The present study demonstrates that exogenous FABP4 induces endothelial cell dysfunction
in vitro, as assessed by the impact on one of their main properties, the vasodilatory mechanisms. We have also shown that this effect is mediated by the interaction of FABP4 with the insulin-signalling pathway in vascular cells. FABP4 alters eNOS activation, as was demonstrated by the reduction of eNOS phosphorylation at Ser
1177 and NO production. In addition, the reduction of IRS1 phosphorylation at Tyr
989 and Akt phosphorylation at Ser
473 suggests that the effect observed for eNOS activation is due to the interference of FABP4 in the insulin-signalling pathway in endothelial cells. Our results support a direct effect of extracellular FABP4 on vascular cells and, therefore, a putative effect of circulating FABP4 on peripheral tissues. This observation is important because FABP4 is recognised as a biomarker of cardiometabolic risk but it could be considered as mediator of peripheral tissue damage. Several studies have linked FABP4 levels to obesity, T2D and MS [
4,
8,
11]. Additionally, FABP4 has been associated with the burden of coronary atheromatosis, but a causal role has not yet been established. The only report that showed a direct effect of FABP4 on cells demonstrated that FABP4 reduces the contractile capacity of cardiomyocytes [
15]. We have previously reported that plasma FABP4 levels are associated with endothelial dysfunction in diabetic patients [
16]. Our present data supports a causal role of FABP4 in the dysfunction of the vascular wall. These findings are in accordance with a previous study suggesting that an elevated expression of intracellular FABP4 in vascular endothelial cells contributes to endothelial dysfunction both
in vivo and
in vitro[
22]. In contrast, a recent study showed that FABP4/5 inhibitors ameliorate dyslipidaemia but not insulin resistance in diet-induced obese mice [
23]. Our results show that exogenous FABP4 alters the insulin-signalling pathway at its early activation steps. Additionally, it has already been shown that FABP4 interferes with the insulin receptor [
24]. Moreover, FABP4 could be phosphorylated on Tyr
19 in response to insulin stimulation [
25]. Our results support these previous observations and extend them to the exogenous FABP4, which has a greater clinical implication. Along with a decrease in eNOS expression, in our hands, FABP4 increased the expression of vascular cell adhesion protein 1 (VCAM1), E-selectin and leukocyte adhesion to endothelial cells (data not shown), suggesting a more global effect of FABP4 on endothelial function. In our experiments, HUVECs were stimulated with 600nM insulin because that was the dose with which we observed an increase in the phospho-eNOS produced and because other authors also obtained maximal nitric oxide production with similar dose [
26].
The mechanisms that FABP4 utilises to interfere with the cell are currently being investigated. Although previous studies showed that Heart-FABP (62 % homology with FABP4) has the capability of binding to a membrane receptor described in cardiac cells [
27], this observation has neither been confirmed nor extended to other FABP family members. It has been shown that heart FABP levels in serum could represent a useful biomarker for myocardial function in pre-diabetic patients [
28]. It is not known whether FABP4 interacts directly with the insulin receptor or if it interacts with other components of the cell membrane and then secondarily modifies the insulin cascade. It is also unknown whether circulating FABP4 can be internalised into the cell to act by intracellular mechanisms. We have observed that the amount of intracellular FABP4 increases after FABP4 incubations with respect to non-treated cells, which suggests that FABP4 could be internalised by endothelial cells. FABP4 is an intracellular long-chain fatty acid transporter [
1,
29]. Although this function has not yet been demonstrated for circulating FABP4, we cannot exclude that the effects associated with extracellular FABP4 could be mediated by fatty acids, which are molecules known to play a role in the dysfunction of the insulin-signalling pathway. This finding could be important evidence that FABP4 plays a pivotal role in impairing the insulin signalling pathway.
Our findings suggest that high levels of FABP4 in the plasma are not just a clinical manifestation of insulin resistance but also a causative factor of the development of insulin resistance at the vascular level leading to NO metabolism alteration.