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Endothelial cell (EC) dysfunction precedes the onset of cardiovascular diseases (CVD), main complications of diabetes. Sphingolipids (SL) are essential components of cell membranes and bioactive lipids regulating different cellular functions in health and diseases. Dysregulation of SL, such as ceramides (Cer) accrual, has been implicated in diabetes and CVD, although specific mechanisms remain poorly understood. Whereas Cer accrual impairs endothelial nitric oxide synthase (eNOS) activity, low ceramides disrupt endothelial signal transduction and vascular tone regulation. To date, how Cer changes in diabetic EC in vivo is unknown. Metformin (Met), first-line therapy for diabetes, was shown to lower Cer in liver and muscle in mice. This study investigates the role of sphingolipid metabolism in diabetes and whether Met directly regulates this pathway.We discovered that Met can directly downregulate serine palmitoyltransferase activity, catalyzing the first step of the de novo biosynthesis of SL, thus refraining Cer from accrual. To assess this effect in vivo , we used non-obese diabetic mice, a model of type 1 diabetes, and nondiabetic mice as control. Diabetic mice were treated with Met (300mg/Kg/d) or vehicle for 5 weeks. Diabetic mesenteric arteries showed an impaired vasodilation to acetylcholine and exaggerated response to vasoconstrictors. Met treatment significantly improved endothelial function, by restoring acetylcholine-vasodilation and increasing phosphorylation of vasodilator stimulated phosphoprotein, index of eNOS-cGMP signaling. Interestingly, whereas Cer and sphingomyelins (SM) increased in the heart and liver, they were significantly decreased in myocardial EC, suggesting that in diabetes SL are differentially regulated. Met refrained Cer/SM from accrual in the heart and liver, while further decreased Cer/SM in EC. In conclusions, Met prevents Cer accrual in the heart and liver, thus limiting its deleterious effects. Contrary to the current believe, Cer are suppressed in diabetic EC in vivo , and might contribute to the progression of dysfunction. Most likely, Met downregulation of sphingolipid de novo biosynthesis, already suppressed in diabetic EC, does not underlie beneficial effects of Met on the endothelium.

Worldwide >1.6 billion of people are overweight or obese, a condition causally linked to type 2 diabetes (T2D), metabolic syndrome, cardiovascular diseases. In addition to precede the onset of diabetic CV complications, growing evidence implicate endothelial dysfunction to contribute to metabolic disorders. The sphingolipid ceramides, important membrane components and intracellular signaling molecules, have been implicated in insulin resistance and diabetes, and its vascular complications. However, mechanisms disrupting sphingolipid homeostasis and its impact on vascular and metabolic dysfunctions remain poorly understood. Recently, we discovered that Nogo-B, a membrane protein of the endoplasmic reticulum, inhibits sphingolipid de novo biosynthesis via serine palmitoyltransferase, first and rate liming enzyme, to impact vascular functions. Thus, the aim of this study was to investigate how Nogo-B regulation of sphingolipid signaling in the endothelium impacts metabolic and vascular homeostasis in obesity-T2D model.We discovered that in diabetic resistance arteries Nogo-B was overexpressed and correlated with a marked decrease of ceramides and sphingomyelins, vascular dysfunction, and hypertension. Interestingly, systemic, and endothelial genetic deletion of Nogo-B protected the mice from vascular dysfunctions and hypertension in diabetes, by preserving local sphingolipid metabolism and signaling. These findings, the first to investigate local mechanisms of sphingolipid metabolism derangement and its pathological implications, are a paradigm shift for the long-standing belief that vascular ceramide accrual underlies diabetic dysfunctions. Furthermore, our data also show that S1P secreted by the endothelium can downregulate hepatic gluconeogenesis, and that Nogo-B-mediated suppression of sphingolipid de novo biosynthesis can remove S1P brake on liver glucose production and exacerbates diabetes. Our findings suggest that suppression of endothelial sphingolipid signaling via Nogo-B underlies both vascular and metabolic dysfunctions in diabetes.

