The current study provides definitive evidence that a loss of insulin signaling in the vascular endothelium without changes in systemic metabolism promotes early events in atherogenesis and accelerates the progression of advanced atherosclerotic disease. These outcomes support the hypothesis previously examined by us (Jiang et al., 1999
) and others (Steinberg et al., 1996
), proposing that in patients with obesity or type 2 diabetes, insulin resistance in endothelial cells may contribute to increased risk for development of cardiovascular disease.
Our results show that systemic insulin action in EIRAKO mice is no different than in control mice. Thus, endothelial insulin resistance did not cause changes in whole-body glucose tolerance, circulating insulin concentrations, or insulin sensitivity in EIRAKO mice compared to controls. This is consistent with what has previously been reported for VENIRKO mice (Vicent et al., 2003
) and with findings in transgenic mice with a mutant insulin receptor targeted to endothelium (Duncan et al., 2008
). Furthermore, the similarity in cholesterol and triglyceride content in lipoproteins between EIRAKO mice and their controls shows that insulin signaling in endothelial cells does not significantly alter regulation of lipoprotein metabolism in the liver or adipose tissue. This lack of differences in systemic insulin sensitivity or lipoprotein profile between control and EIRAKO mice is essential in order for us to conclude that the accelerated atherosclerosis in EIRAKO mice is a direct result of the loss of insulin action on the endothelium.
Leukocyte rolling on and adhesion to endothelial cells in vivo
were increased in both EIRAKO mice, which are apoE null, and VENIRKO mice, which are apoE wild-type. These findings correspond with results showing upregulation of VCAM-1 expression in endothelial cells isolated from EIRAKO mice, and decreased VCAM-1 protein expression in endothelial cells treated with insulin. The functional significance of this finding was demonstrated by experiments in vivo
showing that a VCAM-1 blocking antibody reduced leukocyte-endothelial cell adhesion in EIRAKO mice to below control levels. Epidemiological evidence supports that upregulation of VCAM-1 in association with insulin resistance is clinically important, because in patients with type 2 diabetes, soluble VCAM-1 concentrations are independently associated with cardiovascular mortality (Jager et al., 2000
). However, previously published mechanistic studies on the effect of insulin on the expression of factors that promote adhesion have been contradictory. Several studies, among them work by De Catarina et al. (Madonna et al., 2004
), have shown that insulin can increase the expression of ICAM-1 and VCAM-1 in endothelial cells, although other studies have shown that insulin inhibits expression of ICAM-1 ((Aljada et al., 2000
)) and P-selectin (Booth et al., 2001
). We are not aware of previously published evidence demonstrating regulation of VCAM-1 by insulin in vivo
In the current study, NO-mediated vasodilator function was impaired in EIRAKO mice, and loss of insulin-stimulated phosphorylation of Akt and eNOS was clearly demonstrated in the aorta in vivo
and in isolated endothelial cells. NO synthesized by eNOS decreases both leukocyte rolling and adhesion (Lefer et al., 1999
), and NO has been shown to inhibit expression of VCAM-1 and other factors promoting leukocyte adhesion through deactivation of NFκB (De Caterina et al., 1995
; Khan et al., 1996
). Therefore, decreased endothelium-derived NO could result in increased expression of adhesion molecules, or promote atherosclerotic plaque development and complexity through other mechanisms. For example, impaired NO production could increase the number of intraplaque vascular smooth muscle cells, as shown by our data from brachial artery immunohistochemistry, because NO inhibits vascular smooth muscle cell migration (Sarkar et al., 1996
). However, NO production could not account for insulin-stimulated downregulation of VCAM-1 in our cell culture studies, as L-NAME was unable to block these effects of insulin. Therefore, it is likely that the regulation of VCAM-1 by insulin is mediated by another mechanism, perhaps through the nuclear factor forkhead box O1 (FoxO1/FKHR). Transcriptional activity of FoxO1 is suppressed by insulin (Nakae et al., 1999
; Tang et al., 1999
) and FoxO1 activation upregulates VCAM-1 expression (Abid et al., 2006
). Such mechanisms may be similar to or separate from the pro-inflammatory and pro-atherosclerotic changes observed in whole-body Akt1−/−
mice (Fernandez-Hernando et al., 2007
). These questions are currently being investigated in our laboratory.
