Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease. To elucidate the molecular basis of NAFLD we performed an exome-wide association study of liver fat content. Three variants were associated with increased liver fat at the exome-wide significance level: two in PNPLA3, an established locus for NAFLD, and one (Glu167Lys) in TM6SF2, a gene of unknown function. The Glu167LysTM6SF2 variant was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and lower levels of LDL-cholesterol, triglycerides and alkaline phosphatase in 3 independent populations (n>80,000). Recombinant Glu167LysTM6SF2 produced 50% less protein than wild-type TM6SF2 when expressed in cultured hepatocytes. Adeno-associated virus-mediated shRNA knockdown of Tm6sf2 in mice increased liver triglyceride content 3-fold and decreased VLDL secretion by 50%. Taken together, these data indicate that TM6SF2 activity is required for normal VLDL secretion, and that impaired TM6SF2 function causally contributes to NAFLD.

A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3), is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2–3 fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 mRNA. Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). Conclusion These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver.

A missense mutation (E167K) in TM6SF2 (transmembrane 6 superfamily member 2), a polytopic protein of unknown function, is associated with the full spectrum of fatty liver disease. To investigate the role of TM6SF2 in hepatic triglyceride (TG) metabolism, we inactivated the gene in mice. Chronic inactivation of Tm6sf2 in mice is associated with hepatic steatosis, hypocholesterolemia, and transaminitis, thus recapitulating the phenotype observed in humans. No dietary challenge was required to elicit the phenotype. Immunocytochemical and cell fractionation studies revealed that TM6SF2 was present in the endoplasmic reticulum and Golgi complex, whereas the excess neutral lipids in the Tm6sf2−/− mice were located in lipid droplets. Plasma VLDL-TG levels were reduced in the KO animals due to a 3-fold decrease in VLDL-TG secretion rate without any associated reduction in hepatic apoB secretion. Both VLDL particle size and plasma cholesterol levels were significantly reduced in KO mice. Despite levels of TM6SF2 protein being 10-fold higher in the small intestine than in the liver, dietary lipid absorption was only modestly reduced in the KO mice. Our data, taken together, reveal that TM6SF2 is required to mobilize neutral lipids for VLDL assembly but is not required for secretion of apoB-containing lipoproteins. Despite TM6SF2 being located in the endoplasmic reticulum and Golgi complex, the lipids that accumulate in its absence reside in lipid droplets.

A sequence variation (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is strongly associated with fatty liver disease (FLD), but the underlying mechanism remains obscure. Here we used knock-in (KI) mice (Pnpla3148M/M) to examine the mechanism responsible for accumulation of triglyceride (TG) and PNPLA3 in hepatic lipid droplets (LDs). No differences were found between Pnpla3148M/M and Pnpla3+/+ mice in hepatic TG synthesis, utilization, or secretion. These results are consistent with TG accumulation in the Pnpla3148M/M mice being caused by impaired TG mobilization from LDs. Sucrose feeding, which is required to elicit fatty liver in KI mice, led to a much larger and more persistent increase in PNPLA3 protein in the KI than in the WT mice. Inhibition of the proteasome (bortezomib), but not macroautophagy (3-methyladenine), markedly increased PNPLA3 levels in WT mice, coincident with the appearance of ubiquitylated forms of the protein. Bortezomib did not increase PNPLA3 levels in Pnpla3148M/M mice, and only trace amounts of ubiquitylated PNPLA3 were seen in these animals. Conclusion: These results are consistent with the notion that the 148M variant disrupts ubiquitylation and proteasomal degradation of PNPLA3, resulting in accumulation of PNPLA3-148M and impaired mobilization of TG from LDs.

Fatty liver disease (FLD) is a disorder in which accumulation of triglycerides (TGs) in the liver can lead to inflammation, fibrosis, and cirrhosis. Previously, we identified a variant (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) that is strongly associated with FLD, but the mechanistic basis for the association remains elusive. Although PNPLA3 has TG hydrolase activity in vitro, inactivation or overexpression of the WT protein in mice does not cause steatosis. In contrast, expression of two catalytically defective forms of PNPLA3 (I148M or S47A) in sucrose-fed mice causes accumulation of both PNPLA3 and TGs on hepatic lipid droplets (LDs). To determine if amassing PNPLA3 on LDs is a cause or consequence of steatosis, we engineered a synthetic isoform of PNPLA3 that uncouples protein accumulation from loss of enzymatic activity. Expression of a ubiquitylation-resistant form of PNPLA3 in mice caused accumulation of PNPLA3 on hepatic LDs and development of FLD. Lowering PNPLA3 levels by either shRNA knockdown or proteolysis-targeting chimera (PROTAC)-mediated degradation reduced liver TG content in mice overexpressing PNPLA3(148M). Taken together, our results show that the steatosis associated with PNPLA3(148M) is caused by accumulation of PNPLA3 on LDs.