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

Abstract

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.

FIGURE 1

Validation of the PNPLA3 antibody in HEK293 cells and in mice overexpressing human PNPLA3. Western blot analysis using antibodies detecting (A) human PNPLA3 or (B) the FLAG‐tag in lysates from HEK293 cells transfected with plasmids, as indicated in the figure. (C–H) Immunohistochemistry analyses of human PNPLA3 levels on sections from transgenic mice expressing human PNPLA3 (magnification ×40). (C, E, G) Mice fed a chow diet or (D, F, H) a high‐sucrose diet were administered saline solution (C,D), control ASO (E, F), or ASO targeting human PNPLA3 (G, H). The size bar (in red) indicates 50 μm. (I) Quantification of PNPLA3 expression determined by immunohistochemistry as the fractional area of the image containing a signal from the PNPLA3 antibody. Boxes reflect the interquartile range, with the horizontal line indicating median PNPLA3 expression. Statistical analysis was performed using Wilcoxon tests; n = 4 mice/treatment group. ASO, antisense oligonucleotide; h, human; m, mouse; PNPLA3, patatin‐like phospholipase domain‐containing 3.

FIGURE 2

Immunohistochemistry analysis of PNPLA3 in liver biopsies from subjects with low and high steatosis grade. (A–H) Representative images showing PNPLA3 protein levels obtained with the antibody from R&D Systems (AF5208) at two magnification levels in an individual without any signs of steatosis (A,B), in an individual with steatosis grade 1 (C,D), 2 (E,F) or 3 (G,H). The size bar in the left panel indicates 200 μm and in the right panel 50 μm. The blue boxes (A,C,E,G) indicate the area magnified. PNPLA3, patatin‐like phospholipase domain‐containing 3.

FIGURE 3

PNPLA3 levels in relation to disease severity in patients with NAFLD. Hepatic PNPLA3 protein levels in biopsy samples of patients with NAFLD from follow‐up 1 grouped by histologic assessment. Hepatic PNPLA3 protein levels versus (A) steatosis grade, (B) presence of lobular inflammation, (C) presence of hepatocellular ballooning, and (D) presence of significant fibrosis. Statistical analysis was performed using Wilcoxon/Kruskal‐Wallis rank‐sum tests. NAFLD, nonalcoholic fatty liver disease; PNPLA3, patatin‐like phospholipase domain‐containing 3.

FIGURE 4

Hepatic PNPLA3 protein levels, hepatic lipid fractional area, and lipid droplet size in liver biopsies divided by PNPLA3 I48M  genotype. PNPLA3 I148M genotype versus (A) hepatic PNPLA3 protein levels, (B) hepatic lipid levels, and (C) average hepatic lipid droplet size. PNPLA3 I148M risk allele carriers CG (light blue triangle), GG (dark blue square), noncarriers CC (gray circles). Statistical analysis was performed comparing risk allele carriers to noncarriers by Wilcoxon rank‐sum tests. PNPLA3, patatin‐like phospholipase domain‐containing 3.

FIGURE 5

PNPLA3 protein levels in patients with NAFLD with different steatosis grades and PNPLA3 I148M genotype. Hepatic PNPLA3 levels in biopsy samples of patients with NAFLD grouped by PNPLA3 I148M allele variant showing risk allele carriers (CG, light blue triangle; GG, dark blue square) and noncarriers (CC, gray circles) and by steatosis grade. Statistical analysis was performed comparing PNPLA3 148M risk allele carriers to noncarriers by Wilcoxon rank‐sum tests at each steatosis grade. NAFLD, nonalcoholic fatty liver disease; PNPLA3, patatin‐like phospholipase domain‐containing 3.