Patatin‐like phospholipase domain‐containing protein 3 and Liver Disease: Opportunities to Unravel Mechanisms Underlying Statistical Associations
Authors: Jérôme Boursier, Anna Mae Diehl
Journal: Hepatology (2014)

Potential conflict of interest: Nothing to report. See Article on Page 108 Nonalcoholic fatty liver disease (NAFLD) is a complex disease that results from interactions between susceptible polygenic backgrounds and environmental factors. Genome‐wide association studies have made it possible to study relationships between a given phenotype and millions of single‐nucleotide polymorphisms.1 Using this hypothesis‐free approach, the association between fatty liver and the rs738409 polymorphism of patatin‐like phospholipase domain‐containing protein 3 (PNPLA3; I148M) was discovered in 20082 and quickly confirmed by several population‐based studies around the world.3 In addition to liver steatosis, PNPLA3 148M variant has been associated with the full spectrum of NAFLD lesions (i.e., steatohepatitis, cirrhosis, and hepatocellular carcinoma [HCC]).4 The association between PNPLA3 148M and liver steatosis or fibrosis has also been demonstrated in other causes of chronic liver diseases (CLDs), such as chronic viral hepatitis, alcoholic liver disease, and hemochromatosis.5 Therefore, the aggregate data reveal a strong association between the PNPLA3 148M variant and the pathogenesis of CLDs. Beyond the simple statistical association, the challenge is now to decipher the pathophysiological mechanisms linking PNPLA3 148M to the severity of CLDs. However, 6 years after the discovery of PNPLA3 148M, mechanisms driving related liver pathobiology remain largely elusive. PNPLA3 is primarily expressed in the liver and adipose tissue, where it partitions between membranes and lipid droplets.7 PNPLA3, alternatively referred to as adiponutrin, hydrolyzes acylglycerols with maximal activity against the three major glycerolipids, triacylglycerol (TAG), diacylglycerol, and monoacylglycerol.8In vitro studies indicate that the 148M variant restricts access of substrate to the catalytic site of the enzyme, thus suggesting a basis for the association between variant PNPLA3 and hepatic triglyceride (TG) accumulation.7 However, this loss‐of‐function theory has been challenged by in vivo studies, because PNPLA3 knockout mice failed to demonstrate significant increases in liver fat.9 Conversely, PNPLA3 was shown to have lysophosphatidic acid acyltransferase activity, and this activity is increased in the 148M variant, providing a potential alternative mechanism for increased cellular lipid accumulation.11 However, these results were not replicated in other studies, and this increase‐in‐function theory has been challenged because simply overexpressing human PNPLA3 in mouse liver did not increase steatosis.12 On the other hand, overexpression of the human 148M variant did recapitulate the fatty liver phenotype in mice, and metabolic studies suggested a complex mechanism involving increased formation of fatty acids and TAG, impaired hydrolysis of TAG, and relative depletion of TAG long‐chain polyunsaturated fatty acids.12 In this issue of Hepatology, Smagris et al. present a new PNPLA3148M/M knock‐in (KI) mouse model where isoleucine at residue 148 of the endogenous murine PNPLA3 gene was replaced with methionine, thus reproducing the PNPLA3 148M human variant.13 This model was developed to address limitations of previous studies that relied upon overexpression of the human 148M variant, because human PNPLA3 is only 76% identical to the mouse protein, and constitutive overexpression of high levels of PNPLA3 does not appropriately model the physiological changes in PNPLA3 levels that follow feeding. Another PNPLA3S47A/A model, where the catalytic serine in the patatin domain was replaced by alanine, was also developed to evaluate the effect of an obligate loss‐of‐function mutation in PNPLA3. Under chow diet conditions, liver lipid levels were not different between wild‐type (WT) PNPLA3+/+ and KI PNPLA3148M/M mice. However, after 4 weeks of high‐sucrose diet (HSD), PNPLA3148M/M mice showed a significant increase in liver TAG content, with marked liver steatosis and larger lipid droplets. This phenotype was not explained by modification in body composition, adipose tissue morphology, plasma lipid levels, or insulin resistance. In addition, messenger RNA (mRNA) expression of genes involved in fatty acid synthesis, TAG lipolysis, fatty acid oxidation, hepatic inflammation, or fibrosis was not significantly different between PNPLA3+/+ and PNPLA3148M/M mice. Finally, liver steatosis in PNPLA3148M/M mice was not associated with any obvious alteration in the spectrum of fatty acids or hepatic glycerolipids. Similar results were obtained in PNPLA3S47A/A mice, proving that PNPLA3 catalytic activity was not required for the steatotic phenotype. In both PNPLA3148M/M mice and PNPLA3S47A/A mice, increased PNPLA3 protein was found in liver lipid droplets. Because comparative gene identification 58 (CGI‐58), a cofactor of the TG lipase, adipose triglyceride lipase (ATGL), was also increased, the investigators postulated that PNPLA3 may sequester CGI‐58 on the lipid