Studies On The Role Of Sphingosine Kinase In The Regulation Of Glucose Homeostasis

Extensive attention has been paid to the effect of sphingolipid metabolites as well as related metabolic enzymes on regulation of glucose metabolism. Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P). These metabolites have been implicated in glucose metabolism, such as glucose uptake, insulin resistance or insulin secretion. The balance between cellular levels of Cer/Sp and S1P determines whether cells survive or die, which is termed as"sphingolipid rheostat". Generally, Cer and Sp, the precursor of S1P, are associated with cell growth arrest and apoptosis, whereas S1P has been implicated in cellular proliferation and survival. Numerous studies identify ceramides as potential contributors to insulin resistance induced by saturated FFAs,TNF-αor glucocorticoids. Sphingosine kinase (SPK) is the key enzyme in"sphingolipid rheostat"as it forms an essential checkpoint that regulates the relative levels of S1P, Sp, and Cer. As for the possible effect of SPK1 on glucose metabolism, a systemic research of it has been made in the present study.First of all, we have prepared and purified Ad-SPK1----recombinant adenovirus harbering SPK1 gene. Then we evaluated the effects of SPK1/S1P signaling molecules on glucose uptake and insulin sensitivity in vitro. When infected with Ad-SPK1, or treated directly by S1P, SMMC-7721 hepatoma cells and C2C12 myoblasts increased significantly glucose uptake. And these cells had a much more significant glucose uptake when stimulated by insulin. To further validate the action of SPK1 on glucose uptake, we applied DMS, a potent inhibitor of SPK1, and SPK1-specific siRNA to block the activity or expression of SPK1 in both cell lines. It showed that the treated cells had a remarkable decrease in both basal and insulin-induced glucose uptake. Further study has found that insulin can enhance SPK activity in cells. Thus, our studies represent a key finding in that the SPK1/S1P seems to correlate with insulin signaling pathway and have a significant insulin-sensitizing effect.Secondly, based upon in vitro cell experiments, we investigated the role of SPK1 in in vivo glucose utilization in the streptozotocin (STZ)-induced type 1 diabetic rat model and spontaneous KK-Ay type 2 diabetic mice model using a adenoviral- mediated gene transfer approach. It was demonstrated for the first time that adenoviral-mediated gene transfer of SPK1 can markedly improve blood glucose level and lipid profiles of whether type 1 or type 2 diabetic animals. Besides, Ad-SPK1 transfection also protected against chromic hyperglycemia-resulted organ injury,such as hepatic and cardiac myocyte injury. Also, SPK1 gene transfection could enhance Akt/GSK-3βcascades of insulin signal transduction downstream of the insulin receptor. Our studies reveal that SPK1 is a key factor in regulation of glucose metabolism and homeostasis and of clinical importance in the management of diabetes.Furthermore, to further elucidate the essential role of SPK in glucose metabolism, and determine whether SPK can serve as a novel target for screening and identifying anti-diabetic drugs or not, we analyzed the effect of glimepiride, third-generation sulfonylurea, which is the most effective and widely used anti-diabetic drug in managing the type 2 diabetes mellitus, on SPK activity in vitro and in vivo. It showed that glimepiride could increase SPK activity in a dose-dependent manner in both 7721 and C2C12 cell line. Glimepiride also markedly enhanced muscle, liver, and fat SPK activity of KK-Ay type 2 diabetic mice. These results indicate that glimepiride exerts its anti-diabetic effect possible via SPK/S1P pathway.Combining the above results and the important role of lipid kinase in glucose homeostasis, we believe that:①SPK1 might be another lipid kinase connecting closely with glucose homeostasis besides PI-3K;②"Sphingolipid rheostat"might be the regluator of glucose homeostasis.③Our findings also provide a new target for the prevention and treatment of diabetes and diabetic complications.