Spatholobus suberectus Exhibits Antidiabetic Activity In Vitro and In Vivo through Activation of AKT-AMPK Pathway

Abstract

Glucose deposition in peripheral tissue is an important parameter for the treatment of type 2 diabetes mellitus. The aim of this study was to investigate the effects of Spatholobus suberectus (Ss) on glucose disposal in skeletal muscle cells and additionally explore its in vivo antidiabetic potential. Treatment of ethanolic extract of S. suberectus (EeSs) significantly enhanced the glucose uptake, mediated through the enhanced expression of GLUT4 in skeletal muscle via the stimulation of AKT and AMPK pathways in C2C12 cells. Moreover, EeSs have potential inhibitory action on α-glucosidase activity and significantly lowered the postprandial blood glucose levels in STZ-induced diabetic mice, associated with increased expression of GLUT4 and AKT and/or AMPK-mediated signaling cascade in skeletal muscle. Furthermore, administration of EeSs significantly boosted up the antioxidant enzyme expression and also mitigated the gluconeogenesis enzyme such as PEPCK and G-6-Pase enzyme expression in liver tissue of STZ-induced diabetic mice model. Collectively, these findings suggest that EeSs have a high potentiality to mitigate diabetic symptoms through stimulating glucose uptake in peripheral tissue via the activation of AKT and AMPK signaling cascade and augmenting antioxidant potentiality as well as blocking the gluconeogenesis process in diabetic mice.

Figure 1

A classical feature of Spatholobus suberectus and HPLC chromatogram of the extracts monitored at 280 nm. A classical feature of Spatholobus suberectus bark (a) is shown. The total polyphenolic and flavonoid content (b) and the HPLC chromatogram of the major compounds (c) were analyzed as described in detail in Materials and Methods. (1) Catechin and (2) epicatechin. TPC: total polyphenolic contents; TFC: total flavonoid contents; GAE: gallic acid equivalent; AeSs: aqueous extract of S. suberectus; EeSs: ethanolic extract of S. suberectus.

Figure 2

α-Glucosidase inhibitory activity of Spatholobus suberectus. Results are mean ± standard deviation (SD) of triplicate experiments. ^∗∗p < 0.01 versus control using Student's t-test. AeSs: aqueous extract of S. suberectus; EeSs: ethanolic extract of S. suberectus.

Figure 3

Cell viability and glucose uptake associated with signaling activity of Spatholobus suberectus in C2C12 cells. C2C12 cells were seeded at a density of 2 × 10⁵ cells per well (96-well plate), and then the MTT assay (a) was performed. Differentiated C2C12 cells were incubated with AeSs and EeSs with or without insulin at indicated concentration for 24 hr and the glucose uptake (b) was measured as described in Materials and Methods. (c) Differentiated C2C12 cells were collected and mRNA levels of genes were determined by RT-PCR; (d and e) protein extracts were prepared and subjected to western blot assay using indicated primary antibody; beta-actin levels were used as a control for equal loading (d and e). ^∗∗p < 0.01 versus control; ^∗p < 0.05 versus control using Student's t-test. NT: not treated; Ros: rosiglitazone-treated; AeSs: aqueous extract of S. suberectus; EeSs: ethanolic extract of S. suberectus.

Figure 4

In vivo antidiabetic activity of Spatholobus suberectus. STZ-induced diabetic mice were treated with EeSs or acarbose at 200 mg/kg dose and oral glucose tolerance test was monitored at various time intervals (a) after an oral load of glucose (2 gm/kg). After treatment, the skeletal muscle tissues were excised and RT-PCR analysis (b) was performed, followed by western blot analysis (c and d). NT: not treated; SDC: STZ-induced diabetic control mice; SDAC: STZ-induced diabetic acarbose-treated mice; SDEeSs: STZ-induced diabetic EeSs-treated mice. ^#p < 0.05 versus NT; ^∗p < 0.05 versus SDC group using ANOVA followed by LSD test.

Figure 5

Effects of Spatholobus suberectus on antioxidant and gluconeogenesis-related enzymes. STZ-induced diabetic mice were treated with EeSs or acarbose at 200 mg/kg; after treatment, the liver tissue was excised. This was followed by RT-PCR analysis of antioxidant (a) and gluconeogenesis-related enzymes (b). NT: not treated; SDC: STZ-induced diabetic control mice; SDAC: STZ-induced diabetic acarbose-treated mice; SDEeSs: STZ-induced diabetic EeSs-treated mice.