Distinct regulation of adiponutrin/PNPLA3 gene expression by the transcription factors ChREBP and SREBP1c in mouse and human hepatocytes

Background & Aims

The adiponutrin/PNPLA3 (patatin-like phospholipase domain-containing protein 3) variant I148M has recently emerged as an important marker of human fatty liver disease. In order to understand the role of the adiponutrin/PNPLA3 protein, we investigated the regulation of its expression in both human and mouse hepatocytes.

Methods

Adiponutrin/PNPLA3 and lipogenic enzyme expression was determined by real-time PCR analysis in a wide panel of analysis in vivo in the mouse liver and in vitro in murine hepatocytes and human hepatocyte cell lines infected with ChREBP or SREBP1c-expressing adenoviruses.

Results

We show that in the mouse liver, adiponutrin/PNPLA3 gene expression is under the direct transcriptional control of ChREBP (carbohydrate-response element-binding protein) and SREBP1c (sterol regulatory element binding protein1c) in response to glucose and insulin, respectively. In silico analysis revealed the presence of a ChoRE (carbohydrate response element) and of a SRE (sterol response element) binding site on the mouse adiponutrin/PNPLA3 gene promoter. Point mutation analysis in reporter gene assays identified the functional response of these two binding sites in the mouse adiponutrin/PNPLA3 promoter. In contrast, in human immortalized hepatocytes and in HepG2 hepatoma cells, only SREBP1c was able to induce adiponutrin/PNPLA3 expression, whereas ChREBP was unable to modulate its expression.

Conclusions

All together, our results suggest that adiponutrin/PNPLA3 is regulated by two key factors of the glycolytic and lipogenic pathways, raising the question of its implication in the metabolism of carbohydrates and lipids.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease in western countries [1]. It includes a large panel of liver disorders ranging from benign fatty liver (hepatic steatosis) to a progressive form with an inflammatory response known as steatohepatitis or NASH, and may ultimately evolve in fibrosis and cirrhosis [2]. NAFLD is a component of the metabolic syndrome and is associated with obesity and type-2 diabetes [2], [3]. Although the mechanisms leading to NAFLD are still unclear, the dysregulation of lipid metabolism was involved [4]. It was estimated that 30% of the TG content in NAFLD livers came from de novo lipogenesis, underlying the importance of this pathway in the etiology of NAFLD [5], [6].

Hepatic de novo lipogenesis is controlled by transcription factors SREBP1c (Sterol regulatory Element-binding protein1c) and ChREBP (Carbohydrate response Element-Binding Protein), which mediate, respectively, the genic effects of insulin and glucose on glycolytic and lipogenic genes [7], [8], [9], [10]. Insulin enhances SREBP1c gene expression and its cleavage, therefore, favoring its binding to sterol-response element (SRE) in the promoters of target genes [7]. On the other hand, ChREBP acts by forming a heterotetramer with Max-like protein X (Mlx) resulting in an efficient binding to ChoRE sequences and functional activity [11], [12]. Inhibition of ChREBP or SREBP1c in the liver of obese ob/ob mice leads to an improvement of hepatic steatosis [13], [14], supporting their role as key determinants of the lipogenic pathway under both physiological and pathophysiological conditions.

Recently, the adiponutrin/PNPLA3 gene has emerged as a new marker of human hepatic steatosis [15]. Initially discovered in the adipose tissue, adiponutrin/PNPLA3 is regulated by the nutritional status in mouse. Its expression is decreased upon fasting and induced upon high-carbohydrate diet feeding [16], [17], [18], [19]. Glucose and insulin regulate adiponutrin/PNPLA3 expression in mouse adipocytes and the human adipose tissue [16], [20], [21], [22]. Adiponutrin/PNPLA3 belongs to a large family of PNPLA (patatin-like Phospholipase domain containing) enzymes [23], [24], which share a common “patatin-like” domain, harboring a lipase/esterase activity. Among them, ATGL/PNPLA2 (Adipose Triglyceride Lipase), which is the closest protein related to adiponutrin/PNPLA3, increases TG hydrolysis and fat mobilization in adipose cells, a function that remains uncertain for adiponutrin/PNPLA3 [24]. A single nucleotide polymorphism (SNP) I148M in the adiponutrin/PNPLA3 gene was associated with increased hepatic fat in different ethnic groups susceptible to NAFLD [25] and with an increased liver inflammation [25], [26]. Lastly, overexpression of this variant in human hepatoma cells leads to increased TG accumulation [27], suggesting that adiponutrin/PNPLA3 could participate in liver lipid homeostasis. Considering these findings, we aimed at investigating the molecular mechanisms controlling adiponutrin/PNPLA3 expression in both mouse liver and human hepatocyte cell lines.