A Sequence Variation (I148M) in PNPLA3 Associated with Nonalcoholic Fatty Liver Disease Disrupts Triglyceride Hydrolysis

Abstract

Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that ∼90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.

PNPLA3 gene in liver diseases

“…(SREBP-1c) and carbohydrate response element binding protein (ChREBP) [74][75][76]. In human stellate cells, PNPLA3 is also downregulated by intracellular retinol levels [72].…”

“…In human stellate cells, PNPLA3 is also downregulated by intracellular retinol levels [72]. At the cellular level, PNPLA3 is located in endoplasmic reticulum and lipid droplet membranes [76,77].…”

“…PNPLA3 exhibits predominantly hydrolase (lipase) activity in vitro against triglycerides (TG) and retinyl esters in hepatic stellate cells [76,[78][79][80] (Fig. 1).…”

“…In mice, He et al showed that the I148M substitution seems to abolish hydrolase activity by reducing substrate access to the enzyme's active site and so promotes TG accumulation suggesting a loss of function [76]. Using recombinant human PNPLA3 and PNPLA3-I148M proteins, studies showed that fatty acid release increased linearly with increased TG and was reduced by the I148M substitution, indicating a loss of function [78,82].…”

“…Using recombinant human PNPLA3 and PNPLA3-I148M proteins, studies showed that fatty acid release increased linearly with increased TG and was reduced by the I148M substitution, indicating a loss of function [78,82]. Nevertheless, He et al also observed that an overexpression of wild-type (WT) PNPLA3 in mouse liver created no obvious phenotype and, more specifically, did not reduce hepatic TG content [76]. This could suggest a gain of function and a lipogenic role for PNPLA3 that would be enhanced by the presence of the I148M mutation.…”