TM6SF2 E167K variant decreases PNPLA3-mediated PUFA transfer to promote hepatic steatosis and injury in MASLD

Article information

Clin Mol Hepatol. 2024;30(4):863-882

Publication date (electronic) : 2024 July 26

doi : https://doi.org/10.3350/cmh.2024.0268

Baokai Sun ¹^,^*, Xiaoqian Ding ¹^,^*, Jie Tan ¹, Jie Zhang ¹, Xueru Chu ¹, Shuimi Zhang ¹, Shousheng Liu ², Zhenzhen Zhao ², Shiying Xuan^,¹

, Yongning Xin^,¹

, Likun Zhuang^,³

¹Department of Infectious Diseases, Qingdao Municipal Hospital, Qingdao University, Qingdao, China

²Clinical Research Center, Qingdao Municipal Hospital, Qingdao University, Qingdao, China

³Central Laboratories, Qingdao Municipal Hospital, Qingdao University, Qingdao, China

Corresponding author : Likun Zhuang Central Laboratories, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China Tel: +86-053288905295, Fax: +86-053288905295, E-mail: zlk0823@163.comz

Yongning Xin Department of Infectious Diseases, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China Tel: +86-053267757325, Fax: +86-053267757325, E-mail: xinyongning9812@163.com

Shiying Xuan Department of Infectious Diseases, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China Tel: +86-053288905831, Fax: +86-053288905831, E-mail: xuanshiyingshili@163.com

*These authors contributed equally to this work.

Editor: Silvia Sookoian, CONICET (National Scientific and Technical Research Council), Argentina

Received 2024 April 16; Revised 2024 July 5; Accepted 2024 July 26.

Abstract

Backgrounds/Aims

Transmembrane 6 superfamily member 2 (TM6SF2) E167K variant is closely associated with the occurrence and development of metabolic dysfunction-associated steatotic liver disease (MASLD). However, the role and mechanism of TM6SF2 E167K variant during MASLD progression are not yet fully understood.

Methods

The Tm6sf2^167K knock-in (KI) mice were subjected to high-fat diet (HFD). Hepatic lipid levels of Tm6sf2^167K KI mice were detected by lipidomics analysis. Thin-layer chromatography (TLC) was used to measure the newly synthesized triglyceride (TG) and phosphatidylcholine (PC).

Results

The TM6SF2 E167K variant significantly aggravated hepatic steatosis and injury in HFD-induced mice. Decreased polyunsaturated PC level and increased polyunsaturated TG level were found in liver tissue of HFD-induced Tm6sf2^167K KI mice. Mechanistic studies demonstrated that the TM6SF2 E167K variant increased the interaction between TM6SF2 and PNPLA3, and impaired PNPLA3-mediated transfer of polyunsaturated fatty acids (PUFAs) from TG to PC. The TM6SF2 E167K variant increased the level of fatty acid-induced malondialdehyde and reactive oxygen species, and decreased fatty acid-downregulated cell membrane fluidity. Additionally, the TM6SF2 E167K variant decreased the level of hepatic PC containing C18:3, and dietary supplementation of PC containing C18:3 significantly attenuated the TM6SF2 E167K-induced hepatic steatosis and injury in HFD-fed mice.

Conclusions

The TM6SF2 E167K variant could promote its interaction with PNPLA3 and inhibit PNPLA3-mediated transfer of PUFAs from TG to PC, resulting in the hepatic steatosis and injury during MASLD progression. PC containing C18:3 could act as a potential therapeutic supplement for MASLD patients carrying the TM6SF2 E167K variant.

Keywords: Transmembrane 6 superfamily member 2; Patatin-like phospholipase domain-containing protein 3; Polyunsaturated fatty acids; Hepatic steatosis; Metabolic dysfunction-associated steatotic liver disease

Graphical Abstract

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as nonalcoholic fatty liver disease (NAFLD), is a liver disease associated with metabolic disorders, and is primarily characterized by excessive accumulation of fat within hepatocytes [1]. The natural progression of MASLD includes steatosis, metabolic dysfunctionassociated steatohepatitis (MASH), liver fibrosis/cirrhosis, and MASH-related hepatocellular carcinoma (HCC) [2]. MASLD is one of the most prevalent chronic liver diseases, and its incidence rate has reached about 30% worldwide due to the widespread prevalence of obesity and metabolic syndrome [3]. MASLD has become one of the major causes of end-stage liver disease, posing a serious threat to global public health [4,5]. Therefore, it is of paramount importance to understand the pathogenesis of MASLD, identify its early diagnostic indicators, and develop effective treatment strategies.

In recent years, the roles of genetic factors in the occurrence and development of MASLD have garnered increasing attention [6,7]. An exome-wide association study by Kozlitina et al. [8] revealed a significant positive correlation between the TM6SF2 E167K (rs 58542926 c. 499 G>A, p. Glu 167 Lys) variant and MASLD occurrence. Subsequent studies have confirmed that the TM6SF2 E167K variant is a risk factor for the occurrence of MASLD and is closely associated with the development of MASH-associated liver cirrhosis and HCC [9,10]. TM6SF2 is primarily expressed in hepatic parenchymal cells, where it is predominantly located in the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment [11]. It has been revealed that TM6SF2 is involved in the regulation of hepatic lipid metabolism with opposing effects on the secretion of triglyceride (TG)-rich lipoproteins and hepatic TG content [11,12]. Namely, the knockdown or knockout of TM6SF2 in the liver of mice results in reduced very-low-density lipoprotein (VLDL) secretion and elevated hepatic TG content [8,13]. In a study on the impact of the TM6SF2 E167K variant on glucose metabolism, the TM6SF2 E167K variant has been shown to affect hepatic steatosis induced by high-fat diet (HFD) in male mice, but the mechanism remains unclear [14]. Some studies have suggested that the TM6SF2 E167K variant results in decreased protein stability and expression level of TM6SF2, and the TM6SF2 E167K variant might be a loss-of-function mutation that leads to hepatic lipid accumulation through the inactivation of TM6SF2 [8,9,15]. Nevertheless, some studies have shown that overexpression of TM6SF2 in the mouse liver does not decrease hepatic TG content or increase plasma TG level [16]. Therefore, a single theory based on functional loss is insufficient to fully explain the pathogenic mechanism of the TM6SF2 E167K variant during the occurrence of MASLD.

Luukkonen et al. [17] found a phenotype characterized by decreased hepatic phosphatidylcholine (PC) content and increased hepatic TG content in the TM6SF2 E167K variant carriers, suggesting that decreased hepatic PC content might be a characteristic of the TM6SF2 E167K variant carriers. The hepatic PC content is also decreased during the progression of MASLD [18,19]. PC plays a crucial role in hepatic lipid transport and serves as a key component in the assembly of VLDL particles [20,21]. Deficiency in PC leads to the degradation of VLDL particles in the liver, resulting in impaired secretion of TG and subsequently triggering hepatic steatosis [22,23]. Therefore, we hypothesized that the decrease in hepatic PC content might be involved in the elevation of hepatic TG content in MASLD patients carrying the TM6SF2 E167K variant. We discovered a potential interaction between patatin-like phospholipase domain-containing protein 3 (PNPLA3) and TM6SF2 through bioinformatics predictions and the string software (https://cn.string-db.org). PNPLA3 is considered to act as an acyltransferase that transfers polyunsaturated fatty acids (PUFAs) from TG to soluble phospholipids such as PC, or as a TG-hydrolyzing enzyme that hydrolyzes PUFAs from TG [24,25]. The deletion of PNPLA3 results in a decrease in polyunsaturated PC content and an increase in polyunsaturated TG content in mouse livers [24,26]. Therefore, we speculated that PNPLA3 might be involved in the TM6SF2 E167K-regulated lipid metabolism in the progression of MASLD.

