Discussion
The main findings of our cross-sectional human study are: (1) patients with MetS and T2DM show increased plasma ANGPTL4 levels compared with Controls, (2) patients with MetS+I show higher levels of ANGPTL4 compared with patients with MetS−I, (3) plasma CRP, as a marker of low-grade inflammation, positively correlates with plasma ANGPTL4, independent of age, waist circumference, glucose and lipid variables.
Thus far, the expression of ANGPTL4 in numerous tissues including adipose tissue and liver was thought to be mainly regulated by PPARs. Consistent with a role of PPARs, fatty acids increase ANGPTL4 expression in vitro in numerous cell types.1 ,6–8 Furthermore, in vivo modulation of plasma FFA levels also modulates plasma ANGPTL4 levels,9 ,10 with a positive correlation observed between the change in FFA levels and change in ANGPTL4 levels.10 In our previous study, we postulated that patients with T2DM may have higher ANGPTL4 levels than healthy participants due to elevated plasma FFA.10 Although in the present paper we do observe increased plasma ANGPTL4 levels in patients with T2DM compared with Controls, plasma FFA levels were not different between both groups. In addition, plasma ANGPTL4 levels were elevated in patients with MetS compared with Controls, whereas their plasma FFA levels did not differ from the healthy Controls. In fact, in this cross-sectional study we were unable to detect a correlation between plasma FFA levels and plasma ANGPTL4 levels, which is likely related to the matching on visceral obesity and the subsequent limited variation in FFA between groups.
Therefore, we hypothesized that additional factors such as inflammation may contribute to the increased ANGPTL4 levels in patients with MetS and T2DM. Indeed, we did observe a progressive increase in the plasma ANGPTL4 in males according to inflammatory status (healthy Controls < patients with MetS−I < patients with MetS+I and patients with T2DM). Furthermore, in all groups combined, plasma ANGPTL4 showed a positive correlation with CRP, an established marker for low-grade inflammation.
To get more insight into the causal relationship between inflammation and ANGPTL4 expression we treated human macrophages with various PRR ligands. Macrophages were used because of their sensitivity to inflammatory stimuli, not because they are suspected to be solely responsible for the changes in plasma ANGPTL4 levels observed in vivo. Of all ligands tested, agonists for TLR3 (ie, poly(I:C)) and TLR4 (ie, LPS) consistently induced the expression and release of ANGPTL4 in different types of human macrophages. It remains unclear which intracellular pathway activated by TLR activation is responsible for induction of ANGPTL4. Also, it is unsure whether FFA, known to activate PRRs such as TLR4 in adipocytes and macrophages,11 ,12 may increase ANGPTL4 expression via TLR4 activation. It should be noted that the similarity in plasma FFA levels between the various groups despite major differences in plasma ANGPTL4 levels, suggests that plasma FFA are not a major determinant of ANGPTL4 in the population studied. Since CRP itself is a marker rather than a mediator of inflammation,18 the nature of the inflammatory stimuli that cause an increase in plasma ANGPTL4 in humans evidently still has to be identified. Also, it would be of interest to study the response of ANGPTL4 to inflammatory stimuli in other cell types, including human adipocytes.
We hypothesized that the inflammation induced increase in plasma ANGPTL4 in patient with MetS and T2DM may induce further dyslipidemia. Indeed, taken all groups together, we observed a modest negative correlation between ANGPTL4 and plasma HDL-cholesterol (r=−0.143; p<0.009). Some limitations do apply to our study. First of all, we previously showed that the ANGPTL4 ELISA measures full-length ANGPTL4 and the C-terminal truncated fragment of ANGPTL4 but not the N-terminal truncated fragment. Only full-length and the N-terminal fragment of ANGPTL4 influence plasma lipids. It is unclear to what extent the plasma concentrations of C-terminal and full-length ANGPTL4 reflect the concentration of N-terminal ANGPTL4. The mechanism underlying the relationship between inflammation and plasma ANGPTL4 levels is unknown as yet, and probably involves causal stimuli other than CRP that merely represents a marker of inflammation. Further studies into the causal relationships are thus required. Also, it is unclear why CRP is lower in T2DM compared with patients with MetS+I. It should be noted that patients with well-controlled diabetes with an HbA1c between 6.5% and 8.5%, a BMI between 25–32 kg/m2, BP <150/85 mm Hg, and no history of diabetes-related complications were included, who were treated with drugs such as statins with pleotropic anti-inflammatory effects. Furthermore, higher CRP in patients with MetS+I may be explained by a higher percentage of smokers in that group. Also, from the current study set up it is unclear whether the relationship between ANGPTL4 and CRP in humans is solely explained by activation of macrophages. Other cell types and tissues may also be involved.
In conclusion, we provide a novel link between inflammation and ANGPTL4. An increased inflammatory state in patients with MetS and T2DM was associated with higher plasma ANGPTL4 levels. Additionally, a positive correlation was observed between plasma CRP and ANGPTL4. Although in vitro studies confirmed that PRR ligands increase ANGPTL4 expression, further studies into the precise mechanisms underlying the relationship between inflammation and ANGPTL4 in humans are warranted.