Molecular Mechanism Of Pomalidomide And Afatinib Regulating GLUT4 Transport

Type 2 diabetes mellitus（T2DM）is a multi-gene inherited complex disease,usually manifested by insulin resistance and insufficient insulin secretion.GLUT4 protein,as the main protein of skeletal muscle cells and adipose cells,can rapidly regulate blood glucose level under the action of insulin to maintain the relative stability of blood glucose in the body,and is an important target for the treatment of type 2 diabetes.In order to further explore the molecular mechanisms of GLUT4 expression and transport in L6 cells,based on the existing drug screening system,we screened Promalidomide（POM）and Afatinib that could promote GLUT4 expression.Then,we explored the molecular mechanisms of GLUT4 expression and transport induced by POM and Afatinib.First,we studied the molecular mechanisms of POM on glucose uptake and GLUT4expression and transport in L6 cells in vitro,and examined the role of POM on glucose and lipid metabolism and GLUT4 expression in vivo.The results showed that POM promoted GLUT4 expression and plasma membrane fusion in L6 cells.POM promoted glucose uptake in L6 cells,which was inhibited in GLUT4-ko-L6 cells,suggesting that POM and Afatinib regulated glucose uptake in L6 cells mainly through GLUT4.Western blotting showed that POM up-regulated the phosphorylation of PKC and AMPK in L6cells,but not Akt.In addition,POM promoted glucose uptake in a Ca²⁺dependent manner.In vivo experiment,through the establishment of KK-Ay type 2 diabetic model mice,via intragastric administration for four weeks,POM reduced KK-Ay type 2 diabetes mice in blood sugar,and improved KK-Ay mice in sugar metabolism.POM also reduced the degree of insulin resistance and serum lipid level in mice.Although POM had no significant effect on the body weight of in adipose tissue in KK-Ay mice,RT-PCR technology was used to detect cellular inflammatory factors in mouse adipose tissue,and the results showed that POM could downregulate the expression of IL-1β,IL-6 and TNF-αin adipose tissue.HE staining and immunofluorescence staining showed that POM could improve hepatic steatosis in mice,such as hepatocyte enlargement,fat vacuole,fat cell hypertrophy and compensatory hypertrophy of islet cells.Western blotting showed that POM up-regulated GLUT4 expression and phosphorylation of PKC and AMPK in skeletal muscle and adipose tissue.Secondly,the glucose uptake kit detected that Afatinib could increase glucose uptake in L6 cells,and real-time imaging and Western blot showed that Afatinib could promote GLUT4 expression and fusion with plasma membrane.In GLUT4-ko-L6 cells,glucose uptake induced by Afatinib was significantly inhibited,suggesting that Afatinib regulates glucose uptake by L6 cells mainly through GLUT4.At the protein level,Afatinib up-regulated the phosphorylation of PKC and AMPK in L6 cells,but not Akt.At the same time,after exposed to AMPK inhibitor Compound C and PKC inhibitors G（?）6983respectively,the role of Afatinib raised GLUT4 was significantly suppressed,which showed that Afatinib regulated the expression and transport of GLUT4 mainly through PKC and AMPK signaling pathways.In addition,intracellular Ca²⁺was stained with Fluo-4 AM fluorescent dye,and the intracellular Ca²⁺level of myc GLUT4-m Orange-L6cells stimulated by Afatinib was detected by confocal laser microscopy.Glucose uptake of L6 cells treated with Afatinib under different external calcium conditions was detected by glucose uptake kit.The results showed that Afatinib promoted glucose uptake in Ca²⁺dependent manner.In conclusion,POM and Afatinib up-regulate GLUT4 expression,translocation and glucose uptake in L6 cells through PKC and AMPK.Meanwhile,POM and Afatinib regulate glucose uptake in L6 cells and are affected by calcium.In KK-Ay mice,POM can up-regulate GLUT4 expression and phosphorylation of PKC and AMPK in skeletal muscle and adipose tissue,and improve glucose and lipid metabolism in KK-Ay mice.Therefore,POM and Afatinib are promising multitarget drugs for the treatment of type 2 diabetes.