Liver transcriptomics highlights interleukin-32 as novel NAFLD-related cytokine and candidate biomarker.

OBJECTIVE: Efforts to manage non-alcoholic fatty liver disease (NAFLD) are limited by the incomplete understanding of the pathogenic mechanisms and the absence of accurate non-invasive biomarkers. The aim of this study was to identify novel NAFLD therapeutic targets andbiomarkers by conducting liver transcriptomic analysis in patients stratified by the presence of the PNPLA3 I148M genetic risk variant. DESIGN: We sequenced the hepatic transcriptome of 125 obese individuals. 'Severe NAFLD' was defined as the presence of steatohepatitis, NAFLD activity score ≥4 or fibrosis stage ≥2. The circulating levels of the most upregulated transcript, interleukin-32 (IL32), were measured by ELISA. RESULTS: Carriage of the PNPLA3 I148M variant correlated with the two major components of hepatic transcriptome variability and broadly influenced gene expression. In patients with severe NAFLD, there was an upregulation of inflammatory and lipid metabolism pathways. IL32 was the most robustly upregulated gene in the severe NAFLD group (adjusted p=1×10-6), and its expression correlated with steatosis severity, both in I148M variant carriers and non-carriers. In 77 severely obese, and in a replication cohort of 160 individuals evaluated at the hepatology service, circulating IL32 levels were associated with both NAFLD and severe NAFLD independently of aminotransferases (p<0.01 for both). A linear combination of IL32-ALT-AST showed a better performance than ALT-AST alone in NAFLD diagnosis (area under the curve=0.92 vs 0.81, p=5×10-5). CONCLUSION: Hepatic IL32 is overexpressed in NAFLD, correlates with hepatic fat and liver damage, and is detectable in the circulation, where it is independently associated with the presence and severity of NAFLD.

MBOAT7 is anchored to endomembranes by six transmembrane domains.

Membrane bound O-acyltransferase domain- containing 7 (MBOAT7, also known as LPIAT1) is a protein involved in the acyl chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 is a susceptibility risk genetic locus for non-alcoholic fatty liver disease (NAFLD) and mental retardation. Although it has been shown that MBOAT7 is associated to membranes, the MBOAT7 topology remains unknown. To solve the topological organization of MBOAT7, we performed: A) solubilization of the total membrane fraction of cells overexpressing the recombinant MBOAT7-V5, which revealed MBOAT7 is an integral protein strongly attached to endomembranes; B) in silico analysis by using 22 computational methods, which predicted the number and localization of transmembrane domains of MBOAT7 with a range between 5 and 12; C) in vitro analysis of living cells transfected with GFP-tagged MBOAT7 full length and truncated forms, using a combination of Western Blotting, co-immunofluorescence and Fluorescence Protease Protection (FPP) assay; D) in vitro analysis of living cells transfected with FLAG-tagged MBOAT7 full length forms, using a combination of Western Blotting, selective membrane permeabilization followed by indirect immunofluorescence. All together, these data revealed that MBOAT7 is a multispanning transmembrane protein with six transmembrane domains. Based on our model, the predicted catalytic dyad of the protein, composed of the conserved asparagine in position 321 (Asn-321) and the preserved histidine in position 356 (His-356), has a lumenal localization. These data are compatible with the role of MBOAT7 in remodeling the acyl chain composition of endomembranes.

Human Multilineage 3D Spheroids as a Model of Liver Steatosis and Fibrosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries. Despite the high prevalence of NAFLD, the underlying biology of the disease progression is not clear, and there are no approved drugs to treat non-alcoholic steatohepatitis (NASH), the most advanced form of the disease. Thus, there is an urgent need for developing advanced in vitro human cellular systems to study disease mechanisms and drug responses. We attempted to create an organoid system genetically predisposed to NAFLD and to induce steatosis and fibrosis in it by adding free fatty acids. We used multilineage 3D spheroids composed by hepatocytes (HepG2) and hepatic stellate cells (LX-2) with a physiological ratio (24:1). HepG2 and LX-2 cells are homozygotes for the PNPLA3 I148M sequence variant, the strongest genetic determinant of NAFLD. We demonstrate that hepatic stellate cells facilitate the compactness of 3D spheroids. Then, we show that the spheroids develop accumulations of fat and collagen upon exposure to free fatty acids. Finally, this accumulation was rescued by incubating spheroids with liraglutide or elafibranor, drugs that are in clinical trials for the treatment of NASH. In conclusion, we have established a simple, easy to handle, in vitro model of genetically induced NAFLD consisting of multilineage 3D spheroids. This tool may be used to understand molecular mechanisms involved in the early stages of fibrogenesis induced by lipid accumulation. Moreover, it may be used to identify new compounds to treat NASH using high-throughput drug screening.

