Patatin-like Phospholipase Domain-containing 3 I148M Research Articles

Role of PNPLA3 in Hepatic Stellate Cells and Hepatic Cellular Crosstalk.

Since its discovery, the patatin-like phospholipase domain containing 3 (PNPLA3) (rs738409 C>G p.I148M) variant has been studied extensively to unravel its molecular function. Although several studies proved a causal relationship between the PNPLA3 I148M variant and MASLD development and particularly fibrosis, the pathological mechanisms promoting this phenotype have not yet been fully clarified. We summarise the latest data regarding the PNPLA3 I148M variant in hepatic stellate cells (HSCs) activation and macrophage biology or the path to inflammation-induced fibrosis. Elegant but contradictory studies have ascribed PNPLA3 a hydrolase or an acyltransferase function. The PNPLA3 I148M results in hepatic lipid accumulation, which predisposes the hepatocyte to lipotoxicity and lipo-apoptosis, producing DAMPs, cytokines and chemokines leading to recruitment and activation of macrophages and HSCs, propagating fibrosis. Recent studies showed that the PNPLA3 I148M variant alters HSCs biology via attenuation of PPARγ, AP-1, LXRα and TGFβ activity and signalling. The advent of refined techniques in isolating HSCs has made PNPLA3's direct role in HSCs for liver fibrosis development more apparent. However, many other mechanisms still need detailed investigations.

PNPLA3 allele frequency has no impact on biliary bile acid composition or disease course in patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease that leads to bile duct strictures, cholestasis, and biliary cirrhosis. PNPLA3 (patatin-like phospholipase domain containing 3), regulates cellular lipid synthesis by converting lysophosphatidic acid into phosphatidic acid. Isoleucine mutation to methionine at position 148 (I148M) causes a loss of this function. Only two studies, with contradictory results, have evaluated the role of PNPLA3 in PSC. The rs738409(G) variant of PNPLA3 has been associated with an increased risk for transplantation in male patients with dominant strictures (DS). The study aimed to evaluate the PNPLA3 allele frequency effect on the clinical outcomes, progression, and prognosis of PSC. Furthermore, we analyzed the impact of PNPLA3 on phospholipid and bile acid composition to evaluate the effect of the PNPLA3 status on UDCA response. We recruited 560 patients prospectively and collected clinical and laboratory data as well as liver histology and imaging findings. PNPLA3 (CC, CG, GG) alleles were analyzed with TaqManTM. We also analyzed bile acids (BA), cholesterol and phospholipids and individual BA from a sample aspirated during endoscopic retrograde cholangiography (ERC). Among the recruited patients, 58.4%, 35.7% and 5.9% had the wild (CC), heterozygous (CG) and homozygous (GG) alleles, respectively. The PNPLA3 haplotype did not impact bile composition or individual BA. In addition, we found no differences in age at diagnosis, disease progression, liver fibrosis or survival between the cohorts. The PNPLA3 I148M variant had no significant impact on on bile composition, including UDCA content, clinical outcomes, progression of liver fibrosis, hepatobiliary cancer risk, liver transplantation, or overall survival.

Hepatic patatin-like phospholipase domain-containing 3 levels are increased in I148M risk allele carriers and correlate with NAFLD in humans.

In nonalcoholic fatty liver disease (NAFLD) the patatin‐like phospholipase domain‐containing 3 (PNPLA3) rs738409 variant is a contributor. In mice, the Pnpla3 148M variant accumulates on lipid droplets and probably leads to sequestration of a lipase cofactor leading to impaired mobilization of triglycerides. To advance our understanding of the localization and abundance of PNPLA3 protein in humans, we used liver biopsies from patients with NAFLD to investigate the link to NAFLD and the PNPLA3 148M genotype. We experimentally qualified an antibody against human PNPLA3. Hepatic PNPLA3 protein fractional area and localization were determined by immunohistochemistry in biopsies from a well‐characterized NAFLD cohort of 67 patients. Potential differences in hepatic PNPLA3 protein levels among patients related to degree of steatosis, lobular inflammation, ballooning, and fibrosis, and PNPLA3 I148M gene variants were assessed. Immunohistochemistry staining in biopsies from patients with NAFLD showed that hepatic PNPLA3 protein was predominantly localized to the membranes of small and large lipid droplets in hepatocytes. PNPLA3 protein levels correlated strongly with steatosis grade (p = 0.000027) and were also significantly higher in patients with lobular inflammation (p = 0.009), ballooning (p = 0.022), and significant fibrosis (stage 2–4, p = 0.014). In addition, PNPLA3 levels were higher in PNPLA3 rs738409 148M (CG, GG) risk allele carriers compared to 148I (CC) nonrisk allele carriers (p = 0.0029). Conclusion: PNPLA3 protein levels were associated with increased hepatic lipid content and disease severity in patients with NAFLD and were higher in PNPLA3 rs738409 (148M) risk allele carriers. Our hypothesis that increased hepatic levels of PNPLA3 may be part of the pathophysiological mechanism of NAFLD is supported.

