Downregulation of PHGDH expression and hepatic serine level contribute to the development of fatty liver disease

Highlights

• PHGDH is an enzyme catalyzing the committed step of serine biosynthesis pathway.

• Hepatic expression of PHGDH and serine concentration are decreased in steatosis animal models.

• There is a significant negative correlation between the serine concentration and liver fat fraction in patients with NAFLD.

• Transgenic overexpression of PHGDH in mice attenuates HFD-induced fatty liver accompanied by increased SIRT1 activity.

Abstract

Objective

Supplementation with serine attenuates alcoholic fatty liver by regulating homocysteine metabolism and lipogenesis. However, little is known about serine metabolism in fatty liver disease (FLD). We aimed to investigate the changes in serine biosynthetic pathways in humans and animal models of fatty liver and their contribution to the development of FLD.

Methods

High-fat diet (HFD)-induced steatosis and methionine-choline-deficient diet-induced steatohepatitis animal models were employed. Human serum samples were obtained from patients with FLD whose proton density fat fraction was estimated by magnetic resonance imaging. 3-Phosphoglycerate dehydrogenase (Phgdh)-knockout mouse embryonic fibroblasts (MEF) and transgenic mice overexpressing Phgdh (Tg-phgdh) were used to evaluate the role of serine metabolism in the development of FLD.

Results

Expression of Phgdh was markedly reduced in the animal models. There were significant negative correlations of the serum serine with the liver fat fraction, serum alanine transaminase, and triglyceride levels among patients with FLD. Increased lipid accumulation and reduced NAD⁺ and SIRT1 activity were observed in Phgdh-knockout MEF and primary hepatocytes incubated with free fatty acids; these effects were reversed by overexpression of Phgdh. Tg-Phgdh mice showed significantly reduced hepatic triglyceride accumulation compared with wild-type littermates fed a HFD, which was accompanied by increased SIRT1 activity and reduced expression of lipogenic genes and proteins.

Conclusions

Human and experimental data suggest that reduced Phgdh expression and serine levels are closely associated with the development of FLD.

Introduction

Non-alcoholic fatty liver disease (NAFLD) has become one of the most common liver diseases, affecting up to 25% of the population in Western countries [1]. The incidence of this disease is much higher among obese subjects [2]. The spectrum of the disease extends from simple steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis. NASH-related hepatocellular carcinoma is a major indication for liver transplantation in the United States [3]. Chronic excessive use of alcohol can lead to alcohol-related liver disease (ALD), one of the most important public health problems worldwide. Although the pathogenic mechanisms of ALD and NAFLD are not completely understood, they share common factors such as oxidative stress, mitochondrial and endoplasmic reticulum stress, as well as systemic inflammatory mediators [[4], [5], [6]]. Hepatic lipid accumulation caused by abnormal lipid metabolism is the earliest phenotype of both NAFLD and ALD. Hepatocellular lipid metabolism is tightly regulated by complex metabolic processes, including hepatic lipid uptake, oxidation and excretion, and de novo lipid synthesis and degradation [7,8]. Intrinsic or extrinsic factors affecting any of these processes can lead to the development of fatty liver disease (FLD).

Serine plays an important role in cellular metabolic processes that link glycolysis with one-carbon metabolism to maintain redox balance and proliferation. Serine is synthesized de novo in mammalian cells by channeling the glycolytic intermediate 3-phosphoglycerate into the serine synthesis pathway. This pathway is catalyzed by 3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH), with PHGDH playing a key role [9]. Changes in PHGDH enzyme activity or gene expression have been reported in some diseases. Upregulation of PHGDH and serine synthesis pathway flux have been observed in many tumors in which PHGDH activity is required for proliferation and survival [10]. Expression of enzymes of the serine synthesis pathway, including PHGDH, is also increased in the lungs of humans with pulmonary fibrosis to promote collagen production [11]. A systems biology approach, based on the employment of genome-scale metabolic models of hepatocytes, proposed that the serine deficiency in NASH is partly due to downregulation of genes involved in serine biosynthesis [12]. However, no experimental or clinical studies have been conducted with regard to the questions whether PHGDH expression or serine metabolism is altered in FLD, or how their alteration would affect development of the disease.

Activating transcription factor 4 (ATF4) and the protooncogene c-Myc are anabolic transcription factors responsible for the expression of genes involved in the uptake and synthesis of certain nonessential amino acids [13,14]. Expression of Phgdh is regulated transcriptionally by ATF4 and c-Myc. Recently, nuclear factor erythroid 2-related factor (NRF2) was found to regulate the gene expression of key serine/glycine biosynthesis enzymes, including Phgdh, Psat1, and Shmt2, via ATF4 to promote glutathione and nucleotide production [15]. Changes in NRF2 activity in FLD are variable. A high-fat diet (HFD) and methionine-choline-deficient (MCD) diet induce oxidative stress and inflammation, resulting in activation of NRF2, which is responsible for adaptation to oxidative stress and protection against NASH. Therefore, mice lacking NRF2 develop more severe NASH than do wild-type mice fed a HFD [16,17]. On the other hand, HFD- or alcohol-induced downregulation of Nrf2 and its target genes contributes to the development of FLD by lowering the thresholds for the unfolded protein response and inflammation, increasing lipogenesis, and inhibiting the ability to protect against oxidative stress [18,19]. AMPK-NRF2 axis is an important intracellular signaling pathway leading to ARE-dependent transcriptional activation. AMPK induces nuclear translocation of NRF2 in several mechanisms. AMPK phosphorylates NRF2 directly at the Ser558 residue located in the canonical nuclear export signal. Therefore, mutation of Nrf2 (S550A) or the treatment with nuclear export inhibitor fails to accumulate NRF2 in the nucleus following AMPK activation [20]. Indirect activation mechanisms are better understood. Activation of AMPK induces Akt-mediated inhibitory phosphorylation of GSK3β at Ser9 and consequently NRF2 activation by nuclear translocation [21]. Therefore, reduced AMPK activity in FLD suppresses NRF2 nuclear translocation, which is required for gene transactivation mediated by antioxidant response elements [22].

We and others have reported that supplementation with serine attenuates the development of FLD by activating homocysteine metabolism and decreasing the production of reactive oxygen species via the glutathione antioxidant system [23,24]. In this study, we investigated the role of serine metabolism in the development of FLD and found that hepatic expression of Phgdh and the serine concentration were decreased in animal models of FLD. There was a significant negative correlation between the serum serine concentration and liver fat fraction in patients with NAFLD. An AMPK-dependent decrease in the expression and nuclear translocation of NRF2 was responsible for the downregulation of PHGDH. Transgenic mice overexpressing Phgdh (Tg-Phgdh) were resistant to HFD-induced fatty liver, which was accompanied by increased SIRT1 activity and reduced expression of genes and proteins associated with lipogenesis. We conclude that reduced PHGDH expression and serine concentration are closely associated with the development of FLD. This suggests that serine metabolism is critical in maintaining cellular homeostasis of hepatic lipid metabolism following metabolic challenge.