The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation

Abstract

A sequence variation (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is strongly associated with fatty liver disease, but the underlying mechanism remains obscure. In this study, we used knock-in (KI) mice (Pnpla3^148M/M ) to examine the mechanism responsible for accumulation of triglyceride (TG) and PNPLA3 in hepatic lipid droplets (LDs). No differences were found between Pnpla3^148M/M and Pnpla3^+/+ mice in hepatic TG synthesis, utilization, or secretion. These results are consistent with TG accumulation in the Pnpla3^148M/M mice being caused by impaired TG mobilization from LDs. Sucrose feeding, which is required to elicit fatty liver in KI mice, led to a much larger and more persistent increase in PNPLA3 protein in the KI mice than in wild-type (WT) mice. Inhibition of the proteasome (bortezomib), but not macroautophagy (3-methyladenine), markedly increased PNPLA3 levels in WT mice, coincident with the appearance of ubiquitylated forms of the protein. Bortezomib did not increase PNPLA3 levels in Pnpla3^148M/M mice, and only trace amounts of ubiquitylated PNPLA3 were seen in these animals.

Conclusion: These results are consistent with the notion that the 148M variant disrupts ubiquitylation and proteasomal degradation of PNPLA3, resulting in accumulation of PNPLA3-148M and impaired mobilization of TG from LDs. (Hepatology 2017;66:1111-1124).

Figure 1

³H‐H₂O and ¹⁴C‐palmitate incorporation in Pnpla3^+/+(WT) and Pnpla3^148/M/M (KI) mice. (A‐C) Incorporation of ³H‐H₂O into (A) fatty acids, (B) TG, and (C) PA in liver, white adipose tissue (WAT), kidney, and blood of 11‐ to 13‐week‐old female PNPLA3‐WT and PNPLA3‐KI mice (n = 10 per group). Mice were fed an HSD for 4 weeks and then feeding was synchronized for 3 days (protocol 1). During the last fasting cycle, the fast was extended to 24 hours. Mice were then stagger‐fed at 4‐hour intervals before being injected intraperitoneally with ³H‐H₂O (50 mCi in 250 μL 0.9% NaCl). After 1 hour, the mice were killed and tissues were processed as described in Materials and Methods. Bars represent the mean ± standard error of the mean (SEM). (D) Incorporation of ¹⁴C‐palmitate into hepatic TG in 12‐week old female WT and KI mice (10/group). Mice were handled as described in panel A but were injected through the tail vein with ¹⁴C‐palmitate‐BSA (200 μCi) and killed after 1 hour. Each bar represents the mean ± SEM. Levels were compared among lines using a Student t test. The experiment was repeated once, and the results were similar.

Figure 2

VLDL‐TG secretion (A) and fatty acid oxidation (B) in Pnpla3^+/+ and Pnpla3^148M/M mice. (A) Male WT and KI mice (n = 5 per group, age 14‐15 weeks) were fed an HSD for 7 days and then feedings were synchronized (protocol 2). After a 4‐hour fast, Triton WR‐1339 (500 μg/g) was injected into the tail veins. Blood was collected at the indicated time points, and plasma TG levels were measured. Mean plasma TG levels (± SEM) at each point are shown. Rates of VLDL‐TG secretion were determined by least square regression of plasma TG levels plotted against time. Slope estimates were determined from the linear portion of each graph and compared using a Student t test (P = 0.52). The experiment was repeated once, and the results were similar. (B) Male mice (n = 3 per group, age 11‐12 weeks) were fed an HSD for 4 weeks and the feedings were synchronized for 3 days (protocol 1). The mice were killed at the end of the last refeeding cycle and primary hepatocytes were isolated. Oxygen consumption was measured in the primary hepatocytes using Seahorse XF‐24 analyzer as described in Materials and Methods. Cells were treated with either etomoxir (300 μM), an inhibitor of carnitine palmitoyl transferase‐1 or with BSA‐palmitate (200 μM). Values represent the mean ± SEM. **P < 0.01, ***P < 0.001.

Figure 3

Triglyceride mobilization from livers of fasting Pnpla3^+/+ and Pnpla3^148M/M mice. Male mice (n = 3 per group, age 11‐12 weeks) were fed a chow diet for 3 days (12 hours of fasting [8 am to 8 pm] and 12 hours of feeding [8 pm to 8 am]). The mice were fasted 24 hours in the last cycle and then refed with a chow diet. Mice were sacrificed at the indicated time points and livers were collected. Hepatic lipids were measured as described in Materials and Methods. The experiment was repeated once, and the results were similar.