PNPLA3 overexpression results in reduction of proteins predisposing to fibrosis.

Liver fibrosis is a pathological scarring response to chronic hepatocellular injury and hepatic stellate cells (HSCs) are key players in this process. PNPLA3 I148M is a common variant robustly associated with liver fibrosis but the mechanisms underlying this association are unknown. We aimed to examine a) the effect of fibrogenic and proliferative stimuli on PNPLA3 levels in HSCs and b) the role of wild type and mutant PNPLA3 overexpression on markers of HSC activation and fibrosis.Here, we show that PNPLA3 is upregulated by the fibrogenic cytokine transforming growth factor-beta (TGF-ß), but not by platelet-derived growth factor (PDGF), and is involved in the TGF-ß-induced reduction in lipid droplets in primary human HSCs. Furthermore, we show that retinol release from human HSCs ex vivo is lower in cells with the loss-of-function PNPLA3 148M compared with 148I wild type protein. Stable overexpression of PNPLA3 148I wild type, but not 148M mutant, in human HSCs (LX-2 cells) induces a reduction in the secretion of matrix metallopeptidase 2 (MMP2), tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2), which is mediated by retinoid metabolism. In conclusion, we show a role for PNPLA3 in HSC activation in response to fibrogenic stimuli. Moreover, we provide evidence to indicate that PNPLA3-mediated retinol release may protect against liver fibrosis by inducing a specific signature of proteins involved in extracellular matrix remodelling.

Cys Site-Directed Mutagenesis of the Human SLC1A5 (ASCT2) Transporter: Structure/Function Relationships and Crucial Role of Cys467 for Redox Sensing and Glutamine Transport.

The human plasma membrane transporter ASCT2 is responsible for mediating Na- dependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in P. pastoris and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2.

The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS: We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS: The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS: We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

The rs2294918 E434K variant modulates patatin-like phospholipase domain-containing 3 expression and liver damage.

UNLABELLED: The patatin-like phosholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is a major determinant of hepatic fat and predisposes to the full spectrum of liver damage in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate whether additional PNPLA3 coding variants contribute to NAFLD susceptibility, first in individuals with contrasting phenotypes (with early-onset NAFLD vs. very low aminotransferases) and then in a large validation cohort. Rare PNPLA3 variants were not detected by sequencing coding regions and intron-exon boundaries either in 142 patients with early-onset NAFLD nor in 100 healthy individuals with alanine aminotransferase <22/20 IU/mL. Besides rs738409 I148M, the rs2294918 G>A polymorphism (E434K sequence variant) was over-represented in NAFLD (adjusted P = 0.01). In 1,447 subjects with and without NAFLD, the 148M-434E (P < 0.0001), but not the 148M-434K, haplotype (P > 0.9), was associated with histological NAFLD and steatohepatitis. Both the I148M (P = 0.0002) and E434K variants (P = 0.044) were associated with serum ALT levels, by interacting with each other, in that the 434K hampered the association with liver damage of the 148M allele (P = 0.006). The E434K variant did not affect PNPLA3 enzymatic activity, but carriers of the rs2294918 A allele (434K) displayed lower hepatic PNPLA3 messenger RNA and protein levels (P < 0.05). CONCLUSIONS: Rare loss-of-function PNPLA3 variants were not detected in early-onset NAFLD. However, PNPLA3 rs2294918 E434K decreased PNPLA3 expression, lessening the effect of the I148M variant on the predisposition to steatosis and liver damage. This suggests that the PNPLA3 I148M variant has a codominant negative effect on triglycerides mobilization from lipid droplets, mediated by inhibition of other lipases.

DEPDC5 variants increase fibrosis progression in Europeans with chronic hepatitis C virus infection.

