Angiopoietin-like protein 4 (ANGPTL4) is an inhibitor of endothelial lipase (EL) while the ANGPTL4/8 complex has reduced EL-inhibitory activity

Abstract

We previously demonstrated that angiopoietin-like protein 8 (ANGPTL8) forms ANGPTL3/8 and ANGPTL4/8 complexes that increase with feeding to direct fatty acids (FA) toward adipose tissue through differential modulation of lipoprotein lipase (LPL) activity. Each complex correlated inversely with high density lipoprotein cholesterol (HDL) in control subjects. We thus investigated ANGPTL3/8 and ANGPTL4/8 levels in type 2 diabetes patients, who can present with decreased HDL. While ANGPTL3/8 levels in type 2 diabetes patients were similar to those previously observed in normal controls, ANGPTL4/8 levels were roughly twice as high as those in control subjects. Concentrations of ANGPTL3/8 and ANGPTL4/8 in type 2 diabetes patients were inversely correlated with HDL, with the correlation being significant for ANGPTL4/8. We therefore measured the ability of the various ANGPTL proteins and complexes to inhibit endothelial lipase (EL), the enzyme which hydrolyzes phospholipids (PL) in HDL. While confirming ANGPTL3 as an EL inhibitor, we found that ANGPTL4 was a more potent EL inhibitor than ANGPTL3. Interestingly, we observed that while ANGPTL3/8 had increased EL-inhibitory activity compared to ANGPTL3 alone, ANGPTL4/8 exhibited decreased potency in inhibiting EL compared to ANGPTL4 alone. Together, these results show for the first time that ANGPTL4 is a more potent EL inhibitor than ANGPTL3 and suggest a possible reason for why ANGPTL4/8 levels are correlated inversely with HDL.

Keywords: Angiopoietin-like protein; Endothelial lipase; HDL; Phospholipids; Postprandial state; Triglycerides.

Figure 1

ANGPTL3 and ANGPTL3/8 inhibition of EL. (A) The ability of ANGPTL3 or ANGPTL3/8 to inhibit EL at 37 °C was assessed using an EL stable expression cell line and phospholipase A₁ selective substrate. Fluorescence was monitored with an excitation wavelength of 485 nm and an emission wavelength of 516 nm. Readings were taken at 1 and 30 min, with the 1-minute reading subtracted from the 30-minute reading to correct for background fluorescence. Results are shown as the mean ± SD (n = 6 from 3 independent experiments). (B) The ability of ANGPTL3 or ANGPTL3/8 to inhibit EL was assessed as in Figure 1A except at 22 °C. Results are shown as the mean ± SD (n = 6 from 3 independent experiments).

Figure 2

ANGPTL4 and ANGPTL4/8 inhibition of EL. (A) The ability of ANGPTL4 or ANGPTL4/8 to inhibit EL at 37 °C was assessed using an EL stable expression cell line and phospholipase A₁ selective substrate. Fluorescence was monitored with an excitation wavelength of 485 nm and an emission wavelength of 516 nm. Readings were taken at 1 and 30 min, with the 1-minute reading subtracted from the 30-minute reading to correct for background fluorescence. Results are shown as the mean ± SD (n = 4 from 2 independent experiments). (B) The ability of ANGPTL4 or ANGPTL4/8 to inhibit EL was assessed as in Figure 2A except at 22 °C. Results are shown as the mean ± SD (n = 8 from 4 independent experiments).

Figure 3

ANGPTL4 and ANGPTL4/8 inhibition of recombinant EL protein activity. The ability of ANGPTL4 or the ANGPTL4/8 complex to inhibit human recombinant EL protein activity was assessed using phospholipase A₁ selective substrate. Fluorescence was monitored with an excitation wavelength of 485 nm and an emission wavelength of 516 nm. Readings were taken at 1 and 30 min, with the 1-minute reading subtracted from the 30-minute reading to correct for background fluorescence. Results are shown as the mean ± SD (n = 4 from 2 independent experiments).