Atypical angiopoietin-like protein that regulates ANGPTL3

Abstract

Angiopoietin-like proteins (ANGPTLs) play major roles in the trafficking and metabolism of lipids. Inactivation of ANGPTL3, a gene located in an intron of DOCK7, results in very low levels of LDL-cholesterol (C), HDL-C and triglyceride (TAG). We identified another ANGPTL family member, ANGPTL8, which is located in the corresponding intron of DOCK6. A variant in this family member (rs2278426, R59W) was associated with lower plasma LDL-C and HDL-C levels in three populations. ANGPTL8 is expressed in liver and adipose tissue, and circulates in plasma of humans. Expression of ANGPTL8 was reduced by fasting and increased by refeeding in both mice and humans. To examine the functional relationship between the two ANGPTL family members, we expressed ANGPTL3 at physiological levels alone or together with ANGPTL8 in livers of mice. Plasma TAG level did not change in mice expressing ANGPTL3 alone, whereas coexpression with ANGPTL8 resulted in hypertriglyceridemia, despite a reduction in circulating ANGPTL3. ANGPTL8 coimmunoprecipitated with the N-terminal domain of ANGPTL3 in plasma of these mice. In cultured hepatocytes, ANGPTL8 expression increased the appearance of N-terminal ANGPTL3 in the medium, suggesting ANGPTL8 may activate ANGPTL3. Consistent with this scenario, expression of ANGPTL8 in Angptl3(-/-) mice failed to promote hypertriglyceridemia. Thus, ANGPTL8, a paralog of ANGPTL3 that arose through duplication of an ancestral DOCK gene, regulates postprandial TAG and fatty acid metabolism by controlling activation of its progenitor, and perhaps other ANGPTLs. Inhibition of ANGPTL8 provides a new therapeutic strategy for reducing plasma lipoprotein levels.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Domain structure and sequence similarity of ANGPTL3, ANGPTL4, and ANGPTL8. (A) Domain organization of ANGPTL4, ANGPTL3, and ANGPTL8. (B) Chromosomal location and exon-intron structure of ANGPTL3 and ANGPTL8. (C) Multiple sequence alignment of a conserved N-terminal motif in three ANGPTLs. A conserved acidic residue at amino acid 40 in ANGPTL4 is indicated by an asterisk. Positions with semi-invariant, uncharged, and hydrophobic residues are highlighted in black, yellow, and gray, respectively. National Center for Biotechnology Information gene identifier (NCBI gi) numbers are shown on the right (where available). Consensus was computed on the PROMALS3D alignment of all sequences.

Fig. 2.

Tissue distribution of ANGPTL8 expression in humans and genetic association of sequence variation in ANGPTL8 with plasma lipoprotein levels. (A) ANGPTL8 mRNA levels in human tissues were determined by real-time PCR, and normalized to expression levels of 36B4. Levels were expressed as a ratio relative to liver expression, which was set to 1. (B) Distribution of ANGPTL8 in human plasma. Plasma lipoproteins were isolated by ultracentrifugation and equal proportions of each fraction were subjected to immunoblot analysis with antibodies to ANGPTL8 and ApoE. (C) Mean (± SE) LDL-C and HDL-C values in the DHS and ARIC participants.

Fig. 3.

Overexpression of human ANGPTL8 and ANGPTL3 in livers of wild-type and Angptl3−/− mice. (A) Plasma levels of TAG, cholesterol, and NEFA were measured in 10-wk-old C57Bl6/J male mice (n = 4–5 per group) 3 d after being injected with adenovirus. (B) Immunoblot analysis of plasma ANGPTL8. (C) Human and mouse ANGPTL3 were measured by ELISA, as described in Materials and Methods. (D) In an independent experiment, ANGPTL3 was immunoprecipitated from pooled plasma samples (n = 3 per group) of mice infected with the indicated viruses using a mouse anti-human ANGPTL3 mAb. Immunoprecipates were subjected to immunoblotting with an anti-ANGPTL3 polyclonal antibody. (E) Coimmunoprecipitation of ANGPTL3 with ANGPTL8-FLAG from plasma of mice expressing human ANGPTL8-FLAG and ANGPTL3 (n = 6 per group). The immunoprecipitated proteins were blotted with polyclonal antibodies to human ANGPTL8 and the N terminus of human ANGPTL3 (Left). Plasma levels of ANGPTL3 and ANGPTL8 (input) were assessed by ELISA and by immunoblotting, respectively (Right). (F) Wild-type and Angptl3−/− mice (n = 3–5 per group) were infected with recombinant ANGPTL8 adenoviruses. After 3 d, the mice were fasted for 4 h, killed, and plasma levels of TAG, cholesterol and NEFA were measured. The experiments were all performed three times with similar results and representative data are shown.

Fig. 4.

ANGPTL8 promotes cleavage of ANGPTL3 in cultured hepatocytes. ANGPTL8-FLAG was expressed alone or together with either ANGPTL3 (A) or ANGPTL6 (B) in HepG2 cells as described in Materials and Methods. Immunoblot analysis was performed to detect ANGPTL8, ANGPTL3, and ANGPTL6. (C) Cells are required for cleavage of ANGPTL3. Recombinant mouse ANGPTL3 protein (1 μg/mL) was added to the conditioned medium of HuH7 cells infected with ANGPTL8 or control adenoviruses. ANGPTL3 was also added to the same medium in the absence of cells. Cells were grown for 16 h at 37 °C, 5% CO2. Medium was subjected to immunoblot analysis with anti-ANGPTL8 and anti-mANGPTL3 antibodies as described in Materials and Methods. The asterisk represents a nonspecific band.

Fig. 5.

Regulation of ANGPTL8 mRNA in mouse tissues. (A) C57BL/6J mice (n = 5 per group) were fasted for 12 h, or fasted for 12 h and then refed with a high-carbohydrate diet for 12 h. Messenger RNA samples from liver were pooled and analyzed by Real-time PCR. Values are expressed as ratios compared with levels in the ad libitum fed group. (B) Expression of ANGPTL8 in livers from SREBP-1a, SREBP-1c, SREBP-2 transgenic (Tg) mice. Fatty acid synthase (FAS) and HMG-CoA reductase (HMGCR) mRNA levels were used as positive controls. (C) ANGPTL8 and FAS in livers of mice treated with the LXR agonist T0901317 (0.015%) for 4 d. (D) Liver-specific Scap−/− mice (4 mice per group) and littermate controls were fed ad libitum, fasted for 24 h, or fasted for 24 h and then refed for 12 h with a high-carbohydrate diet. (E) Immunoblot analysis of ANGPTL8 in plasma from humans after 12-h fasting and 6 h after ingestion of a meal. Fibronectin was used as a loading control.