Piero Pingitore

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.

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.

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.

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

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. (Hepatology 2016;63:787–798)

PNPLA3 overexpression results in reduction of proteins predisposing to fibrosis

Abstract 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-β), but not by platelet-derived growth factor (PDGF), and is involved in the TGF-β-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.

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

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 β‐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 β‐catenin pathway. (Hepatology 2016;63:418–427)

DEPDC5 variants increase fibrosis progression in Europeans with chronic HCV infection

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 β‐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 β‐catenin pathway. (Hepatology 2016;63:418–427)

Causal relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty liver

Nonalcoholic fatty liver disease is epidemiologically associated with hepatic and metabolic disorders. The aim of this study was to examine whether hepatic fat accumulation has a causal role in determining liver damage and insulin resistance.

Telomerase reverse transcriptase germline mutations and hepatocellular carcinoma in patients with nonalcoholic fatty liver disease

In an increasing proportion of cases, hepatocellular carcinoma (HCC) develops in patients with nonalcoholic fatty liver disease (NAFLD). Mutations in telomerase reverse transcriptase (hTERT) are associated with familial liver diseases. The aim of this study was to examine telomere length and germline hTERT mutations as associated with NAFLD‐HCC. In 40 patients with NAFLD‐HCC, 45 with NAFLD‐cirrhosis and 64 healthy controls, peripheral blood telomere length was evaluated by qRT‐PCR and hTERT coding regions and intron–exon boundaries sequenced. We further analyzed 78 patients affected by primary liver cancer (NAFLD‐PLC, 76 with HCC). Enrichment of rare coding mutations (allelic frequency <0.001) was evaluated by Burden test. Functional consequences were estimated in silico and by over‐expressing protein variants in HEK‐293 cells. We found that telomere length was reduced in individuals with NAFLD‐HCC versus those with cirrhosis (P = 0.048) and healthy controls (P = 0.0006), independently of age and sex. We detected an enrichment of hTERT mutations in NAFLD‐HCC, that was confirmed when we further considered a larger cohort of NAFLD‐PLC, and was more marked in female patients (P = 0.03). No mutations were found in cirrhosis and local controls, and only one in 503 healthy Europeans from the 1000 Genomes Project (allelic frequency = 0.025 vs. <0.001; P = 0.0005). Mutations with predicted functional impact, including the frameshift Glu113Argfs*79 and missense Glu668Asp, cosegregated with liver disease in two families. Three patients carried missense mutations (Ala67Val in homozygosity, Pro193Leu and His296Pro in heterozygosity) in the N‐terminal template‐binding domain (P = 0.037 for specific enrichment). Besides Glu668Asp, the Ala67Val variant resulted in reduced intracellular protein levels. In conclusion, we detected an association between shorter telomeres in peripheral blood and rare germline hTERT mutations and NAFLD‐HCC.