Gangliosides are sialic acid-containing glycosphingolipids that are present on all mammalian plasma membranes where they participate in recognition and signaling activities. We have established mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide ␣2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. Our results show that GM3 ganglioside is a negative regulator of insulin signaling, making it a potential therapeutic target in type 2 diabetes.

Sphingosine-1-phosphate (S1P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S1P 1 receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. To understand the physiologic functions of the two other widely expressed S1P receptors, we generated S1P 2 and S1P 3 null mice. Neither the S1P 2 null mice nor the S1P 3 null mice exhibited significant embryonic lethality or obvious phenotypic abnormalities. To unmask possible overlapping or collaborative functions between the S1P 1 , S1P 2 , and S1P 3 receptors, we examined embryos with multiple S1P receptor mutations. We found that S1P 1 S1P 2 double null and S1P 1 S1P 2 S1P 3 triple null embryos displayed a substantially more severe vascular phenotype than did embryos with only S1P 1 deleted. We also found partial embryonic lethality and vascular abnormalities in S1P 2 S1P 3 double null embryos. Our results indicate that the S1P 1 , S1P 2 , and S1P 3 receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development. Sphingosine-1 phosphate (S1P)1 is a sphingolipid metabolite that is present at high levels in the blood (1-3). Through the interaction with a family of five G-protein-coupled receptors (S1P 1-5 ), originally known as EDG receptors, sphingosine-1-phosphate triggers diverse cellular responses, including cytoskeletal changes, proliferation, and migration (1, 4 -8). The S1P 1 , S1P 2 , and S1P 3 receptors are widely expressed, including on embryonic endothelial cells (Table I) (9 -14). S1P 4 and S1P 5 receptor expression is more restricted and found on the cells of the immune and nervous systems (15, 16). The S1P 1 receptor couples selectively to the G i signaling pathway, whereas the S1P 2 and S1P 3 receptors both couple to the G i , G q , and G 12/13 pathways (2,(17)(18)(19)(20). In addition to these five S1P receptors, GPR3, GPR6, GPR12, and GPR63 have been characterized as G-protein-coupled receptors that interact with sphingosine-1-phosphate (21-23).The major physiological effects of S1P receptor signaling defined thus far have been localized to the immune and vascular systems. A global deletion of the S1P 1 receptor in mice results in lethality beginning at E12.5 due to severe hemorrhage as the result of deficient coverage of vessels by vascular smooth muscle cells, a process that occurs during the last stages of angiogenesis and is necessary for stabilizing the vascular system (14). Through analysis of endothelial cell-specific S1P 1 receptor knock-out mice, we have shown that the S1P 1 receptor functions within endothelial cells to regulate vascular smooth muscle cell coverage (24). The function of the S1P 1 receptor in the developing vasculature is also essential for proper limb development (25). Deletion of the S1P 1 receptor in T-cells has re...

The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S1P1 in adult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, was enhanced in mice with endothelial cell–specific deletion of S1pr1 and suppressed in mice with endothelial cell–specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. The chaperones ApoM+HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; therefore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM+HDL-S1P, but not to albumin-S1P, promoted the formation of a cell surface S1P1–β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-1 abundance. Although S1P bound to either chaperone induced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelial cell–specific deletion of S1pr1 in the hypercholesterolemic Apoe−/− mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM+HDL to act as a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL.