Preconditioning L6 Muscle Cells with Naringin Ameliorates Oxidative Stress and Increases Glucose Uptake.

R Dhanya,K B Arun,P Jayamurthy,P Nisha,V M Nisha,H P Syama,T R Santhosh Kumar,M.Faadiel Essop

doi:10.1371/journal.pone.0132429

Abstract

Enhanced oxidative stress contributes to pathological changes in diabetes and its complications. Thus, strategies to reduce oxidative stress may alleviate these pathogenic processes. Herein, we have investigated Naringin mediated regulation of glutathione (GSH) & intracellular free radical levels and modulation of glucose uptake under oxidative stress in L6 cell lines. The results from the study demonstrated a marked decrease in glutathione with a subsequent increase in free radical levels, which was reversed by the pretreatment of Naringin. We also observed that the increased malondialdehyde level, the marker of lipid peroxidation on induction of oxidative stress was retrieved on Naringin pretreatment. Addition of Naringin (100 μM) showed approximately 40% reduction in protein glycation in vitro. Furthermore, we observed a twofold increase in uptake of fluorescent labeled glucose namely 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2 - NBDG) on Naringin treatment in differentiated L6 myoblast. The increased uptake of 2-NBDG by L6 myotubes may be attributed due to the enhanced translocation of GLUT4. Our results demonstrate that Naringin activate GSH synthesis through a novel antioxidant defense mechanism against excessive Reactive Oxygen Species (ROS) production, contributing to the prevention of oxidative damage in addition to its effect on glycemic control.

Highlights

Diabetes mellitus is often defined as a hyperglycemic condition arising due to insulin resistance or impaired insulin secretion
Dulbecco’s modified Eagle’s media (DMEM), antibiotic-antimycotic mix, insulin, rosiglitazone, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Naringin and Tertiary butyl hydrogen peroxide (TBHP) were purchased from Sigma—Aldrich Chemicals (St Louis, MO, USA); Monoclonal anti-GLUT4 antibody, secondary anti-mouse immunoglobulin (IgG; Fab specific)–fluorescein isothiocyanate (FITC) antibody produced in goat were purchased from Santa Cruz Biotechnology, USA; Foetal bovine serum (FBS) was purchased from Gibco-BRL (Auckland, NZ); Horse serum was purchased from PAN Biotech (Aidenbach, Germany); 2-(7-Nitrobenz-2-oxa1,3-diazol-4-yl) amino-2-deoxy-D-glucose (2-NBDG) was purchased from Molecular Probe (Invitrogen Life Technologies, Carlsbad, CA, USA); BCA protein assay kit was procured from Pierce Biotechnology, Rockford, USA; All other chemicals used were of standard analytical grade
To investigate the effect of Naringin on oxidative stress associated with diabetes mellitus we induced stress in L6 skeletal muscle cells by using TBHP

Summary

Introduction

Diabetes mellitus is often defined as a hyperglycemic condition arising due to insulin resistance or impaired insulin secretion. The oxidative stress induced pathways is known to be associated with the onset of diabetes and its complications, which is the real cause of the morbidity and mortality associated with diabetes [1]. Oxidative stress is reported to be responsible for the impaired secretion of insulin and glucose utilization in peripheral tissues in diabetic conditions [2]. The inhibition of ROS generation or its neutralization is reported to prevent the development of diabetic complications [3,4,5,6,7]. Evans et al. PLOS ONE | DOI:10.1371/journal.pone.0132429. PLOS ONE | DOI:10.1371/journal.pone.0132429 July 6, 2015

Objectives

Methods

Results