Effects And Mechanisms Of Neohesoeridin On Glucose And Lipid Metabolism In Vitro And In Vivo

ObjectiveThe incidence of obesity and diabetes has been growing at an alarming rate, which represent major health threats to the world population today. Due to insufficient efficacy and adverse reactions of current medications regulating glucose and lipid metabolism, there have been persistent efforts to identify potential compounds or find out a new target that can improve glucose and lipid metabolism. It has great significance finding substances from natural products to prevent and control diseases related to abnormalities of glucose and lipid metabolism and further studying its mechanisms.Numerous studies have indicated the safty and a variety of pharmacological effects of citrus flavonoids. Neohesperidin is a major flavanone glycoside, which is rich in citrus flavedo and albedo. For the first time, we systematically studied the glucose and lipid regulatory effects and mechanisms of neohesperidin, and with the efficacy explicitly illustrated, the key related signaling molecules were been looked up. Using cell and animal models of obesity and diabetes, we researched the effect of neohesperidin on glucose and lipid metabolism regulation, and further clarified on cell and animal levels its molecular mechanisms of action, especially that related to FGF21and AMPK.This study could provide ideas and methods to prevent glucose and lipid metabolic disorder diseases, and provide experimental basis and clues to clarify the related molecular mechanism of FGF21and AMPK. It also has important guiding significance in theory and practice of prevention and control for chronic metabolic diseases.Methods 1. Effects of neohesperidin on glucose and lipid metabolism:1) Using glucose oxidase method according to the protocol provided by the manufacturer and the SRB assay, the effect of neohesperidin on glucose consumption was tested in HepG2cells.2) FFAs-induced lipid accumulated HepG2cells were treated with neohesperidin or other agents for24h. Then oil red staining analysis was performed, and cellular triglycerides and total cholesterols were determined.3) While a group of mice were fed with standard chow diet, the other mice were kept on a high-fat diet (HFD) for19weeks to reach an obesity state with glucose and lipid metabolism disorder before experiments were started. We detected the effects of neohesperidin on glucose and lipid metabolism in diet-induced obesity (DIO) mice. After2weeks administration, glucose tolerance test (GTT) was performed. After8weeks administration, fasted or random blood glucose was measured with a One-Touch Ultra glucometer. Body weight, waist circumference, body length, food intake, total fat, triglycerides, total cholesterols, free fatty acid, LDL-C, HDL-C, apoA-I and apoB were measured. Then oil red staining was carried out, and triglycerides and total cholesterols of liver were analysed.4) After2weeks administration, the level of glucose, triglycerides and total cholesterols in plasma were determined in db/db mice.5) C57mice were injected by STZ after8weeks treatment and then continued with the same administration for3weeks, and then blood glucose levels were measured.6) After2weeks administration of neohesperidin in an acute hyperlipidemia C57mouse model induced by egg yolk emulsion, triglycerides, total cholesterols, LDL-C and HDL-C were detected.7) After2weeks administration of neohesperidin in normal C57mice, glucose tolerance and blood glucose levels were tested.8) Effects of neohesperidin on blood glucose levels in amylum-loaded mice were observed comparing with naringin and hesperidin. 2. Possible mechanisms of neohesperidin regulating glucose and lipid metabolism:1) Western blotting was used to detect the expression of p-AMPK protein after treatment of neohesperidin, and then RT-PCR was used to detect the expression of hmg-coa, srebp1c,fas and cptl mRNA expression.2) The effects of neohesperidin on PPAR-a and FGF21protein and mRNA expression were detected using western blotting and RT-PCR.3) The effect of neohesperidin on a-glycosidase activity was tested.Results1. Neohesperidin regulated glucose and lipid metabolism in vivo and in vitro:1) Neohesperidin, hesperidin and naringin concentration-dependently stimulated glucose consumption