LASS2 regulates hepatocyte steatosis by interacting with NDUFS2/OXPHOS related proteins

https://doi.org/10.1016/j.bbrc.2020.02.166Get rights and content

Highlights

  • LASS2 play a protective role in FFAs-induced hepatocellular steatosis.

  • LASS2 regulate hepatic steatosis via AMPK activation.

  • LASS2 might interact with NDUFS2/OXPHOS related proteins.

  • The production of mtROS may be related to the interaction between LASS and NDUFS2.

Abstract

LAG1 longevity assurance homolog 2 (LASS2), a highly conserved transmembrane protein, has been reported to be associated with nonalcoholic fatty liver disease (NAFLD). However, the effect of LASS2 on energy homeostasis and its mechanism remains unknown. In this study, we found lower expression levels of LASS2 in the livers of mice with liver steatosis induced by a high-fat diet (HFD) and free fatty acids (FFAs)-treated hepatocytes. In FFAs-treated Hepa1-6 cells and mouse primary hepatocytes (MPHs), LASS2 overexpression significantly decreased intracellular lipid content compared with the control cells. LASS2 overexpression also significantly upregulated lipolysis-related proteins, such as ATGL and HSL, and inhibited lipogenesis-related proteins, such as SREBP1 and FAS. In addition, the phosphorylation levels of AMPK and ACC increased significantly. On the contrary, LASS2 knockdown in FFAs-treated hepatocytes aggravated lipid accumulation via facilitating lipogenesis and inhibiting lipolysis. Further, co-IP and LC-MS analysis found that LASS2 might interacted with NDUFS2 to inhibit lipogenesis. The production of mitochondrial reactive oxygen species (mtROS) may be related to the interaction between LASS2 and NDUFS2. Collectively, we are the first time to showed that LASS2 might promote the phosphorylation of AMPK via mtROS produced by interaction with NDUFS2/OXPHOS, thus inhibiting lipogenesis.

Introduction

There is now increasing evidence that nonalcoholic fatty liver disease (NAFLD) is not only a prevalent chronic liver disease worldwide, but associated with other metabolic diseases [1]. For example, NAFLD can increase the risk of type 2 diabetes mellitus (T2DM) [2], and can also develop into cirrhosis or hepatocellular carcinoma [3]. It has also been reported that NAFLD increases the risk of cardiovascular disease [4] and chronic kidney disease [5]. With the rising prevalence of NAFLD and its related disease, the global medical and economic burden is greatly increased. Although in the past few decades, much widespread attention has focused on NAFLD, and there have been many studies, but its pathogenesis remains unclear, and further research is needed.

Ceramides is a member of the sphingolipids family, which can promote cell proliferation, differentiation and apoptosis, and play an crucial role in maintaining cell homeostasis [6]. De novo ceramide is catalyzed by ceramide synthases (CerSs), which is a key enzyme of sphingolipid metabolism [7]. CerSs, also known as LAG1 longevity assurance homolog (LASS), contains six isoforms (CerS1∼6 or LASS1∼LASS6) in mammals [8], each of which has different fatty acid (FA) substrate preferences [9]. CerS2/LASS2, synthesizing C22–C24 ceramides, is far more abundant in the liver than other isoforms. LASS2 displays complex regulation patterns and has the genomic characteristics of the “housekeeper” gene, which is not found in other members of the family [8,10]. Previous studies have demonstrated that LASS2 is involved in lipid metabolism, insulin resistance (IR), hepatic steatosis [8,11], oxidative stress [12], and endoplasmic reticulum stress [13], all of which are related to the occurrence and development of NAFLD. However, the role of LASS2 in the NAFLD remains controversial. Recently, Bauer et al. reported that Drosophila CerS shlank (Drosophila encodes for only one CerS gene) might have a double role as an enzyme and transcriptional regulator, sensing lipid levels and regulating genes related lipid metabolism according to energy requirements [14]. In addition, they indicated that LASS2/CerS2 seems to have similar ability in mammals [14], However, it has not been confirmed. Therefore, we speculated whether LASS2, as a transmembrane protein, is involved in the regulation of lipid metabolism or energy homeostasis in addition to the role of enzyme. In the present study, we reported for the first time that LASS2 play a protective role in FFAs-induced hepatocellular steatosis via regulating AMP-activated protein kinase (AMPK) activation. The mechanism underlying may be an increase in mtROS mediated by binding of NDUFS2/OXPHOS related proteins.

Section snippets

Animal study

Six-week old male C57BL/6J, ob/ob, db/db, KK-Ay, APOE−/- mice were purchased from BeiJing HFK Bioscience co., LTD. Mice were maintained on a standard low-fat chow diet (CHOW, fat, 10%; protein, 20%; carbohydrates, 70%) for one week adaptive feeding with a 12-h light/dark cycle. C57BL/6J mice were fed either a CHOW diet (CHD) or High-fat diet (HFD, protein, 20%; fat, 45%; carbohydrates, 35%) for 16 weeks. The tissues were obtained and stored at −80 °C for further analysis.

Adenoviruses mediated expression vectors

Adv-mLASS2-GFP and

Down-regulation of LASS2 expression in liver tissue of mice with hepatosteatosis and hepatocytes with steatosis

To characterize the role of LASS2 on lipid metabolism, we evaluated the LASS2 expression in the liver of mice with hepatosteatosis and hepatocytes with steatosis. The LASS2 protein relative levels of APOE−/- mice, ob/ob mice, db/db mice, KK-Ay mice were reduced by 68%, 32%, 39%, and 65%, respectively, compared to C57BL/6J mice (Fig. 1A), and LASS2 mRNA levels were also reduced in obese mice (Fig. 1B). We further discovered the LASS2 protein levels, but not mRNA levels in liver of C57BL/6J mice

Discussion

In recent years, LASS2, as an enzyme involved in de novo ceramides, has been paid more attention on lipid metabolism disorder. Although there are controversies, current studies have shown that LASS2 play an important role in hepatic steatosis [8,11,22,23]. Some studies have found that LASS2-induced specific ceramides with long-acyl chains affect insulin signaling or lipid metabolism [[24], [25], [26]]. As a transmembrane protein, whether LASS2 regulates lipid metabolism by interacting with

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by research grants from the National Natural Science Foundation of China (grant no. 81960494, 81570752).

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