LZP is required for hepatic triacylglycerol transportation through maintaining apolipoprotein B stability

Abstract

The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.

Fig 1. LZP-deficient mice exhibit lower serum triacylglycerol (TG).

(A) Growth curve of wild type (WT) and Lzp knockout (KO) mice fed with chow diet (CHD) or high fat diet (HFD) for 10 weeks. Body weight was measured weekly (n = 7). (B, C) Representative whole body (B) and liver (C) images of WT and Lzp KO mice fed with CHD or HFD. (D) The liver/body ratios of WT and Lzp KO mice fed with CHD or HFD (n = 7). (E-F) The images, weights of subcutaneous white adipose tissue (gWAT) and inguinal WAT (iWAT) and, as well as their body ratios (n = 7). (G-H) Quantifications of serum and liver TG from 4-month-old mice that have been fed with CHD or HFD (n = 11–13). (I) Images of H&E staining (left panel, scale bar represents 10 μm), Oil Red O staining (middle panel, scale bar represents 10 μm), and fluorescence staining with Bodipy493 (right panel, scale bar represents 50 μm) on liver sections from 4-month-old WT and Lzp KO mice fed with CHD or HFD. (J) Transmission electron micrographs of liver sections from WT and Lzp KO mice fed with HFD. Scale bar represent 5μm. Data were expressed as means ± SEM, and statistically analyzed by student’s t-test or one-way ANOVA, *P < 0.05, **P < 0.01. Numerical values for each of the experiments represented are available in S3 Table.

Fig 2. LZP deficiency causes hepatic TG accumulation.

(A) TG contents of isolated hepatocytes was measured (left panel) and LZP expression were analyzed by Western blotting assay (right panel). Primary hepatocytes isolated from WT or Lzp KO mice were transfected with 3 μg plasmids containing mLZP-myc or empty vehicle for 24 h, and then followed 0.2 mM OA or PA treatments for 16 h. Finally, the cells were harvested for TG contents quantification and LZP expression analysis. (B) Images of Oil Red O (ORO) staining of hepatocytes isolated from WT and Lzp KO mice. The hepatocytes were transfected with 3 μg plasmids containing mLZP-myc or empty vehicle for 24 h, then followed 16 h treatments of 0.2 mM OA or PA to enhance lipid formation. Scale bar represents 50μm. (C) Images of immunofluorescence staining of LZP (red), LDs (stained with Bodipy 493, green), and nucleus (blue) of hepatocytes isolated from WT and Lzp KO mice. The hepatocytes were treated as above. Scale bar, 20μm. (D-E) Quantification of the cellular TG contents of L02 or HepG2 cells. Both cells stably expressed LZP or GFP were treated with 0.25 mM OA or PA for 16 h, and their ectopic expressions of LZP were validated by Western blot assay. (F-G) Oil Red O (ORO) staining of L02 cells or HepG2 cells, which stably expressed LZP or GFP were treated with 0.25mM OA or PA overnight. Scale bar, 20μm. (H, I) Immunofluorescence staining of LZP (red) and lipid droplets (green) of L02 or HepG2 cells, which were transfected with plasmids containing LZP-myc or empty vehicle, followed by 0.25mM OA or PA treatments overnight. The photos were taken by Nikon & A1Si laser scanning confocal microscope. Scale bar, 10 μm(H) or 20 μm(I). Data were expressed as means ± SEM, and statistically analyzed by student’s t-test, *P < 0.05, **P < 0.01. Numerical values for each of the experiments represented are available in S4 Table.

Fig 3. LZP is required for TG secretion.

(A) Both LZP and apoB were measured by Western blot assay in livers, serum, and VLDL particles from WT and KO mice. VLDL were isolated via isopycnic KBr gradient method. (B) Lzp deficiency decreased apoB level in hepatocyte and serum. The key proteins involved in VLDL assembly, transportation, and secretion were analyzed with indicated antibodies (n = 3). (C) LZP deficiency decreased TG secretion. WT and Lzp^-/- mice were injected with 500 mg/kg Triton WR-1339 after 8 h fasting. Blood samples were collected at the indicated time points for TG detection (n = 3). Data were expressed as means ± SEM, and statistically analyzed by student’s t-test or one-way ANOVA, *P < 0.05, **P < 0.01. Numerical values for each of the experiments represented are available in S6 Table.

