Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Nov 8;11(11):2702.
doi: 10.3390/nu11112702.

Curcumin Ameliorates Nonalcoholic Fatty Liver Disease Through Inhibition of O-GlcNAcylation

Affiliations
Free PMC article

Curcumin Ameliorates Nonalcoholic Fatty Liver Disease Through Inhibition of O-GlcNAcylation

Da Eun Lee et al. Nutrients. .
Free PMC article

Abstract

The cause of progression to non-alcoholic fatty liver disease (NAFLD) is not fully understood. In the present study, we aimed to investigate how curcumin, a natural phytopolyphenol pigment, ameliorates NAFLD. Initially, we demonstrated that curcumin dramatically suppresses fat accumulation and hepatic injury induced in methionine and choline-deficient (MCD) diet mice. The severity of hepatic inflammation was alleviated by curcumin treatment. To identify the proteins involved in the pathogenesis of NAFLD, we also characterized the hepatic proteome in MCD diet mice. As a result of two-dimensional proteomic analysis, it was confirmed that thirteen proteins including antioxidant protein were differentially expressed in hepatic steatosis. However, the difference in expression was markedly improved by curcumin treatment. Interestingly, eight of the identified proteins are known to undergo O-GlcNAcylation modification. Thus, we further focused on elucidating how the regulation of O-linked β-N-acetylglucosamine (O-GlcNAc) modification is associated with the progression of hepatic steatosis leading to hepatitis in MCD diet mice. In parallel with lipid accumulation and inflammation, the MCD diet significantly up-regulated hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) via ER stress. Curcumin treatment alleviates the severity of hepatic steatosis by relieving the dependence of O-GlcNAcylation on nuclear factor-κB (NF-κB) in inflammation signaling. Conversely, the expressions of superoxide dismutase 1 (SOD1) and SIRT1 were significantly upregulated by curcumin treatment. In conclusion, curcumin inhibits O-GlcNAcylation pathway, leading to antioxidant responses in non-alcoholic steatohepatitis (NASH) mice. Therefore, curcumin will be a promising therapeutic agent for diseases involving hyper-O-GlcNAcylation, including cancer.