Growing evidence correlate the accrual of the sphingolipid ceramide in plasma and cardiac tissue with heart failure (HF). Regulation of sphingolipid metabolism in the heart and the pathological impact of its derangement remain poorly understood. Recently, we discovered that Nogo-B, a membrane protein of endoplasmic reticulum, abundant in the vascular wall, down-regulates the sphingolipid de novo biosynthesis, via serine palmitoyltransferase (SPT), first and rate liming enzyme, to impact vascular functions and blood pressure. Nogo-A, a splice isoform of Nogo, is transiently expressed in cardiomyocyte (CM) following pressure overload. Cardiac Nogo is upregulated in dilated and ischemic cardiomyopathies in animals and humans. However, its biological function in the heart remains unknown.We discovered that Nogo-A is a negative regulator of SPT activity and refrains ceramide de novo biosynthesis in CM exposed to hemodynamic stress, hence limiting ceramide accrual. At 7 days following transverse aortic constriction (TAC), SPT activity was significantly upregulated in CM lacking Nogo-A and correlated with ceramide accrual, particularly very long chain ceramides, which are the most abundant in CM, resulting in the suppression of “beneficial” autophagy. At 3 months post-TAC, mice lacking Nogo-A in CM showed worse pathological cardiac hypertrophy and dysfunction, with 50% mortality rate. Mechanistically, Nogo-A refrains ceramides from accrual, therefore preserves the “beneficial” autophagy, mitochondrial function, and metabolic gene expression, limiting the progression to HF under sustained stress.

Ceramides are sphingolipids that modulate a variety of cellular processes via 2 major mechanisms: functioning as second messengers and regulating membrane biophysical properties, particularly lipid rafts, important signaling platforms. Altered sphingolipid levels have been implicated in many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus–related conditions; however, molecular mechanisms by which ceramides impact endothelial functions remain poorly understood. In this regard, we generated mice defective of endothelial sphingolipid de novo biosynthesis by deleting the Sptlc2 (long chain subunit 2 of serine palmitoyltransferase)—the first enzyme of the pathway. Our study demonstrated that endothelial sphingolipid de novo production is necessary to regulate (1) signal transduction in response to NO agonists and, mainly via ceramides, (2) resting eNOS (endothelial NO synthase) phosphorylation, and (3) blood pressure homeostasis. Specifically, our findings suggest a prevailing role of C16:0-Cer in preserving vasodilation induced by tyrosine kinase and GPCRs (G-protein coupled receptors), except for Gq-coupled receptors, while C24:0- and C24:1-Cer control flow-induced vasodilation. Replenishing C16:0-Cer in vitro and in vivo reinstates endothelial cell signaling and vascular tone regulation. This study reveals an important role of locally produced ceramides, particularly C16:0-, C24:0-, and C24:1-Cer in vascular and blood pressure homeostasis, and establishes the endothelium as a key source of plasma ceramides. Clinically, specific plasma ceramides ratios are independent predictors of major cardiovascular events. Our data also suggest that plasma ceramides might be indicative of the diseased state of the endothelium.

Coronary artery disease is a leading cause of myocardial infarction (MI) worldwide. Alterations in sphingolipid levels have been linked to atherosclerosis although specific molecular mechanisms are poorly understood. Recently, we discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular functions by inhibiting serine palmitoyltransferase (SPT), the rate-limiting enzyme of the de novo sphingolipid biosynthesis. Mice lacking Nogo-B are resistant to hypertension and heart failure. Here, we employed a novel model of coronary atherosclerotic lesions induced by hypercholesterolemia and hypertension, well-known risk factors for atherosclerosis. To this aim, transverse aortic constriction (TAC) surgery was performed in mice lacking endothelial Nogo-B in ApoE -/- background and ApoE -/- mice as control. ApoE -/- mice developed coronary atherosclerotic lesions within 6 weeks following TAC, (without the need of long-term high-cholesterol diet) and 70% of the mice died of MI at 6-week post-TAC. On the contrary, mice lacking Nogo-B specifically in endothelial cells were markedly resistant to the development of coronary atherosclerotic lesions and MI (20%). Mechanistically, in the absence of endothelial Nogo-B, the biosynthesis of sphingolipids, particularly S1P, is upregulated, protecting the endothelium from hypertension and hypercholesterolemia-triggered vascular inflammation and atherogenesis. This study identifies an important and novel role of endothelial Nogo-B-dependent regulation of sphingolipid de novo biosynthesis in the coronary atherosclerosis, a primary cause of myocardial infarction.