The effect of high glucose on insulin resistance and mitochondrial calcium homeostasis.

<p>A: The effect of 33mM glucose (HG) on Akt phosphorylation was performed by western blotting method. B&C: The effect of HG on mitochondrial calcium was assessed using the adenoviruses expressing the low-affinity mitochondrial probe. D&G: The effect of HG on cytosolic calcium was evaluated by adenoviruses expressing the cytosolic probe. Data are shown as a mean ± SEM of at least three separate experiment. NG: normal glucose, HG: high glucose, # <0.05, * <0.01, NS = not significance.</p

The effect of MCU inhibition on the phosphorylation of MAPK and NF-κB pathway.

<p>HepG2 cells were treated with HG for 24 h. After treatment, cells were lysed and protein extracts were immunoblotted with specific antibodies. The effect of MCU inhibition on JNK (A), P38 (B), ERK (C) and IKKα-β (D) phosphorylation has been demonstrated. Data are shown as a mean ± SEM of at least three separate experiment. MCU knockdown cells, SC: Scramble, NG: normal glucose, HG: high glucose, # <0.05, * <0.01, * * <0.001.</p

The effect of MCU inhibition on ROS production in HepG2 cells.

<p>HepG2 cells were treated with HG for 24 h. A: H₂O₂ levels were measured using flow cytometry with DCFH-DA. B: Mitochondrial ROS level using MitoSOX red dye. Data are shown as a mean ± SEM of at least three separate experiment.: MCU knockdown cells, SC: Scramble, NG: normal glucose, HG: high glucose, # <0.05, * <0.01, * * <0.001.</p

Importance of MCU inhibition in high glucose-induced pro-inflammatory and coagulative responses.

<p>HepG2 stable cells were generated by infecting the cells with the supernatants of lentiviruses expressing MCU shRNA. Real-time PCR and western blotting were used to detect MCU mRNA and protein levels in HepG2 stable cells, respectively. A: Protein levels of MCU, B: mRNA level of MCU, C) mitochondrial calcium concentration in MCU-KD cells. D: the effect of MCU inhibition on the mRNA expression of TNF-α (E), IL-6 (F), PAI-1 (G), FGA (H), and FGB(I) were measured using real time PCR. Data are shown as a mean ± SEM of at least three separate experiment. MCU-KD: MCU knockdown cells, SC: Scramble, NG: normal glucose, HG: high glucose, # <0.05, * <0.01, * * <0.001.</p

Proposed scheme of interplay between Ca²⁺, ATP and V_m in the β-cell.

<p>The oxidation of glucose that enters the β-cell hyperpolarises the mitochondrial membrane (ΔΨ_m) thereby leading to the elevation of cytosolic ATP/ADP ratio, closing of K_ATP channels, depolarisation of the plasma membrane (V_m) and Ca²⁺ entry. Elevated cytosolic [Ca²⁺] triggers a number of ATP-dependent processes including insulin secretion and Ca²⁺ removal into the ER and extracellular medium. By entering mitochondria via MCU, Ca²⁺ potentiates oxidative metabolism to counter-balance ATP expenditure. Ca²⁺ exits mitochondria via NCLX.</p

Chronic exposure to high-glucose and high-FFA medium impairs Ca²⁺ entry into mitochondria.

<p>β-Cells were pre-cultured in FFA-free medium containing 11 mM glucose (“control”) or medium containing 17 mM glucose and 0.5 mM palmitate (“FFA⁺”) for 48–72 h. <b><i>A</i></b>: The cells were voltage-clamped at −70 mV and five depolarising bursts were applied at 4 min⁻¹, as indicated in V_m trace (above). [Ca²⁺]_cyt and [Ca²⁺]_mit were monitored with Fura-Red and 2mt8RP, respectively. <b><i>B</i></b>: Peak [Ca²⁺]_mit induced by a single burst related to the respective peak [Ca²⁺]_cyt (Δ[Ca²⁺]_mit/Δ[Ca²⁺]_cyt), measured in control (blue columns, n = 10) and FFA⁺ (white columns, n = 9) cells. *Differences are significant with p<0.05 (*) or p<0.01 (**).</p

Effect of the NCLX silencing on [Ca²⁺]_cyt and [Ca²⁺]_mit dynamics.

