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Protective and Antioxidant Effects of Insulin on Rat Brain Cortical Neurons in an in vitro Model of Oxygen and Glucose Deprivation

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Abstract

Insulin is one of the most promising protectors in the treatment of neurodegenerative and other diseases associated with brain injuries. In these diseases, brain insulin levels, in contrast to its blood levels, are as a rule heavily reduced, which, along with the development of insulin resistance, leads to impaired insulin signaling in neurons. The aim of this work was to study the protective and antioxidant effects of insulin on cultured rat brain cortical neurons using oxygen–glucose deprivation followed by a restoration of oxygen and glucose supply to neurons as an in vitro model of ischemia–reperfusion brain injury. OGD exposure for 1 or 3 h with subsequent incubation of cultured cortical neurons in complete (oxygen- and glucose-containing) growth medium decreased neuronal viability and increased the production of reactive oxygen species, while the preincubation of neurons with insulin at micromolar concentrations had protective and antioxidant effects. One-hour OGD followed by incubation in complete growth medium led to downregulation of protein kinase B (Akt) activity [decreased pAkt(Ser473)/Akt ratio] and upregulation of glycogen synthase kinase-3beta (GSK-3beta) activity, which is one of the main Akt targets, as manifested in a decreased pGSK-3beta(Ser9)/GSK-3beta ratio. In contrast, preincubation with insulin upregulated Akt and downregulated GSK-3beta activities. Apparently, these effects of insulin significantly contribute to its neuroprotective action, because GSK-3beta activation leads to mitochondrial dysfunction and neuronal death. Insulin was also shown to increase the neuronal activity of protein kinase regulated by extracellular signals (ERK1/2), which was diminished by OGD and subsequent exposure to complete growth medium containing glucose and oxygen.

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REFERENCES

  1. Chen Y, Guo Z, Mao YF, Zheng T, Zhang B (2018) Intranasal insulin ameliorates cerebral hypometabolism, neuronal loss, and astrogliosis in streptosotocin-induced Alzheimer’s rat model. Neurotox Res 33: 716–724. https://doi.org/10.1007/s12640-017-9809-7

    Article  CAS  PubMed  Google Scholar 

  2. Song Y, Ding W, Bei Y, Xiao Y, Tong HD, Wang LB, Ai LY (2018) Insulin is a potential antioxidant for diabetes-associated cognitive decline via regulating Nrf2 dependent antioxidant enzymes. Biomed Pharmacother 104: 474–484. https://doi.org/10.1016/j.biopha.2018.04.097

    Article  CAS  PubMed  Google Scholar 

  3. Fine JM, Stroebel BM, Faltesek KA, Terai K, Haase L, Knutzen K.E, Kosyakovsky J, Bowe TJ, Fuller AK, Frey WH, Hanson LR (2020) Intranasal delivery of low-dose insulin ameliorates motor dysfunction and dopaminergic cell death in a 6-OHDA rat model of Parkinson’s Disease. Neurosci Lett 714: 134567. https://doi.org/10.1016/j.neulet.2019.134567

    Article  CAS  PubMed  Google Scholar 

  4. Milstein JL, Ferris HA (2021) The brain as an insulin-sensitive metabolic organ. Mol Metab 52:101234. https://doi.org/10.1016/j.molmet.2021.101234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Claxton A, Baker LD, Hanson AJ, Trittschuh EH, Collerton B, Morgan A, Callaghan M, Arbuckle M, Behl C, Craft S (2015) Long-acting insulin Detemir improves cognition for adults with mild cognitive impairment or early-stage Alzheimer’s disease dementia. J Alzheimers Dis 44: 897–906. https://doi.org/10.3233/JAD-141791

