Nutr Res Pract.  2019 Jun;13(3):196-204. 10.4162/nrp.2019.13.3.196.

Free fatty acid-induced histone acetyltransferase activity accelerates lipid accumulation in HepG2 cells

• Chung S
• Hwang JT
• Park JH
• Choi HK

• KMID: 2453283
• DOI: http://doi.org/10.4162/nrp.2019.13.3.196

Abstract

BACKGROUND/OBJECTIVES
Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disease triggered by epigenetic alterations, including lysine acetylation at histone or non-histone proteins, affecting the stability or transcription of lipogenic genes. Although various natural dietary compounds have anti-lipogenic effects, their effects on the acetylation status and lipid metabolism in the liver have not been thoroughly investigated.
MATERIALS/METHODS
Following oleic-palmitic acid (OPA)-induced lipid accumulation in HepG2 cells, the acetylation status of histone and non-histone proteins, HAT activity, and mRNA expression of representative lipogenic genes, including PPARγ, SREBP-1c, ACLY, and FASN, were evaluated. Furthermore, correlations between lipid accumulation and HAT activity for 22 representative natural food extracts (NExs) were evaluated.
RESULTS
Non-histone protein acetylation increased following OPA treatment and the acetylation of histones H3K9, H4K8, and H4K16 was accelerated, accompanied by an increase in HAT activity. OPA-induced increases in the mRNA expression of lipogenic genes were down-regulated by C-646, a p300/CBP-specific inhibitor. Finally, we detected a positive correlation between HAT activity and lipid accumulation (Pearson's correlation coefficient = 0.604) using 22 NExs.
CONCLUSIONS
Our results suggest that NExs have novel applications as nutraceutical agents with HAT inhibitor activity for the prevention and treatment of NAFLD.

Keyword

Histone acetyltransferases; lipogenesis; non-alcoholic fatty liver disease; lipid metabolism; HepG2 cell

MeSH Terms

Acetylation
Dietary Supplements
Epigenomics
Hep G2 Cells*
Histone Acetyltransferases*
Histones*
Lipid Metabolism
Lipogenesis
Liver
Lysine
Metabolic Diseases
Non-alcoholic Fatty Liver Disease
RNA, Messenger
Sterol Regulatory Element Binding Protein 1
Histone Acetyltransferases
Histones
Lysine
RNA, Messenger
Sterol Regulatory Element Binding Protein 1

Figure

• 

  Fig. 1 Oleic and Palmitic acids (OPA)-induced activation of HAT increased non-histone and histone acetylation. (A), (B). Total acetylation of non-histone proteins and histone actylation were increased following OPA treatment. HepG2 cells were treated with either 1X or 2X OPA for 24 hr, in the presence or absence of TSA, HDAC inhibitor. Either total proteins or histone proteins were extracted from the cells, and the acetylation status was determined as the indicated antibodies. (B). (C). The HAT activity was measured in an NE from HepG2 cells. Activity in the OPA-treated group is presented as a percentage relative to that in the control group, which was incubated without OPA. The values presented are the means ± SD of three independent experiments. *P < 0.05 (left panel). To confirm nuclear fraction, western blotting was carried out. HDAC1 and tubulin were used as nuclear and cytosolic markers, respectively (right panel). 1XOPA, 400 µM OA+100 µM PA; 2XOPA, 800 µM OA+200 µM PA; TSA, trichostatin A; HDAC, histone deacetylases.

• 

  Fig. 2 mRNA expression of lipogenic genes is dependent on HAT activity. (A) mRNA expression of genes related lipid metabolism was measured by qRT-PCR. Oleic and palmitic acid (OPA) was treated for 24hr in HepG2 cells, RNA was extracted, and then 2 µg RNA was synthased to cDNA. All experiments were carried out three times independently and data are expressed as mean ± SD. *P < 0.05 (student t-test). (B) C-646, a specific inhibitor of p300/CBP, blocked the OPA-induced mRNA expression of lipogenic genes. HepG2 cells were treated with OPA in the presence or absence of C-646 for 18 h. mRNA expression of ACLY, FASN, SREBP-1c, and PPARγ was measured by qRT-PCR. The values presented are the means±SD of three independent experiments. Data are expressed as mean±SD. *P < 0.05 (student t-test). PPAR-γ, peroxisome proliferator-activated receptor-γ; SREBP1c, sterol regulatory element-binding protein-1c; ACLY, ATP citrate lyase; FASN, fatty acid synthase.

• 

  Fig. 3 Effect of natural extracts (NExs) on lipid accumulation in HepG2 cells. (A) HepG2 cells were treated oleic acid only or co-treated with oleic acid and NExs for 24h. Cells were stained with Oil red O staining as described in Materials and Methods and were viewed by microscopy (upper panel). Quantitative Oil red O dye as fat drops and data are expressed as mean ± SD. ##control group, **P < 0.01, *P < 0.05 (student t-test) (low panel). (B) Cytotoxicity test of NExz in HepG2 cells. HepG2 cells were treated with the various NExs in the presence or absence of oleic and palmitic acids (OPA) for 24 h. The cell viability was measured using a WST-1 assay in HepG2 cells. All data are expressed as mean ± SD from the samples of each group. There was no statistical significance.

• 

  Fig. 4 Effect of natural extracts (NExs) on histone acetyl transferase activity. (A) HAT activity assays were performed with NExs as described in Materials and Methods. The result was represented with relative percentage compared to a positive control which was incubated without a NEx. Values are means ± SD for three independent experiments. ##control group, **P < 0.01, *P < 0.05 (student t-test). Epigallocatechin gallate (EGCG) was used as a negative control.

• 

  Fig. 5 There are positive correlation between lipid accumulation and HAT activity. To validate the correlation between HAT activity and lipid accumulation, Pearson's correlation value was calculated based on the experimental data from Oil red O (Fig. 3A) and HAT assay (Fig. 4) following NExs treatment with oleic and palmitic acids (OPA). Pearson's correlation value is 0.604.

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