in HepG2cells. Neohesperidin exerted a stronger effect on glucose consumption comparing with hesperidin at the same concentration.2) Neohesperidin decreased lipid accumulations in FFA-induced lipid accumulated HepG2cells, and decreased triglyceride and total cholesterol contents in FFA-induced lipid accumulated HepG2cells in a concentration-dependent manner.3) Neohesperidin could regulate glucose and lipid metabolism in diet-induced obesity mice, resulting in improved glucose tolerance, lowered blood glucose, body weight, adipose tissue mass and FER, decreased triglycerides and total cholesterols in liver, gastrocnemius muscle and plasma, and reduced ratios of LDL-C/HDL-C and apoB/apoA-I.4) Neohesperidin lowered plasma glucose and triglyceride contents in db/db diabetic mice.5) Neohesperidin decreased blood glucose in STZ-induced diabetic mice.6) Neohesperidin reduced plasma triglycerides, total cholesterols, LDL-C contents and the ratio of LDL-C/HLD-C, increased plasma HDL-C contents in an acute hyperlipidemia C57mouse model induced by egg yolk emulsion. Neohesperidin exerted a stronger effect on plasma HDL-C contents comparing with the same dosage of hesperidin and naringin.7) Neohesperidin improved glucose tolerance, but had no effects on normal blood glucose in C57mice.8) Neohesperidin dose-dependently improved amylum tolerance in C57mice.2. Mechanisms of neohesperidin regulating glucose and lipid metabolism:1) Effects of neohesperidin on AMPK and its signaling pathways:Neohesperidin upregulated p-AMPK expression in HepG2cells and FFA-induced lipid accumulated HepG2cells. Furthermore, neohesperidin inhibited the mRNA expression of srebplc, fas and hmg-coa, increased that of cptl in FFA-induced lipid accumulated HepG2cells. In the same way, neohesperidin stimulated p-AMPK expression and regulated mRNA expression of srebplc and cptl in the liver of DIO mice.2) Effects of neohesperidin on PPAR-a and FGF21expression:Neohesperidin increased protein and mRNA expression of PPAR-a and FGF21in FFA-induced lipid accumulated HepG2cells; it upregulated PPAR-a protein and gene expression, increased fgf21mRNA expression, and elevated plasma FGF21levels.3) Neohesperidin inhibited a-glucosidase activity.ConclusionNeohesperidin can regulate glucose and lipid metabolism in cell and animal models of obesity and diabetes, and the mechanisms may be related to the activation of AMPK and PPAR-a and the gene expression and secretion of FGF21.1. In the in vitro study, neohesperidin improves glucose metabolism in HepG2cells and decreases lipid accumulation in FFA-induced lipid accumulated HepG2cells and the mechanisms are associated with stimulated AMPK activity in FFA-induced lipid accumulated HepG2cells. Furthermore, neohesperidin inhibits the mRNA expression of srebplc, fas and hmg-coa, increases that of cptl, all these are related to stimulated PPAR-a protein and gene expression, and increased FGF21protein and gene expression.2. Neohesperidin improves glucose tolerance, lowers blood glucose, body weight, adipose tissue mass and FER, decreases triglycerides and total cholesterols in liver, gastrocnemius muscle and plasma, reduces the ratios of LDL-C/HDL-C and apoB/apoA-I in diet-induced obesity mice. The mechanisms of these effects are related to a stimulation of AMPK protein activity, an inhibition of mRNA expression of srebplc, and and increase of mRNA expression of cptl. Furthermore, the effects are also associated with the enhanced PPAR-a protein and gene expression, the increased fgf21mRNA expression in liver, and the elevated plasma FGF21levels.3. Neohesperidin lowers plasma glucose and triglycerides in db/db mice, and decreases blood glucose in STZ-induced diabetic mice.4. Neohesperidin reduces plasma triglycerides, total cholesterols, LDL-C contents and the ratio of LDL-C/HLD-C, and increases plasma HDL-C contents in an acute hyperlipidemia C57mouse model induced by egg yolk emulsion. Neohesperidin exerts a stronger effect on plasma HDL-C contents comparing with the same dosage of hesperidin and narigin.5. Neohesperidin improves glucose tolerance, but has no effects on normal blood glucose in C57mice.6. Neohesperidin dose-dependently improves amylum tolerance in C57mice, and this action may be associated with an inhibition of a-glucosidase activity.