Association of PNPLA3 I148M with Liver Disease Biomarkers in Latinos

Introduction: Liver disease accounts for approximately 2 million deaths per year worldwide. The majority of liver diseases are due to complications of cirrhosis, viral hepatitis, and hepatocellular carcinoma. Increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) may indicate liver disease. Moreover, there are additional noninvasive liver fibrosis indices that help to estimate liver damage, including AST-to-ALT ratio, AST-to-platelet ratio index (APRI), fibrosis-4 (FIB-4) score, and nonalcoholic fatty liver disease (NAFLD) fibrosis score. The aims of the present study were to (1) perform an association analysis of the patatin-like phospholipase domain containing 3 (PNPLA3) I148M (rs738409) variant with ALT, AST, and various liver fibrosis indices, and (2) determine whether there are gender-related differences in these associations. Methods: We obtained demographic, anthropometric, and metabolic phenotypes from Latino adult participants (n = 503, 64% female, 36.4 ± 0.5 years) from the Arizona Insulin Resistance (AIR) registry. SNP genotyping of I148M was performed using the TaqMan allelic discrimination assay. We used linear regression for the association analyses of the genotypes with ALT, AST, and the various liver fibrosis indices. We included genotype, age, body mass index, and alcohol status in the linear regression model. Results: The variant I148M was in Hardy-Weinberg equilibrium, with genotype distribution: non-risk CC 118, heterozygous CG 246, and risk GG 139. The G allele was significantly associated with increased ALT and AST levels (p = 7.8 × 10^–7 and p = 9.7 × 10^–6, respectively). Moreover, we showed that the G allele was significantly associated with higher APRI (p = 3.7 × 10^–7) and FIB-4 score (p = 4.1 × 10^–3). When we analyzed the data by gender, we observed similar significant trends for ALT, AST, and APRI (all, p < 0.01). In females, the G allele was significantly associated with increased FIB-4 score (p = 6.9 × 10^–3), which was not observed in the males (p > 0.05). There was no association of the I148M variant with AST/ALT ratio nor NAFLD risk score, whether analyzed in all adults or by gender. Discussion/Conclusion: Our findings provide additional evidence of an association of PNPLA3 I148M with several liver disease biomarkers in male and female Latinos residing in the Southwest of the United States.

The pharmacogenomics of asparaginase‐mediated toxicities in acute lymphoblastic leukemia (ALL) patients

