Intranasal administration of insulin to rats with forebrain ischemia and reperfusion decreases the intensity of autophagy and apoptosis in hippocampus and frontal brain cortex, possible mechanism of unsulin action

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Abstract

Rat forebrain ischemia and subsequent three-day reperfusion were found to result in an increase in the levels of autophagy marker LC3B-II and glial fibrillary acidic protein (GFAP) and activation of caspase-3 in the hippocampus and frontal cortex. At the same time, intranasal administration of 0.5 IU insulin to rats with forebrain ischemia and reperfusion (before ischemia and daily during reperfusion) markedly and significantly diminished the level of LC3B-II and caspase-3 activity in the hippocampus and frontal cortex. It demonstrates the ability of insulin to inhibit the activation of autophagy and apoptosis in forebrain structures during ischemia and reperfusion. It was not possible to find out a significant decrease in the level of GFAP in these brain structures under the influence of insulin administration to animals. Intranasal administration of insulin has been found to activate the protein kinase Akt (which activates the mTORC1 complex, known to inhibit autophagy processes) and to inhibit the protein kinase AMPK (initiating autophagy processes) in the hippocampus and cerebral cortex of rats with forebrain ischemia and reperfusion. These effects of insulin apparently underly its ability to diminish the autophagic and apoptotic neuronal death. The data on the modulation by insulin, administered intranasally to rats with forebrain ischemia and reperfusion, of Akt and AMPK activities are in agreement with more detailed studies of the possible mechanism of the neuroprotective action of insulin, which we previously made in vitro on cortical neurons under oxidative stress conditions.

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I. O. Zakharova

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS

Email: avrova@iephb.ru
Russian Federation, St. Petersburg

L. V. Bayunova

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS

Email: avrova@iephb.ru
Russian Federation, St. Petersburg

D. K. Avrova

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS

Email: avrova@iephb.ru
Russian Federation, St. Petersburg

N. F. Avrova

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS

Author for correspondence.
Email: avrova@iephb.ru
Russian Federation, St. Petersburg

