Upstream stimulating factor 1 suppresses autophagy and hepatic lipid droplet catabolism by activating mTOR

Abstract

Previous studies indicate that the transcription factor upstream stimulating factor 1 (USF1) is involved in the regulation of lipid and glucose metabolism. However, the role of USF1 in lipid-induced autophagy remains unknown. Interestingly, we found that USF1 overexpression suppresses autophagy-related gene expression in HepG2 cells. Further assays confirmed that USF1 could transcriptionally activate mTOR expression, thereby suppressing rapamycin-induced autophagy in HepG2 cells. Moreover, pharmacological activation of autophagy with rapamycin decreases the numbers and sizes of lipid droplets (LDs) in HepG2 cells exposed to an oleate/palmitate mixture. Of note, USF1 upregulation decreases colocalization of LDs and autophagosomes. In conclusion, our data provide evidence that USF1 contributes to abnormal lipid accumulation in the liver by suppressing autophagy via regulation of mTOR transcription.

Keywords: USF1; autophagy; lipid droplet catabolism; mTOR.

Figure 1

Characteristics of the 5‐week and 10‐week HFD‐fed animal model. (A,B) Measurement of the body and liver weights in the HFD‐fed mice (n = 5). (C) Oil red O and H&E staining in the livers of HFD‐fed mice (n = 5). Data represent the mean ± SEM, n = 5 mice. *P < 0.05; **P < 0.01 versus control. The bar represents 200 μm.

Figure 2

Expression changes in autophagy‐associated genes in the 5‐week and 10‐week HFD‐fed mice. (A,B,C and D) Real‐time PCR results showing the relative expression patterns of autophagy‐associated genes, including beclin1, Vps34, Atg12 and Atg4B in the livers of 5‐week and 10‐week HFD‐fed mice (n = 5). (E) Western blot assay was carried out to evaluate the expression of Beclin1 and p62 in the livers of 5‐week and 10‐week HFD‐fed mice (n = 3). Data represent the mean ± SEM, n = 5 mice. *P < 0.05; **P < 0.01 versus control.

Figure 3

Increased expression of USF1 in 10‐week HFD‐fed mice. Real‐time PCR and Western blots showing the expression of USF1 in the livers of 5‐week (A,B) and 10‐week (C,D) HFD‐fed mice (n = 5). Data represent the mean ± SEM, n = 5 mice. *P < 0.05; **P < 0.01 versus control.

Figure 4

Activation of USF1 inhibits autophagy in hepatocytes. (A,B) Real‐time PCR showing the relative expression patterns of autophagy‐associated genes, including Bnip3, Atg12, beclin1, Bnip3 l and Atg4b, in HepG2 cells transfected with Ad‐USF1 or siUSF1 (n = 6). (C) GFP‐LC3 transfection assay images showing the number of punctate structures. (D) Electron microscopy examination of the autophagosomes in HepG2 cells treated with rapamycin in the absence or presence of USF1. White arrows indicate autophagosomes. Data represent the mean ± SEM, n = 6 independent experiments. *P < 0.05; **P < 0.01 versus control. The bar represents 10 μm.

Figure 5

USF1 mediates autophagy by transcriptionally activating mTOR. (A, B) Western blots showing the expression of mTOR, p‐S6K, p‐S6/S6 and LC3‐II/LC3‐I in HepG2 cells transfected with Ad‐USF1 or siUSF1 (n = 6). (C) Schematic analysis of the conserved E‐box elements in the mTOR promoter region of USF1 in humans and mice. (D) Promoter reporter analysis and (E) ChIP assay in HepG2 cells transfected with Ad‐USF1 and siUSF1. (F) Western blot assay showed that the expression and activity of mTOR were increased in the livers of 10‐week HFD‐fed mice. Data represent the mean ± SEM, n = 6 independent experiments. *P < 0.05; **P < 0.01 versus control.

Figure 6

Increased USF1 promotes hepatic lipid accumulation. (A, B) Real‐time PCR and Western blot analyses of lipid metabolism genes after adenovirus‐mediated overexpression of USF1 in HepG2 cells. (C, D) Real‐time PCR and Western blot analyses of lipid metabolism genes in HepG2 cells transfected with siUSF1. Data represent the mean ± SEM, n = 6 independent experiments. *P < 0.05; **P < 0.01 versus control.

Figure 7

Suppression of autophagy by USF1 promotes lipid accumulation. (A,B and C) Immunofluorescence/BODIPY 493/503, Oil red O staining and electron microscopy images showing the LDs and autophagosomes in HepG2 cells. (D) Western blot analyses show enhanced LC3II/LC3I and decreased S6K phoshorylation changes after rapamycin treatment, but the effects were largely reversed by the upregulation of USF1. (E) Lipid levels in HepG2 cells transfected with USF1 in the presence or absence of rapamycin. (F) Relative lipid contents were determined in HepG2 cells treated with Fato in the presence or absence of Ad‐USF1. Data represent the mean ± SEM, n = 6 independent experiments. *P < 0.05; **P < 0.01 versus control. The bar represents 10 μm. White arrows indicate autophagosomes and red arrows indicate lipid droplets.