The bacterial enzyme L‐asparaginase (ASNase) is an essential component of effective combination chemotherapy for the treatment of acute lymphoblastic leukemia (ALL). However, the development of immune‐mediated toxicities to this agent can increase the risk of leukemia relapse in children, whereas the high risk of liver injury limits the use of ASNase in adults despite its benefit on treatment outcome. The overarching goal of our studies is to understand the underlying molecular mechanisms of common asparaginase‐mediated toxicities that lead to poor treatment outcome by integrating genome‐wide association studies (GWAS) and experimental validations using genetic and pharmacological models.In the first example, we focused on ASNase‐induced immunotoxicity. We identified by GWAS a higher risk of ASNase immunotoxicity among carriers of the nuclear factor of activated T‐cells, cytoplasmic, calcineurin‐dependent 2 (NFATC2) rs6021191 variant, which leads to elevated expression of this transcription factor involved in the regulation of immune responses. Consistent with our GWAS results, we found the NFATc2‐deficient mice were protected from ASNase‐induced immunotoxicity. Furthermore, we have demonstrated that the pharmacological inhibition of the NFAT pathway in wild‐type mice yields similar protection as NFATc2 deficiency. Current research aims to develop a selective inhibitor of calcineurin‐mediated NFATc2 activation that is expected to attenuate ASNase‐induced immunotoxicity in pediatric ALL patients.In another example, we focused on ASNase‐induced hepatotoxicity, including steatosis. Our group led the first GWAS of ASNase hepatotoxicity and identified an association between the patatin‐like phospholipase domain‐containing 3 (PNPLA3) I148M variant and alanine aminotransferase (ALT) levels after ASNase therapy. The PNPLA3 I148M variant is associated with the development of non‐alcoholic liver disease (NAFLD) and our results are the first to link the variant to drug‐induced liver injury. Consistent with our GWAS, mice treated with ASNase develop fatty livers after a single dose. Additional studies elucidating the mechanism of the toxicity have identified a novel mechanism of drug‐induced liver injury involving ASNase‐induced adipose tissue lipolysis, systemic free fatty acid mobilization, hepatic triglyceride accumulation, and fatty acid‐induced liver injury. Our lab has further demonstrated that pharmacological inhibition of adipose triglyceride lipase (Atgl), which is a key enzyme involved in adipose lipolysis, protects from ASNase‐mediated liver injury. We believe inhibition of ASNase‐induced steatosis will help expand ASNase therapy to adult ALL patients.In summary, our studies are clinically relevant and highly translational. Our ultimate goal is to improve ALL survival by mitigating ASNase‐induced toxicities and ensuring that patients can benefit from the therapeutic effect of ASNase on leukemias.Support or Funding InformationThis study was supported by the University of Pittsburgh School of Pharmacy and the NIH Grant RO1 CA216815.

The role of genetic and epigenetic factors in non alcoholic fatty liver disease (NAFLD) pathogenesis

Background and aimNonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in developed countries, as well as the most common indication for hepatic liver failure in these countries. In this study, we aimed to determine the clinical prognostic significance of the patatin-like phospholipase domain containing 3 (PNPLA3) I148M polymorphism as the genetic dimension in the pathogenesis of NAFLD, the miRNA-122 level as the epigenetic dimension, and to use the miRNA-122 level in NAFLD patients as a biological marker. Material and metodThis study included 40 patients with NAFLD diagnosis who underwent various tests and 20 control groups. At the beginning of the study, the patient's was obtained written informed consent. Age and gender, bady mass ındex (BMI), insulin resistance (IR), aspartate aminotransferase (AST), alanin aminotransferase (ALT) levels were recorded in the patient and control group. ResultsMiRNA-122 level, PNPLA3 I148M polymorphism and various parameters were compared. No statistically significant results were found between miRNA-122 levels when the patient and control group were compared as a result of our study (p = .090). No significant results were found between patient and control group for PNPLA3 I148M polymorphism (p = .087). ConclusionIn the light of this obtained data, progress in NAFLD management will benefit us from further scientific studies in terms of providing new diagnostic biomarkers and therapeutic targets.

A Microphysiological System for Studying Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments. There is an urgent need for better understanding of the genetic and molecular pathways that underlie NAFLD/NASH, and currently available preclinical models, be they in vivo or in vitro, do not fully represent key aspects of the human disease state. We have developed a human in vitro co‐culture NASH model using primary human hepatocytes, Kupffer cells and hepatic stellate cells, which are cultured together as microtissues in a perfused three‐dimensional microphysiological system (MPS). The microtissues were cultured in medium containing free fatty acids for at least 2 weeks, to induce a NASH‐like phenotype. The co‐culture microtissues within the MPS display a NASH‐like phenotype, showing key features of the disease including hepatic fat accumulation, the production of an inflammatory milieu, and the expression of profibrotic markers. Addition of lipopolysaccharide resulted in a more pro‐inflammatory milieu. In the model, obeticholic acid ameliorated the NASH phenotype. Microtissues were formed from both wild‐type and patatin‐like phospholipase domain containing 3 (PNPLA3) I148M mutant hepatic stellate cells. Stellate cells carrying the mutation enhanced the overall disease state of the model and in particular produced a more pro‐inflammatory milieu. Conclusion: The MPS model displays a phenotype akin to advanced NAFLD or NASH and has utility as a tool for exploring mechanisms underlying the disease. Furthermore, we demonstrate that in co‐culture the PNPLA3 I148M mutation alone can cause hepatic stellate cells to enhance the overall NASH disease phenotype.