droplet, resulting in a reduction of ATGL activity. To directly examine this hypothesis, PNPLA3 and CGI‐58 were determined in liver lipid droplets of PNPLA3148MTg mice expressing mutant human PNPLA3. Despite having increased PNPLA3, lipid‐droplet–associated CGI‐58 in transgenic mice was no different than in WT mice, suggesting that CGl‐58 alterations are not required for the development of steatosis associated with mutant PNPLA3. Therefore, as with earlier studies in rodent models, the current work failed to uncover a mechanism for PNPLA3‐related steatosis. This latest disappointment emphasizes the limitations of our current, state‐of‐the‐art approaches that attempt to model human mutations in mice. Genetically modified animals do not always faithfully recapitulate human physiology. For example, PNPLA3 expression in humans is highest in the liver, whereas in mice hepatic expression is low and adipose tissue expression dominates.14 Moreover, because homology of PNPLA3 is not perfect between mice and humans, whether the I148M substitution has a similar impact on the PNPLA3 protein function between these two species may be questionable. Nonetheless, a potentially important caveat from the current study is that different outcomes were observed when mutant mice were fed high‐fat diets (HFDs) versus HSDs. On an HFD, the liver TAG content was not significantly different between PNPLA3+/+ and PNPLA3148M/M mice, whereas the liver TAG content was differentially increased in PNPLA3148M/M mice on an HSD. Moreover, the HFD increased lipid droplet PNPLA3 content 7‐fold less than the HSD. This lack of effect of HFD on PNPLA3‐related pathology confirms results obtained in a mouse model overexpressing human mutant PNPLA3.12 They are also in line with human cross‐sectional data in Hispanic children carrying PNPLA3 148M, which showed that liver steatosis was associated with dietary carbohydrate intake.16 Taken together, these data suggest that PNPLA3 148M induces an abnormal response to high‐carbohydrate diet and de novo lipogenesis. PNPLA3 is regulated transcriptionally by liver X receptor and sterol regulatory element‐binding protein 1c (SREBP‐1c) and, posttranscriptionally, by fatty acids.14 However, Smagris et al. found no differences in mRNA levels of SREBP‐1c and its target genes and no differences in fatty acid profile between WT and PNPLA3148M/M or PNPLA3S47A/A mice. PNPLA3 accumulated in liver lipid droplets of both KI mice strains, suggesting that PNPLA3 may interact with a lipid‐droplet–localized cofactor to increase TAG storage. Interestingly, a very recent in vitro study suggested that PNPLA3 remodels newly synthesized TAG in a fatty‐acid–selective manner.17 The 148M variant preferentially localized to lipid droplets and was associated with defective remodeling activity, potentially explaining TAG accumulation in lipid droplets. Many mechanistic studies about PNPLA3 have focused on lipid metabolism and steatosis in hepatocytes. However, PNPLA3 148M has also been linked to steatohepatitis, liver fibrosis, and HCC in human population‐based studies, suggesting that PNPLA3 might have a more widespread role in the modulation of liver metabolism than its classical TAG lipase/acyltransferase function. Recent metabolic profiling of Huh‐7 cells subsequent to PNPLA3 silencing and overexpression of WT or mutant PNPLA3 supports this concept.18 Knocking down PNPLA3 in this hepatoma cell line caused the amino acid and carbohydrate metabolome to resemble a catabolic response. Overexpressing PNPLA3 148M mirrored the metabolic changes observed during gene silencing, but in the opposite direction. Interestingly, overexpression of the 148M variant was also associated with an increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction, two processes that have been linked with the severity of liver lesions observed in NAFLD. It also worth noting that PNPLA3 expression is not confined to hepatocytes. In mice, PNPLA3 has been demonstrated in hepatic stellate cells (HSCs).14 Given that this cell subtype is closely linked to fibrosis in CLDs, it would be of interest to evaluate PNPLA3 in HSCs. Indeed, recent work has shown that PNPLA3 mRNA and protein levels are higher in human HSC than hepatocytes.19 Interestingly, WT PNPLA3 had retinyl‐palmitate lipase activity and promoted the extracellular release of retinol by hydrolyzing retinyl palmitate into retinol and palmitic acid, with a concomitant reduction in cellular lipid droplet content. The PNPLA3 148M variant demonstrated markedly reduced enzymatic activity. These results suggest novel mechanisms by which the PNPLA3 variant might impact liver biology and support further research in this area. In conclusion, the accumulating data suggest a much more complex role for PNPLA3 than initially suspected because none of the pathologies associated with variant PNPLA3 are easily explained by “simple” changes in its TAG hydrolase activity. Given the robust association between variant PNPLA3 and the full spectrum of fatty liver disease in humans, there is little doubt that future work aimed at deciphering the role of PNPLA3 and its 148M variant will lead to critical insights into liver disease pathophysiology.