    Article  CAS  PubMed  Google Scholar 

  6. Craft S, Claxton A, Baker LD, Hanson AJ, Collerton B, Trittschuh EH, Dahl D, Caulder E, Neth B, Montine TJ, Jung Y, Maldjian J, Whitlow C, Friedman S (2017) Effects of regular and long-acting insulin on cognition and Alzheimer’s disease biomarkers: A pilot clinical trial. J Alzheimers Dis 57: 1325–1334. https://doi.org/10.3233/JAD-161256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Avgerinos KI, Kalaitzidis G, Malli A, Kalaitzoglou D, Myserlis PG, Lioutas VA (2018) Intranasal insulin in Alzheimer’s dementia or mild cognitive impairment. A systematic review. J Neurol 265: 1497–1510. https://doi.org/10.1007/s00415-018-8768-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Novak P, Maldonado DAP, Novak V (2019) Safety and preliminary efficacy of intranasal insulin for cognitive impairment in Parkinson disease and multiple system atrophy: A double-blinded placebo-controlled pilot study. PLoS One 14: e0214364. https://doi.org/10.1371/journal.pone.0214364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lochhead JJ, Kellohen KL, Ronaldson PT, Davis TP (2019) Distribution of insulin in trigeminal nerve and brain after intranasal administration. Sci Rep 9: 2621. https://doi.org/10.1038/s41598-019-39191-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fan LW, Carter K, Beatt A, Pang Y (2019) Rapid transport of insulin to the brain following intranasal administration in rats. Neural Regen Res 14: 1046–1051. https://doi.org/10.4103/1673-5374.250624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hallschmid M (2021) Intranasal insulin. J Neuroendocrinol 33: e12934. https://doi.org/10.1111/jne.12934

    Article  CAS  PubMed  Google Scholar 

  12. Shpakov AO, Derkach KV, Berstein LM (2015) Brain signaling systems in the Type 2 diabetes and metabolic syndrome: promising target to treat and prevent these diseases. Future Sci OA 1: FSO25. https://doi.org/10.4155/fso.15.23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferré P, Birnbaum MJ (2004) AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428: 569–574. https://doi.org/10.1038/nature02440

    Article  CAS  PubMed  Google Scholar 

  14. Rizk NN, Myatt-Jones J, Rafols J, Dunbar JC (2007) Insulin-like growth factor-1 (IGF-1) decreases ischemia-reperfusion induced apoptosis and necrosis in diabetic rats. Endocrine 31: 66–71. https://doi.org/10.1007/s12020-007-0012-0

    Article  CAS  PubMed  Google Scholar 

  15. Jiang LH, Yuan XL, Yang NY, Ren L, Zhao FM, Luo BX, Bian YY, Xu JY, Lu DX, Zheng YY, Zhang CJ, Diao YM, Xia BM, Chen GJ (2015) Daucosterol protects neurons against oxygen-glucose deprivation/reperfusion-mediated injury by activating IGF1 signaling pathway. Steroid Biochem Mol Biol 152: 45–52. https://doi.org/10.1016/j.jsbmb.2015.04.007

    Article  CAS  Google Scholar 

  16. Gong P, Zou Y, Zhang W, Tian Q, Han S, Xu Z, Chen Q, Wang X, Li M (2021) The neuroprotective effects of Insulin-like growth factor-1 via the Hippo/YAP signaling pathway are mediated by the PI3K/AKT cascade following cerebral ischemia/reperfusion injury. Brain Res Bull 177: 373–387. https://doi.org/10.1016/j.brainresbull.2021.10.017

    Article  CAS  PubMed  Google Scholar 

  17. Lioutas VA, Alfaro-Martinez F, Bedoya F, Chung CC, Pimentel DA, Novak V (2015) Intranasal insulin and insulin-like growth factor-1 as neuroprotectants in acute ischemic stroke. Transl Stroke Res 6: 264–275. https://doi.org/10.1007/s12975-015-0409-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zorina II, Galkina OV, Bayunova LV, Zakharova IO (2019) Effect of insulin on lipid peroxidation and glutathione levels in a two-vessel occlusion model of rat forebrain ischemia followed by reperfusion. J Evol Biochem Physiol 35: 333–335. https://doi.org/10.1134/S0022093019040094

    Article  Google Scholar 

  19. Zorina II, Fokina EA, Zakharova IO, Bayunova LV, Shpakov AO (2020) Characteristics of changes in lipid peroxidation and Na+/K+-ATPase activity in the cortex of old rats in conditions of two-vessel cerebral ischemia/reperfusion. Adv Geront 10: 156–161. https://doi.org/10.1134/s2079057020020162

    Article  Google Scholar 

  20. Mielke JG, Wang YT (2005) Insulin exerts neuroprotection by counteracting the decrease in cell-surface GABA receptors following oxygen-glucose deprivation in cultured cortical neurons. J Neurochem 92: 103–113. https://doi.org/10.1111/j.1471-4159.2004.02841.x

    Article  CAS  PubMed  Google Scholar 

  21. Sun X, Yao H, Douglas RM, Gu XQ, Wang J, Haddad GG (2010) Insulin/PI3K signaling protects dentate neurons from oxygen-glucose deprivation in organotypic slice cultures. J Neurochem 112: 377–388. https://doi.org/10.1111/j.1471-4159.2009.06450.x