We observed a dramatic increase in mononuclear cell adhesion when mononuclear cells from wild-type mice were injected into EIRAKO mice. In contrast, no abnormality of mononuclear cell adhesion to endothelial cells was observed when mononuclear cells from EIRAKO mice were injected into control littermates or wild-type mice. These data show that increased leukocyte-endothelial cell interaction in EIRAKO mice is caused by endothelial cell dysfunction rather than changes in monocyte function. Similarly, replacement of bone marrow in apoE null mice with bone marrow from EIRAKO mice or their controls showed that insulin receptor downregulation in bone marrow-derived cells was not sufficient to accelerate atherosclerosis. We therefore conclude that the vascular inflammation and accelerated atherosclerosis observed in EIRAKO mice is a result of the loss of insulin signaling in endothelial cells, with no significant contribution from the downregulation of insulin signaling in leukocytes.
A previous study has shown that the loss of insulin receptors in macrophages or leukocytes may decrease atherosclerosis in apoE null mice (Baumgartl et al., 2006
). Furthermore, replacing the bone marrow in apoE null mice with transplants from insulin receptor substrate-2 (IRS-2)/apoE double knockout mice decreased atherosclerosis even though IRS-2/apoE double knockout mice had increased atherosclerosis compared to apoE null controls (Baumgartl et al., 2006
; Gonzalez-Navarro et al., 2008
). Another study showed that transplantation of bone marrow from insulin receptor null mice to lethally irradiated LDL receptor null mice had no effect after 8 weeks of an atherogenic diet, and demonstrated a modest increase after 12 weeks (Han et al., 2006
), as well as increased apoptosis of macrophages and an increased rate of necrotic core formation in atherosclerotic plaques. Therefore, even though decreased expression of insulin receptor in mononuclear cells in EIRAKO mice did not appear to affect increased leukocyte-endothelial interaction or atherosclerotic development, complete loss of insulin signaling in leukocytes, or loss of insulin action in certain subsets of bone-marrow derived cells, may decrease the progression of atherosclerosis while promoting necrotic core formation in more advanced plaques.
In vascular tissue from obese rats and patients with diabetes, the major insulin signaling pathway represented by PI3K and Akt is affected selectively by insulin resistance, whereas the pathway represented by mitogen-activated protein kinase (MAPK) is intact (Jiang et al., 1999
). We have therefore proposed that in the normal, insulin-sensitive state, insulin action on the endothelium is mainly mediating anti-atherogenic effects by activating the PI3K/Akt pathway (Jiang et al., 1999
). However, in the insulin resistant state, often associated with hyperinsulinemia, pro-atherogenic insulin action on endothelium may be mediated by MAPK pathways (Rask-Madsen and King, 2007
). By using a mouse model with a null mutation of the insulin receptor gene in endothelial cells, our study may underestimate the contribution of selective insulin resistance in the endothelium in disease states because potential pro-atherosclerotic insulin signaling mediated by MAPK is lost in the mouse model, while it may be functional in human disease. Future studies will be needed to determine whether enhancement of the insulin receptor substrate/PI3K/Akt signaling pathway in endothelium can delay atherosclerosis development.
In summary, we have demonstrated that insulin action on the endothelium is mainly anti-atherogenic and that loss of insulin signaling in endothelial cells promotes initiation of atherosclerosis and its progression, and increases the complexity of advanced atherosclerotic lesions. The findings further indicate that insulin plays a quantitatively prominent role in maintaining normal endothelial function, and that endothelial insulin resistance may accelerate atherosclerosis by causing impaired activation of eNOS, increased endothelial expression of VCAM-1, and increased leukocyte interaction with endothelial cells. These results provide the rationale for identifying interventions which can decrease the risk of atherosclerotic disease in patients with insulin resistance or diabetes by alleviating insulin resistance in the endothelium.