Type 2 diabetes; Uncoupling protein-2 (UCP2).

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Palmitate-induced ROS are mitochondrial. Mitochondrial and cytoplasmic superoxide levels were estimated from MitoSOX (5 µM, panels A and C) and DHE (100 µM, panels B and C) oxidation rates, respectively, in INS-1E cells exposed for 24 h at 11 mM glucose to BSA-conjugated palmitate or BSA alone. (Panels A and B) Probes were injected at times indicated by the arrows and background-corrected fluorescence was recorded at 28-s intervals; for clarity, only a selection of measurements is shown. Relative fluorescence units (RFU) were normalised to cell number using mean INS-1E viability data (Fig. 5). Probe oxidation rates (inset panels) were calculated from the slopes of the progress curves; except for the first 4 measurements after probe addition, all data were included in these calculations. Black and grey symbols (both main and inset panels) reflect BSA control and palmitate-exposed cells, respectively. Data are mean±SEM from 3–4 independent exposures that involved 7–8 replicates per treatment. Statistical significance of rate differences was tested by unpaired Student's t-tests. * Differs from the equivalent palmitate condition (P<0.05). (Panel C) Probe oxidation rates were determined ±15 µM antimycin A (striped and white bars, respectively). Data are mean±SEM from 3–11 independent exposures that involved 7–8 replicates per treatment. Statistical significance of rate differences was tested by two-way ANOVA with Tukey's post-hoc analysis. ⁎, ⁎⁎ Differs from the equivalent antimycin A condition (P<0.05 and P<0.01, respectively).
Fig. 2
Fig. 2
UCP2 protein in INS-1E cells is not affected by palmitate and/or palmitoleate. (Panel A) Typical Western blot showing cross-reactivity of UCP2 antibodies with partially purified recombinant human UCP2 (Recombinant Ucp2) and INS-1E proteins separated by SDS-PAGE (see Experimental section). Proteins were isolated from cells exposed for 24 h at 11 mM glucose to BSA-conjugated palmitate (PA) and/or palmitoleate (POA), BSA alone (BSA) or serum-supplemented growth medium (serum). (Panel B) Typical blots showing data from cells transfected with Ucp2-targeted or scrambled siRNA oligonucleotides (Ucp2 and Scr, respectively) before fatty acid exposure. (Panel C) Typical relation between signal intensity and amount of recombinant UCP2 as determined for each individual experiment (cf. panels A and B) to allow comparison of UCP2 levels between different samples. Membrane images were analysed with ImageQuant software using its 1D gel analysis feature: background in defined lanes was subtracted by the rolling ball function, bands reflecting known hUCP2 amounts were boxed, and by applying the quality calibration function the presented relation was generated. (Panel D) UCP2 content approximated as picograms per 50 µg total extracted protein. Data represent mean±SEM from 3 independent fatty acid exposures. Data were analysed statistically by one-way ANOVA with Tukey’s post-hoc analysis revealing no significant differences between conditions.
Fig. 3
Fig. 3
UCP2 knockdown does not change the effect of palmitate on mitochondrial ROS. MitoSOX oxidation rates were determined (see Fig. 1) in non-transfected INS-1E cells (NT) or cells transfected with scrambled or Ucp2-targeted siRNA oligonucleotides. Cells were exposed for 24 h at 11 or 4 mM glucose (panels A and B, respectively) to BSA-conjugated palmitate or BSA alone (grey and black bars, respectively). Data represent mean±SEM of 4–11 separate exposures with 3–8 replicates per condition. Statistical significance of rate differences was tested – separately at high and low glucose – by two-way ANOVA with Sidak's post-hoc analysis. ⁎⁎ Differs from the equivalent BSA condition (P < 0.01).
Fig. 4
Fig. 4
UCP2 knockdown amplifies attenuation by palmitoleate of palmitate-induced mitochondrial ROS. MitoSOX oxidation rates were determined (see Fig. 1) in non-transfected INS-1E cells (NT) or cells transfected with scrambled or Ucp2-targeted siRNA oligonucleotides. Cells were exposed for 24 h at 11 or 4 mM glucose (panels A and B, respectively) to BSA-conjugated palmitoleate (white bars), palmitoleate plus palmitate (grey bars) or to BSA alone (black bars). Data represent mean±SEM of 3–5 separate exposures with 3–8 replicates per condition. Statistical significance of rate differences was tested – separately at high and low glucose – by two-way ANOVA with Tukey's post-hoc analysis. ⁎, ⁎⁎⁎ Differs from the equivalent Ucp2-targeted condition (P<0.05 and P<0.001, respectively).
Fig. 5
Fig. 5
Effect of UCP2 knockdown on the viability of NEFA-exposed INS-1E cells. Viability was determined as described in ‘experimental’ using non-transfected cells (black bars) and cells transfected with scrambled or Ucp2-targeted siRNA oligonucleotides (white and grey bars, respectively). Cells were exposed for 24 h at 11 and 4 mM glucose (panels A and B, respectively) to BSA-conjugated palmitate and/or palmitoleate, or to BSA alone. Absolute cell numbers (cf. Fig. 6) were expressed as a percentage of control values obtained with cells grown in standard serum-supplemented growth medium. Values are mean±SEM of 4 independent experiments with 3–5 replicates per treatment. Statistical significance of mean differences was tested – separately at high and low glucose – by two-way ANOVA with Tukey's post-hoc analysis. ⁎, ⁎⁎, ⁎⁎⁎, ⁎⁎⁎⁎ Differs from equivalent palmitate condition (P<0.05, P<0.01, P<0.001, P<0.0001, respectively).
Fig. 6
Fig. 6
INS-1E cell viability correlates inversely with mitochondrial ROS. Absolute cell number (cf. Fig. 5) was plotted as a function of the MitoSOX oxidation rate (cf. Figs. 3 and 4). Data reflect the behaviour of non-transfected (NT) cells and that of cells transfected with scrambled or Ucp2-targeted siRNA oligonucleotides after 24-h exposure at low (G4) and high (G11) glucose to BSA-conjugated palmitate and/or palmitoleate or to BSA alone.
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