Impaired hepatic lipid synthesis from polyunsaturated fatty acids in TM6SF2 E167K variant carriers with NAFLD

Background

Carriers of the transmembrane 6 superfamily member 2 E167K gene variant (TM6SF2^EK/KK) have decreased expression of the TM6SF2 gene and increased risk of NAFLD and NASH. Unlike common ‘obese/metabolic’ NAFLD, these subjects lack hypertriglyceridemia and have lower risk of cardiovascular disease. In animals, phosphatidylcholine (PC) deficiency results in a similar phenotype. PCs surround the core of VLDL consisting of triglycerides (TGs) and cholesteryl-esters (CEs). We determined the effect of the TM6SF2 E167K on these lipids in the human liver and serum and on hepatic gene expression and studied the effect of TM6SF2 knockdown on hepatocyte handling of these lipids.

Methods

Liver biopsies were taken from subjects characterized with respect to the TM6SF2 genotype, serum and liver lipidome, gene expression and histology. In vitro, after TM6SF2 knockdown in HuH-7 cells, we compared incorporation of different fatty acids into TGs, CEs, and PCs.

Results

The TM6SF2^EK/KK and TM6SF2^EE groups had similar age, gender, BMI and HOMA-IR. Liver TGs and CEs were higher and liver PCs lower in the TM6SF2^EK/KK than the TM6SF2^EE group (p <0.05). Polyunsaturated fatty acids (PUFA) were deficient in liver and serum TGs and liver PCs but hepatic free fatty acids were relatively enriched in PUFA (p <0.05). Incorporation of PUFA into TGs and PCs in TM6SF2 knockdown hepatocytes was decreased (p <0.05). Hepatic expression of TM6SF2 was decreased in variant carriers, and was co-expressed with genes regulated by PUFAs.

Conclusions

Hepatic lipid synthesis from PUFAs is impaired and could contribute to deficiency in PCs and increased intrahepatic TG in TM6SF2 E167K variant carriers.

Introduction

Common non-alcoholic fatty liver disease (NAFLD) is associated with features of the metabolic syndrome, such as hypertriglyceridemia, and increased risk of cardiovascular disease (CVD).¹ In 2014, a non-synonymous adenine-to-guanine substitution (rs58542926) replacing glutamate at residue 167 with lysine (E167K) in the transmembrane 6 superfamily member 2 (TM6SF2) protein was shown to increase liver fat content[2], [3] and the risk of liver fibrosis.⁴ In these and several subsequent studies, variant allele carriers (TM6SF2^EK/KK) were found to be neither more obese nor more insulin-resistant than non-carriers (TM6SF2^EE).[2], [5], [6], [7], [8], [9], [10], [11], [12] In contrast to most cases with ‘obese/metabolic’ NAFLD, TM6SF2 variant allele carriers have normal[5], [6], [10], [11] or decreased[2], [8], [9], [10], [12] plasma TG concentrations and a reduced risk of CVD.[12], [13]

The exact function of the TM6SF2 variant is unknown. In hepatocytes, TM6SF2 minor allele carriage is associated with decreased TM6SF2 protein expression.² TM6SF2 siRNA inhibition in hepatocytes increases cellular TG concentrations and reduces TG secretion, while overexpression of TM6SF2 has the opposite effect.³ In mice, hepatic knockdown of TM6SF2 increases liver triglycerides (TG) and cholesteryl esters (CE), the main constituents of the hydrophobic core of very low-density lipoprotein (VLDL) particles.²

Hepatic steatosis, reduced hepatic TG secretion, and protection from CVD characterize animals with deletion of genes that are involved in phosphatidylcholine (PC) synthesis such as CTP:phosphocholine cytidylyltransferase alpha (CTα) and phosphatidylethanolamine N-methyltransferase (PEMT).[14], [15] A similar phenotype also results from deletion of lysophosphatidylcholine acyltransferase 3 (LPCAT3), which decreases hepatic concentrations of polyunsaturated PCs.¹⁶ PCs are the only phospholipids required for assembly of VLDL particles.¹⁵ PC deficiency increases intrahepatic degradation of VLDL particles and thereby reduces their secretion.¹⁵ Dietary restriction of choline, a precursor of PC, increases liver fat in humans.¹⁷ Consumption of a methionine-choline-deficient diet, a widely used in vivo experimental model for NAFLD and NASH, reduces hepatic PC synthesis and plasma TGs and increases liver TGs without inducing insulin resistance.[15], [18] In humans, a polymorphism in PEMT, which reduces PC synthesis, may be more frequent in participants with NAFLD[19], [20], [21] and in participants with non-alcoholic steatohepatitis (NASH)²² than in those without, and characterizes participants who develop NAFLD on a choline-depleted diet.²³ Since these changes resemble those of carriers of the TM6SF2 E167K gene variant,[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13] we hypothesized that reduced PC synthesis might characterize carriers of the E167K variant.

In the present study, we profiled hepatic gene expression and measured concentrations of TGs, CEs, PCs and free fatty acids (FFA) in human liver biopsy samples from carriers (TM6SF2^EK/KK) and non-carriers (TM6SF2^EE) of the TM6SF2 E167K variant. Since the in vivo results seemed to support our hypothesis, we directly measured incorporation of saturated (16:0, palmitic acid), monounsaturated (18:1, oleic acid), and polyunsaturated fatty acids (20:4, arachidonic acid) into TG, CE, and PC pools in TM6SF2 knockdown HuH-7 cells. In addition, we measured circulating concentrations and composition of TGs, CEs and PCs in carriers and non-carriers of the gene variant.