Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop ”

Letter by Rask-Madsen et al Regarding Article, “Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop” To the Editor: In an article published online on December 5, Viswambharan et al presents interesting data on atherosclerosis in mice with overexpression of the insulin receptor in vascular endothelial cells. Their work adds to a growing literature on the importance of insulin signaling in endothelial cells with impact on blood flow regulation, whole-body metabolism, angiogenesis, and atherogenesis. Viswambharan et al ascribe increased atherosclerosis in these mice to overactive signaling through Akt, an important pathway activated by insulin. However, it is important to note that gain of insulin-stimulated Akt signaling in endothelial cells may not model any common human condition. In many tissues, insulin-stimulated phosphatidylinositol 3-kinase/Akt signaling is impaired, whereas other signaling pathways through MAPK (mitogen-activated protein kinase) or SREBP1c (sterol regulatory element binding protein-1c) remain sensitive to insulin. To describe this phenomenon, we proposed the term selective insulin resistance in 1999. This concept has been embraced by many groups working on insulin signaling in endothelial cells and other cell types. According to this concept, insulin-stimulated Akt activation is impaired in endothelial cells in obesity or diabetes mellitus. Hyperinsulinemia develops as compensatory mechanism to overcome insulin resistance but because certain signaling pathways remain intact, there can actually be supranormal activation of MAPK or other pathways. However, MAPK signaling was not studied by Viswambharan et al. We have previously published that knockout of the insulin receptor targeted to vascular endothelium increases atherosclerosis by 3-fold in animals followed for 12 months. This general finding was actually confirmed by Gage et al. We have further shown that activation of the β isoform of protein kinase C in endothelial cells can induce insulin resistance in these cells. Others have shown that loss off Akt1 or its downstream target FoxO increases atherosclerosis; in fact, mice with FoxO (forkhead box O) 1/3/4 knockout in endothelial cells have one of the most dramatic phenotypes in the literature on mouse atherosclerosis. There is, therefore, an emerging consensus that loss of insulin signaling and, more specifically, insulin-stimulated Akt/FoxO signaling, accelerates atherosclerosis. Viswambharan et al imply inconsistencies in this paradigm by citing 2 studies as evidence for the association between unrestrained Akt activity and atherosclerosis. However, these studies do not measure insulin signaling. The authors also argue for the clinical relevance of their study by citing the PROactive study (PROspective pioglitAzone Clinical Trial In macroVascular Events). The rationale for this clinical trial, however, is to normalize insulin signaling in patients with insulin resistance, as opposed to achieving supranormal insulin signaling. Although ablation of all insulin signaling in endothelial cells can answer the question of how loss of all insulin signaling, on the balance, affects atherosclerosis development, we have been aware that this model does not accurately model clinically relevant selective insulin resistance. We, therefore, generated Apoe (apolipoprotein E)-null mice with overexpression of insulin receptor substrate-1 in endothelial cells, which causes selective gain of phosphatidylinositol 3-kinase/Akt signaling without increasing MAPK activity. Two different high-fat diets increased atherosclerosis in Apoe-null mice, and this increase was partly prevented by endothelial cell overexpression of insulin receptor substrate-1. We also identified endothelin receptor type B as a target of insulin signaling through Akt. By itself, insulin has a modest effect on NO production through modification of endothelial NO synthase (eNOS) phosphorylation. However, exposure of endothelial cells to insulin upregulates endothelin receptor type B, which activates eNOS in endothelial cells by increasing intracellular Ca2+. In keeping with the effect of insulin on endothelin receptor type B expression and endothelin-1 on eNOS activation, insulin and endothelin-1 cause a >3-fold higher NO production than endothelin-1 alone. This interplay between endothelin-1 and insulin signaling greatly potentiates the ability of insulin to activate eNOS. Furthermore, endothelial cell insulin receptor substrate-1 overexpression in mice maintains its ability to reduce atherosclerosis even in mice with mutant eNOS, which cannot be phosphorylated on Ser1177. Why, then, is there an apparent discrepancy between the findings in the study by Viswambharan et al and mouse models with loss of insulin signaling or gain of insulin-stimulated insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling? One answer is that vascular effects of augmenting insulin signaling may not be reciprocal to loss of insulin signaling. The authors, in fact, have made this point by showing that overexpression of the insulin receptor, in the current article, and expression of a dominant-negative insulin receptor mutant, in their previous article that was not discussed, both increase expression of NOX2 (NADPH oxidase 2). If both observations are correct, they strongly suggest that the mechanism proposed by Viswambharan et al is incomplete and need to include additional signaling, for example, induction of NOX2 by MAPK during supranormal insulin signaling. Dissecting insulin signaling in endothelial cells by genetic manipulation of the mouse will never, of course, be perfectly accurate in modeling endothelial cell insulin resistance in man. However, with our current knowledge of abnormal insulin resistance, we need to take the complexities of signaling described above into consideration. Further studies of mice with overexpression of the insulin receptor mice may show how supranormal insulin signaling affects other pathways than phosphatidylinositol 3-kinase/Akt signaling and provide evidence for how insulinsensitive pathways in selective insulin resistance can promote atherosclerosis.