Our study aimed to comprehensively investigate the role of the TM6SF2 E167K variant in hepatic lipid regulation and inflammation during the progression of MASLD by utilizing a Tm6sf2^167K knock-in (KI) mouse model and lipidomics. We also aimed to investigate the mechanism through which the TM6SF2 E167K variant regulates the function of PNPLA3, leading to a decrease in polyunsaturated PC level and an increase in polyunsaturated TG level. Furthermore, we identified the downregulation of the level of PC containing C18:3 in liver tissue of Tm6sf2^167K KI mice fed with HFD. We further validated the significant role of plasma PC (16:0/18:3) in indicating the occurrence of human MASLD, and revealed the inhibitory effect of PC containing C18:3 on hepatic steatosis in mice. Through this study, we provide new scientific insights into the mechanism underlying the pathogenesis, and the exploration of novel biomarkers for MASLD patients carrying the TM6SF2 E167K variant.

MATERIALS AND METHODS

Animals

A Tm6sf2^167K KI mouse model was created using CRISPR/Cas9 technology on the C57BL/6J background (Nanjing Institute of Biotechnology, Nanjing, China). An sgRNA targeting the mouse Tm6sf2 gene was constructed using the Cas9/RNA system gene-targeting technique (sgRNA is detailed in Supplementary Table 1). Simultaneously, a donor vector carrying the Tm6sf2-E167K fragment was constructed and co-injected with the Cas9 system into mouse zygotes. Sequencing was performed to validate the genotypes, and primer information is shown in Supplementary Table 2. To generate a mouse model of MASLD, 8-weekold male mice from both the Tm6sf2^167K/K (mutant) homozygous group and the Tm6sf2^167E/E (wild type, WT) control group were randomly divided into two groups, and fed either a control diet (CD) or a HFD (60% fat; Research Diets, New Brunswick, NJ, USA) (n=6 per group) [16]. The Tm6sf2^167E/E and Tm6sf2^167K/K mice were created by mating Tm6sf2167E/K heterozygous mice. To perform the experiment with PC containing C18:3 (PC18:3n-3, ZL-159048, STANDARDS, Shanghai, China), 8-week-old male Tm6sf2^167E/E and Tm6sf2^167K/K mice were subjected to dietary intervention using PC containing C18:3. PC containing C18:3 was dissolved using soybean oil (S110245; Aladdin, Shanghai, China) at a concentration of 25 mg/mL. The regular (once every 3 days) and quantitative (90 mg/kg PC containing C18:3) gavage was performed for 16weeks. Soybean oil served as the control reagent (n=5-6 per group) [27]. All mice were housed under constant temperature (22°C) and humidity (40–60%) conditions with a light/dark cycle of 12 hours. Sixteen weeks later, the mice were fasted for 6 hours and then euthanized. All mice were simultaneously subjected to experimental treatments to minimize potential confounders. All animal experiments were approved by the Medical Animal Protection Committee of Qingdao University.

Cell culture

293T cell line was purchased from Procell (Wuhan, China). 293T cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (Biological Industries, Beit-Haemek, Israel). Mouse primary hepatocytes were obtained from the 8-week-old male Tm6sf2^167E/E and Tm6sf2^167K/K mice. The extraction steps for mouse primary hepatocytes could be found in the previous studies [28,29]. Mouse primary hepatocytes were cultured in high-glucose DMEM medium containing 2% fetal bovine serum, 100 nM dexamethasone (Solarbio, Beijing, China), and 100 nM insulin (Solarbio). Cells were incubated at 37°C in a humidified incubator with 5% CO₂. Cells were treated with 0.5 mM free fatty acids (FFAs, oleic acid: palmitic acid=2:1) for 24 hours to induce cellular lipid degeneration. Oleic acid and palmitic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Human samples

Peripheral blood samples of 143 patients, who met the diagnostic criteria for MASLD [1], were collected in this study at Qingdao Municipal Hospital. Blood cells were used to detect the TM6SF2 rs58542926 genotype, with details about the primers provided in Table S3. A total of five healthy controls (HC) and 10 MASLD patients with the TM6SF2 E167K variant were identified. Additionally, six HC and 11 MASLD patients without the TM6SF2 E167K variant were randomly selected as controls. Twenty-one MASLD patients underwent magnetic resonance imaging (MRI) proton density fat fraction (PDFF) examination to assess hepatic fat content. The basic information and clinical characteristics of the participants are outlined in Table S4. Informed consent was obtained from all the participants. The entire study was conducted in accordance with the Declarations of Helsinki and Istanbul. This study was approved by the Ethics Committee of Qingdao Municipal Hospital.

Statistical analysis

Quantitative data were presented as mean and standard deviation (SD). Differences between the two groups were evaluated using the two-tailed Student’s t test or Mann– Whitney U test. Pearson correlation analysis was used to assess the correlation between two parameters. The receiver operator characteristic (ROC) curve analysis was used to determine the area under the curves (AUC) value, sensitivity, and specificity. Statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA). P-values lower than 0.05 were considered statistically significant.

Additional Materials and Methods were described in Supplementary information.

RESULTS

The TM6SF2 E167K variant exacerbates HFDinduced hepatic steatosis and injury in mice

To investigate the role of the TM6SF2 E167K variant during MASLD progression, we utilized CRISPR/Cas9 technology to change the 499th nucleotide of the mouse Tm6sf2 gene coding sequence from G to A and substitute lysine (K) for glutamate (E) (Fig. 1A). Genetic sequencing confirmed the point mutation at the 499th nucleotide of the Tm6sf2 gene in Tm6sf2^167K KI mice (Fig. 1B). The Tm6sf2^167E/E and Tm6sf2^167K/K mice were fed with HFD for 16 weeks to establish the MASLD model (Fig. 1C). There were no significant changes in the Tm6sf2 mRNA or TM6SF2 protein levels between the Tm6sf2^167E/E and Tm6sf2^167K/K mice fed with either CD or HFD (Fig. 1D and E). A 16-week HFD resulted in significant increases in body weight, liver weight, and hepatic index in the Tm6sf2^167K/K mice compared with the Tm6sf2^167E/E mice (Fig. 1F–I). Additionally, hematoxylin and eosin (H&E) staining and Oil Red O staining showed that the Tm6sf2^167K/K mice displayed a more severe degree of steatosis compared with the Tm6sf2^167E/E mice under HFD (Fig. 1J). The TG content in liver tissue was higher in the Tm6sf2^167K/K mice than that in the Tm6sf2^167E/E mice fed with HFD (Fig. 1K). In contrast, the plasma TG and total cholesterol (TC) levels showed no significant differences between the Tm6sf2^167E/E and Tm6sf2^167K/K mice (Supplementary Fig. 1). Then primary hepatocytes were isolated from the Tm6sf2^167E/E and Tm6sf2^167K/K mice and treated with FFAs. The results of both Oil Red O staining and TG detection showed that primary hepatocytes from the Tm6sf2^167K/K mice displayed a more significant accumulation of lipids compared with the Tm6sf2^167E/E mice (Supplementary Fig. 2). Taken together, these results indicate that the TM6SF2 E167K variant significantly increases the sensitivity of mouse liver to HFD, making it more prone to developing hepatic steatosis. Moreover, the MASLD activity score in the Tm6sf2^167K/K mice was higher than that in the Tm6sf2^167E/E mice under HFD (Fig. 1L). Simultaneously, the Tm6sf2^167K/K mice fed with HFD had significantly elevated plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels compared with the Tm6sf2^167E/E mice (Fig. 1M and N), suggesting that the TM6SF2 E167K variant could also exacerbate hepatic injury under HFD stimulation.