The PNPLA3 I148M variant promotes lipid-induced hepatocyte secretion of CXC chemokines establishing a tumorigenic milieu.

The I148M variant of the Patatin-like phospholipase domain-containing 3 (PNPLA3) protein is associated with an increased risk for liver inflammation and hepatocellular carcinoma (HCC), but the underlying mechanism is unknown. We hypothesized that enhanced CXC chemokine secretion mediates hepatic inflammation that accelerates development of HCC. Expandable primary human (upcyte®) hepatocytes and human PLC/PRF/5 hepatoma cells were lentivirally transduced with both PNPLA3 I148M variants and stimulated with lipids. Cytokine levels in culture supernatant and patient sera (n = 80) were analyzed by ELISA. Supernatants were assessed in transmigration experiments, tube formation, and proliferation assays. In vitro, lipid stimulation of transduced hepatocytes dose-dependently induced the production of interleukin-8 and CXCL1 in hepatocytes carrying the PNPLA3 148M variant. In line, sera from PNPLA3 148M-positive patients with alcoholic liver cirrhosis contained higher levels of interleukin-8 and CXCL1 than patients with wild-type PNPLA3. Supernatants from lipid-stimulated hepatocytes with the PNPLA3 148M variant induced enhanced migration of white blood cells, angiogenesis, and cell proliferation in comparison with supernatants from wild-type hepatocytes via CXC receptors 1 and 2. Increased production of interleukin-8 and CXCL1 by hepatocytes carrying the PNPLA3 148M variant contributes to a pro-inflammatory and tumorigenic milieu in patients with alcoholic liver disease. KEY MESSAGES: The PNPLA3 148M variant is associated with cirrhosis and hepatocellular carcinoma. Lipid stimulation of hepatocytes with this variant induces IL-8 and CXCL1. Supernatants from hepatocytes with this variant promote migration and angiogenesis. Sera from patients with this variant contained enhanced levels of IL-8 and CXCL1. The PNPLA3 148M variant contributes to a tumorigenic milieu via IL-8 and CXCL1.

1896-P: Effect of PNPLA3 I148M Genotypes on Palmitate-Induced Hepatic Inflammation and Apoptosis

Background: GWAS has revealed that rs738409 C&amp;gt;G patatin-like phospholipase domain-containing 3(PNPLA3) is strongly associated with nonalcoholic fatty liver disease(NAFLD), but the mechanism remains obscure. Our study aims to investigate differences in mechanism for genotypes of PNPLA3 I148M in palmitate-induced inflammation and apoptosis. Method: We constructed point mutation PNPLA3 148 I/I,148 I/M 148 M/M HepG2 cell lines via CRISPR-associated (Cas) 9 system. Cells were exposed to palmitate to induce lipid accumulation. Oil red O staining was used to explore the lipid deposition. Levels of inflammatory and apoptotic factors were determined by real-time PCR and western blotting. Result: Lipid accumulation was remarkably increased in cells induced by PA, it is remarkably higher in 148M/M cell than in 148I/I HepG2 cell (P&amp;lt;0.05 PA vs. BSA). Expression of apoptotic and inflammatory markers BIP, p-PERK, CHOP, p-JNK increased significantly in 148M/M and 148I/M hepG2 cells (P&amp;lt;0.05). Compared with 148I/I cells, The mRNA expression of TNF-α and IL-8 was higher in 148M/M hepG2 cells induced by palmitate. Conclusion: Different genotypes of PNPLA3 I148M have different effects on PA-induced lipid deposition, PNPLA3 148M may play an important role in pathogenesis of NAFLD through ER stress CHOP and IRE1α-JNK pathway. Disclosure Y. Chen: None. H. Liang: None. X. Yan: None. X. Xu: None.

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

ObjectiveNonalcoholic 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. MethodsWe 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. ResultsASO-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. ConclusionThis 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.

