Studies On The Antidiabetic Activities Of Orientin And A Flavonoid-rich Extract From Flowers Of Wisteria Sinensis

Type 2 diabetes(T2DM)is a metabolic disorder syndrome influenced by interactions between genetic and environmental factors and is expected to affect more than 400 million adults worldwide by 2030,which is characterized by impaired insulin regulation and function.Insulin plays a crucial role in maintaining glucose and lipid homeostasis in tissues such as skeletal muscle,adipose tissue,and liver.In the setting of insulin resistance(IR),functional deficits in glucose transport,glycogen synthesis,gluconeogenesis,and lipolysis in insulin target tissues lead to hyperglycemia,a typical indicator of T2 DM.At present,there are many types of medicines for the treatment of diabetes,but most of them have drug resistance and side effects.Therefore,the search for anti-diabetic drugs with lower toxicity and high efficiency is in line with the growing demand of today’s society.In the early stage of the experiment,insulin-resistant 3T3-L1 adipocytes were established and used to evaluate the effect of drugs on glucose metabolism at the cellular level in the state of IR.In vitro experiments showed that orientin significantly enhanced the glucose uptake capacity of cells,increased the phosphorylation level of AMPK and the protein level of IRS-1,and prevented the serine phosphorylation of IRS-1 induced by insulin resistance.Furthermore,Compound C(an AMPK inhibitor)prevented the effect of orientin on insulin signaling molecules including p-IRS-1,IRS-1 and GLUT-4.This result indicated that orientin-induced activation of AMPK can regulate the expression of target proteins of the insulin pathway.KK-Ay mice are genetically deficient mice which were used to study the effects of orientin in vivo.Oral administration of orientin could effectively relieve symptoms of IR including abnormally elevated glucose levels,impaired oral glucose tolerance,hyperinsulinemia,and defective function of pancreatic islets β cell.Western blotting was used to detect the expression of target proteins in insulin-sensitive organs(liver and white adipose tissue),and the results were similar with in vitro experiments that orientin activated AMPK pathway and insulin signaling pathway.In addition,orientin slowed down the progression of obesity and improve dyslipidemia,hepatic steatosis and adipocyte hypertrophy.In order to elucidate the anti-obesity effect of orientin,we demonstrated that orientin inhibited lipid droplet accumulation in 3T3-L1 cells in a dose-dependent manner(2.5-10 μM)by Oil Red O staining experiments.The results of q-PCR combined with Western blotting showed that orientin caused the down-regulation of adipogenesis transcription factors and adipocyte-specific genes,and activated the AMPK/ACC signaling pathway.Similarly,the treatment of orientin promoted the phosphorylation of AMPK and ACC and reduced the expression of SREBP1-c in the adipose and liver of mice.In conclusion,orientin-induced activation of AMPK pathway can maintain the homeostasis of glucose and lipid metabolism.The antidiabetic effect of orientin mainly comes from the inhibition of obesity and insulin resistance.GLUT4 is one of 13 sugar transporter proteins(GLUT1-GLUT12,and HMIT)encoded in the human genome,which plays a key role in regulating whole body glucose homeostasis.GLUT4 expression is exquisitely regulated in muscle and this regulation is critical for insulin-stimulated glucose uptake.The research group established a rat skeletal muscle L6 cell line stably expressing IRAP-m Orange in the early stage.IRAP is a reporter molecule that indirectly reflects the translocation of GLUT4.The fluorescence intensity changes on the surface of the cell membrane were observed by laser confocal microscopy,which can efficiently screen active drugs that can stimulate GLUT4 translocation.In vitro,a Flavonoid-Rich extract from flowers of Wisteria Sinensis(WS-FRE)remarkably stimulated the translocation of GLUT4(Glucose transporter 4)to the plasma membrane.Mechanism analysis revealed that WS-FRE enhanced glucose uptake via activation of Akt and GLUT4.In vivo,4-week treatment of WS-FRE alleviated the typical symptoms including hyperglycemia,glucose intolerance,hyperinsulinemia and dyslipidemia.Histopathological examinations revealed that WS-FRE relieved hepatic steatosis,adipocyte hypertrophy and pancreatic islet lesion to a certain extent in diabetic mice.Western blot analysis indicated that WS-FRE resulted in the suppression of IRS-1 serine phosphorylation and an elevation of PI3 K p85,Akt phosphorylation and GLUT4 expression in insulin target tissues,which contributed to improve insulin resistance in peripheral tissues,thereby stabilizing glucose metabolism in diabetic mice.Diverse anti-diabetic effects of WS-FRE may be attributed to regulation of GLUT4,IRS-1 serine phosphorylation,PI3 K phosphorylation and Akt phosphorylation.