Figure 1.

The TM6SF2 E167K variant exacerbates HFD-induced hepatic steatosis and injury in mice. (A) Strategy of constructing Tm6sf2^167K KI mouse model using CRISPR/Cas9 technology. (B) Gene sequencing of Tm6sf2^167E/E and Tm6sf2^167K/K mice. (C) Mice fed with CD and HFD diets for 16 weeks to establish MASLD mouse model. (D) Relative expression level of Tm6sf2 mRNA in liver tissue of the Tm6sf2167E/E and Tm6sf2^167K/K mice after MASLD modeling, β-Actin served as an internal reference. (E) WB results of TM6SF2 protein in liver tissue of the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling, β-Actin was used as a loading control. (F–M) Body size and liver morphology (F), body weight (G), liver weight (H), hepatic index (I), H&E staining and Oil Red O (J, scale: 50 μm), hepatic TG content (K), MASLD activity score (L), plasma ALT level (M) and plasma AST level (N) of the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. Data are presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05, **P<0.01; NS, no significance. TM6SF2 transmembrane 6 superfamily member 2; HFD, high-fat diet; KI, knock-in; CD, control diet; MASLD, metabolic dysfunction-associated steatotic liver disease; WB, western blotting; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

The TM6SF2 E167K variant causes the accumulation of PUFA-containing TG and downregulation of PUFA-containing PC in liver tissue of HFD-fed mice

To further investigate the impact of the TM6SF2 E167K variant on hepatic lipids composition, we used liquid chromatography-mass spectrometry (LC-MS) to conduct untargeted lipidomics analysis of liver tissue from the Tm6sf2^167E/E and Tm6sf2^167K/K mice fed with CD or HFD. The results revealed that the quality-control (QC) samples clustered together and were located in the middle of each group, indicating good experimental reproducibility and distinct separation among the groups (Fig. 2A). A total of 27 lipid subclasses and 1231 lipid molecules were identified in the lipidomics analysis (Fig. 2B). Quantitative analysis of the identified lipids subtypes revealed that TG and PC were the two most predominant subtypes in terms of content (Supplementary Fig. 3). Hepatic TG content was significantly higher in the Tm6sf2^167K/K mice than in the Tm6sf2^167E/E mice under HFD (Fig. 2C). Under both the CD and HFD conditions, hepatic PC content was significantly lower in the Tm6sf2^167K/K mice compared with the Tm6sf2^167E/E mice (Fig. 2D). To further analyze the impact of the TM6SF2 E167K variant on the hepatic contents of unsaturated TG and PC, lipids were categorized into three groups, namely saturated fatty acid (SFA)-lipids, monounsaturated fatty acid (MUFA)-lipids, and polyunsaturated fatty acid (PUFA)-lipids. Under HFD conditions, compared with the Tm6sf2^167E/E mice, the Tm6sf2^167K/K mice showed a significant increase in the hepatic contents of monounsaturated TG and polyunsaturated TG, whereas the level of polyunsaturated PC was noticeably reduced (Fig. 2E and F). Furthermore, by analyzing the ratio of PC to TG in different groups, we also found that the HFD-induced Tm6sf2^167K/K mice displayed a significant decrease in the ratio of polyunsaturated PC to TG compared with the Tm6sf2^167E/E mice (Fig. 2G). Additionally, under HFD conditions, the Tm6sf2^167K/K mice had notably higher percentages of monounsaturated TG in monounsaturated lipids and polyunsaturated TG in polyunsaturated lipids (Fig. 2H), but lower percentage of monounsaturated or polyunsaturated PC in total monounsaturated or polyunsaturated lipids compared with the Tm6sf2^167E/E mice (Fig. 2I). These findings collectively suggest that the TM6SF2 E167K variant might influence the transfer of hepatic unsaturated fatty acids (particularly PUFAs) between TG and PC, resulting in an increase in polyunsaturated TG content and a decrease in polyunsaturated PC content in the liver of HFD-induced mice.

Figure 2.

The TM6SF2 E167K variant causes the accumulation of PUFA-containing TG and downregulation of PUFA-containing PC in liver tissue of HFD-fed mice. (A) PCA score plots of untargeted lipidomics in liver tissue of the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. (B) Statistical graphs of lipid subclasses and lipid molecule quantities detected in untargeted lipidomics analysis of mouse liver tissue. (C, D) Relative levels of TG (C) and PC (D) in liver tissue of the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. (E) Relative levels of SFA-TG, MUFA-TG and PUFA-TG in liver tissue of the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. The lipids were divided into three categories based on the number of unsaturated bonds: SFA-lipids (containing only SFAs), MUFAlipids (containing one or more MUFAs and no PUFAs) and PUFA-lipids (containing one or more PUFAs). (F) Relative levels of SFA-PC, MUFA-PC, and PUFA-PC in liver tissue of the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. (G) The ratio of hepatic PC and TG in the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling in the three groups. (H) The percentages of SFA-TG, MUFA-TG and PUFA-TG in the three groups from both the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. (I) The percentages of SFA-PC, MUFA-PC and PUFA-PC in the three groups from both the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. n=6 per group. Data are presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05; NS, no significance. TM6SF2 transmembrane 6 superfamily member 2; PUFA, polyunsaturated fatty acid; TG, triglyceride; PC, phosphatidylcholine; HFD, high-fat diet; MUFA, monounsaturated fatty acid; SFA, saturated fatty acid; MASLD, metabolic dysfunction-associated steatotic liver disease.

