ACACA reduces lipid accumulation through dual regulation of lipid metabolism and mitochondrial function via AMPK- PPARα- CPT1A axis

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is a multifaceted metabolic disorder, whose global prevalence is rapidly increasing. Acetyl CoA carboxylases 1 (ACACA) is the key enzyme that controls the rate of fatty acid synthesis. Hence, it is crucial to investigate the function of ACACA in regulating lipid metabolism during the progress of NAFLD.

Methods: Firstly, a fatty liver mouse model was established by high-fat diet at 2nd, 12th, and 20th week, respectively. Then, transcriptome analysis was performed on liver samples to investigate the underlying mechanisms and identify the target gene of the occurrence and development of NAFLD. Afterwards, lipid accumulation cell model was induced by palmitic acid and oleic acid (PA ∶ OA molar ratio = 1∶2). Next, we silenced the target gene ACACA using small interfering RNAs (siRNAs) or the CMS-121 inhibitor. Subsequently, experiments were performed comprehensively the effects of inhibiting ACACA on mitochondrial function and lipid metabolism, as well as on AMPK- PPARα- CPT1A pathway.

Results: This data indicated that the pathways significantly affected by high-fat diet include lipid metabolism and mitochondrial function. Then, we focus on the target gene ACACA. In addition, the in vitro results suggested that inhibiting of ACACA in vitro reduces intracellular lipid accumulation, specifically the content of TG and TC. Furthermore, ACACA ameliorated mitochondrial dysfunction and alleviate oxidative stress, including MMP complete, ATP and ROS production, as well as the expression of mitochondria respiratory chain complex (MRC) and AMPK proteins. Meanwhile, ACACA inhibition enhances lipid metabolism through activation of PPARα/CPT1A, leading to a decrease in intracellular lipid accumulation.

Conclusion: Targeting ACACA can reduce lipid accumulation by mediating the AMPK- PPARα- CPT1A pathway, which regulates lipid metabolism and alleviates mitochondrial dysfunction.

Keywords: ACACA; AMPK/PPARα/CPT1A; Mitochondrial dysfunction; NAFLD.

Fig. 1

Long-term high-fat diet exacerbates lipid accumulation in the liver of mice. A Representative image of different groups with H&E staining, Scale bars, 200 μm. B Body weight and liver weight change of different groups (n = 8/group). C The content of TG/TC in liver tissue at different points. D The representative images and the quantification of Oil Red O staining (right). Scale bars, 10 μm. Data are the mean ± SEM from 8 independent experiments. ^*P < 0.05 and ^**P < 0.01 compared with the LFD group

Fig. 2

Dynamic analysis of transcriptome data A Volcano polt of different genes at different time points. B Heatmap showing the GSVA-enriched pathways related to lipid metabolism and mitochondrial function. C Venn plot showing the DEGs among groups. D Radial heatmap illustrating the expression of 46 DEGs obtained as in C. E The top 20 of bubble plot of KEGG enrichment for 46 DEGs

Fig. 3

PAOA-mediated intracellular lipid accumulation. A RT-qPCR shown the mRNA expression changes of ACACA in livers. B Western blot analysis of protein levels of ACACA in livers and protein expression was normalized to β-actin. C The effect of different concentrations of PAOA on cell viability after 24 h of incubation. D Representative images of oil red O staining of cells. Scale bars, 200 μm. E The mRNA level of ACACA in cells. Data are the mean ± SEM from 3 independent experiments. ^*P < 0.05 and ^**P < 0.01 compared with the LFD group or Control group

Fig. 4

A Representative image and the quantitative fluorescence intensity of Nile red staining. Blue recaptures the nucleus, Red presents the lipid droplet. Scale bars, 50 μm. B, C The content of TG\TC in cells. Data are the mean ± SEM from 3 independent experiments. Values with different letters are significantly different in the groups (^*P < 0.05 and ^**P < 0.01 compared with the Control group, ^#P < 0.05 and ^##P < 0.01 compared with the PAOA group)

Fig. 5

Inhibition of ACACA alleviated PAOA-induced intracellular oxidative stress A, B Representative image and the quantitative fluorescence intensity of ROS. Scale bars, 20 μm. C The activity of GSH in cells. Data are the mean ± SEM from 3 independent experiments. Values with different letters are significantly different in the groups (^*P < 0.05 and ^**P < 0.01 compared with the Control group, ^#P < 0.05 and ^##P < 0.01 compared with the PAOA group)

Fig. 6

Inhibition of ACACA ameliorates PAOA-induced intracellular mitochondrial dysfunction. A Representative image of MMP with high connotation live cell imaging. Blue recaptures the nucleus, Red presents JC-10 polymer and green presents JC-10 monomer. Scale bars, 50 μm. B The quantitative fluorescence intensity of JC-10. C The content of ATP in cells. Data are the mean ± SEM from 3 independent experiments. Values with different letters are significantly different in the groups (^*P < 0.05 and ^**P < 0.01 compared with the Control group, ^#P < 0.05 and ^##P < 0.01 compared with the PAOA group)

Fig. 7

Effect of ACACA on protein abundance of targets involved in lipid metabolism and mitochondrial dysfunction in cells. A, B Western blot analysis of protein in cells and protein expression was normalized to β-actin. Data are the mean ± SEM from 3 independent experiments. Values with different letters are significantly different in the groups (^*P < 0.05 and ^**P < 0.01 compared with the Control group, ^#P < 0.05 and ^##P < 0.01 compared with the PAOA group)

Fig. 8

ACACA reduces lipid accumulation through dual regulation of lipid metabolism and mitochondrial function via AMPK- PPARα- CPT1A axis