Diabetes, whether due to insulin shortage or insulin resistance, is associated with many complications, among which are breakdowns in the vasculature's functions and regenerative abilities (angiogenesis). Researchers led by Dr. Clay Semenkovich of the Washington University School of Medicine, St. Louis, Missouri, have dissected a key insulin-regulated pathway that helps maintain vasculature function and integrity (Wei et al. 2011). Their findings may lead to novel targets for treating secondary complications associated with diabetes, including impaired wound healing and neuropathy.
Although insulin had been known to regulate the activity of the protein fatty-acid synthase (FASN) in major fat-producing tissues, such as the liver and white adipose tissue, its effects on FASN in endothelial tissues, which line the vasculature, had been unclear. Semenkovich and his team aimed to clarify those effects. First, they assessed FASN levels in endothelia from C57BL/6 mice with streptozotocin (STZ)-induced diabetes (an insulin-deficient model) and diabetic B6.BKS(D)-Leprdb/J ("db/db", 000697) mice (an insulin-resistant model). They found that FASN levels are low in both models. Additionally, they found that insulin induces FASN production in cultured human endothelial cells. These results led them to conclude that insulin regulates FASN not only in major fat-producing tissues but also in vascular endothelial tissues.
Semenkovich and his team then investigated the effect of FASN-deficiency in live mice. To circumvent the embryonic lethality of FASN-deficiency, they produced vascular endothelial cell-specific FASN-deficient mice by breeding mice that carry loxP sites flanking Fasn exons 4-8 to B6.Cg-Tg(Tek-cre)12Flv/J (004128) mice, which express Cre recombinase in endothelial and hematopoietic cells under the control of the Tie-2 promoter. These "FASTie" mice express low levels of FASN in lung and aortic endothelia, and in bone marrow. The researchers found that though the low FASN levels do not affect the total amount of endothelial nitric oxide synthase (Nos3; eNOS) in endothelial cells of these mice, it disrupts eNOS localization and activity in the membranes of those cells.
The major function of eNOS is to regulate blood vessel permeability and vascular tone. To perform this function, it must be located in the vascular endothelial cell caveolae – small invaginations in endothelial cell membranes – and, to be localized there, it must be palmitoylated. If unpamitoylated, eNOS is not tethered to the membrane caveolae and cannot efficiently make nitrous oxide (NO), which is required to inhibit smooth muscle contraction. Untethered eNOS can produce superoxide leading to oxidative stress, thus compromising endothelial integrity. Semenkovich and his team found that FASN, whose major product is palmitate, physically interacts with and palmitoylates eNOS. Disrupting FASN expression, either in vitro or in vivo, decreases eNOS palmitoylation. Substantiating this, the researchers found that treating human vascular endothelial cells with insulin, which increases FASN, increases eNOS palmitoylation.
Not surprisingly, Semenkovich and his team found that defective FASN-eNOS signaling increases the permeability of cultured endothelial cells and of FASTie mouse blood vessels. They suspected that this hyperpermeability – along with the oxidative stress induced by unpalmitoylated and mislocalized eNOS – mediates inflammation. Indeed, they found that cultured endothelial cells from FASTie mice express more inflammatory molecules and contain more inflammatory cells than comparable cultures from wild-type mice. Additionally, FASTie mice treated with lipopolysaccharide (LPS) exhibit greater lung inflammation and are more likely to die from proinflammatory responses than wild-type mice.
In addition to being at risk for infections, people with diabetes commonly develop peripheral vascular disease, which may necessitate limb amputation. Semenkovich and his team found that this could be due to diabetes-mediated FASN disruption. They demonstrated that FASN-deficient human endothelial cells have reduced wound-healing capacity and that aortic rings from FASTie mice exhibit reduced vessel outgrowth. Additionally, following femoral artery ligation, FASTie mice exhibit prolonged tissue necrosis and diminished capillary formation, indicating impaired angiogenesis.
Finally, Semenkovich and his team substantiated that defects in the FASN-eNOS palmitoylation pathway are present in mouse models of diabetes. They assayed eNOS in the lungs from mice with STZ-induced (insulin-deficient) diabetes and from db/db mice, which have insulin resistant diabetes, and found that eNOS palmitoylation is abnormally low in both models.
In summary, the Semenkovich team elucidated a biochemical pathway critical to maintaining endothelial integrity. They showed that insulin, known to regulate FASN activity in major fat-producing tissues such as the liver and white adipose tissue, also regulates endothelial FASN activity. They found that endothelial FASN physically interacts with eNOS, palmitoylates it, and localizes it to endothelial cell caveolae, where it regulates blood vessel permeability. Either insulin resistance or insulin deficiency lead to peripheral vascular disease by disrupting endothelial cell integrity, increasing blood vessel permeability and impairing angiogenesis. These findings suggest that targeting the insulin-FASN-eNOS pathway may lead to new therapies for treating severe complications associated with diabetes.
Wei X, Schneider JG, Shenouda SM, Lee A, Towler DA, Chakravarthy MV, Vita JA, Semenkovich CF. 2011. De novo lipogenesis maintains vascular homeostasis through endothelial nitric-oxide synthase (eNOS) palmitoylation. J Biol Chem 286:2933-45. [PMID:21098489]