The TM6SF2 E167K variant enhances the interaction between TM6SF2 and PNPLA3 proteins in the ER

To further explore the molecular mechanism by which the TM6SF2 E167K variant regulated the levels of polyunsaturated TG and polyunsaturated PC, we used the string protein interaction network to screen for the functionally related proteins with TM6SF2 (Supplementary Fig. 4A). It has previously been shown that the deletion of PNPLA3 resulted in a decrease in hepatic polyunsaturated PC content and an increase in polyunsaturated TG content in mice [24,26]. This phenomenon is highly consistent with the alterations in hepatic polyunsaturated PC and TG levels induced by the TM6SF2 E167K variant that we identified in mice. We subsequently analyzed the relative expression levels of TM6SF2 and PNPLA3 genes in the livers of 94 NAFLD patients using the GEO database dataset (GSE174478), and found a strong positive correlation between the expression levels of TM6SF2 and PNPLA3 (Supplementary Fig. 4B). Next, we extracted primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice and treated them with FFAs. Immunofluorescence (IF) analysis showed a significant colocalization of TM6SF2 and PNPLA3 proteins, and the TM6SF2 E167K variant increased the co-localization of these proteins (Fig. 3A). Co-immunoprecipitation (Co-IP) results showed an interaction between TM6SF2 and PNPLA3 proteins under conditions with and without FFAs, and the TM6SF2 E167K variant significantly enhanced the interaction between TM6SF2 and PNPLA3 (Fig. 3B and Supplementary Fig. 5A). We overexpressed Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids in 293T cells. IF results also showed a co-localization between TM6SF2 and PNPLA3 proteins and the TM6SF2 E167K variant increased the co-localization (Supplementary Fig. 6). Co-IP analysis also indicated an interaction between TM6SF2 and PNPLA3, which was strengthened by the TM6SF2 E167K variant (Fig. 3C and Supplementary Fig. 5B). TM6SF2 is primarily distributed in the ER and PNPLA3 is distributed in the lipid droplets (LDs) [11,30]. We next explored whether the TM6SF2 E167K variant could affect the distribution of PNPLA3 in the LDs. Firstly, we extracted protein of the ER and LDs from primary hepatocytes of the Tm6sf2^167E/E and Tm6sf2^167K/K mice. Western blotting (WB) analysis indicated that the TM6SF2 E167K variant significantly enhanced PNPLA3 enrichment in the ER (Fig. 3D and Supplementary Fig. 5C) and reduced its distribution in the LDs in primary hepatocytes with or without FFA treatment (Fig. 3E and Supplementary Fig. 5D). In cells overexpressing Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids, the TM6SF2 E167K variant also enhanced PNPLA3 enrichment in the ER (Fig. 3F and Supplementary Fig. 5E) and vreduced its distribution in the LDs in cells with or without FFA treatment (Fig. 3G and Supplementary Fig. 5F). We further used the ER marker protein ERP72 and the LDs marker protein PLIN2 to fluorescently label the ER and LDs in primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice, respectively. IF results showed that the TM6SF2 E167K variant enhanced the colocalization of PNPLA3 with the ER, while conversely reducing its colocalization with the LDs in hepatocytes treated with FFAs (Fig. 3H and I). These results suggest that the TM6SF2 E167K variant could enhance the interaction between TM6SF2 and PNPLA3 proteins in the ER to cause the depletion of PNPLA3 in the LDs, which might influence PNPLA3’s biological functions within the LDs.

Figure 3.

The TM6SF2 E167K variant enhances the interaction between TM6SF2 and PNPLA3 proteins. (A) IF analysis for the co-localization of TM6SF2 and PNPLA3 proteins from primary hepatocytes of the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. Scale bar: 20 μm. (B) Co-IP analysis for the interaction between TM6SF2 and PNPLA3 proteins from primary hepatocytes of the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. (C) Co-IP analysis for the interaction between TM6SF2 and PNPLA3 in 293T cells co-transfected with Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids and PNPLA3^148I plasmids. (D, E) WB analysis for the PNPLA3 expression in the ER (D) and LDs (E) from primary hepatocytes of the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. ERP72 and PLIN2 were employed as marker proteins for the ER and LDs. (F, G) WB analysis for the PNPLA3 expression in the ER (F) and LDs (G) in 293T cells co-transfected with Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids and PNPLA3^148I plasmids under conditions with and without FFAs. ERP72 and PLIN2 were employed as marker proteins for the ER and LDs. (H, I) IF analysis for the colocalization of PNPLA3 with the ER (H) and LDs (I) from primary hepatocytes of the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. Scale bar: 20 μm. IF, immunofluorescence; TM6SF2 transmembrane 6 superfamily member 2; FFAs, free fatty acids; Co-IP, co-immunoprecipitation; PNPLA3, patatin-like phospholipase domain-containing protein 3; WB, western blotting; ER, endoplasmic reticulum; LDs, lipid droplets.

We also examined whether the PNPLA3 I148M variant affects the interaction between TM6SF2 and PNPLA3 proteins. Co-IP results showed that the PNPLA3 I148M variant did not cause significant changes in the interaction between TM6SF2 and PNPLA3 proteins (Supplementary Fig. 7). These results suggest that the PNPLA3 I148M variant could not affect the interaction between TM6SF2 and PNPLA3 proteins.

The TM6SF2 E167K variant impairs PNPLA3-mediated transfer of PUFAs from TG to PC

Previous studies have suggested that PNPLA3 is an enzyme involved in regulating lipids metabolism and could catalyze the transfer of PUFAs from TG to PC [24]. Based on our findings that the TM6SF2 E167K variant induces changes in the levels of PUFAs between TG and PC in the liver, it is reasonable to speculate that the TM6SF2 E167K variant might affect the transfer of PUFAs from TG to PC mediated by PNPLA3. We utilized alkynyl-labeled linoleic acid (C18:2) as a substrate and examined the changes in linoleic acid incorporated into TG or PC. The labeled lipids were separated on a thin-layer chromatography (TLC) plate and analyzed by fluorescence imaging to realize rapid analysis of newly synthesized lipids (Fig. 4A). Primary hepatocytes were extracted from the Tm6sf2^167E/E and Tm6sf2^167K/K mice and treated with FFAs and alkynyl-labeled linoleic acid. The results of linoleic acid incorporation assays showed that the newly synthesized PC/TG ratio in primary hepatocytes from the Tm6sf2^167K/K mice was significantly decreased compared with that in the Tm6sf2^167E/E mice, and the difference in the newly synthesized PC/TG ratio in primary hepatocytes between the two groups was significantly abolished after knockdown of PNPLA3 (Fig. 4B–D and Supplementary Fig. 8A). We also performed a confirmatory validation in 293T cells transfected with TM6SF2^167E or TM6SF2^167K plasmids. Upon overexpression of TM6SF2^167E or TM6SF2^167K plasmids, PNPLA3 knockdown was performed to validate the reversibility (Fig. 4E and Supplementary Fig. 8B). We observed a significant decrease in the PUFA-containing PC/TG ratio of newly synthesized lipids in cells with the TM6SF2 E167K variant compared with the control group (Fig. 4F and G), and the difference disappeared upon knockdown of PNPLA3 (Fig. 4H and I). These results indicate that the TM6SF2 E167K variant affects the function of PNPLA3, resulting in an elevation of polyunsaturated TG level and a reduction in polyunsaturated PC level.

Figure 4.

The TM6SF2 E167K variant impairs PNPLA3-mediated transfer of PUFAs from TG to PC. (A) Scheme for tracing fatty acids using click chemistry. Lipids were separated by TLC and visualized by fluorescence imaging. (B) WB analysis for PNPLA3 in primary hepatocytes, which were transfected with si-NC or si-Pnpla3 under conditions with and without FFAs, from the Tm6sf2^167E/E and Tm6sf2^167K/K mice. (C) TLC analysis of newly synthesized TG and PC in primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice. Primary hepatocytes were transfected with si-NC or si-Pnpla3 under conditions with and without FFAs. (D) Quantitative analysis of results from (C): Bar graphs represent the ratio of newly synthesized PC to TG. (E) WB analysis for 293T cells with TM6SF2 overexpression and PNPLA3 knockdown. (F, H) TLC analysis of newly synthesized TG and PC in 293T cells overexpressed with TM6SF2167E or TM6SF2167K plasmids, and transfected with si-NC (F) or si-PNPLA3 (H). (G, I) Quantitative analysis of results from (F, H): Bar graphs represent the ratio of newly synthesized PC to TG. Data are presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05, ***P<0.001; NS, no significance. TM6SF2 transmembrane 6 superfamily member 2; PNPLA3, patatin-like phospholipase domain-containing protein 3; PUFA, polyunsaturated fatty acid; TG, triglyceride; PC, phosphatidylcholine; TLC, thin-layer chromatography; WB, western blotting; FFAs, free fatty acids.

