Early septic shock induces loss of oxidative phosphorylation yield plasticity in liver mitochondria

Abstract

We aimed to study the change in mitochondrial oxidative phosphorylation efficiency occurring at the early stage of septic shock in an experimental model. Thirty-six male Wistar rats were divided into two groups. In the first group, a cecal ligation and puncture (CLP) was carried out to induce septic shock for 5 h. The second group includes sham-operated rats and constitutes the control group. Blood gas analysis, alanine amino transferase, and lactic acid dosages were assayed 5 h after surgery. Liver mitochondria were isolated for in vitro functional characterization, including mitochondrial respiratory parameters, oxidative phosphorylation efficiency, oxi-radical production, membrane potential, and cytochrome c oxidase activity and content. Liver interleukin 1β (IL-1β) and tumor necrosis α mRNA levels were determined. Septic shock induced a severe hypotension occurring 180 min after CLP in association with a metabolic acidosis, an increase in plasma alanine amino transferase, liver IL-1β gene expression, and mitochondrial reactive oxygen species production. The rates of mitochondrial oxygen consumption and the activity and content of cytochrome c oxidase were significantly decreased while no alterations in the oxidative phosphorylation efficiency and inner membrane integrity were found. These results show that contrary to what was expected, liver mitochondria felt to adjust their oxidative phosphorylation efficiency in response to the decrease in the mitochondrial oxidative activity induced by CLP. This loss of mitochondrial bioenergetics plasticity might be related to mitochondrial oxidative stress and liver cytokines production.

Time course of liver mitochondrial function and intrinsic changes in oxidative phosphorylation in a rat model of sepsis

“…However, contrary to what could be expected, this was not associated with an increase in oxidative phosphorylation efficiency ATP/O, which could have compensated for energy production (ATP generation) from mitochondrial respiratory chain oxidative loss. This result reinforces and extends previous data [25] on the failure of the mitochondrial oxidative phosphorylation system to adjust its intrinsic coupling efficiency in early septic shock. Furthermore, in line with previous findings that ROS [40], sepsis [41], or endotoxemia [42] negatively affects ATP synthase activity, the late inhibition and depletion of ATP synthase subunit β observed in the present study 36 h after the onset of CLP may also explain mitochondrial uncoupling of ATP production from oxygen consumption.…”

“…Liver mitochondria were prepared using a differential centrifugation protocol [25]. Briefly, liver was immediately dissected and cut up finely with sharp scissors and diluted 1:10 ( w / v ) in ice-cold isolation medium consisting of 250 mM sucrose, 20 mM Tris/HCl, and 1 mM EGTA, pH 7.3.…”

“…To measure oxidative phosphorylation efficiency, oxygen consumption and ATP synthesis were performed at 37 °C in respiratory medium supplemented with glucose (20 mM), hexokinase (3 U mL −1 ) as previously described [25, 26]. Respiratory substrates were succinate (5 mM) in the presence of rotenone (5 μM) or pyruvate (5 mM) plus malate (2.5 mM).…”

“…The rate of mitochondrial ROS production (H 2 O 2 ) was measured at 37 °C in the respiratory medium following the rate of appearance of resorufin from Amplex Red with a Kontron SFM25 fluorescence spectrophotometer (excitation at 563 nm, emission at 587 nm) as described previously [25, 29]. Reaction conditions were 0.1 mg mL −1 of mitochondrial protein, 1 U mL −1 of horseradish peroxidase, and 10 μM Amplex Red.…”

“…Cytochrome c oxidase subunit 1 (CcO1), and ATP synthase subunit β (ATP synthase β) were detected in liver homogenates by Western blot using standard techniques as described previously [25, 30]. The primary antibodies employed were a mouse anti-mouse polyclonal anti-CcO 1 (1:1000 dilution; Invitrogen, Cergy Pontoise, France), a mouse anti-mouse polyclonal anti-ATP synthase β (1:1000 dilution; Life Technologies, Courtaboeuf, France), and a rabbit anti-rabbit monoclonal anti-β-actin antibody (1:1000 dilution, Sigma Aldrich, Saint-Quentin, Fallavier, France).…”