Effects Of The Three Different Structural Sphingolipids From The Sea Cucumber Cucumaria Frondosa On Adipogenesis And Lipid Metabolism

Sphingolipids, which mainly consist of cerebroside, ceramide and long chain base (LCB), are one of the most important lipids in the body wall of the sea cucumber. Cerebroside consists of a ceramide that binds with a glycosyl, while ceramide is composed by a long-chain fatty acid and a LCB, which are connected by amide linkage. Researches showed that sphingolipids from the sea cucumber possessed the bioactivities of anti-cancer, anti-alzheimer disease and regulating energy metabolism. Disorders of adipogenesis and lipid metabolism in adipocytes are critical factors leading to obesity. The effect of sphingolipids from the sea cucumber has been primarily researched, but its mechanism is still unknown and the active fragment is not illuminated. Therefore, cerebroside, ceramide and LCB were obtained from the sea cucumber Cucumaria frondosa (Cf-Cb、Cf-Cm and Cf-LCB) and its effects on the adipogenesis and lipid metabolism were researched. The underlying mechanisms and the active fragment in the sphingolipids were further clarified. The results are as follows:3T3-L1 preadipocytes were induced to mature adipocytes using the standard cocktail method. Oil-red O staining results showed that Cf-Cb、Cf-Cm and Cf-LCB significantly inhibited the differentiation of 3T3-L1 cells and the inhibitory effect was stronger at the early stage of differentiation. C/EBPa and PPARy are the most important transcriptional factors in the process of adipogenesis. qRT-PCR and western blotting results indicated that Cf-Cb、Cf-Cm and Cf-LCB down-regulated both the mRNA and protein expressions of C/EBPa and PPARy. WNT/β-catenin pathway is the well-established negative regulators of adipogenesis. qRT-PCR results showed that Cf-Cb、Cf-Cm and Cf-LCB significantly enhanced the mRNA expressions of FZ1、LRP5、LRP6、β-catenin、CCND1 and c-myc, but had no effect on that of Wnt10b and GSK3β. Western blotting results further indicated that the protein expressions of the key factors of WNT/β-catenin pathway, LRP6, total and nuclear β-catenin levels were increased by Cf-Cb、Cf-Cm and Cf-LCB, indicating that the WNT/β-catenin pathway was activated. Furthermore, when cells were treated with 21H7, the inhibitor of P-catenin, adipogenesis and the expressions of C/EBPa and PPARγ were enhanced, while total and nuclear β-catenin levels were reduced. Cf-Cb、Cf-Cm and Cf-LCB significantly reversed the effects of 21H7, further demonstrating their promoting effect on WNT/β-catenin pathway. In conclusion, Cf-Cb、Cf-Cm and Cf-LCB inhibited the adipogenesis through activating the WNT/β-catenin pathway.The effects of Cf-Cb、Cf-Cm and Cf-LCB on the lipid metabolism were researched using the 3T3-L1 mature adipocytes. Results showed that Cf-Cb、Cf-Cm and Cf-LCB reduced the TG content in adipocytes and increased the FFA content in the culture supernatant, indicating that Cf-Cb、Cf-Cm and Cf-LCB can inhibit lipid accumulation and accelerate the lipidolysis process. Western blotting results further showed that Cf-Cb、Cf-Cm and Cf-LCB markedly down-regulated the expression of the lipogenic factors, GPAT and FAS, while up-regulated that of the lipidolysis related factors, HSL and CPT-1. AMPK is an important regulator of the intracellular energy metabolism. Results indicated that Cf-Cb、Cf-Cm and Cf-LCB significantly enhanced the phosphorylation of AMPK and ACC, suggesting that Cf-Cb、Cf-Cm and Cf-LCB can activate the AMPK pathway. Further studies showed that Compound C, a well-known AMPK inhibitor, increased the intracellular TG content and GPAT and FAS expressions, while reduced the FFA content, HSL and CPT-1 expression and AMPK and ACC phosphorylation. Cf-Cb、Cf-Cm and Cf-LCB significantly improved the effects of Compound C, further proving that they can activate the AMPK pathway. These results indicated that Cf-Cb、Cf-Cm and Cf-LCB regulated the lipid metabolism of adipocytes by promoting the AMPK pathway.The effects of cerebroside,ceramide and LCB from the C. frondosa on adipocyte differentiation and lipid metabolism were compared using two systems:the three kinds of sphingolipids whose concentrations are the same and the three kinds of sphingolipids that contain the same content of LCB. Results showed that when cells were treated with the same concentrations of Cf-Cb、Cf-Cm and Cf-LCB,the effects of Cf-LCB on the proliferation and differentiation of 3T3-L1 cells, WNT/β-catenin pathway, lipid metabolism and AMPK pathway were better that the others. When cells were treated with Cf-Cb、Cf-Cm and Cf-LCB that contains the same content of LCB, their effects were almost the same. Therefore, the active fragment of the three kinds of sphingolipids maybe the LCB.These researches systematically studied the anti-adipogenic effect of sphingolipids from the sea cucumber and the underlying mechanisms were also clarified. This paper provides a scientific basis for the utilization of sphingolipids from the sea cucumber on the prevention and treatment of obesity.