Title:Pharmacological Activation of AMPK Prevents Drp1-mediated Mitochondrial Fission and Alleviates Hepatic Steatosis In vitro
Volume: 24
Issue: 12
关键词:
肝脂肪变性，AMPK，线粒体动力学，ROS， MMP， L-02细胞。
摘要:
Background: The incidence of non-alcoholic fatty liver disease (NAFLD) is
increasing worldwide. Adenosine monophosphate-activated protein kinase (AMPK)
activation is beneficial for NAFLD treatment. Recent studies show the excessive
fission of mitochondria during NAFLD progression, so targeting mitochondria dynamics
may be a possible target for NAFLD. Still, little is known about whether AMPK
regulates mitochondrial dynamics in hepar.
Objective: This study investigated whether AMPK activation alleviates hepatic
steatosis by regulating mitochondrial dynamics mediated by GTPase dynamin-related
protein 1 (Drp1).
Methods: Human hepatocyte line L-02 cells were cultured and subjected to palmitic
acid (PA) treatment for 24 h to establish a hepatic steatosis model in vitro, which was
pre-treated with different tool drugs. Hepatocyte function, hepatocyte lipid content,
mitochondrial reactive oxygen species (ROS) production, and mitochondrial
membrane potential (MMP) were examined. The expression levels of genes and
proteins associated with mitochondrial dynamics were assessed using reverse
transcription-quantitative PCR and western blotting.
Results: The results indicated that 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside
(AICAR), an AMPK activator, improved hepatocyte function, as demonstrated
by decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
activity (P<0.05 or P<0.01). In addition, AICAR decreased total cholesterol (TC) and
triglyceride (TG) content and lipid deposition in hepatocytes (P<0.01); decreased
ROS production; improved MMP (P<0.01); reduced fission-1 (Fis1) and mitochondrial
fission factor (Mff) mRNA expression; and downregulated p-Drp1 (Ser 616) protein
expression. In contrast, AICAR increased mitochondrial fusion factor mitofusin-1
(Mfn1) and mitofusin-2 (Mfn2) mRNA expression and upregulated p-Drp1 (Ser 637)
protein expression. Mdivi-1, a Drp-1 inhibitor, was used to confirm whether
mitochondrial dynamics regulated by Drp1-mediated the role of AICAR. Similar to
AICAR, Mdivi-1 improved hepatocyte function and MMP significantly, decreased ROS
production and lipid deposition, downregulated Fis1 and Mff mRNA expression,
downregulated p-Drp1 (Ser 616) protein expression, and enhanced Mfn1 and Mfn2
mRNA and p-Drp1 (Ser 637) protein expression. However, Compound C, an AMPKspecific
inhibitor, had less impact on the protective effect of Mdivi-1.
Conclusion: The results demonstrated that AMPK activation has a protective effect on
hepatic steatosis in vitro, largely dependent on the inhibition of Drp1-mediated
mitochondrial fission.
