MCU-knockdown attenuates high glucose-induced inflammation through regulating MAPKs/NF-κB pathways and ROS production in HepG2 cells

Abstract

Mitochondrial Ca2+ is a key regulator of organelle physiology and the excessive increase in mitochondrial calcium is associated with the oxidative stress. In the present study, we investigated the molecular mechanisms linking mitochondrial calcium to inflammatory and coagulative responses in hepatocytes exposed to high glucose (HG) (33mM glucose). Treatment of HepG2 cells with HG for 24 h induced insulin resistance, as demonstrated by an impairment of insulin-stimulated Akt phosphorylation. HepG2 treatment with HG led to an increase in mitochondrial Ca2+ uptake, while cytosolic calcium remained unchanged. Inhibition of MCU by lentiviral-mediated shRNA prevented mitochondrial calcium uptake and downregulated the inflammatory (TNF-α, IL-6) and coagulative (PAI-1 and FGA) mRNA expression in HepG2 cells exposed to HG. The protection from HG-induced inflammation by MCU inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). Importantly, MCU inhibition in HepG2 cells abrogated the phosphorylation of p38, JNK and IKKα/IKKβ in HG treated cells. Taken together, these data suggest that MCU inhibition may represent a promising therapy for prevention of deleterious effects of obesity and metabolic diseases.

Fig 1. The effect of high glucose on insulin resistance and mitochondrial calcium homeostasis.

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.

Fig 2. Importance of MCU inhibition in high glucose-induced pro-inflammatory and coagulative responses.

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.

Fig 3. The effect of MCU inhibition on ROS production in HepG2 cells.

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.

Fig 4. The effect of MCU inhibition on the phosphorylation of MAPK and NF-κB pathway.

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.