Fig 4. LZP interacts with apoB.

(A) Co-localization of LZP with Calnexin (ER marker), ADRP (lipid marker), or GS28 (Golgi marker) in hepatocytes from WT mice. Scale bar, 20 μm. (B) LZP expression levels in cytoplasm, nucleus, and different organelle isolated from livers of WT mice. (C) Co-localization of LZP and apoB in hepatocytes from WT mice or in HepG2 cells overexpressed LZP. Scale bar, 20 μm. (D) Co-immunoprecipitation of LZP and apoB in hepatocytes or in human HepG2 cells overexpressed LZP. (E) In vitro co-immunoprecipitation of commercial apoB and purified LZP. (F) Co-localization of the ectopic LZP (red) and MTTP (green) (Upper panel) or endogenous BiP (green) (lower panel) in hepatocytes. Scale bar, 20 μm. (G) Co-immunoprecipitations of endogenous LZP and apoB, MTTP, or BIP in hepatocytes isolated from WT mice.

Fig 5. LZP maintains apoB stability.

(A) LZP deficiency decreased the cellular and secreted apoB levels from hepatocytes, whilst LZP overexpression recovered apoB level. (B-C) Ectopic LZP expression increased cellular and secreted apoB levels from both L02 (B) and HepG2 cells (C). (D) LZP deficiency decreased the half-life of apoB in hepatocytes. Hepatocytes were treated with 100 μg/ml CHX for the indicated times. (E) Ectopic LZP extended half-life of apoB in HepG2 cells. HepG2 cells overexpressed GFP or LZP were treated with 100 μg/ml CHX for the indicated times. The apoB’s integral optical density of each time point was normalized by 0 time and value was labeled at the upper of band. (F) Blocking ER-Golgi trafficking with brefeldin A (BFA) reduced apoB and LZP in cell culture medium. (G) MG132, not CQ, blocked the degradation of apoB in hepatocytes. Hepatocytes were treated with 20μM MG132 or 50μM CQ for 24h. (H) MG132 blocked the degradation of cellular and secreted apoB as well as LZP from HepG2-LZP cells. Numerical values for each of the experiments represented are available in S8 Table.

Fig 6. LZP attenuates AMFR-dependent apoB degradation.

(A) LZP deficiency increased ubiquitination of apoB in hepatocytes, while ectopic LZP overexpression decreased it. Hepatocyte from WT or Lzp KO mice were transfected with mLZP-myc and HA-ub for 24 h, then treated with 20 μM MG132 for another 6 h. (B) Ectopic LZP inhibited apoB ubiquitination in HepG2 cells, while the AMFR overexpression increased apoB ubiquitination. (C) Co-immunoprecipitation of LZP and AMFR with indicated antibodies in HepG2 cells ectopically expressed AMFR and LZP. (D) LZP deficiency enhanced the apoB-AMFR interaction in liver. (E) Overexpressed LZP did not decrease the apoB ubiquitination level in the absence of AMFR in HepG2 cells. (F) ERAD inhibitor Eeyarestatin I (Eey) increased the intracellular and secreted apoB levels in both HepG2-GFP and HepG2-LZP cells. The cells were treated with Eey for 24h.

Fig 7. LZP highly expresses at livers of obese mice and patients.

(A-B) Lzp mRNAs (A) and protein levels (B) in livers from mice fed with CHD or HFD, as well as db/db mice. (C-D) Both OA and PA increased hepatic Lzp mRNA (C) and protein levels (D). The hepatocytes separated from 10 weeks old mice were treated with OA or PA for 16 h, and then were harvested for real-time PCR and Western blot assays. (E) Both OA and PA increased LZP in HepG2 cells with ectopic LZP. (F) The analysis of Lzp mRNA levels in livers of obese, steatosis, and NASH patients using the dataset (GSE48452) excluded surgery patients. (G) Schematic illustration of the role of LZP in hepatic lipid metabolism. LZP interacts with apoB to attenuate its ubiquitination mediated by AMFP and subsequent proteasome degradation and to assistant VLDL assembly and secretion. Data were expressed as means ± SEM, and statistically analyzed by one-way ANOVA, *P < 0.05, **P < 0.01. Numerical values for each of the experiments represented are available in S9 Table.