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 approximately 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.

Figures

FIGURE 1.

Structural model of wild type and mutant (I148M) PNPLA3. The domain structure of PNPLA3, showing the patatin-like domain (black) and locations of the catalytic dyad (Ser47 and Asp166) and the I148M substitution associated with increased hepatic triglyceride content (26), is shown. Structure models of normal (Ile148) and mutant (Met148) PNPLA3 are shown in the left and right panels, respectively. Protein traces are rainbow-colored from N to C terminus (blue to red) with side chains of catalytic dyad residues (positions 47 and 166) shown. The dots indicate a space-filling model corresponding to van der Waals atomic radii. Oxygen and sulfur atoms are colored red and yellow, respectively. The model was built using heartleaf horsenettle (Solanum cardiophllum) patatin (Protein Data Bank code 1oxw) as a template. Images were prepared in PyMOL (19).

FIGURE 2.

Effect of I148M substitution on triglyceride hydrolysis in vitro. FLAG-tagged human wild type and mutant (I148M) PNPLA3 were partially purified from Sf9 cells using nickel affinity chromatography as described under “Experimental Procedures.” A, a total of 20, 40, or 80 μg of protein was incubated at 37 °C for 15 min with 3H-triolein emulsions (60 μm of [9,10-3H]triolein). Lipids were extracted with butanol and separated by TLC, and the free fatty acid bands were excised and quantitated by scintillation counting. B, emulsions of radiolabeled triolein were incubated with 40 μg of partially purified recombinant PNPLA3 for the times indicated. The free fatty acid release was quantitated as described in A. C, partially purified wild type PNPLA3, PNPLA3-I148M and a 1:1 mixture of wild type and mutant PNPLA3 were incubated with [3H]triolein emulsions, and free fatty acid release was measured as described in A. Proteins were examined by immunoblotting using an anti-FLAG epitope antibody (Sigma). Each experiment was repeated twice, and similar results were obtained. WT, wild type.

FIGURE 3.

Adenovirus-mediated expression of PNPLA3 in the livers of mice. A, 12-week-old male C57BL/6J mice (n = 6 mice/group) were injected with 1.25 × 1011 recombinant adenovirus particles expressing no insert (Vector) or V5-tagged versions of wild type PNPLA3 (WT) PNPLA3-I148M, PNPLA3-S453I, or untagged PNPLA3-S47A. Three days after injection, Oil Red O staining was performed on liver sections as described under “Experimental Procedures.” Pictures were taken by a Leica microscope at ×40 magnification. B, immunoblot analysis of PNPLA3-V5 expression in lysates from livers of mice injected with recombinant adenoviruses. Representative blots from two mice in each group are shown. C, lipids were extracted from the livers and assayed using enzymatic kits as described under “Experimental Procedures.” Values are means ± S.D. p values were calculated using analysis of variance and corrected for multiple testing using the Bonferroni procedure. *, p < 0.0001 for the triglyceride and cholesterol ester (vector versus S47A and versus I148M). This experiment was repeated three times, and the results were similar.

FIGURE 4.

Triglyceride hydrolase activity of PNPLA3 in HuH-7 cells. A, HuH-7 cells were infected with adenoviruses encoding wild type or mutant forms of human PNPLA3. After 48 h, the medium was changed to DMEM plus 1 μCi [14C]palmitate. After 4 h, the medium was changed to DMEM plus 10% FCS, and the cells were harvested after the indicated time intervals. Lipids were extracted from the cells, fractionated by TLC, and visualized using a Storm 820 PhosphorImager (Amersham Biosciences). The activity of each band was quantitated using ImageQuant TL analysis software (Molecular Dynamics). The relative amount of labeled triglyceride was expressed as a fraction of the value obtained at the zero time point from cells expressing wild type PNPLA3. B, HuH-7 cells were infected with adenoviruses encoding wild type or mutant forms of human PNPLA3. After 48 h, the medium was changed to DMEM plus 10% FCS plus 1 μCi of [14C]palmitate. After 24 h, the medium was changed to DMEM plus 10% FCS plus triacsin C (5 μm). Cells were harvested after the indicated time intervals, and lipids were analyzed by TLC as described above. These experiments were repeated twice, and the results were similar.

FIGURE 5.

Subcellular localization of PNPLA3 in cultured hepatoma (HuH-7) cells. A, HuH-7 cells were grown in the absence and presence of 400 μm oleate for 24 h and MG132 (2.5 μm) for 12 h. Membranes and lipid droplets were isolated by density gradient ultracentrifugation as described under “Experimental Procedures.” One-twentieth of the total volume of each fraction was analyzed by SDS-PAGE and immunoblotted for PNPLA3, ADRP, and calnexin. A rabbit polyclonal antibody to the last 20 amino acids of human PNPLA3 was used to detect endogenous PNPLA3. B, postnuclear supernatants (PNS) prepared from HuH-7 cells stably expressing PNPLA3-V5 were subjected to ultracentrifugation at 100,000 × g to separate the cytoplasm (C) and membranes (M) as described under “Experimental Procedures.” One-twentieth of the total volume of each fraction was analyzed by SDS-PAGE, and immunoblotting using a V5 monoclonal antibody and rabbit anti-calnexin polyclonal antibody. C and D, membrane fractions from HuH-7 cells expressing wild type PNPLA3, PNPLA3-I148M (C), or truncated PNPLA3 (D) were suspended in 450 μl of 10 mm Tris, pH 7.4. Membranes were repelleted by centrifugation at 100,000 × g for 1 h at 4 °C and resuspended in the indicated buffers as described under “Experimental Procedures.” Pellet (P) and supernatant (S) fractions were subjected to 10% SDS-PAGE and analyzed by immunoblotting. E, lipid droplets were isolated from HuH-7 cells stably expressing recombinant PNPLA3 and PNPLA3-I148M. The postnuclear supernatant was adjusted to a sucrose concentration of 20%, applied to the bottom of a discontinuous sucrose gradient, and centrifuged at 28,000 × g as described under “Experimental Procedures.” A total of 20 μg of protein from the PNS and 17 μg from the lipid droplet fractions were subjected to immunoblotting using antibodies against V5 and ADRP, a lipid droplet marker. There experiments were repeated at least twice, and the results were similar.

FIGURE 6.

Immunolocalization of recombinant human PNPLA3 to lipid droplets in oleate-treated HuH-7 cells. A, HuH-7 cells grown on glass coverslips were infected with a recombinant adenovirus encoding wild type PNPLA3. Cells were cultured for 16 h in DMEM plus 10% FCS with or without oleate-conjugated albumin (400 μm), fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100, and stained with an anti-V5 antibody and a goat anti-mouse antibody conjugated to Alexa Fluor 568. Lipid droplets were visualized using 1 μg/ml boron dippyrromethane (BODIPY). B, HuH-7 cells were grown on glass coverslips and infected with a control recombinant adenovirus (Vector) or with adenoviruses encoding either wild type PNPLA3 or PNPLA3-I148M or transfected with a plasmid encoding PNPLA3-S47A. All constructs contained a V5 epitope tag at the C terminus. The cells were cultured in medium containing oleate-conjugated albumin (400 μm) for 16 h and processed for immunofluorescence as described under “Experimental Procedures.”