Fig 2: Secretion of ANGPTL8. (A) Western blots showing conditioned media (lanes 1–6) and cell lysates (lanes 7–12) from 293T cells transfected with full-length (F) or truncated (T) ANGPTL3 and/or with ANGPTL8. Blots on the right show media after 10-fold concentration using Millipore centrifugal filter units. (B) Western blots against ANGPTL8 showing cell lysates, conditioned media, and 10× concentrated conditioned media from HepG2 cells transfected with full-length ANGPTL3 and/or ANGPTL8. (C) Western blots showing cell lysate (lanes 1–3) and conditioned media (lanes 5–7) from 293T cells transfected with ANGPTL8 alone (lanes 1 and 5), ANGPTL8 alone and incubated in ANGPTL3 conditioned media for 48 h (lanes 2 and 6), or co-transfected with ANGPTL8 and ANGPTL3 (lanes 3 and 7). (D) Western blots showing conditioned media (lanes 1–6) and cell lysates (lanes 7–12) from 293T cells transfected with full-length ANGPTL3 (F), the C-terminal domain of ANGPTL3 (C) and/or with ANGPTL8. All western blots were performed with antibodies against the V5 tag for ANGPTL8 and against the Strep tag for ANGPTL3.

Fig 3: Enhancement of LPL inhibition by ANGPTL8. Lipase activity of LPL conditioned media incubated with the conditioned media of cells expressing ANGPTL3 alone (ANGPTL3), ANGPTL8 alone (ANGPTL8), both ANGPTL3 and ANGPTL8 (A3 + A8 co-transfected), a mixture of ANGPTL3 and ANGPTL8 conditioned media (A3 + A8 mixed) or a mixture of ANGPTL3 and ANGPTL8 conditioned media that was incubated for 30 min at 37 °C before being added to LPL (A3 + A8 mixed and pre-incubated). (A) Lipase activity of LPL after incubation with the indicated concentrations of mouse ANGPTL3 and/or mouse ANGPTL8 conditioned media for 30 min at 37 °C as measured by fluorescence assay. Points represent mean (±SEM) of 3 independent experiments each done in duplicate. (B) Lipase activity of LPL after incubation with the indicated concentrations of mouse ANGPTL3 and/or mouse ANGPTL8 conditioned media for 30 min at 37 °C as measured by the lipolysis of radiolabeled chylomicrons. Points represent mean (±SEM) of 3 independent experiments each done in quadruplicate. (C) Lipase activity of LPL after incubation with the indicated concentrations of human ANGPTL3 and/or human ANGPTL8 conditioned media for 30 min at 37 °C as measured by fluorescence assay. Points represent mean (±SEM) of 3 independent experiments each done in duplicate. (D) Lipase activity of GPIHBP1-bound LPL (see Figure 1) incubated with the indicated concentrations of ANGPTL3 and/or ANGPTL8 conditioned media for 30 min at 37 °C and then washed. Points represent mean (±SEM) of 3 independent experiments each done in triplicate. For each graph, activity was normalized to control treated LPL. **p < 0.01; ***p < 0.001 compared to ANGPTL3 alone.

Fig 4: Overexpression of ANGPTL3 in ANGPTL8-deficient mice. 14–15 week old male wild-type and Angptl8-/- mice (n = 3–5/group) were injected via the tail vein with adenovirus expressing either Strep-tagged mouse ANGPTL3 (A3) or GFP. After 4 days, mice were fasted for 20 h and then re-fed for 4 h. (A) Western blot of liver lysates from Angptl8-/- mice using an antibody against the Strep-tag. (B) Total plasma ANGPTL3 levels as measured by ELISA. Bar graphs show mean ± SEM. C. Plasma triglyceride levels (mean ± SEM; ***p < 0.001; ns, not significant).

Fig 5: ANGPTL3 inhibition of LPL. (A) Lipase activity of LPL conditioned media incubated with control media or the indicated concentrations of ANGPTL3 for 10 or 30 min at 37 °C. Activity was normalized to control treated LPL. Points represent mean (±SEM) of 2 independent experiments each done in duplicate. (B) GPIHBP1-expressing RHMVECs were incubated with LPL for 3.5 h at 4 °C. After washing off unbound LPL, cells were incubated with the indicated concentrations of ANGPTL3 or control media for 30 min at 37 °C and then washed again. Surface-bound LPL was then released from cells with 100 U/ml heparin and immediately assayed for lipase activity. Activity was normalized to control treated LPL. Points represent mean (±SEM) of 3 independent experiments each done in triplicate. (C) Plasma ANGPTL3 concentrations (mean ± SEM) in fasted and fed male C57BL/6 mice (n = 6/group; **p < 0.01).