Figure 4

Dysregulation of PNPLA3‐148M expression in LDs of Pnpla3^+/+ and PNPLA3^148M/M mice upon fasting (A) and refeeding (B). (A) Male WT and KI mice (n = 3 per group, age 10‐12 weeks) were fed an HSD for 3 days (protocol 1). At the end of the last feeding cycle, mice were fasted and killed at the indicated time points. Lipid droplets and mRNA were isolated from the liver as described in Materials and Methods. Real‐time polymerase chain reaction was used to quantify selected mRNAs using 36B4 mRNA as a normalization standard. The level of PNPNLA3 mRNA in WT mice at time 0 was set at 1. LD proteins were precipitated using acetone, and immunoblotting was performed. Proteins (3 μg) were size‐fractionated by way of 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and incubated with the following antibodies: anti‐mPNPLA3 (19A6), anti‐mATGL, and anti‐mPLIN2 (see Materials and Methods for types of antibodies and dilutions). Signals were quantified using a LICOR system. (B) PNPLA3 accumulation on LDs after sucrose feeding. Male WT and KI mice (n = 3 per group, age 12‐14 weeks) were synchronized on a chow diet for 3 days (protocol 2). At the end of the last fasting cycle, the mice were fed an HSD for the indicated times and then killed. Livers were processed as described in Materials and Methods. The experiment was repeated once, and the results were similar. Values represent the mean ± SEM. Levels were compared among lines using a Student t test. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Figure 5

Ubiquitylation of PNPLA3 in vivo in PNPLA3Tg^WT/+ and PNPLA3Tg^148M/+ mice. (A) Male PNPLA3 Tg mice (n = 3 per group, age 11‐13 weeks) were fed an HSD for 3 days (protocol 1). At 8 am the mice were injected with bortezomib (1 mg/kg) and vehicle control (0.9% NaCl plus 5% ethanol) through the tail vein. After 8 hours, the mice were killed and livers were collected and processed as described in Materials and Methods. Each bar represents the mean ± SEM. (B) Pnpla3^+/+ and Pnpla3^148M/M male mice (n = 3 per group, age 12‐14 weeks) were treated as described in panel A, except that the mice were killed after 5 hours. Liver samples were pooled and LDs were isolated as described in Materials and Methods. PNPLA3 was immunoprecipitated from the LD fraction, and immunoblot analysis was performed after the proteins (10% of input) were size‐fractionated by way of 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis using an anti‐mPNPLA3 and anti‐ubiquitin mAb (see Materials and Methods for antibody type and dilution). Signals were quantified using a LICOR system. Each bar represents the mean ± SEM. Levels were compared among lines using a Student t test. *P < 0.05. **P < 0.01. ****P < 0.0001. *Nonspecific band. The experiment was repeated twice, and the results were similar.

Figure 6

Ubiquitylated PNPLA3 in LDs of Pnpla3^+/+, Pnpla3^47A/A, and Pnpla3^148M/M mice. (A) Pnpla3^+/+, Pnpla3^S47A/+, and Pnpla3^S47A/A female mice (n = 8 per group, age 13‐16 weeks) were fed an HSD for 4 weeks and then feedings were synchronized for 3 days (protocol 1). Mice were killed after the last refeeding cycle, and the livers were collected for lipid analyses and LD isolation. (B) Pnpla3^+/+, Pnpla3^148M/M, and Pnpla3^47A/A female mice (n = 3 per group per time point, age 11‐13 weeks) were fed an HSD for 3 days, and the feedings were then synchronized (protocol 1). After the last refeeding cycle, mice were injected with bortezomib as described in Figure 5. Mice were killed after 5 hours, and the livers were collected and pooled. LDs were isolated and samples were processed exactly as described in Figure 5B. Each bar represents the mean ± SEM. Levels were compared among lines using a Student t test. **P < 0.01. ***P < 0.001. ****P < 0.0001. The data are representative of three independent experiments.

Figure 7

Hepatic PNPLA3 levels in 3‐MA–treated mice expressing human PNPLA3‐WT and ‐148M. Male PNPLA3Tg^WT/+ and PNPLA3Tg^148M/+ mice (n = 3 per group, age 11‐13 weeks) were fed an HSD for 1 week. Feeding was synchronized (protocol 1), and at the end of the third feeding cycle, the mice were treated with 3‐MA (15 mg/kg) and fasted for 3 hours before being killed. LDs were harvested from the liver and immunoblotting was performed as described in Materials and Methods. Phospho‐S6 ribosomal protein was used as a positive control. The data are representative of two independent experiments. Each bar represents the mean ± SEM. Levels were compared among lines using a Student t test. *P < 0.05. ***P < 0.001.