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

Dhanya R, Arun KB, Nisha VM, Syama HP, Nisha P, Santhosh Kumar TR, Jayamurthy P - PLoS ONE (2015)

Bottom Line: Thus, strategies to reduce oxidative stress may alleviate these pathogenic processes.Addition of Naringin (100 μM) showed approximately 40% reduction in protein glycation in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Agroprocessing and Natural Products Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Industrial Estate, Pappanamcode, Thiruvananthapuram-695019, Kerala, India.

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.

No MeSH data available.


Related in: MedlinePlus

Intracellular ROS production and Fluorescence intensity analysis in L6 myoblast.(A) Fluorescence images (20 X magnifications) of untreated cell (i); Figures (ii), (iii) & (iv) represents cells induced with TBHP at 1, 10 & 100 μM; (v) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control. (B) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 3h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control; #P≤0.05 versus TBHP.(C) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 24h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Scale bar corresponds to 87 μM. TBHP: Tertiary butyl hydrogen peroxide; Na1+TBHP, Na2+TBHP & Na3+TBHP represents relative fluorescence intensity analysis of cells pretreated with Naringin (1, 10 & 100 μM) followed by induction of TBHP; Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test.* P≤0.05 versus control; #P≤0.05 versus TBHP.
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pone.0132429.g002: Intracellular ROS production and Fluorescence intensity analysis in L6 myoblast.(A) Fluorescence images (20 X magnifications) of untreated cell (i); Figures (ii), (iii) & (iv) represents cells induced with TBHP at 1, 10 & 100 μM; (v) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control. (B) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 3h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control; #P≤0.05 versus TBHP.(C) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 24h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Scale bar corresponds to 87 μM. TBHP: Tertiary butyl hydrogen peroxide; Na1+TBHP, Na2+TBHP & Na3+TBHP represents relative fluorescence intensity analysis of cells pretreated with Naringin (1, 10 & 100 μM) followed by induction of TBHP; Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test.* P≤0.05 versus control; #P≤0.05 versus TBHP.

Mentions: To investigate the effect of Naringin on oxidative stress associated with diabetes mellitus we induced stress in L6 skeletal muscle cells by using TBHP. Induction of free radicals with TBHP at three different concentrations (1, 10 & 100 μM) for 3 h revealed that cells generated significant levels of intracellular reactive oxygen species as compared to control (Fig 2A), (P≤0.05). TBHP at 100 μM (Fig 2A (iv)) showed a significant increase in intracellular ROS and this concentration was used to induce stress condition in further studies. Pretreatment of Naringin for 3 & 24 h at different concentrations (1μM, 10μM & 100 μM), dose dependently reduced ROS concentration as shown in Fig 2B & 2C (P≤0.05). The fluorescence intensity of the images was analyzed by BD Image Data Explorer software and has been illustrated in Fig 2A(v), 2B(vi) & 2C(vi).


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

Dhanya R, Arun KB, Nisha VM, Syama HP, Nisha P, Santhosh Kumar TR, Jayamurthy P - PLoS ONE (2015)

Intracellular ROS production and Fluorescence intensity analysis in L6 myoblast.(A) Fluorescence images (20 X magnifications) of untreated cell (i); Figures (ii), (iii) & (iv) represents cells induced with TBHP at 1, 10 & 100 μM; (v) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control. (B) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 3h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control; #P≤0.05 versus TBHP.(C) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 24h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Scale bar corresponds to 87 μM. TBHP: Tertiary butyl hydrogen peroxide; Na1+TBHP, Na2+TBHP & Na3+TBHP represents relative fluorescence intensity analysis of cells pretreated with Naringin (1, 10 & 100 μM) followed by induction of TBHP; Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test.* P≤0.05 versus control; #P≤0.05 versus TBHP.
© Copyright Policy
Related In: Results  -  Collection

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pone.0132429.g002: Intracellular ROS production and Fluorescence intensity analysis in L6 myoblast.(A) Fluorescence images (20 X magnifications) of untreated cell (i); Figures (ii), (iii) & (iv) represents cells induced with TBHP at 1, 10 & 100 μM; (v) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control. (B) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 3h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. *P≤0.05 versus control; #P≤0.05 versus TBHP.(C) Figure (i), (ii), (iii), (iv) & (v) represents fluorescence images of untreated cells, cells induced with TBHP (100 μM) and cells pretreated with Naringin (1, 10 & 100 μM) for 24h respectively; (vi) Represents the relative fluorescence intensity analysis by BD Image Data Explorer software. Scale bar corresponds to 87 μM. TBHP: Tertiary butyl hydrogen peroxide; Na1+TBHP, Na2+TBHP & Na3+TBHP represents relative fluorescence intensity analysis of cells pretreated with Naringin (1, 10 & 100 μM) followed by induction of TBHP; Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test.* P≤0.05 versus control; #P≤0.05 versus TBHP.
Mentions: To investigate the effect of Naringin on oxidative stress associated with diabetes mellitus we induced stress in L6 skeletal muscle cells by using TBHP. Induction of free radicals with TBHP at three different concentrations (1, 10 & 100 μM) for 3 h revealed that cells generated significant levels of intracellular reactive oxygen species as compared to control (Fig 2A), (P≤0.05). TBHP at 100 μM (Fig 2A (iv)) showed a significant increase in intracellular ROS and this concentration was used to induce stress condition in further studies. Pretreatment of Naringin for 3 & 24 h at different concentrations (1μM, 10μM & 100 μM), dose dependently reduced ROS concentration as shown in Fig 2B & 2C (P≤0.05). The fluorescence intensity of the images was analyzed by BD Image Data Explorer software and has been illustrated in Fig 2A(v), 2B(vi) & 2C(vi).

Bottom Line: Thus, strategies to reduce oxidative stress may alleviate these pathogenic processes.Addition of Naringin (100 μM) showed approximately 40% reduction in protein glycation in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Agroprocessing and Natural Products Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Industrial Estate, Pappanamcode, Thiruvananthapuram-695019, Kerala, India.

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.

No MeSH data available.


Related in: MedlinePlus