Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation

https://doi.org/10.1016/j.bbrc.2015.03.095Get rights and content

Highlights

  • PPARγ promotes MGAT1 expression in human primary hepatocytes.

  • PPARγ directly regulates MGAT1 promoter activity.

  • Human MGAT1 promoter has at least two PPARγ-binding elements.

  • Inhibition of MGAT1 expression attenuates hepatic lipid accumulation in humans.

Abstract

Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans.

Introduction

With the rise in obesity, the incidence of fatty liver disease has gradually increased and is now a significant healthcare issue. Hepatic steatosis, characterized by an increase in intrahepatic triacylglycerol (TG), is an important marker of metabolic dysfunction and is associated closely with insulin resistance and dyslipidemia [1], [2]. Because hepatic steatosis appears in the early stage of metabolic disease, the early detection and management of the disease, as well as understanding the mechanism of hepatic steatosis, are extremely important. To date, the exact etiology of hepatic steatosis has not been defined, but transcription factors such as sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate response element-binding protein (ChREBP) that control the expression of lipogenic genes have been found to be important in hepatic lipid accumulation [3], [4].

In a past decade, peroxisome proliferator-activated receptor γ (PPARγ) has been described as a protein that is involved in hepatic steatosis [5], [6], [7]. PPARγ is a ligand-activated transcription factor that mainly controls adipogenesis [8]. Although PPARγ expression in hepatic tissue is low (about 10–30% of that in adipose tissue [9]), it is significantly increased in a rodent model of obesity and has an important role in fatty liver formation [6], [10], [11]. Under a high-fat diet, PPARγ stimulates the expression of genes involved in TG synthesis, such as adipose differentiation-related protein (ARDP) and fat-specific protein 27 (FSP27), and increases fatty acid uptake by the induction of fatty acid binding protein (FABP) and CD36 in the liver [12].

Previously, we investigated the role of PPARγ in hepatic steatosis, identifying monoacylglycerol O-acyltransferase 1 (MGAT1) as a novel therapeutic target gene for hepatic steatosis in mice [10]. Subsequently, other groups also reported the beneficial metabolic effects of hepatic MGAT1 suppression in mouse models [13], [14]. MGAT is an enzyme that catalyzes monoacylglycerol (MAG) and fatty acyl CoAs to form diacylglycerol (DAG), which is then acylated to form TG by diacylglycerol acyltransferase (DGAT) [15]. In mice, there are two subtypes of MGATs (MGAT1 and MGAT2), but only MGAT1 is associated with hepatic steatosis [10]. However, in humans, there is one more subtype, designated MGAT3, which is mainly expressed in gastrointestinal tracts along with MGAT2 [16]. Interestingly, the mouse gene corresponding to human MGAT3 has not been found; it appears to be a pseudogene [17]. It was reported that the human liver exhibits MGAT activity; the expression levels of MGAT2 and MGAT3 are higher than that of MGAT1, and the hepatic expression of MGAT3 is highly correlated with total MGAT activity [18]. However, the regulation of the expression of each subtype of MGAT, and its association with diet-induced non-alcoholic fatty liver diseases, remain uncertain. For these reasons, although the inhibition of MGAT1 expression resulted in a dramatic reduction of hepatic steatosis in mouse models, it is important to investigate whether human MGATs are regulated under conditions of pathological lipid accumulation, in order to develop a therapeutic agent.

Therefore, we investigated the differences among MGATs gene and their roles in human hepatocyte models. In particular, we focused on the responsiveness of the MGAT gene by PPARγ that is known as a regulator of murine MGAT1 gene. In this study, we report that among the MGAT family, MGAT1 is the most sensitive target gene of PPARγ, suggesting its role in human fatty liver formation as well as its potential as a target for therapeutics.

Section snippets

Analysis of gene expression by quantitative reverse transcription polymerase chain reaction (RT-PCR)

Human primary hepatocytes were purchased from BD Biosciences (Metabolism-Qualified Cat. No.454543), and RNA was isolated using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. For quantitative RT-PCR, cDNA was synthesized from 5 μg of total RNA using a random hexamer and SuperScript reverse transcriptase III (Invitrogen). RT-PCR was performed using the following primers: MGAT1, 5′-CTCGG GCCGA TGTCC ATTGG A-3′, 5′-GGGTA TGCCA GTCAA AGTAA AGC-3′; MGAT2, 5′-CCTTC GGGGA

Human MGAT1 is upregulated by PPARγ in human primary hepatocytes

MGAT genes show a variety of subtypes in different species. In human, unlike in mouse or rat, there are three subtypes of MGAT enzymes, MGAT1, MGAT2, and MGAT3 (Fig. 1A). In a phylogenetic tree with inferred evolutionary relationships, MGAT3 shares higher sequence homology with the diacylglycerol O-acyltransferase 2 (DGAT2) enzyme than with MGAT1 or MGAT2 [19]. It is considered that MGAT3 has a significant DGAT activity [18], [19], suggesting that it plays a different role in lipid-accumulating

Discussion

Metabolic syndrome is characterized by abdominal obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density cholesterol levels, and is closely associated with diabetes mellitus and cardiovascular disease. Hepatic steatosis is defined as intrahepatic TG content with 5% or more of liver volume or liver weight, and appears in the early phase of metabolic syndrome [23]. Because hepatic lipid accumulation affects systemic insulin sensitivity and

Conflict of interest

None.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) Grants 2011-0030086, funded by the Korean government, Ministry of Science, ICT and Future Planning (MSIP).

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