The TM6SF2 E167K variant leads to an increase in MDA and ROS levels, and a decrease in cell membrane fluidity of fatty acid-treated hepatic cells

PUFAs are particularly susceptible to the attack by oxygen free radicals, which induce lipids peroxidation stress and cellular damage [31]. ROS could induce lipid peroxidation, and MDA was a product of lipid peroxidation [32,33]. We treated primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice with FFAs and assessed the impact of the TM6SF2 E167K variant on the levels of intracellular ROS and MDA. Specifically, under FFA treatment conditions, both the ROS and MDA levels in primary hepatocytes from the Tm6sf2^167K/K mice were significantly elevated compared with those in the Tm6sf2^167E/E mice (Fig. 5A and B). In cells overexpressing Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids, we found that the TM6SF2 E167K variant could significantly increase the levels of ROS and MDA under FFA treatment (Fig. 5C and D). These findings indicate that the TM6SF2 E167K variant might exacerbate intracellular lipids peroxidation level of FFA-treated hepatocytes. We also investigated cell-membrane fluidity using a lipophilic pyrene probe called pyrene decanoic acid (PDA) (Fig. 5E). Our findings showed that FFA treatment significantly reduced cell-membrane fluidity in both mouse primary hepatocytes and 293T cells. The TM6SF2 E167K variant showed a noticeable enhancement of FFA-induced reduction in cell-membrane fluidity (Fig. 5F and G). Furthermore, the electron microscopy results of liver tissue in the HFDtreated mice showed that the Tm6sf2^167K/K mice exhibited more severe disruption and fragmentation of the ER, suggesting that the E167K variant could increase the sensitivity of the ER to HFD-induced lipid stress in hepatocytes (Fig. 5H). These data suggest that the TM6SF2 E167K variant significantly enhances lipid-induced MDA and ROS levels and exacerbates damage to the cell membrane of hepatocytes.

Figure 5.

The TM6SF2 E167K variant leads to an increase in MDA and ROS levels, and a decrease in cell-membrane fluidity of fatty acid-treated hepatic cells. (A, B) The ROS (A) and MDA (B) levels of primary hepatocytes treated with 0.5 mM FFAs for 24 hours in the Tm6sf2^167E/E and Tm6sf2^167K/K mice. (C, D) The ROS (C) and MDA (D) levels in 0.5 mM FFA-treated 293T cells overexpressing TM6SF2^167E or TM6SF2^167K plasmids. (E) Pattern diagram for detecting cellular membrane fluidity. Relative membrane fluidity (relative fluorescence units, RFUs) is a ratio of excimer to monomer fluorescence. (F) The membrane fluidity of primary hepatocytes treated with 0.5 mM FFAs for 24 hours in the Tm6sf2^167E/E and Tm6sf2^167K/K mice. (G) The membrane fluidity in 0.5 mM FFA-treated 293T cells overexpressing TM6SF2167E or TM6SF2167K plasmids. (H) Transmission electron microscopy images of liver tissue from the Tm6sf2^167E/E and Tm6sf2^167K/K mice fed with HFD. The red arrows indicate the ER. Scale bar: 500 nm. Data are presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05, **P<0.01; NS, no significance. TM6SF2 transmembrane 6 superfamily member 2; MDA, malondialdehyde; ROS, reactive oxygen species; FFAs, free fatty acids.

The TM6SF2 E167K variant decreases the hepatic level of PC containing C18:3, and dietary intervention with PC containing C18:3 attenuates the TM6SF2 E167K-induced hepatic steatosis and injury in HFD-fed mice

Using volcano plot analysis, we identified differentially expressed lipid molecules in the liver tissue between the Tm6sf2^167E/E and Tm6sf2^167K/K mice (Fig. 6A and B). Figure 6C shows the levels of differentially expressed phospholipid molecules downregulated by the TM6SF2 E167K variant or HFD in the liver tissue of mice. Here, we focused on PC containing C18:3, namely PC (16:0/18:3) +H and PC (18:3/20:4) +H for further analysis (Fig. 6D and E), which are two molecules with clear chemical structure and high abundance. The level of PC (16:0/18:3) was lower in the Tm6sf2^167K/K mice fed with either CD or HFD (Fig. 6D). Therefore, we speculated that the decreased level of PC containing C18:3 might represent a phenotype of MASLD with the TM6SF2 E167K variant and its deficiency might exacerbate hepatic steatosis.

Figure 6.

The TM6SF2 E167K variant causes a decrease in the level of PC containing C18:3 in liver. (A, B) Volcano plots of untargeted lipidomics data in liver tissue of the Tm6sf2^167E/E and Tm6sf2^167K/K mice fed with CD or HFD diet. Green points indicate significantly downregulated lipids (FC<0.65, P<0.05), while red points indicate significantly up-regulated lipids (FC>1.54, P<0.05). (C) Hierarchical clustering analysis of untargeted lipidomics in liver tissue from the Tm6sf2^167E/E and Tm6sf2^167K/K mice fed with CD or HFD diet. Each row represents a differential lipid molecule. Red represents relatively high expression level, while blue represents relatively low expression level. (D, E) The relative levels of PC (16:0/18:3) +H and PC (18:3/20:4) +H in liver tissue of the Tm6sf2^167E/E and Tm6sf2^167K/K mice. n=6 per group. Data are presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05, **P<0.01; NS, no significance. TM6SF2 transmembrane 6 superfamily member 2; PC, phosphatidylcholine; CD, control diet; HFD, high-fat diet.

To investigate whether the supplementation of PC containing C18:3 through diet could affect HFD-induced hepatic steatosis, we introduced dietary intervention with PC containing C18:3 in the HFD-induced Tm6sf2^167E/E or Tm6sf2^167K/K mice (Fig. 7A). Our results showed that the intervention with PC containing C18:3 abolished the increase in body weight, liver weight, hepatic index, hepatic MDA level, hepatic steatosis and hepatic TG content, and eliminated the reduction in membrane fluidity of hepatocytes caused by TM6SF2 E167K variant in HFD-fed mice (Fig. 7B–I). We also found that intervention with PC containing C18:3 abolished the increase in hepatic injury indicators including MASLD activity score, plasma ALT level, and plasma AST level induced by TM6SF2 E167K variant in HFD-fed mice (Fig. 7J–L). These findings suggest that PC containing C18:3 could attenuate HFD-induced hepatic steatosis and injury, and this attenuation effect is significantly dependent on the TM6SF2 E167K variant.

Figure 7.

