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Dysregulated Sphingolipid De Novo Biosynthesis Contributes to Diabetic Cardiomyopathy and Vascular Dysfunction - Role of Metformin
Martina Smimmo1,2, Onorina L. Manzo1, Luisa Rubinelli1, Jason Pierce3, Valentina Vellecco2, Besim Ogretem3, Maria Rosaria Bucci2, Annarita Di Lorenzo11 Department of Pathology and Laboratory Medicine, Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, USA
2 Department of Pharmacy, University of Naples Federico II, Naples, Italy
3Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, USA
Background: Diabetes significantly increases the risk of people for cardiovascular diseases, and dysregulation of sphingolipid metabolism has been implicated in these conditions, although specific mechanisms remain poorly understood. Sphingolipids (SL) are essential components of cell membranes and bioactive lipids, and dysregulation of this pathway has been implicated in diabetes and cardiovascular diseases. Sphingolipid ceramides are increased in the heart of diabetic mice, contributing to lipotoxicity. and implicated in diabetic cardiomyopathy.
In vitro high glucose boosts ceramide de novo biosynthesis in endothelial cells (EC). Ceramide accrual impairs endothelial nitric oxide synthase (eNOS) activity, whereas ceramide suppression impairs endothelial signal transduction and vascular tone regulation, suggesting that maintaining ceramide homoeostasis is crucial to preserve vascular health. However, there is a lack of understanding of these mechanisms in vivo, specifically whether the accrual or decrease of ceramide is causal of endothelial dysfunction.
Aims and Methods: Recent studies have shown that metformin, a first-line therapy for diabetes, can lower ceramides in liver and muscle in obese mice. Considering this scientific evidence, here we investigate how diabetes impact sphingolipid metabolism in EC of blood vessels and cardiomyocytes, and whether a mechanism of action of metformin directly regulates this pathway. To this aim, we used non-obese diabetic mice, a well-established model of type 1 diabetes. Diabetic mice were treated with metformin (300mg/Kg/d) or vehicle for 4 weeks.
Results: Diabetic mice presented both diastolic and systolic dysfunction that correlated with high levels of ceramides and sphingomyelins in the heart, supporting the cardiomyopathy associated to lipotoxicity. Interestingly, metformin treatment improved both diastolic and systolic dysfunction of diabetic mice, which correlated with decreased ceramides to the levels of not-diabetic mice. It is noteworthy to mention, that both groups were maintained on high glycemia (>400 mg/dL) for 4 weeks, thus arguing against a role of glucose on metformin-improved cardiomyopathy. Metformin also improved the changes in the expression of genes included CD36, IL-6, IL-1 and LDLr compared to diabetic mice treated with vehicle. Next, we tested the hypothesis that metformin can directly inhibit the first and rate-limiting enzyme of the sphingolipid de novo biosynthesis, serine palmitoyltransferase (SPT). Data of SPT assay in cardiac microsomes revealed that metformin can directly downregulates SPT activity already at 0.3 M, which is below the reported concentration of circulating metformin at the dose used in this study.
We then investigated the role of this pathway in the vasculature. Diabetic mesenteric arteries showed an impaired vasodilation to acetylcholine and exaggerated response to vasoconstrictors, supporting previous studies. Interestingly, contrary to in vitro studies, ceramides and sphingomyelins were significantly decreased in myocardial endothelial cells isolated from diabetic mice, suggesting that suppression and not accrual of ceramides are implicated in diabetic endothelial dysfunction. Mechanistically, in diabetes Nogo-B expression is significantly upregulated and decreases SPT activity. Notably, metformin treatment significantly improved endothelial function, by restoring acetylcholine-vasodilation and increasing phosphorylation of vasodilator stimulated phosphoprotein, index of eNOS-cGMP signaling. This effect correlated with further decreased of endothelial sphingolipids without affecting Nogo-B or SPT expression. Our findings also revealed that in diabetes there is a cell-type specific regulation of sphingolipid de novo pathway, specifically in EC versus cardiomyocytes.
Conclusions: This study identified the inhibition of SPT activity as a novel mechanism of action of metformin, preventing ceramide and sphingomyelin accrual in the heart and preventing diastolic and systolic dysfunction. Findings from this study also argue against the longstanding believe that ceramide accrual is causal of endothelial dysfunction, but demonstrated that diabetes suppresses sphingolipid de novo biosynthesis in EC via Nogo-B.
This research was in funded by R01 HL126913 from National Heart, Lung, and Blood Institute.
Speakers
Martina Smimmo