UNLABELLED: Chronic hepatitis C virus (HCV) infection may progress to cirrhosis and hepatocellular carcinoma (HCC). Recently, two genetic variants, DEPDC5 rs1012068 and MICA rs2596542, were associated with the onset of HCC in Asian subjects with chronic HCV infection. The aim of the present study was to analyze whether DEPDC5 and MICA genetic variants were associated with liver disease progression in European subjects with chronic HCV infection. In a Northern Italian discovery cohort (n = 477), neither DEPDC5 rs1012068 nor MICA rs2596542 were associated with HCC (n = 150). However, DEPDC5 rs1012068 was independently associated with cirrhosis (n = 300; P = 0.049). The association of rs1012068 with moderate to severe fibrosis was confirmed in an independent cross-sectional German cohort (n = 415; P = 0.006). Furthermore, DEPDC5 rs1012068 predicted faster fibrosis progression in a prospective cohort (n = 247; P = 0.027). Next, we examined the distribution of nonsynonymous DEPDC5 variants in the overall cross-sectional cohort (n = 912). The presence of at least one variant increased the risk of moderate/severe fibrosis by 54% (P = 0.040). To understand the molecular mechanism underlying the genetic association of DEPDC5 variants with fibrosis progression, we performed in vitro studies on immortalized hepatic stellate cells (LX-2). In these cells, down-regulation of DEPDC5 resulted in increased expression of ß-catenin and production of its target matrix metallopeptidase 2 (MMP2), a secreted enzyme involved in fibrosis progression. CONCLUSION: DEPDC5 variants increase fibrosis progression in European subjects with chronic HCV infection. Our findings suggest that DEPDC5 down-regulation may contribute to HCV-related fibrosis by increasing MMP2 synthesis through the ß-catenin pathway.

PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells.

Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.

Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function.

The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.

Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.

The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni(2+)-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [(3)H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na(+). Internal Na(+) slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [(3)H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [(3)H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na(+) on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism.

Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5).

The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na(+)-glutamineex/glutaminein transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K(+) gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na(+). Internal Na(+) exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.

PSD3 downregulation confers protection against fatty liver disease.

Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.

LPIAT1/MBOAT7 contains a catalytic dyad transferring polyunsaturated fatty acids to lysophosphatidylinositol.

Human membrane bound O-acyltransferase domain-containing 7 (MBOAT7), also known as lysophosphatidylinositol acyltransferase 1 (LPIAT1), is an enzyme involved in the acyl-chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 rs641738 variant has been associated with the entire spectrum of fatty liver disease (FLD) and neurodevelopmental disorders, but the exact enzymatic activity and the catalytic site of the protein are still unestablished. Human wild type MBOAT7 and three MBOAT7 mutants missing in the putative catalytic residues (N321A, H356A, N321A + H356A) were produced into Pichia pastoris, and purified using Ni-affinity chromatography. The enzymatic activity of MBOAT7 wild type and mutants was assessed measuring the incorporation of radiolabeled fatty acids into lipid acceptors. MBOAT7 preferentially transferred 20:4 and 20:5 polyunsaturated fatty acids (PUFAs) to lysophosphatidylinositol (LPI). On the contrary, MBOAT7 showed weak enzymatic activity for transferring saturated and unsaturated fatty acids, regardless the lipid substrate. Missense mutations in the putative catalytic residues (N321A, H356A, N321A + H356A) result in a loss of O-acyltransferase activity. Thus, MBOAT7 catalyzes the transfer of PUFAs to lipid acceptors. MBOAT7 shows the highest affinity for LPI, and missense mutations at the MBOAT7 putative catalytic dyad inhibit the O-acyltransferase activity of the protein. Our findings support the hypothesis that the association between the MBOAT7 rs641738 variant and the increased risk of NAFLD is mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling. Taken together, the increased knowledge of the enzymatic activity of MBOAT7 gives insights into the understanding on the basis of FLD.

Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing PNPLA3 levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in PNPLA3 which, when co-inherited, reduces PNPLA3 mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the PNPLA3 gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and PNPLA3. These pathways may be exploited by drug modalities to "tune down" the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.

The role of PNPLA3 in health and disease.

The human patatin-like phospholipase domain-containing 3 (PNPLA3) gene encodes for a protein of 481 amino-acids. The variant rs738409 is a cytosine to guanine substitution, encoding for the isoleucine to methionine substitution at position 148 (I148M) of the protein. This variant is strongly associated with the entire spectrum of liver disease. Although this variant is one of the best characterized and deeply studied, the mechanism behind the PNPLA3 and the liver disease is still not well defined. Functionally, it has become clear that the PNPLA3 protein is an enzyme with lipase activity towards triglycerides and retinyl esters, and acyltransferase activity on phospholipids. The aim of this review is to collect the latest data, obtained by in vitro and in vivo experiments, on the functional aspects of the PNPLA3 protein. Defining the precise role of PNPLA3 in the liver lipid metabolism, in order to develop novel therapies for the treatment of liver disease, will be the key of future research.