Keywords: NAFLD and SOD1; O-GlcNAcylation; curcumin; liver.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of curcumin on lipid accumulation in methionine and choline-deficient (MCD) diet mice. Mice were fed a normal diet (ND), MCD diet, or MCD/curcumin (100 mg/kg) diet for 3 weeks. (A) Representative pictures of liver sections from these groups were assessed by hematoxylin-eosin (H&E) staining, Oil Red O staining, and immunohistochemical (IHC) analysis against carbohydrate response element-binding protein (ChREBP) (original magnification 200×). (B) and (C), Hepatic expression levels of phosphorylated AMP-activated protein kinase (p-AMPK), phosphorylated Liver kinase B1 (p-LKB1), and phosphorylated Acetyl-CoA carboxylase (p-ACC) along with ChREBP, Sterol regulatory element-binding protein (SREBP) and Fatty acid synthase (FAS) were measured by immunoblot assay. The levels of proteins were normalized to endogenous β-actin protein for each group. Data are mean ± SD (n = 7/group). * p < 0.05 vs. ND diet group, and # p < 0.05 vs. MCD diet group.
Figure 2
Figure 2
Curcumin modulated the nuclear factor-κB (NF-κB) pathway to alleviate inflammation. Liver tissues were prepared from the mice fed ND, MCD diet, or MCD/curcumin (100 mg/kg) diet. (A) The mRNA levels of hepatic Toll-like receptor 4 (TLR4), Tumor necrosis factor-alpha (TNF-α) and Interleukin 6 (IL-6) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). (B) Representative immunoblot analyses of phosphorylated IκB kinase α/β (p-IKKα/β), p-IκBα, and p-NF-κB p65. (C) Liver sections were subjected to immunoblot and IHC analyses (original magnification 200×) for α-smooth muscle actin (α-SMA). Data are mean ± SD (n = 7/group). * p < 0.05 vs. ND diet group, and # p < 0.05 vs MCD diet group.
Figure 3
Figure 3
Verification of differentially expressed proteins. Liver tissue extractions were supplied from mice fed ND, MCD diet, or MCD/curcumin (100 mg/kg) diet. (A) The hepatic protein expression levels of Cytokeratin 8 (CK8), Cytokeratin 18 (CK18), AnnexinA5 (ANXA5) and peroxiredoxin 6 (Prx6) were determined by immunoblotting using equally identified amounts of total liver protein by immunoblotting. The levels of proteins were normalized to endogenous β-actin protein for each group. Data were obtained three times of independent experiments. (B) Liver sections were analyzed by the IHC test with antibody against CK18 (original magnification 200×). (C) O-GlcNAc (O-linked β-N-acetylglucosamine) was immunoprecipitated with anti-O-GlcNAc antibody and then immunoblotted against CK8 and CK18, separately. Data are mean ± SD (n = 7/group). * p < 0.05 vs. ND diet group, and # p < 0.05 vs MCD diet group.
Figure 4
Figure 4
O-GlcNAcylation and the proteins associated with HBP. (A) Hepatic expression levels of inositol requiring enzyme 1α (IRE1α), O-GlcNAc transferase (OGT), O-GlcNAcase (OGA) and total O-GlcNAcylated proteins by immunoblot analysis in ND, MCD diet, or MCD/curcumin (100 mg/kg) diet mice. In the graphs, the intensity of proteins was determined by densitometry. Data were obtained from three times of independent experiments. (B) The levels of protein expression associated with the hexosamine biosynthetic pathway (HBP) were determined by qRT-PCR analysis. The levels of X-box binding protein 1 (XBP1) and glutamine:fructose-6-phosphate amidotransferase (GFAT1) mRNAs are shown as fold increase. Gene expression levels were normalized to 18s rRNA as an internal standard, and the data are presented as the means ± SD (n = 7/group). * p < 0.05 vs. ND diet group, and # p < 0.05 vs. MCD diet group. (C) The left panel shows that p65 immunoprecipitates were immunoblotted against acetyl lysine and O-GlcNAc, separately. Similarly, on the right panel, hepatic expression levels of ChREBP were measured, and ChREBP was also immunoprecipitated and then immunoblotted against O-GlcNAc.
Figure 5
Figure 5
Regulation of O-GlcNAcylation and antioxidant protein in AML12 cells. (A) AML12 cells were either untreated or pretreated with 250 μM palmitate (Pal) and incubated with curcumin (3 μM), OSMI-1 (20 μM), Azaserine (100 μM) or Thiamet-G (200 μM) for 12 h. The levels of ROS in cells were measured via DCFDA fluorescence. (B) AML12 cells were treated with Pal (250 μM) and different concentrations of curcumin (0.3 or 3 μM) to analyze the level of O-GlcNAcylated proteins by immunoblot assay. (C) The cells were either untreated or pretreated with 250 μM Pal and incubated with Azaserine (100 μM) or OSMI-1 (20 μM) for 12h. Levels of SIRT1 and SOD1 expression were measured by immunoblot assay. (D) AML12 cells were transfected with control or SIRT1 siRNA for 24 h, and then the cells were treated with or without OSMI-1 (20 μM), followed by incubation with 250 μM Pal for 12 h. Levels of IRE1α, SIRT1, and SOD1 expression were measured by immunoblot analysis. All expression levels were normalized relative to β-actin. The data from three independent experiments are presented as the means ± SD. * p < 0.05.
Figure 6
Figure 6
Curcumin prevents inflammation by inhibiting O-GlcNAcylation and activates antioxidant proteins. (A) Liver lysate was prepared from mice fed with ND, MCD diet, or MCD/curcumin (100 mg/kg) diet. Hepatic expression levels of SIRT1 or SOD1 were measured by immunoblot analysis. The levels of proteins were normalized to endogenous β-actin protein for each group. The relative amounts of the proteins are expressed as fold-increases. Data were obtained from three times of independent experiments. * p < 0.05 vs. ND diet group, and # p < 0.05 vs. MCD diet group. (B) IHC analysis of O-GlcNAcylation, SIRT1 (original magnification 400×), OGT, F4/80, or SOD1 (original magnification 200×) in livers of ND, MCD diet, or MCD/curcumin diet mice. Data are mean ± SD (n = 7/group). * p < 0.05 vs. ND diet group, and # p < 0.05 vs. MCD diet group. (C) The proposed representation showed that curcumin up-regulates antioxidant expression but down-regulates the NF-κB pathway by blocking O-GlcNAcylation signaling pathway. Curcumin-induced SOD1 down-regulates ROS stress and inflammatory signaling. Arrow and bar represent activation and suppression, respectively.

Similar articles

See all similar articles

References

    1. Perla F.M., Prelati M., Lavorato M., Visicchio D., Anania C. The Role of Lipid and Lipoprotein Metabolism in Non-Alcoholic Fatty Liver Disease. Children. 2017;4:46 doi: 10.3390/children4060046. - DOI - PMC - PubMed
    1. Mencin A.A., Lavine J.E. Advances in pediatric nonalcoholic fatty liver disease. Pediatr. Clin. N. Am. 2011;58:1375–1392. doi: 10.1016/j.pcl.2011.09.005. - DOI - PubMed
    1. Jump D.B., Tripathy S., Depner C.M. Fatty acid-regulated transcription factors in the liver. Annu. Rev. Nutr. 2013;33:249–269. doi: 10.1146/annurev-nutr-071812-161139. - DOI - PMC - PubMed
    1. Cho M.C., Lee K., Paik S.G., Yoon D.Y. Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders. PPAR Res. 2008;2008:679137. doi: 10.1155/2008/679137. - DOI - PMC - PubMed
    1. Angulo P. Nonalcoholic fatty liver disease. N. Engl. J. 2002;346:1221–1231. doi: 10.1056/NEJMra011775. - DOI - PubMed

MeSH terms

Feedback