A Pathophysiologic Approach Combining Genetics and Insulin Resistance to Predict the Severity of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a complex disease dictated by both genetic and environmental factors. While insulin resistance (IR) is a key pathogenic driver, two common genetic variants in patatin‐like phospholipase domain containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) also impart significant risk for disease progression. Traditional approaches to NAFLD risk stratification rely on biomarkers of fibrosis, an end result of disease progression. We hypothesized that by combining genetics and a novel measurement for IR we could predict disease progression by the NAFLD activity score (NAS) and histologic presence of significant fibrosis. A total of 177 patients with biopsy‐proven NAFLD were enrolled in this cross‐sectional study. PNPLA3 I148M and TM6SF2 E167K genotypes were determined by TaqMan assays. The enhanced lipoprotein IR index (eLP‐IR) was calculated from serum biomarkers using nuclear magnetic resonance (NMR) spectroscopy. Multivariate regression models were used to study the relationships between genetics, IR, and histologic features of NAFLD. In the multivariate analysis, the eLP‐IR was strongly associated with histologic features of NAFLD activity and hepatic fibrosis (P < 0.001 to 0.02) after adjustment for potential confounders. PNPLA3 148M and TM6SF2 E167K genotypes were significantly associated with steatosis (P = 0.003 and P = 0.02, respectively). A combination of the eLP‐IR and genetic score was able to predict the presence of NAS ≥3 with an area under the receiver operating characteristic curve (AUROC) of 0.74. Adding age to this model predicted stages 3‐4 liver fibrosis with an AUROC of 0.82. Conclusion: This proof‐of‐concept study supports the hypothesis that genetics and IR are major determinants of NAFLD severity and demonstrates the feasibility of a new risk stratification paradigm using exclusively pathogenic factors.

Relationship between non-alcoholic steatohepatitis, PNPLA3 I148M genotype and bone mineral density in adolescents.

It is uncertain whether non-alcoholic steatohepatitis (NASH) is a risk factor for low bone mineral density (BMD). Our aim was to investigate: (a) associations between NASH and BMD values and (b) associations between PNPLA3 I148M genotypes and BMD, in children with histologically proven non-alcoholic fatty liver disease (NAFLD). BMD area (g/cm2 ) was measured using dual-energy X-ray absorptiometry (DEXA). NASH was diagnosed by a Steatosis, Activity and Fibrosis (SAF) score and FLIP algorithm. Genotyping for patatin-like phospholipase domain containing-3 (PNPLA3) I148M genotype (rs738409) (CC, CG and GG) was undertaken using the TaqMan SNP genotyping allelic discrimination method. Logistic regression was used to test associations [OR (95% CIs)] between low BMD, and both NASH and PNPLA3 I148M genotypes. Thirty-four adolescents (mean age 13.8±1.1years) with histologically confirmed NAFLD were studied. Subjects with NASH (n=25) had a lower BMD (means (SDs) 0.87±0.06 vs 0.97±0.12, P=0.005), compared to subjects without NASH. Subjects with PNPLA3 CG+GG genotypes had a lower BMD compared with subjects with PNPLA3-CC genotype (means (SDs) 0.79±0.20 vs 0.92±0.10, P=0.009). PNPLA3 CG+GG genotypes were independently associated with NASH [OR (95% CIs 1.78, 1.24, 2.99)], and low BMD was associated with both PNPLA3 CG+GG (OR 3.62 (95% CIs 1.21, 5.53), P=0.028) and with SAF score (OR 2.76 (95% CIs 1.12, 5.41), P=0.045). Taken together the independent associations between: (a) low BMD and PNPLA3 CG+GG genotype; (b) low BMD and NASH; and (c) PNPLA3 CG+GG genotype and NASH, provide support for a causal relationship between NASH and low BMD.

