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Insulin signaling pathway assessment by enhancing antioxidant activity due to morin using in vitro rat skeletal muscle L6 myotubes cells

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Abstract

Background

Plant-derived phytochemicals such as flavonoids have been explored to be powerful antioxidants that protect against oxidative stress-related diseases. In the present study, Morin, a flavonoid compound was studied for its antioxidant and antidiabetic properties in relation to oxidative stress in insulin resistant models conducted in rat skeletal muscle L6 cell line model.

Methods

Evaluation of antioxidant property of morin was assayed using in vitro methods such as cell viability by MTT assay, estimation of SOD and CAT activity and NO scavenging activity. The anti-oxidative nature of morin on L6 cell line was conducted by the DCF-DA fluorescent activity. Glucose uptake in morin treated L6 myotubes are accessed by 2-NBDG assay in the presence or absence of IRTK and PI3K inhibitors. Further glycogen content estimation due to the morin treatment in L6 myotubes was performed. Antioxidant and insulin signaling pathway gene expression was examined over RT-PCR analysis.

Results

Morin has a negligible cytotoxic effect at doses of 20, 40, 60, 80, and 100 µM concentration according to cell viability assay. Morin revealed that the levels of the antioxidant enzymes SOD and CAT in L6 myotubes had increased. When the cells were subjected to the nitro blue tetrazolium assay, morin lowered reactive oxygen species (ROS) formation at 60 µM concentration displaying 39% ROS generation in oxidative stress condition. Lesser NO activity and a drop in green fluorescence emission in the DCFDA assay, demonstrating its anti-oxidative nature by reducing ROS formation in vitro. Glucose uptake by the L6 myotube cells using 2-NBDG, and with IRTK and PI3K inhibitors (genistein and wortmannin) showed a significant increase in glucose uptake by the cells which shows the up regulated GLUT-4 movement from intracellular pool to the plasma membrane. Morin (60 µM) significantly enhanced the expression of antioxidant genes GPx, GST and GCS as well as insulin signalling genes IRTK, IRS-1, PI3K, GLUT-4, GSK-3β and GS in L6 myotubes treated cells.

Conclusion

Morin has the ability to act as an anti-oxidant by lowering ROS levels and demonstrating insulin mimetic activity by reversing insulin resistance associated with oxidative stress.

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Abbreviations

2-NBDG:

2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose

CAT:

Catalase

DCF-DA:

Dichlorofluorescein diacetate

DM:

Diabetes mellitus

DMEM:

Dulbecco’s modified eagle’s medium

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

GCS:

Glutamyl cysteine synthetase

GLUT-4:

Glucose transporter type 4

GPx:

Glutathione peroxidase

GS:

Glycogen synthase

GSK-3β:

Glycogen synthase kinase 3β

GST:

Glutathione S-transferase

H2O2 :

Hydrogen peroxide

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

IRS:

Insulin receptor substrate

IRTK:

Insulin receptor tyrosine kinase

IRβ:

Insulin receptor β

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

NBT:

Nitro blue tetrazolium

NCCS:

National center for cell science

PBS:

Phosphate buffer saline

PI3K:

Phosphoinositide 3-kinases

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

T2DM:

Type 2 diabetic mellitus

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Acknowledgements

The authors extend their appreciation to the Researchers Supporting Project (RSP-2021/20), King Saud University, Riyadh, Saudi Arabia.

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Authors and Affiliations

  1. SRM Research Institute, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India

    Praveen Kumar Issac, Ajay Guru & Jesu Arockia Raj

  2. Department of Biotechnology, College of Science and Humanities, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India

    Rupmanjari Karan, Ajay Guru & Jesu Arockia Raj

  3. Department of Biotechnology, School of Bioengineering, Faculty of Engineering and Technology, SRM Institute of Science and Technology, Kattankulathur, 603203, Chennai, Tamil Nadu, India

    R. Pachaiappan

  4. Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia

    Mariadhas Valan Arasu & Naif Abdullah Al-Dhabi

  5. Grassland and Forage Division, National Institute of Animal Science, RDA, Seonghwan-Eup, Cheonan-Si, Chungnam, 330801, Republic of Korea

    Ki Choon Choi

  6. Department of Zoology, Pachaiyappa’s College for Men, Kanchipuram, Tamil Nadu, 631501, India

    Ramasamy Harikrishnan

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  1. Praveen Kumar Issac
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Contributions

PKI, RK, AG, PR, JA conceived the study. PR provided the Morin. PKI, RK, AG performed the experiments. PR, MVA, NAA, KCC, RH, JA provided the reagents for the study. All authors designed the experiments, analysed the data, wrote the manuscript, read and approved the final version of the manuscript.

Corresponding author

Correspondence to Jesu Arockia Raj.

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All authors declare that they have no conflict of interest.

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Issac, P.K., Karan, R., Guru, A. et al. Insulin signaling pathway assessment by enhancing antioxidant activity due to morin using in vitro rat skeletal muscle L6 myotubes cells. Mol Biol Rep 48, 5857–5872 (2021). https://doi.org/10.1007/s11033-021-06580-x

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