Flavonoids extracted from mulberry (Mores alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes

Ethnopharmacological relevance: Mulberry (Mortis alba L.) leaves have been widely applied to controlling blood glucose as a efficacious traditional Chinese medicine or salutary medical supplement. The extracts of mulberry leaf suppress inflammatory mediators and oxidative stress, protect the pancreatic beta-cells and modulate glucose metabolism in diabetic rats. Our previous studies and others have shown that mulberry leaf extract has excellent therapeutic effects on type 2 diabetes mellitus (T2DM), however, the underlying mechanism remains to be studied. Aim of the study: Skeletal muscle insulin resistance (IR) plays an important role in the pathogenesis of T2DM. The aim of this study was to investigate the effects and mechanisms of Mulberry leaf flavonoids (MLF) in L6 skeletal muscle cells and db/db mice. Materials and methods: L6 skeletal muscle cells were cultured and treated with/without MLF for in vitro studies. For in vivo studies, the db/db mice with/without MLF therapy were used. Coomassie brilliant blue staining and alpha-SMA immunofluorescence staining were used to identify the differentiated L6 cells. Glucose level and ATP level of L6 myotubes were performed by optical density detection and cell viability was performed by MTT method. Mitochondrial membrane potential of L6 myotubes was detected by JC-1 fluorescent staining. ROS level of L6 myotubes was detected by DCFH-DA fluorescent staining. The body weight, food intake, and blood glucose of the mice were measured in different treatment days. Oral glucose tolerance test (OGTT), starch glucose tolerance test (SIT) and insulin tolerance test (ITT) were performed in mice. Glycated hemoglobin, glycated serum protein, insulin, liver and muscle glycogen, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) of the mice were detected by corresponding kit. The pathologic change of pancreas and skeletal muscle of mice were performed by H & E staining. Immunohistochemistry staining was used to detect the GLUT4 and p-AMPK expressions in skeletal muscle in mice. GLUT4, CPT-1, NRF1, COXIV, PGC-1 alpha, and p-AMPK expression levels in L6 cells and mice were detected by western bolt assay. Results: MLF and metformin significantly ameliorated muscle glucose uptake and mitochondrial function in L6 muscle cells. MLF also increased the phosphorylation of AMPK and the expression of PGC-1 alpha, and up-regulated the protein levels of m-GLUT4 and T-GLUT4. These effects were reversed by the AMPK inhibitor compound C. In db/db mice, MLF improve diabetes symptoms and insulin resistance. Moreover, MLF elevated the levels of p-AMPK and PGC-1 alpha, raised m-GLUT4 and T-GLUT4 protein expression, and ameliorated mitochondrial function in skeletal muscle of db/db mice. Conclusions: MLF significantly improved skeletal muscle insulin resistance and mitochondria! function in db/db mice and L6 myocytes through AMPK-PGC-1 alpha signaling pathway, and our findings support the therapeutic effects of MLF on type 2 diabetes.