Dietary intervention with PC containing C18:3 significantly attenuates the TM6SF2 E167K-induced hepatic steatosis and injury in HFD-fed mice. (A) PC containing C18:3 dietary intervention in HFD-induced Tm6sf2^167E/E and Tm6sf2^167K/K mice. (B–L) Body size and liver morphology (B), body weight (C), liver weight (D), hepatic index (E), MDA level of liver tissue (F), hepatocyte membrane fluidity (G), H&E and Oil Red O staining (H), hepatic TG content (I), MASLD activity score (J), plasma ALT level (K), plasma AST level (L) in CD or HFD-induced Tm6sf2^167E/E and Tm6sf2^167K/K mice with PC containing C18:3 dietary intervention. Scale bar: 50 μm. Data are presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05, **P<0.01; NS, no significance. PC, phosphatidylcholine; TM6SF2 transmembrane 6 superfamily member 2; HFD, high-fat diet; MDA, malondialdehyde; TG, triglyceride; MASLD, metabolic dysfunction-associated steatotic liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

PC (16:0/18:3) is decreased in the plasma of MASLD patients carrying the TM6SF2 E167K variant

In this study, we identified significantly reduced content of PC (16:0/18:3) in the liver tissue of the HFD-induced Tm6sf2^167K/K mice. To investigate the clinical value of PC (16:0/18:3), we collected plasma samples from 21 MASLD patients and 11 HC. Table S4 presents the specific genotype and relevant clinical information.

We conducted targeted lipidomics analysis on the plasma samples obtained from HC and patients with MASLD. A total of 19 lipid subclasses and 879 lipid molecules were analyzed (Supplementary Fig. 9). The MASLD patients with TM6SF2^167E/K+K/K exhibited a significant decrease in plasma PC level compared with the MASLD patients with TM6SF2^167E/E (Fig. 8A). The MASLD patients with TM6SF2^167E/K+K/K showed a significant decrease in the level of polyunsaturated PC compared with the MASLD patients with TM6SF2^167E/E (Fig. 8B). The plasma TG level and the level of polyunsaturated TG showed no significant differences between the TM6SF2^167E/E and TM6SF2^167E/K+K/K groups (Supplementary Fig. 10). Through volcano plot analysis, we identified a set of lipid molecules with significantly decreased expression in the TM6SF2^167E/K+K/K group (Fig. 8C and D). Figure 8E showed the levels of various PC containing C18:3, and the level of PC (16:0/18:3), which was a differentially low-expressed PC identified in Tm6sf2^167K/K mice, was also lower in the patients with TM6SF2^167E/K+K/K than in those with TM6SF2^167E/E. In the MASLD patients correlation analysis revealed a significant negative correlation between the plasma PC (16:0/18:3) level and the liver fat content (r=−0.6143, P<0.05) (Fig. 8F). In the MASLD patients with TM6SF2^167E/K+K/K, the r value between the plasma PC (16:0/18:3) level and the liver fat content was up to −0.7095 (P<0.05) (Fig. 8G). However, in the MASLD patients with TM6SF2^167E/E, there was no significant correlation between the plasma PC (16:0/18:3) level and the liver fat content (r=−0.2193, P>0.05) (Fig. 8H). The significance of the plasma PC (16:0/18:3) level in predicting the TM6SF2 E167K variant was evaluated using ROC curve analysis. The area under the ROC curve (AUC) was 0.7636 to distinguish MASLD patients with TM6SF2^167E/K+K/K from those with TM6SF2^167E/E (Fig. 8I). The significance of plasma PC (16:0/18:3) level in predicting MASLD was also assessed using ROC curve analysis, and the AUC of plasma PC (16:0/18:3) level in predicting MASLD from HC was 0.7446 (P<0.05) (Fig. 8J).

Figure 8.

PC (16:0/18:3) is decreased in the plasma of MASLD patients carrying the TM6SF2 E167K variant. (A) Plasma PC level of HC and MASLD patients with TM6SF2^167E/E or TM6SF2^167E/K+K/K based on targeted lipidomics analysis. (B) Plasma SFA-PC, MUFA-PC, and PUFA-PC levels of HC and MASLD patients with TM6SF2^167E/E or TM6SF2^167E/K+K/K. (C, D) Volcano plots of targeted lipidomics data in plasma of HC (C) and MASLD patients (D) with TM6SF2^167E/E or TM6SF2^167E/K+K/K. Green dots represent significantly downregulated lipids (P<0.05), and red dots represent significantly upregulated lipids (P<0.05). (E) Changes in the levels of PC containing C18:3 in HC and MASLD patients with TM6SF2^167E/E or TM6SF2^167E/K+K/K. (F–H) Correlation between the plasma PC (16:0/18:3) level and the liver fat content in MASLD patients (F), MASLD patients with TM6SF2^167E/K+K/K (G), and MASLD patients with TM6SF2^167E/E (H). (I) ROC analysis of the plasma PC (16:0/18:3) level for discriminating MASLD patients with TM6SF2^167E/E from MASLD patients with TM6SF2^167E/K+K/K. (J) ROC analysis of the plasma PC (16:0/18:3) level for discriminating HC from MASLD patients. HC-TM6SF2^167E/E (n=6), HC-TM6SF2^167E/K+K/K (n=5), MASLD-TM6SF2^167E/E (n=11), MASLD-TM6SF2^167E/K+K/K (n=10). Data are presented as means and SD. Two-tailed Student’s t test or Mann– Whitney U test was used to compare normally or non-normally distributed data, respectively. Pearson correlation analysis was used to assess the correlation between two parameters. *P<0.05; NS, no significance. PC, phosphatidylcholine; MASLD, metabolic dysfunctionassociated steatotic liver disease; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; HC, healthy controls; TM6SF2 transmembrane 6 superfamily member 2; ROC, receiver operator characteristic.

DISCUSSION

In this study, we found that the TM6SF2 E167K variant promoted hepatic steatosis and injury in mice under HFD induction, which is consistent with the more severe hepatic steatosis observed in MASLD patients carrying the TM6SF2 E167K variant [8,9]. Previous study has shown that the genetic variant TM6SF2 E167K could induce increased hepatocyte fat content by reducing APOB particle secretion [34]. Another research showed that the TM6SF2 E167K variant decreased hepatic TM6SF2 protein stability and expression [8,9,15]. Here, we did not find a significant decrease in TM6SF2 expression in livers from the Tm6sf2^167K/K mice compared with the Tm6sf2^167E/E mice. However, our lipidomics analysis revealed that the Tm6sf2^167K/K mice exhibited a decreased level of polyunsaturated PC and an elevated level of polyunsaturated TG in the liver. PC is a crucial factor in hepatic lipid metabolism, and its deficiency impairs the assembly, maintenance, and secretion of hepatic TGVLDL, resulting in hepatic TG accumulation [19,20]. Therefore, it was hypothesized that the TM6SF2 E167K variant might influence hepatic TG deposition by reducing hepatic PC, especially polyunsaturated PC content. This study showed that dietary intervention with PC containing C18:3 attenuated the TM6SF2 E167K-induced hepatic steatosis and injury in HFD-fed mice. These results revealed a significant role of polyunsaturated PC in liver lipid metabolism and its protective value for MASLD patients carrying the TM6SF2 E167K variant.