Effect of different genotypes of PNPLA3 I148M on hepatocyte proliferation

Objective: To explore the influence of patatin-like phospholipase domain containing-3 (PNPLA3) wild type 148I/I and mutant type 148M/M on HepG2 cell proliferation and the relative mechanisms. Methods: HepG2 cell line stably overexpressing PNPLA3 148I/I, 148M/M and negative control (NC) were set up. Cell counting kit-8 (CCK8) assay was used to measure cell viability. Edu assay was used to determine the ability of cell proliferation. Western blot was used to detect the protein levels in the phosphatidylinositol 3-kinases (PI3K) pathway. Enzyme-linked immunosorbent assay (ELISA) was used to detect proliferation-related PNPLA3 metabolites[arachidonic acid (AA) and lysophosphatidic acid (LPA)]. Quantitative real-time PCR was used to detect the expression level of prostaglandin G/H synthase 2 (PTGS2) and proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) associated with PNPLA3. Results: The cell viability of overexpression of PNPLA3 148M/M group was about 1/3 times higher than that of overexpression of PNPLA3 148I/I group, and the difference was statistically significant[(98.02±1.29)% vs (71.51±2.89)%, P<0.001]. There was no significant difference between overexpression of PNPLA3 148M/M group and negative control group[(98.02±1.29)% vs (100±2.61)%, P=0.181]. The proliferative activity of overexpression of PNPLA3 148M/M group was about 1/3 times higher than that of overexpression of PNPLA3 148I/I group, and the difference was statistically significant(46.46±1.83 vs 35.96±2.65, P=0.001). There was no significant difference between overexpression of PNPLA3 148M/M group and negative control group(46.46±1.83 vs 46.64±7.33, P=0.965). The PGC1α mRNA expression, total PI3K, PThr-308AKT, PSer2448-mammalian target of rapamycin (PSer2448-mTOR) and PGC1α protein expression levels in the overexpression of PNPLA3 148M/M group were higher than those in the overexpression of PNPLA3 148I/I group, but there were no significant differences in AA and LPA levels, as well as PTGS2 mRNA expression levels. Conclusion: PNPLA3 148M/M cell proliferation was stronger than PNPLA3 148I/I.

Genetic Determinants of Circulating Lipoproteins in Nonalcoholic Fatty Liver Disease.

Recent genome-wide association studies have identified 2 genetic polymorphisms in association with nonalcoholic fatty liver disease (NAFLD): patatin-like phospholipase domain containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2), both of which appear to influence the production of very low density lipoprotein (VLDL). The impact of these genetic variations on lipoprotein metabolism in the setting of nonalcoholic steatohepatitis and liver fibrosis are not fully characterized. We measured comprehensive lipoprotein profiles by nuclear magnetic resonance among 170 serially recruited patients in an NAFLD registry, and determined their relationships with PNPLA3 and TM6SF2 genotypes. In this cohort, 72% patients had at least 1 allele of either PNPLA3 I148M or TM6SF2 E167K, and 30% carried 2 alleles. In multivariate models adjusting for histologic features of nonalcoholic steatohepatitis and liver fibrosis, PNPLA3 I148M is associated with a decrease in VLDL particle size. Both PNPLA3 I148M and TM6SF2 E167K genotypes were associated with increases in the size of low density lipoprotein (LDL) and high density lipoprotein particles, phenotypes considered atheroprotective. When adjusted for both genotypes, NAFLD activity score, in particular the degree of hepatic steatosis was strongly associated with increases in the size of VLDL particles, the concentration of LDL, especially small LDL particles, and a decrease in the size of high density lipoprotein particles, all of which are linked with a proatherogenic phenotype. PNPLA3 and TM6SF2 are common genetic variants among NAFLD patients and impact lipoprotein profiles in slightly different ways. The interactions between genotypes, hepatic steatosis, and lipoprotein metabolism shed lights on the pathophysiology of NAFLD, and provide opportunities for personalized treatment in the era of emerging NAFLD therapeutics.

Coding variants in PNPLA3 and TM6SF2 are risk factors for hepatic steatosis and elevated serum alanine aminotransferases caused by a glucagon receptor antagonist.

