A feed-forward loop amplifies nutritional regulation of PNPLA3

Abstract

The upsurge in prevalence of obesity has spawned an epidemic of nonalcoholic fatty liver disease (NAFLD). Previously, we identified a sequence variant (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) that confers susceptibility to both hepatic triglyceride (TG) deposition and liver injury. To glean insights into the biological role of PNPLA3, we examined the molecular mechanisms by which nutrient status controls hepatic expression of PNPLA3. PNPLA3 mRNA levels, which were low in fasting animals, increased approximately 90-fold with carbohydrate feeding. The increase was mimicked by treatment with a liver X receptor (LXR) agonist and required the transcription factor SREBP-1c. The site of SREBP-1c binding was mapped to intron 1 of Pnpla3 using chromatin immunoprecipitation and electrophoretic mobility shift assays. SREBP-1c also promotes fatty acid synthesis by activating several genes encoding enzymes in the biosynthetic pathway. Addition of fatty acids (C16:0, C18:1, and C18:2) to the medium of cultured hepatocytes (HuH-7) increased PNPLA3 protein mass without altering mRNA levels. The posttranslational increase in PNPLA3 levels persisted after blocking TG synthesis with triascin C. Oleate (400 muM) treatment prolonged the half-life of PNPLA3 from 2.4 to 6.7 h. These findings are consistent with nutritional control of PNPLA3 being effected by a feed-forward loop; SREBP-1c promotes accumulation of PNPLA3 directly by activating Pnpla3 transcription and indirectly by inhibiting PNPLA3 degradation through the stimulation of fatty acid synthesis.

Fig. 1.

Expression of PNPLA3 in human tissues (A) and mouse liver (B). (A) Relative levels of PNPLA3 and cyclophilin mRNA were determined by quantitative real-time PCR using cDNA from 48 human tissues (Origene). The cDNAs were standardized using cyclophilin as a calibrator. Each bar represents the mean of triplicate measurements expressed as a fraction of the Ct value obtained from liver, which was set to 1. (B) The relative levels of mRNA from genes expressed predominantly in hepatocytes [albumin (ALB) apolipoprotein B (APOB)] and stellate cells [glial fibrillary acidic protein (GFAP), lecithin retinol acyltransferase (LRAT)]. The hepatocytes and stellate cells were fractionated from mouse liver and mRNA levels were quantitated using quantitative real-time PCR as described in Materials and Methods.

Fig. 2.

Regulation of PNPAL3 expression by SREBPs (A), fasting and refeeding (B), and LXR (C and D). (A) Relative mRNA levels of PNPLA3 (Upper) and fatty acid synthase (FAS) (Lower) in livers of wild-type (WT) mice and mice expressing SREBP-1a, SREBP-1c, or SREBP-2 transgenes (, –46). Mice were fed a high-protein diet for 2 weeks to induce transgene expression and then fasted for 4 h before killing. Equal aliquots of total RNA from five mice in each group were pooled and mRNA levels were assessed by real-time PCR. (B) WT and SCAP knockout (Scap^−/−) mice were fed ad libitum chow diets, fasted for 24 h, or fasted for 24 h and refed a high-carbohydrate diet for 12 h (26). RNA levels were assessed by real-time PCR using pooled samples (five mice/group). (C) Relative levels of mRNA encoding PNPLA3, ABCG5, and APOB in livers of C57BL/6J mice (four mice/group) fed chow diets containing 0.025% of the LXR agonist, T0901317. Pooled hepatic RNA samples were used to probe expression arrays (Affymetrix MG 430 ver. 2, Invitrogen). RNA levels are expressed relative to the level at time 0. (D) WT and SREBP-1c knockout mice were fed chow diets containing 0.025% T0901317 for 12 h and then fasted for 4 h before isolating hepatic mRNA. Equal aliquots of total RNA from five mice in each group were pooled and mRNA levels were assessed by real-time PCR.

Fig. 3.

Identification of SREBP-1c binding sites in Pnpla3. (A) Sequence reads from ChIP-Seq experiments were mapped onto the mouse genome in the University of California Santa Cruz (UCSC) Genome Browser. The numbers of sequence reads corresponding to each position at the Pnpla3 locus are shown for the IgG control and SREBP-1 antibodies. (B) Gene-specific ChIP analysis using liver chromatin enriched by precipitation with an antibody to SREBP-1(solid bars) or control IgG (open bars). (C) ³²P-labeled probe corresponding to the SREBP-1 binding motif from peak 2 (5′-ACTCTCACTGCCC-3′) was incubated with recombinant SREBP-1 (200 ng) and analyzed by electrophoretic mobility shift assay. Increasing concentrations of unlabeled probe (100-, or 300-fold molar excess) for wild-type Pnpla3 (lanes 3 and 4) or for the Ldlr SRE (lanes 7 and 8) were included in the binding reactions as indicated. In lanes 5 and 6, a 100- or 300-fold molar excess of a mutant version of the Pnpla3 site 2 probe was added. In lanes 9 and 10, a mutant oligonucleotide that decreases SREBP-1 binding to Ldlr was added to the reaction. (D) The evolutionary conservation of site 2 sequence for SREBP-1 binding. The boxed sequence matches the recently described SREBP-1 binding motif (32) with a z score of 4.421 (Materials and Methods).

Fig. 4.

Fatty acid-stimulated posttranslational regulation of PNPLA3. (A) Dose–response, (B) time course, and (C) effect of fatty acid saturation and chain length on PNPLA3 protein mass. (A) HuH-7 cells stably expressing PNPLA3-V5 were treated with oleate for 8 h. Immunoblotting of the cell lysates was performed using anti-V5 and calnexin antibodies. (B) Cells were treated with 400 μM oleate for the indicated times before immunoblotting. RNA was extracted from duplicate sets of cells and the abundance of PNPLA3 mRNA was assayed using real-time PCR. (C) Cells transfected with 0.5 μg of pTK-Insig1-myc (34) were treated for 6 h with vehicle or 400 μM palmitate (C16:0), oleate (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidonic acid (C20:4), and eicosapentaenoic acid (C20:5), respectively. Cell lysates were subjected to immunoblotting. (D) Cultured cells were treated with 400 μM oleate plus DMSO or 5 μM triacsin C for the indicated times before immunoblotting.

Fig. 5.

Fatty acids increase half-life of PNPLA3. (A) Cells were treated with BSA or 400 μM oleate for 16 h and then grown in methionine- and cysteine-depleted DMEM. After 1 h, 0.2 mCi/mL ³⁵S-labeled methionine/cysteine was added. One hour later the medium was changed to complete medium and the cells were grown for the indicated times. PNPLA3 was purified from cell lysates using cobalt beads and resolved by SDS/PAGE gels. The gels were dried before autoradiography. (B) The levels of radiolabeled PNPLA3 were measured by densitometry using ImageJ software. The measured values for both treatments were fitted into monoexponential decay model using Microsoft Excel.

Fig. 6.

A feed forward loop for nutritional regulation of PNPLA3. Carbohydrate feeding activates SREBP-1c through the heterodimer LXR/RXR. SREBP-1c transcriptionally activates Pnpla3 as well as several genes encoding enzymes in the fatty acid biosynthetic pathway. Accumulation of fatty acids, specifically C16:0, C18:0, and C18:1, inhibit degradation of PNPLA3.