We also identified several specific downregulated PC molecules containing C18:3 in liver tissue of the Tm6sf2^167K/K mice fed with HFD. We found that PC containing C18:3 was downregulated in plasma of the TM6SF2 E167K variant carriers, and the plasma PC (16:0/18:3) level negatively correlated with the liver fat content in the MASLD patients. Meanwhile, we found that the plasma PC (16:0/18:3) level was able to distinguish MASLD patients from HC, and also distinguish MASLD patients carrying the TM6SF2 E167K variant from non-carriers. Therefore, we hypothesize that the deficiency of PC containing C18:3 is also closely associated with the development of MASLD accelerated by the TM6SF2 E167K variant, and these PC molecules have the potential to be novel disease biomarkers.

Previous studies have suggested that PNPLA3 is an enzyme involved in lipid metabolism, particularly in the conversion of PUFAs from TG to PC [24,26,35]. PNPLA3 also has a predominant lipase activity and I148M mutation results in a loss of function, and the downregulation of PNPLA3 I148M has a beneficial effect against MASLD [36,37]. Knocking out PNPLA3 at the cellular or animal level causes the accumulation of PUFAs in TG and the depletion of PUFAs in phospholipids [24,26]. Here, we confirmed the interaction between TM6SF2 and PNPLA3, and the TM6SF2 E167K variant might inhibit the function of PNPLA3 by increasing binding affinity to PNPLA3. In contrast, the PNPLA3 I148M variant did not have a significant effect on the interaction between TM6SF2 and PNPLA3. It could be inferred that fatty liver would be exacerbated in MASLD patients with TM6SF2 E167K variant, which was independent of PNPLA3 I148M variant. Although both TM6SF2 E167K and PNPLA3 I148M variants could increase the risk of MASLD with late stages [38], previous research has shown that the TM6SF2 E167K variant is unequivocally associated with an increased risk of MASLD-associated hepatic fibrosis, which is independent of PNPLA3 I148M variant [39,40]. Our findings suggest that TM6SF2 and PNPLA3 should be considered as closely related factors during the pathogenesis of MASLD, providing a new direction for exploring the role of genetic factors in MASLD.

Recently, numerous studies have confirmed the involvement of oxidative stress and lipid peroxidation in the development of MASLD based on the classic “two-hit” theory [41,42]. We found that under conditions of lipid stress, the TM6SF2 E167K variant might lead to increased levels of oxidative stress and lipid peroxidation, as well as decreased cellmembrane fluidity. Previous studies have suggested that polyunsaturated PC could inhibit the overexpression of ROS-producing enzymes, remove oxygen free radicals, and reduce the level of lipid peroxidation [27,43,44]. Polyunsaturated PC significantly increases cell-membrane fluidity [43,45]. Thus, we hypothesized that the TM6SF2 E167K variant might increase oxidative stress level and lipid peroxidation level by decreasing the level of polyunsaturated PC. We conducted a dietary intervention on mice using one kind of polyunsaturated PC molecules and found that it significantly reduced the level of lipid peroxidation induced by HFD while increasing the fluidity of cell membranes. These results suggest that the TM6SF2 E167K variant might lead to a downregulation of hepatic polyunsaturated PC level, resulting in increased levels of intracellular oxidative stress, lipid peroxidation, and impaired cell-membrane fluidity to affect the hepatic steatosis and injury.

In summary, the animal experiments on Tm6sf2^167K/K mice indicated that the TM6SF2 E167K variant significantly exacerbated hepatic steatosis and injury. Furthermore, we demonstrated that the TM6SF2 E167K variant inhibited the PNPLA3-mediated transfer of PUFAs from TG to PC by promoting the interaction between TM6SF2 and PNPLA3, resulting in a decrease in polyunsaturated PC content and an increase in polyunsaturated TG content in the liver. Additionally, lipidomics analysis revealed specific downregulation of lipid molecules such as PC (16:0/18:3) in liver tissue of the Tm6sf2^167K/K mice, and the plasma PC (16:0/18:3) could partially distinguish the MASLD patients carrying the TM6SF2 E167K variant. Dietary supplementation of PC containing C18:3 could significantly attenuate the degree of hepatic steatosis and injury induced by the TM6SF2 E167K variant in HFD-fed mice. Through this study, we aimed to provide new scientific insights into the pathogenesis of MASLD and laid the theoretical foundation for exploring novel biomarkers for patients with MASLD.

Notes

Authors’ contribution

Conceptualization: LKZ, YNX and SYX; Methodology: BKS, XQD, JZ and LKZ; Formal Analysis: BKS, XQD, SSL, ZZZ; Investigation: BKS, XQD, JT, XRC and SMZ; Resources: LKZ and YNX; Writing-Original Draft: BKS, XQD and LKZ; Writing - Review & Editing: LKZ, YNX and SYX; Funding Acquisition, LKZ and YNX; Supervision, SSL and SYX.

Conflicts of Interest

The authors have no conflicts to disclose.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China, China (grant number: 32171277, 32270766) and Taishan Scholar Foundation of Shandong Province for LKZ (tsqn202312389).

SUPPLEMENTAL MATERIAL

Supplementary material is available at Clinical and Molecular Hepatology website (http://www.e-cmh.org).

SUPPLEMENTARY MATERIALS AND METHODScmh-2024-0268-Supplementary-Materials-and-Methods.pdf

Supplementary Figure 1.

Plasma TG level (A) and plasma TC level (B) of the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling. Data were presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. NS, no significance. TG, tri-glyceride; TC, total cholesterol; MASLD, metabolic dysfunction-associated steatotic liver disease.

cmh-2024-0268-Supplementary-Fig-1.pdf

Supplementary Figure 2.

Oil Red O staining (A, scale: 50 μm) and cellular TG content (B) of primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. Data were presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05; NS, no significance. TG, triglyceride; FFAs, free fatty acids; BSA, bovine serum albumin.

cmh-2024-0268-Supplementary-Fig-2.pdf

Supplementary Figure 3.

Statistical graphs of lipid subclasses and their relative abundance detected in untargeted lipidomics of liver tissue from the Tm6sf2^167E/E and Tm6sf2^167K/K mice after MASLD modeling (A, B). Data were presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05, **P<0.01; NS, no significance. MASLD, metabolic dysfunction-associated steatotic liver disease.

cmh-2024-0268-Supplementary-Fig-3.pdf

Supplementary Figure 4.

Prediction of the relationship between the TM6SF2 and PNPLA3 protein. (A) The protein-protein interaction network from the string database. Line thickness indicates the strength of association. (B) The correlation between the TM6SF2 and PNPLA3 expression level of NAFLD patients using the GEO database dataset (GSE174478) was assessed by Pearson correlation analysis (n=94). TM6SF2 transmembrane 6 superfamily member 2; PNPLA3, patatin-like phospholipase domain-containing protein 3; NAFLD, nonalcoholic fatty liver disease.

cmh-2024-0268-Supplementary-Fig-4.pdf

Supplementary Figure 5.