LY2409021 is a glucagon receptor antagonist that was associated with hepatic steatosis and elevated aminotransferases in phase 2 diabetes studies. We investigated the relationship between selected genetic variants and hepatic steatosis and elevated alanine aminotransferases (ALTs) associated with LY2409021. Patients participated in a 6‐week placebo‐controlled trial (I1R‐MC‐GLDI [GLDI], n = 246) and a 52‐week placebo‐ and active comparator‐controlled trial (I1R‐MC‐GLDJ [GLDJ], n = 158). GLDJ had endpoints at 6 months, including measures of hepatic fat fraction (HFF) by magnetic resonance imaging. The five genes tested were patatin‐like phospholipase domain containing 3 (PNPLA3) (rs738409 and rs738491), transmembrane 6 superfamily member 2 (TM6SF2) (rs58542926), peroxisome proliferative activated receptor gamma coactivator 1 alpha (PPARGC1A) (rs4361373, rs3774921, rs2970849), adenylate cyclase 3 (ADCY3) (rs713586), and insulin‐like growth factor 1 (IGF‐1) (rs1520220). In GLDI, PNPLA3 I148M (P = 0.001) and TM6SF2 E167K (P = 0.001) were significantly associated with an increase in ALT at 6 weeks for LY2409021 but not for placebo. In GLDJ, PNPLA3 I148M showed the same effect (P = 0.007) on ALT at 6 months but the placebo or sitagliptin did not. In GLDJ, both PNPLA3 and TM6SF2 risk‐allele carriers showed increases in HFF that were numerically greater but not statistically significant. The carriers of PNPLA3 and/or TM6SF2 risk alleles showed significantly increased ALT (GLDI, +13.28 U/L in carriers versus +4.84 U/L in noncarriers, P = 4 × 10–5; GLDJ, +14.6 U/L in carriers versus +1.7 in noncarriers, P = 0.0018) and HFF (GLDJ, +5.35% in carriers versus 2.38% in noncarriers, P = 0.048). Elevation of transaminase and HFF were also noted in the noncarriers but at a significantly lower degree. Conclusion: The carriers of PNPLA3 and/or TM6SF2 variant alleles are at risk for hepatic steatosis and elevated ALT levels caused by LY2409021, a glucagon receptor antagonist. More studies are needed to investigate if our observations are generalizable to hepatic steatosis caused by other medications. (Hepatology Communications 2018;2:561‐570)

PNPLA3 variant and portal/periportal histological pattern in patients with biopsy-proven non-alcoholic fatty liver disease: a possible role for oxidative stress

Pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by predisposing genetic variations, dysmetabolism, systemic oxidative stress, and local cellular and molecular cross-talks. Patatin-like phospholipase domain containing 3 (PNPLA3) gene I148M variant is a known determinant of NAFLD. Aims were to evaluate whether PNPLA3 I148M variant was associated with a specific histological pattern, hepatic stem/progenitor cell (HpSC) niche activation and serum oxidative stress markers. Liver biopsies were obtained from 54 NAFLD patients. The activation of HpSC compartment was evaluated by the extension of ductular reaction (DR); hepatic stellate cells, myofibroblasts (MFs), and macrophages were evaluated by immunohistochemistry. Systemic oxidative stress was assessed measuring serum levels of soluble NOX2-derived peptide (sNOX2-dp) and 8-isoprostaglandin F2α (8-iso-PGF2α). PNPLA3 carriers showed higher steatosis, portal inflammation and HpSC niche activation compared to wild-type patients. DR was correlated with NAFLD activity score (NAS) and fibrosis score. Serum 8-iso-PGF2α were significantly higher in I148M carriers compared to non-carriers and were correlated with DR and portal inflammation. sNox2-dp was correlated with NAS and with HpSC niche activation. In conclusion, NAFLD patients carrying PNPLA3 I148M are characterized by a prominent activation of HpSC niche which is associated with a more aggressive histological pattern (portal fibrogenesis) and increased oxidative stress.

AQP3 is regulated by PPAR\u03b3 and JNK in hepatic stellate cells carrying PNPLA3 I148M

Aquaglyceroporins (AQPs) allow the movement of glycerol that is required for triglyceride formation in hepatic stellate cells (HSC), as key cellular source of fibrogenesis in the liver. The genetic polymorphism I148M of the patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with hepatic steatosis and its progression to steatohepatitis (NASH), fibrosis and cancer. We aimed to explore the role of AQP3 for HSC activation and unveil its potential interactions with PNPLA3. HSC were isolated from human liver, experiments were performed in primary HSC and human HSC line LX2. AQP3 was the only aquaglyceroporin present in HSC and its expression decreased during activation. The PPARγ agonist, rosiglitazone, recovered AQP3 expression also in PNPLA3 I148M carrying HSC. When PNPLA3 was silenced, AQP3 expression increased. In liver sections from patients with NASH, the decreased amount of AQP3 was proportional to the severity of fibrosis and presence of the PNPLA3 I148M variant. In PNPLA3 I148M cells, the blockade of JNK pathway upregulated AQP3 in synergism with PPARγ. In conclusion, we demonstrated profound reduction of AQP3 in HSC carrying the PNPLA3 I148M variant in parallel to decreased PPARγ activation, which could be rescued by rosiglitazone and blockade of JNK.