Deciphering the role of V200A and N291S mutations leading to LPL deficiency.

BACKGROUND AND AIMS: Type I hyperlipoproteinemia is an autosomal recessive disorder of lipoprotein metabolism caused by mutations in the LPL gene, with an estimated prevalence in the general population of 1 in a million. In this work, we studied the molecular mechanism of two known mutations in the LPL gene in ex vivo and in vitro experiments and also the effect of two splice site mutations in ex vivo experiments. METHODS: Two patients with hypertriglyceridemia were selected from the Lipid Clinic in Vienna. The first patient was compound heterozygote for c.680T > C (exon 5; p.V200A) and c.1139+1G > A (intron 7 splice site). The second patient was compound heterozygote for c.953A > G (exon 6; p.N291S) and c.1019-3C > A (intron 6 splice site). The LPL gene was sequenced and post-heparin plasma samples (ex vivo) were used to test LPL activity. In vitro experiments were performed in HEK 293T/17 cells transiently transfected with wild type or mutant LPL plasmids. Cell lysate and media were used to evaluate LPL production, secretion, activity and dimerization by Western blot analysis and LPL enzymatic assay, respectively. RESULTS: Our data show that in both patients, LPL activity is absent. V200A is a mutation that alters LPL secretion and activity whereas the N291S mutation affects LPL activity, but both mutations do not affect dimerization. The effect of these mutations in patients is more severe since they have splice site mutations on the other allele. CONCLUSIONS: We characterized these LPL mutations at the molecular level showing that are pathogenic.

The TM6SF2 E167K genetic variant induces lipid biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids.

There is a high unmet need for developing treatments for nonalcoholic fatty liver disease (NAFLD), for which there are no approved drugs today. Here, we used a human in vitro disease model to understand mechanisms linked to genetic risk variants associated with NAFLD. The model is based on 3D spheroids from primary human hepatocytes from five different donors. Across these donors, we observed highly reproducible differences in the extent of steatosis induction, demonstrating that inter-donor variability is reflected in the in vitro model. Importantly, our data indicates that the genetic variant TM6SF2 E167K, previously associated with increased risk for NAFLD, induces increased hepatocyte fat content by reducing APOB particle secretion. Finally, differences in gene expression pathways involved in cholesterol, fatty acid and glucose metabolism between wild type and TM6SF2 E167K mutation carriers (N = 125) were confirmed in the in vitro model. Our data suggest that the 3D in vitro spheroids can be used to investigate the mechanisms underlying the association of human genetic variants associated with NAFLD. This model may also be suitable to discover new treatments against NAFLD.

Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of advanced chronic liver disease. The progression of NAFLD, including nonalcoholic steatohepatitis (NASH), has a strong genetic component, and the most robust contributor is the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 encoding the 148M protein sequence variant. We hypothesized that suppressing the expression of the PNPLA3 148M mutant protein would exert a beneficial effect on the entire spectrum of NAFLD. METHODS: We examined the effects of liver-targeted GalNAc3-conjugated antisense oligonucleotide (ASO)-mediated silencing of Pnpla3 in a knock-in mouse model in which we introduced the human PNPLA3 I148M mutation. RESULTS: ASO-mediated silencing of Pnpla3 reduced liver steatosis (p = 0.038) in homozygous Pnpla3 148M/M knock-in mutant mice but not in wild-type littermates fed a steatogenic high-sucrose diet. In mice fed a NASH-inducing diet, ASO-mediated silencing of Pnpla3 reduced liver steatosis score and NAFLD activity score independent of the Pnpla3 genotype, while reductions in liver inflammation score (p = 0.018) and fibrosis stage (p = 0.031) were observed only in the Pnpla3 knock-in 148M/M mutant mice. These responses were accompanied by reduced liver levels of Mcp1 (p = 0.026) and Timp2 (p = 0.007) specifically in the mutant knock-in mice. This may reduce levels of chemokine attracting inflammatory cells and increase the collagenolytic activity during tissue regeneration. CONCLUSION: This study provides the first evidence that a Pnpla3 ASO therapy can improve all features of NAFLD, including liver fibrosis, and suppress the expression of a strong innate genetic risk factor, Pnpla3 148M, which may open up a precision medicine approach in NASH.