The quantification of the WB results in Figure 3. (A) The ratio of TM6SF2 to PNPLA3 in IP samples from primary hepatocytes of the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. (B) The ratio of PNPLA3 to Myc-TM6SF2 in IP samples from 293T cells co-expressing Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids and PNPLA3^148I plasmid. (C) The relative expression level of PNPLA3 in the ER of primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. ERP72 acted as the internal reference protein. (D) The relative expression level of PNPLA3 in the LDs of primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice under conditions with and without FFAs. PLIN2 acted as the internal reference protein. (E) The relative expression level of PNPLA3 in the ER of 293T cells co-expressing Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids and PNPLA3^148I plasmid under conditions with and without FFAs. ERP72 acted as the internal reference protein. (F) The relative expression level of PNPLA3 in the LDs of 293T cells co-expressing Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids and PNPLA3^148I plasmid under conditions with and without FFAs. PLIN2 acted as the internal reference protein. Data were presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05. TM6SF2 transmembrane 6 superfamily member 2; PNPLA3, patatin-like phospholipase domain-containing protein 3; FFAs, free fatty acids; ER, endoplasmic reticulum; LDs, lipid droplets.

cmh-2024-0268-Supplementary-Fig-5.pdf

Supplementary Figure 6.

IF analysis for the co-localization of TM6SF2 and PNPLA3 proteins from 293T cells overexpressing Myc-TM6SF2^167E or Myc-TM6SF2^167K plasmids. Scale bar: 50 μm. TM6SF2 transmembrane 6 superfamily member 2; PNPLA3, patatin-like phospholipase domain-containing protein 3.

cmh-2024-0268-Supplementary-Fig-6.pdf

Supplementary Figure 7.

(A) Co-IP experiment was carried out using a specific Myc antibody and normal IgG acted as a negative control in 293T cells overexpressing PNPLA3^148I or PNPLA3^148M plasmids and Myc-TM6SF2^167E plasmid under conditions with and without FFAs. (B) The ratio of PNPLA3 to Myc-TM6SF2 in IP samples from 293T cells overexpressing PNPLA3^148I or PNPLA3^148M plasmids and Myc-TM6SF2^167E plasmid under conditions with and without FFAs. Data were presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. NS, no significance. Co-IP, co-immunoprecipitation; TM6SF2 transmembrane 6 superfamily member 2; PNPLA3, patatin-like phospholipase domain-containing protein 3; FFAs, free fatty acids.

cmh-2024-0268-Supplementary-Fig-7.pdf

Supplementary Figure 8.

The quantification of the WB results in Figure 4. (A) The relative expression level of PNPLA3 in primary hepatocytes from the Tm6sf2^167E/E and Tm6sf2^167K/K mice. Primary hepatocytes were transfected with si-NC or si-Pnpla3 under conditions with and without FFAs. (B) The relative expression level of PNPLA3 in 293T cells overexpressing TM6SF2^167E or TM6SF2^167K, and transfected with si-NC or si-PNPLA3. Data were presented as means and SD. Statistical significance was calculated by two-tailed Student’s t test. *P<0.05. WB, western blotting; TM6SF2 transmembrane 6 superfamily member 2; PNPLA3, patatin-like phospholipase domain-containing protein 3; FFAs, free fatty acids.

cmh-2024-0268-Supplementary-Fig-8.pdf

Supplementary Figure 9.

Statistical graphs of lipid subclasses and lipid molecule quantities detected in targeted lipidomics analysis of the human plasma samples.

cmh-2024-0268-Supplementary-Fig-9.pdf

Supplementary Figure 10.

(A) Plasma TG level of HC and MASLD patients with TM6SF2^167E/E or TM6SF2^167E/K+K/K based on targeted lipidomics analysis. (B) Plasma SFA-TG, MUFA-TG, and PUFA-TG levels of HC and MASLD patients with TM6SF2^167E/E or TM6SF2^167E/K+K/K. Data were presented as means and SD. Two-tailed Student’s t test and Mann-Whitney U tests were used to compare normally and non-normally distributed data, respectively. NS, no significance. TG, triglyceride; HC, healthy controls; MASLD, metabolic dysfunction-associated steatotic liver disease; TM6SF2 transmembrane 6 superfamily member 2; PNPLA3, patatin-like phospholipase domain-containing protein 3; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.

cmh-2024-0268-Supplementary-Fig-10.pdf

Supplementary Figure 11.

WB analysis for TM6SF2 using anti-TM6SF2 antibody (TA335280, origene). Left lane: the lysate of 293T cells overexpressing Myc-NC plasmid; right lane: the lysate of 293T cells overexpressing Myc-TM6SF2^167E plasmid. β-Actin was used as loading control. WB, western blotting; TM6SF2 transmembrane 6 superfamily member 2.

cmh-2024-0268-Supplementary-Fig-11.pdf

Supplementary Table 1.

sgRNA for constructing Tm6sf2^167K KI mice

cmh-2024-0268-Supplementary-Table-1.pdf

Supplementary Table 2.

Primers for sequencing in Tm6sf2^167K KI mice

cmh-2024-0268-Supplementary-Table-2.pdf

Supplementary Table 3.

Primers for identifying TM6SF2 rs58542926 genotype of human samples

cmh-2024-0268-Supplementary-Table-3.pdf

Supplementary Table 4.

Clinical characteristics of the study participants according to the TM6SF2 genotype at rs58542926

cmh-2024-0268-Supplementary-Table-4.pdf

Supplementary Table 5.

Primers for detecting the relative expression level of Tm6sf2

cmh-2024-0268-Supplementary-Table-5.pdf

Supplementary Table 6.

Antibodies used in this study

cmh-2024-0268-Supplementary-Table-6.pdf

Abbreviations

ALT

alanine aminotransferase

APOB

apolipoprotein B

AST

aspartate aminotransferase

AUC

area under the ROC curve

BSA

bovine serum albumin

control diet

Co-IP

co-immunoprecipitation

DAG

diacylglycerol

endoplasmic reticulum

FFAs

free fatty acids

healthy controls

HCC

hepatocellular carcinoma

H&E

hematoxylin and eosin

HFD

high-fat diet

immunofluorescence

knock-in

LC-MS

liquid chromatography-mass spectrometry

LDs

lipid droplets

MASH

metabolic dysfunction-associated steatohepatitis

MASLD

metabolic dysfunction-associated steatotic liver disease

MDA

malondialdehyde

MUFA

monounsaturated fatty acid

NAFLD

nonalcoholic fatty liver disease

phosphatidylcholine

PCR

polymerase chain reaction

PDA

pyrene decanoic acid

PDFF

proton density fat fraction

PNPLA3

patatin-like phospholipase domain-containing protein 3

PUFA

polyunsaturated fatty acid

quality control

ROC

receiver operator characteristic

ROS

reactive oxygen species

standard deviation

SFA

saturated fatty acid

total cholesterol

triglyceride

TLC

thin-layer chromatography

TM6SF2

transmembrane 6 superfamily member 2

VLDL

very low-density lipoprotein

western blotting

wild type

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Notes

Study Highlights

• TM6SF2 E167K variant aggravates hepatic steatosis and injury in MASLD mice.

• TM6SF2 E167K variant decreases polyunsaturated PC level and increases polyunsaturated TG level in liver tissue of MASLD mice.

• TM6SF2 E167K variant increases the interaction between TM6SF2 and PNPLA3 and impairs PNPLA3-mediated transfer of PUFAs from TG to PC in hepatocytes.

• TM6SF2 E167K variant increases MDA and ROS levels, and reduces cell-membrane fluidity in hepatocytes under conditions of lipid stress.

• Dietary supplementation of PC containing C18:3 could significantly attenuate the TM6SF2 E167K-induced hepatic steatosis and injury in HFD-fed mice.