    Article  CAS  PubMed  Google Scholar 

  22. Dichter MA (1978) Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology, and synapse formation. Brain research 149: 279–293. https://doi.org/10.1016/0006-8993(78)90476-6

    Article  CAS  PubMed  Google Scholar 

  23. Mironova EV, Evstratova AA, Antonov SM (2007) A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture. J Neurosci Methods 163: 1–8. https://doi.org/10.1016/j.jneumeth.2007.02.010

    Article  PubMed  Google Scholar 

  24. Hansen MB, Nielsen SE, Berg K (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 119: 203–210. https://doi.org/10.1016/0022-1759(89)90397-9

    Article  CAS  PubMed  Google Scholar 

  25. Zorina II, Bayunova LV, Zakharova IO, Avrova NF (2018) The dependence of the protective effect of insulin on its concentration and modulation of ERK1/2 activity under the conditions of oxidative stress in cortical neurons. Neurochem J 10: 111–116. https://doi.org/10.1134/S1819712417040110

    Article  Google Scholar 

  26. Zakharova I, Sokolova T, Vlasova Y, Bayunova L, Rychkova M, Avrova N (2017) α-Tocopherol at nanomolar concentration protects cortical neurons against oxidative stress. Int J Mol Sci 18: 216. https://doi.org/10.3390/ijms18010216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bonde C, Noraberg J, Noer H, Zimmer J (2005) Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures. Neuroscience 136: 779–794. https://doi.org/10.1016/j.neuroscience.2005.07.020

    Article  CAS  PubMed  Google Scholar 

  28. Zakharova IO, Sokolova TV, Zorina I, Bayunova LV, Rychkova MP, Avrova NF (2018) Protective effect of insulin on rat cortical neurons in oxidative stress and its dependence on modulation of protein kinase B (Akt) activity. J Evol Biochem Physiol 54: 192–204. https://doi.org/10.1134/S0022093018030043

    Article  CAS  Google Scholar 

  29. Bayunova LV, Zorina II, Zakharova IO, Avrova NF (2018) Insulin increases viability of neurons in rat cerebral cortex and normalizes Bax/Bcl-2 atio under conditions of oxidative stress. Bull Exp Bio Med 165: 14–17. https://doi.org/10.1007/s10517-018-4088-8

    Article  CAS  Google Scholar 

  30. Zakharova IO, Sokolova TV, Bayunova LV, Zorina II, Rychkova MP, Shpakov AO, Avrova NF (2019) The protective effect of insulin on rat cortical neurons in oxidative stress and its dependence on the modulation of Akt, GSK-3beta, ERK1/2, and AMPK activities. Int J Mol Sci 20(15): E3702. https://doi.org/10.3390/ijms20153702

    Article  CAS  Google Scholar 

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Funding

This study was implemented within the State assignment to Sechenov Institute of Evolutionary Physiology and Biochemistry no. 075-0152-22-00.

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Authors and Affiliations

  1. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia

    I. O. Zakharova, I. I. Zorina, L. V. Bayunova, A. O. Shpakov & N. F. Avrova

Authors
  1. I. O. Zakharova
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  2. I. I. Zorina
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  3. L. V. Bayunova
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  4. A. O. Shpakov
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  5. N. F. Avrova
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Contributions

Conceptualization (N.F.A., I.O.Z., I.I.Z.); methodology (I.I.Z., I.O.Z.); validation (L.V.B.); data collection (I.O.Z., I.I.Z., L.V.B.); visualization (L.V.B., I.I.Z.); writing the manuscript (N.F.A., A.O.Sh.).

Corresponding author

Correspondence to N. F. Avrova.

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CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

Additional information

Translated by A. Polyanovsky

Russian Text © The Author(s), 2023, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2023, Vol. 59, No. 1, pp. 20–32https://doi.org/10.31857/S0044452923010096.

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Zakharova, I.O., Zorina, I.I., Bayunova, L.V. et al. Protective and Antioxidant Effects of Insulin on Rat Brain Cortical Neurons in an in vitro Model of Oxygen and Glucose Deprivation. J Evol Biochem Phys 59, 20–32 (2023). https://doi.org/10.1134/S0022093023010027

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  • DOI: https://doi.org/10.1134/S0022093023010027

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