References

  1. GBD2019 Stroke Collaborator (2021). Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Neurol 20 (10):795–820. https://doi.org/10.1016/S1474-4422(21)00252-0
  2. Kuriakose D, Xiao Z (2020) Pathophysiology and treatment of stroke: Present status and future perspectives. Int J Mol Sci 21 (20): 7609. https://doi.org/10.3390/ijms21207609
  3. Barthels D, Das H (2020) Current advances in ischemic stroke research and therapies. Biochim Biophys Acta. Mol Basis Dis 1866 (4): 165260. http://doi.org/10.1016/j.bbadis.2018.09.012
  4. Cannistraro RJ, Badi M, Eidelman BH, Dickson DW, Middlebrooks EH, Meschia JF (2019) CNS small vessel disease: A clinical review. Neurology: 92 (24) 1146–1156. http://doi.org/10.1212/WNL.0000000000007654
  5. Han F, Zhang DD, Zhai FF, Xue J, Zhang JT, Yan S, Zhou LX, Ni J, Yao M, Yang M, Li ML, Jin ZY, Dai Q, Zhang SY, Cui LY, Zhu YC (2021) Association between large artery stenosis, cerebral small vessel disease and risk of ischemic stroke. .Sci China Life Sci: 64 (9) 1473–1480. http://doi.org/10.1007/s11427-020-849-x
  6. Markus HS, de Leeuw FE (2023) Cerebral small vessel disease: Recent advances and future directions. Int J Stroke: 18 (1) 4–14. http://doi.org/10.1177/17474930221144911
  7. 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
  8. Tashima T (2020) Shortcut approaches to substance delivery into the brain based on intranasal administration using nanodelivery strategies for insulin. Molecules 25 (21): 5188. https://doi.org/10.3390/molecules25215188
  9. 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-1612566
  10. 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
  11. Novak P, Maldonado DAP, Novak V (2019) Safety and preliminary efficacy of intranasal insulin for cognitive impairment in Parkinson disease and multiple system atropA double-blinded placebo-controlled pilot study. PLoS One 14: e0214364. https://doi.org/10.1371/journal.pone.0214364
  12. Guo Z, Chen Y, Mao YF, Zheng T, Jiang Y, Yan Y, Yin X, Zhang B (2017) Long-term treatment with intranasal insulin ameliorates cognitive impairment, tau hyperphosphorylation, and microglial activation in a streptozotocin-induced Alzheimer’s rat model. Sci Rep 7: 45971. https://doi.org/ 10.1038/srep45971
  13. Kamat PK, Kalani A, Rai S, Tota SK, Kumar A, Ahmad AS (2016) Streptozotocin intracerebroventricular-induced neurotoxicity and brain insulin resistance: a therapeutic intervention for treatment of sporadic Alzheimer’s disease (sAD)-like pathology. Mol Neurobiol 53 (7): 4548–4562. https:/ doi.org/10.1007/s12035-015-9384-y
  14. Zorina II, Zakharova IO, Bayunova LV, Avrova NF (2018) Insulin administration prevents accumulation of conjugated dienes and trienes and tnactivation of Na+, K+-ATPase in the rat cerebral cortex during two-vessel forebrain ischemia and reperfusion. J Evol Biochem Phys 54 (3): 246–249. https://doi.org/10.1371/journal.pone.0214364
  15. Zorina II, Fokina EA, Zakharova IO, Bayunova LV, Shpakov AO (2019) Features of the changes in lipid peroxidation and activity of Na+/K+-ATPase in the brain of the aged rats in the conditions of two-vessel cerebral ischemia/reperfusion. Adv Gerontol. 32 (6): 941–947.
  16. Nabavi SF, Sureda A, Sanches-Silva A, Pandima DK, Ahmed T, Shahid M (2019) Novel therapeutic strategies for stroke: the role of autophagy. Critical Reviews in Clinical Laboratory Sciences 56(3): 182–199. https://doi.org/10.1080/10408363.2019.1575333
  17. Campbell BCV, Khatri P (2020) Stroke. Lancet 396 (10244): 149–142. https://doi.org/10.1016/S0140-6736(20)31179-X
  18. He C, Xu Y, Sun J, Li L, Zhang JH, Wang Y (2022) Autphagy and apoptosis in acute brain injuries: From mechanism to treatment. Antioxid Redox Signal. https://doi.org/10.1089/ars.2021.0094
  19. Zhou H, Wang J, Jiang J, Stavrovskaya IG, Li M, Li W, Wu Q, Zhang X, Luo C, Zhou S, Sirianni AC, Sarkar S, Kristal BS, Friedlander RM, Wang X (2014) N-acetyl-serotonin offers neuroprotection through inhibiting mitochondrial death pathways and autophagic activation in experimental models of ischemic injury. J Neurosci 34: 2967–2978. https://doi.org/10.1523/JNEUROSCI.1948-13.2014
  20. Wang M, Li Y-J, Ding Y, Zhang H-N, Sun T, Zhang K, Yang L, Guo Y-Y, Liu S-B, Zhao M-G, Qu Y-M (2016) Silibinin prevents autophagic cell death upon oxidative stress in cortical neurons and cerebral ischemia-reperfusion injury. Mol Neurobiol 53: 932–943. https://doi.org/10.1007/s12035-014-9062-5
  21. Li L, Tian J, Long MK-W, Chen Y, Lu J, Zhou C, Wang T (2016) Protection against experimental stroke by ganglioside GM1 is associated with the inhibition of autophagy. PLoS One 11: e0144219. https://doi.org/10.1371/journal.pone.0144219
  22. Li X, Wang M, Qin C, Fan W-H, Tian D-S, Liu J-L (2017) Fingolimod suppresses neuronal autophagy through the mTOR/p70S6K pathway and alleviates ischemic brain damage in mice. PloS One 12: e0188748. https://doi.org/10.1371/journal.pone.0188748
  23. Hu Y, Zhou H, Zhang H, Sui Y, Zhang Z, Zou Y, Li K, Zhao Y, Xie J, Zhang L (2022) The neuroprotective effect of dexmedetomidine and its mechanism. Front Pharmacol 13: 965661. eCollection 2022. https://doi.org/10.3389/fphar.2022.965661
  24. Zhang H, Wang X, Chen W, Yang Y, Wang Y, Wan H, Zhu Z (2023) Danhong injection alleviates cerebral ischemia-reperfusion injury by inhibiting autophagy through miRNA-132–3p/ATG12 signal axis. J Ethnopharmacol 300:115724. https://doi.org/10.1016/j.jep.2022.115724