PNPLA3 I148M associations with liver carcinogenesis in Japanese chronic hepatitis C patients.

AimTo investigate associations between patatin-like phospholipase domain-containing 3 (PNPLA3) genotypes and fibrosis and hepatocarcinogenesis in Japanese chronic hepatitis C (CHC) patients.MethodsTwo hundred and thirty-one patients with CHC were examined for PNPLA3 genotypes, liver stiffness measurements (LSM), and hepatocellular carcinoma (HCC) from May 2010 to October 2012 at Fujita Health University Hospital. The rs738409 single nucleotide polymorphism (SNP) encoding for a functional PNPLA3 I148M protein variant was genotyped using a TaqMan predesigned SNP genotyping assay. LSM was determined as the velocity of a shear wave (Vs) with an acoustic radiation force impulse. Vs cut-off values for cirrhosis were set at 1.55 m/s. We excluded CHC patients with a sustained virological response or relapse after interferon treatment.ResultsPNPLA3 genotypes were CC, CG, and GG for 118, 72, and 41 patients, respectively. Multivariable logistic regression analysis selected older age (OR = 1.06; 95% CI: 1.03–1.09; p < 0.0001), higher body mass index (BMI) (OR= 1.12; 95% CI: 1.03–1.22; p = 0.0082), and PNPLA3 genotype GG (OR = 2.07; 95% CI: 0.97–4.42; p = 0.0599) as the factors independently associated with cirrhosis. When 137 patients without past history of interferon treatment were separately assessed, multivariable logistic regression analysis selected older age (OR = 1.05; 95% CI: 1.02–1.09; p = 0.0034), and PNPLA3 genotype GG (OR = 3.35; 95% CI: 1.13–9.91; p = 0.0291) as the factors independently associated with cirrhosis. Multivariable logistic regression analysis selected older age (OR = 1.12; 95% CI: 1.07–1.17; p < 0.0001), PNPLA3 genotype GG (OR = 2.62; 95% CI: 1.15–5.96; p = 0.0218), and male gender (OR = 1.83; 95% CI: 0.90–3.71); p = 0.0936) as the factors independently associated with HCC.ConclusionPNPLA3 genotype I148M is one of risk factors for developing HCC in Japanese CHC patients, and is one of risk factors for progress to cirrhosis in the patients without past history of interferon treatment.

Role of the PNPLA3 I148M polymorphism in nonalcoholic fatty liver disease and fibrosis in Korea.

The role of the patatin-like phospholipase domain-containing 3 (PNPLA3) single-nucleotide polymorphism (SNP), rs738409, in the development and progression of nonalcoholic fatty liver disease (NAFLD) has not been studied in the Korean population. The aim of the study was to investigate the genotype frequency and allele distribution of PNPLA3 rs738409 and the association between the SNP and development of NAFLD and liver fibrosis. A total of 339 Korean adults (155 NAFLD patients and 184 healthy controls) were enrolled. PNPLA3 SNP genotyping was carried out using a TaqMan allelic discrimination assay. Liver fibrosis severity was evaluated by NAFLD fibrosis score (NFS) and BARD score. The frequencies of the PNPLA3 rs738409 genotypes, CC, CG, and GG in the healthy control group were 29.9, 50.0, and 20.1%, respectively, and those in NAFLD patients were 20.0, 48.4, and 31.6%, respectively, showing a higher frequency of the risk allele (G allele) (p = 0.006). Among the NAFLD patients, the CG+GG genotype frequency was significantly higher in patients with advanced fibrosis, defined as NFS ≥ -1.455 or BARD score ≥2, than in patients with mild-to-moderate fibrosis (p = 0.012 and p = 0.046, respectively). In multivariate analysis, the CG+GG genotype was an independent factor for NAFLD development (odds ratio 2.568, 95% CI 1.109-5.945, p = 0.028) and for advanced liver fibrosis according to the criteria of NFS ≥ -1.455 (odds ratio 18.573, 95% CI 2.035-169.526, p = 0.010) or a BARD score ≥2 (odds ratio 4.040, 95% CI 1.084-15.048, p = 0.037). The PNPLA3 rs738409 polymorphism is common and may confer a significant risk of NAFLD and advanced liver fibrosis in the Korean population.

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.