<p>Pancreatic β-cells were infected with lentiviruses delivering nonsense shRNA (“control”) or shRNA against NCLX (“NCLX^-“) for 36–48 h. <b><i>A</i></b>: [Ca²⁺]_cyt and [Ca²⁺]_mit increases in response to 5 depolarising bursts applied at 4 min⁻¹ were measured using Fura-Red and 2mt8RP, respectively. <b><i>B</i></b>: Mean increases in [Ca²⁺]_mit induced by a single depolarising burst or by exposure to 17 mM glucose, related to the respective increases in [Ca²⁺]_cyt (Δ[Ca²⁺]mit/Δ[Ca²⁺]cyt). <b><i>C</i></b>: Glucose-induced changes in [ATP/ADP]_cyt were measured using Perceval (representative for n = 9 control and n = 9 NCLX⁻ cells). <b><i>D</i></b>: Times of half-maximal increase in [ATP/ADP]_cyt in response to 17 mM glucose, in control and NCLX⁻ cells. <b><i>E</i></b>: Mean magnitudes of the second phase of [ATP/ADP]_cyt increase measured in control and NCLX⁻ β-cells. The data were normalised to the width of the range of [ATP/ADP]_cyt change (ΔF_max), measured as the difference in <i>Perceval</i> fluorescence between the peak point at 17 mM glucose and the point corresponding to application of 2 µM FCCP. Differences vs respective NCLX⁻ data are significant with p<0.05 (*) or p<0.01 (**).</p

MCU silencing impairs mitochondrial Ca²⁺ increases.

<p>Pancreatic β-cells were infected with lentiviruses encoding nonsense (“control”) or anti-MCU (“MCU⁻”) shRNA for 72 h. <b><i>A</i></b>: [Ca²⁺]_cyt (Fura-Red) and [Ca²⁺]_mit (2mt8RP) increases were measured in response to 10 depolarising bursts, applied at 4 min⁻¹ by patch pipette (representative traces for n = 12, control, and n = 10, MCU⁻ cells). <b><i>B</i></b>: Mean ratios of maximal increases in [Ca²⁺]_mit to the respective increases in [Ca²⁺]_cyt (Δ[Ca²⁺]_mit/Δ[Ca²⁺]_cyt) measured in control and MCU⁻ β-cells.</p

Chronic glucolipotoxicity slows down the second phase of glucose-induced ATP elevation. <i>A</i>

<p>: Glucose-induced changes in [ATP/ADP]_cyt and [Ca²⁺]_cyt were monitored in control (above) and FFA⁺ (below) cells using Perceval and Fura-Red. <b><i>B</i></b>: Mean time of saturation of the second phase of [ATP/ADP]_cyt increase in control (blue columns, n = 16) and FFA⁺ (white columns, n = 13) cells. <b><i>C</i></b>: Changes in ΔΨ_m measured as mitochondrial TMRE fluorescence, in response to the increase of glucose from 3 to 17 mM, in control and FFA⁺ β-cells. The data are expressed as (F-F_FCCP)/(F₀-F_FCCP), where F₀ and F_FCCP represent TMRE fluorescence intensity in 3 mM glucose and 2 µM FCCP, respectively. <b><i>D</i></b>: Normalised MCU (<i>Ccdc109a</i>), NCLX (<i>Slc24a6</i>) and Pdx1 (<i>Pdx1</i>) mRNA expression levels for control and FFA⁺ cells. *Differences are significant (p<0.05).</p

Glucose induces a biphasic increase in cytosolic ATP/ADP ratio.

<p><b><i>A</i></b>: The effects of high (17mM) glucose on [ATP/ADP]_cyt (reported with <i>Perceval</i>), [Ca²⁺]_cyt (Fura-Red) and V_m were measured in a single β-cell (representative of n = 30 cells). The voltage down-strokes were elicited by 10 ms 10 pA current injections applied every 20 s to monitor the input resistance which increased upon the elevation of [ATP/ADP]_cyt. <b><i>Inset</i></b>: Pseudo-colour images of the patched cell cluster presenting pixel-to-pixel ratios at the time points indicated by arrows (<i>i–vi</i>). ROI is indicated with red oval. Note that a cell expressing high levels of <i>Perceval</i> (just below the ROI) was deliberately excluded from analysis. <b><i>B</i></b>: Characteristic times and amplitudes of glucose-induced [ATP/ADP]_cyt increase in β-cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039722#pone-0039722-g001" target="_blank">Fig. 1A</a>; n = 30). The data were normalised to the width of the range of [ATP/ADP]_cyt change (ΔF_max), measured as the difference in <i>Perceval</i> fluorescence between the peak point at 17 mM glucose and the point corresponding to application of 2 µM FCCP. Depolarisation and onset of electrical activity was taken as zero of the time axis. The change in [ATP/ADP]_cyt (ΔF/ΔF_max) at each point is significant <i>vs</i> every other point (p<0.01, Wilcoxon's paired test).</p