Changes of flavin-containing monooxygenases and trimethylamine-N-oxide may be involved in the promotion of non-alcoholic fatty liver disease by intestinal microbiota metabolite trimethylamine

doi:10.1016/j.bbrc.2022.01.060

Biochemical and Biophysical Research Communications

Volume 594, 26 February 2022, Pages 1-7

https://doi.org/10.1016/j.bbrc.2022.01.060 Get rights and content

Highlights

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Gene differential expression analysis showed that FMO1 played an important role in the development of NAFLD.
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High fat induced the expression of FMO1 and its metabolite TMAO.
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TMA could induce the expression of FMO1 and TMAO.
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The effect of high fat and TMA on TMAO could be blocked by FMO1-siRNA.
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TMAO may act through activating UPR.

Abstract

Evidence shows that trimethylamine (TMA)/trimethylamine-N-oxide (TMAO) is closely related to non-alcoholic fatty liver disease (NAFLD). The conversion of TMA to TMAO is mainly catalyzed by flavin-containing monooxygenases 3 (FMO3) and FMO1. In this study, we explored the role of TMA in the process of NAFLD. The human NAFLD liver puncture data set GSE89632 and rat TMAO gene chip GSE135856 was downloaded for gene differential expression analysis. Besides, oleic acid (OA) combined with palmitate were used to establish high-fat cell model. TMA, TMAO and FMO1-siRNA were used to stimulate L02 cells. Contents of free fatty acid (FFA), triglyceride (TG), TMAO, FMO1 and unfolded protein response (UPR) related proteins GRP78, XBP1, Derlin-1 were detected. Our results showed that FMO1 and PEG10 were important in the progression of NAFLD. Immunohistochemistry showed that FMO1 in NAFLD liver was increased. In addition, the contents of FFA, TG, FMO1 expression, and TMAO were significantly increased after OA + palmitate and TMA stimulation. However, after silencing FMO1 with siRNA, the expressions of these molecules were decreased. Besides, the protein levels of GRP78, XBP1, Derlin-1 were increased after TMAO treatment (all P < 0.05). In Conclusion, high fat and TMA could induce the expression of FMO1 and its metabolite TMAO. When FMO1 is silenced, the effects of high fat and TMA on TMAO are blocked. And the role of TMAO in NAFLD may be through the activation of UPR.

Introduction

Non-alcoholic fatty liver disease (NAFLD) refers to the excessive accumulation of lipid components in liver caused by non-alcoholic factors [1]. The disease spectrum of NAFLD includes non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), cirrhosis, and hepatocellular carcinoma. Intestinal microbiota can break down substances such as choline and L-carnitine in the diet to produce trimethylamine (TMA), which is oxidized to trimethylamine-N-oxide (TMAO) [2]. TMAO is related to metabolic diseases such as diabetes, obesity and NAFLD. Study has confirmed that circulating TMAO level is related to obesity in mice, and inhibition of flavin-containing monooxygenases 3 (FMO3) can reduce the weight of white adipose tissue in mice [3].

The conversion of TMA to TMAO is mainly catalyzed by FMO3 and FMO1 [4]. More than 95% of TMA in the body is excreted in urine in the form of TMAO [2]. The FMO family of humans contains five members FMO1 to FMO5 [5]. The expression of FMOs in liver shows differences in age, sex and species: FMO1 is dominant in human fetal liver, and is inhibited after birth, while FMO5 and FMO3 are dominant in adult liver, FMO1 expression is low [6]. FMO1 is mainly expressed in the liver of male mice, while FMO3 is mainly expressed in female mice [6]. FMO family is mainly involved in the oxidation of drugs or exogenous substances containing weak nucleophiles [7].

The endoplasmic reticulum (ER) is very sensitive to various stimuli. Unfolded or misfolded proteins can aggregate in ER, activating unfolded protein response (UPR), which causes endoplasmic reticulum stress (ERS) [8]. UPR is an adaptive pathway that activates specific protein folding and degradation pathways to restore ER homeostasis [9]. When ER homeostasis cannot be restored, UPR induces changes in cell function or apoptosis [10]. Studies have reported that URP of hepatocytes is closely related to NAFLD [11]. Therefore, both TMAO and UPR can cause lipid metabolism disorders and inflammation in the liver, but the relationship between TMAO and UPR remains unclear.

In this study, we explored the effect of TMA and FMO1 in NAFLD by comparing the difference in gene expression of human NAFLD liver puncture data and rat TMAO gene chip, and establishing a high-fat cell model. TMA, TMAO and FMO1-siRNA were used to explore the mechanism of NAFLD, aim to provide a new idea for the prevention and control of NAFLD.

Section snippets

Chemicals and reagents

Fetal bovine serum (FBS) and DMEM medium were purchased from Gibco (USA). Oleic acid (OA), palmitate, TMA (AR) and TMAO were obtained from Aladdin (China). The cell counting kit 8 (CCK8) was obtained from Dojindo Laboratories (Japan). Free fatty acid (FFA) and triglyceride (TG) detect kits were obtained from Solarbio (China). FMO1 antibody was purchased from Santa Cruz Biotechnology (USA, sc-376924). β-actin, GRP78, XBP1 antibodies were obtained from Proteintech (China) and Derlin-1 antibody

NAFLD samples showed significant differences in gene expression

We downloaded the human NAFLD liver puncture data set GSE89632 from the GEO database, containing 63 liver puncture samples, in which included 24 normal samples, 20 NAFL samples and 19 NASH samples. Differential expression analysis was performed using the limma package of R language. P < 0.05 and |log2FC| ≥1 were used as the threshold value. The results showed that NASH group had 411 differentially expressed genes (DEGs), including 163 up-regulated genes and 248 down-regulated genes. Compared

Discussion

Studies have reported that TMA can be oxidized to TMAO [13]. TMAO is closely related to NAFLD, diabetes and other metabolic diseases. A study finds that the serum TMAO level of NAFLD patients is about 3.9 times higher than that of normal people [14]. The addition of TMAO in the drinking water of mice can lead to fat accumulation in liver [14]. TMAO can also induce inflammation by activating NLRP3 inflammasomes [15]. TMAO plays an important role in metabolism and inflammation, and FMO1 is an

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (82100603).

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¹: These authors contributed equally to this work.

View full text

Changes of flavin-containing monooxygenases and trimethylamine-N-oxide may be involved in the promotion of non-alcoholic fatty liver disease by intestinal microbiota metabolite trimethylamine

Highlights

Abstract

Introduction

Section snippets

Chemicals and reagents

NAFLD samples showed significant differences in gene expression

Discussion

Declaration of competing interest

Acknowledgments

Cell Rep.

Cell Metabol.

Pharmacol. Ther.

J. Hepatol.

Biochem. Pharmacol.

Biochem. Biophys. Res. Commun.

J. Lipid Res.

Biochem. Pharmacol.

Pharmacol. Ther.

J. Hepatol.