Evaluation of therapeutic effect of Premna herbacea in diabetic rat and isoverbascoside against insulin resistance in L6 muscle cells through bioenergetics and stimulation of JNK and AKT/mTOR signaling cascade

Background: Premna herbacea Roxb., a perennial herb is well documented for its therapeutic uses among the traditional health care-givers of Assam, India. Scientific validation on the traditional use of the medicinal plant using modern technology may promote further research in health care. Purpose: This study evaluates the therapeutic potential of methanolic extract of P. herbacea (MEPH) against type 2 diabetes mellitus (T2DM) and its phytochemical(s) in ameliorating insulin resistance (IR), thereby endorsing the plant bioactives as effective anti-hyperglycemic agents. Methods: The anti-diabetic potential of the plant extract was explored both in L6 muscle cells and high fructose high fat diet (HF-HFD) fed male Sprague Dawley (SD) rats. Bioactivity guided fractionation and isolation procedure yielded Verbascoside and Isoverbascoside (ISOVER) as bioactive and major phytochemicals in P. herbacea. The bioenergetics profile of bioactive ISOVER and its anti-hyperglycemic potential was validated in vitro by XFe24 analyzer, glucose uptake assay and intracellular ROS generation by flourometer, FACS and confocal microscopy. The potential of ISOVER was also checked by screening various protein markers via immunoblotting. Results: MEPH enhanced glucose uptake in FFA-induced insulin resistant (IR) L6 muscle cells and decreased elevated blood glucose levels in HF-HFD fed rats. Isoverbascoside (ISOVER) was identified as most bioactive phytochemical for the first time from the plant in the Premna genus. ISOVER activated the protein kinase B/AMPactivated protein kinase signaling cascades and enhanced glucose uptake in IR-L6 muscle cells. ISOVER decreased the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) and increased that of mammalian target of rapamycin (mTOR), thereby attenuating IR. However, molecular docking revealed that ISOVER increases insulin sensitivity by targeting the JNK1 kinase as a competitive inhibitor rather than mTOR. These findings were further supported by the bioenergetics profile of ISOVER. Conclusion: This study for the first time depicts the functional properties of ISOVER, derived from Premna herbacea, in ameliorating IR. The phytochemical significantly altered IR with enhanced glucose uptake and inhibition of ROS through JNK-AKT/mTOR signaling which may pave the way for further research in T2DM therapeutics.