  25. Buckley KM, Hess DL, Sazonova IY, Periyasamy-Thandavan S, John R, Barrett JR, Kirks R, Grace H, Kondrikova G, Johnson MH, Hess DC, Schoenlein PV, Hoda MN, Hill WD (2014) Rapamycin up-regulation of autophagy reduces infarct size and improves outcomes in both permanent MCAL, and embolic MCAO, murine models of stroke. Exp Transl Stroke Med 6: 8. eCollection. https://doi.org/10.1186/2040-7378-6-8
  26. Liu X, Tian F, Wang S, Wang F, Xiong L (2018) Astrocyte Autophagy Flux Protects Neurons Against Oxygen-Glucose Deprivation and Ischemic/Reperfusion Injury. Rejuvenation Res 215: 405–415. https://doi.org/10.1089/rej.2017.1999
  27. Carloni S, Balduini W (2020) Simvastatin preconditioning confers neuroprotection against hypoxia-ischemia induced brain damage in neonatal rats via autophagy and silent information regulator 1 (SIRT1) activation. Exp Neurol 324: 113117. https://doi.org/10.1016/j.expneurol.2019.113117
  28. Fokina EA, Zakharova IO, Bayunova LV, Avrova DK, Ilyasov IO, Avrova NF (2023) Intranasal insulin decreases autophagic and apoptotic death of neurons in the rat hippocampal CA1 region and frontal cortex under forebrain ischemia-reperfusion. J Evol Biochem Physiol 59 (1): 45–56. https://doi.org/10.1134/S0022093023010040
  29. Zakharova IO, Bayunova LV, Avrova DK, Avrova NF (2023) Neuroprotective effect of insulin on rat cortical neurons in oxidative stress is mediated by autophagy and apoptosis inhibition in vitro. J Evol Biochem Phys 59 (5): 1536–1550. https://doi.org/10.1134/S0022093023050071
  30. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. Fourth Edition. Acad. Press. San Diego Calif. USA 1–237.
  31. Молчанова СМ, Москвин АН, Захарова ИО, Юрлова ЛА, Носова ИЮ, Аврова НФ (2005) Влияние двухсосудистой ишемии переднего мозга и введения индометацина и квинакрина на активность Na+, K+-АТФ-азы в разных областях мозга крыс. Ж эвол биохим физиол 41(1): 33–38. [Molchanova SM, Moskvin AN, Zakharova IIu, Iurlova LA, Nosova IIu, Avrova NF (2005) Na, K-atpase activity in different brain regions in cerebral ischemia and influence of quinacrine and indomethacin administration. Zh Evol Biokhim Fiziol 41(1):33–38. (In Russ)].
  32. Sanderson TH, Wider JM (2013) 2-Vessel occlusion/hypotension: A rat model of global brain ischemia. J Vis Exp 76: e50173. https://doi.org/10.3791/50173
  33. Zakharova IO, Sokolova TV, Vlasova YA, Bayunova LV, Rychkova MP, Avrova NF (2017) α-Tocopherol at nanomolar concentration protects cortical neurons against oxidative stress. Int J Mol Sci 18: 216. https://doi.org/10.3390/ijms18010216
  34. Zick Y (2005) Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance Sci STKE268: pe4. https://doi.org/10.1126/stke.2682005pe4
  35. Tanida I, Ueno T, Kominami E (2004) LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 36(12):2503–2518. https://doi.org/10.1016/j.biocel.2004.05.009
  36. Fritzen AM, Frøsig C, Jeppesen J, Jensen TE, Lundsgaard AM, Serup AK, Schjerling P, Proud CG, Richter EA, Kiens B (2016) Role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle. Cell Signal 28(6):663–674. https://doi.org/10.1016/j.cellsig.2016.03.005
  37. Bansal M, Moharir SC, Swarup G (2018) Autophagy receptor optineurin promotes autophagosome formation by potentiating LC3-II production and phagophore maturation. Commun Integr Biol 11 (2): 1–4. https://doi.org/10.1080/19420889.2018.1467189
  38. Ribeiro M, López de Figueroa P, Blanco FJ, Mendes AF, Caramés B (2016) Insulin decreases autophagy and leads to cartilage degradation. Osteoarthritis Cartilage 24:731–739. https://doi.org/10.1016/j.joca.2015.10.017
  39. Pires KM, Torres NS, Buffolo M, Gunville R, Schaaf C, Davis K, Selzman CH, Gottlieb RA, Boudina S (2019) Suppression of cardiac autophagy by hyperinsulinemia in insulin receptor-deficient hearts is mediated by insulin-like growth factor receptor signaling. Antioxid Redox Signal 31(6):444–457. https://doi.org/10.1089/ars.2018.7640
  40. Ding X, Zhang L, Zhang X, Qin Y, Yu K, Yang X (2023) Intranasal insulin alleviates traumatic brain injury by inhibiting autophagy and endoplasmic reticulum stress-mediated apoptosis through the PI3K/Akt/mTOR signaling pathway. Neuroscience 29:23–36. https://doi.org.10.1016/j.neuroscience.2023.08.009
  41. Sanderson TH, Kumar R, Murariu-Dobrin AC, Page AB, Krause GS, Sullivan JM (2009) Insulin activates the PI3K-Akt survival pathway in vulnerable neurons following global brain ischemia. Neurol Res. 31(9):947–958. https://doi.org/10.1179/174313209X382449
  42. Russo V, Candeloro P, Malara N, Perozziello G, Iannone M, Scicchitano M, Mollace R, Musolino V, Gliozzi M, Carresi C (2019) Key role of cytochrome C for apoptosis detection using Raman microimaging in an animal model of brain ischemia with insulin treatment. Appl Spectrosc 73 (10): 1208–1217. https://doi.org/10.1177/0003702819858671
  43. Zhu Y-M, Gao X, Ni Y, Li W, Ken TA, Qiao S-G, Wang C, Xiao-Xuan Xu X–X, Hui-Ling Zhang H-L (2017) Sevoflurane postconditioning attenuates reactive astrogliosis and glial scar formation after ischemia-reperfusion brain injury. Neuroscience 356:125–141. https://doi.org/10.1016/j.neuroscience.2017.05.004
  44. Zhang R, Pei H, Ru L, Li H, Liu G (2013) Bone morphogenetic protein 7 upregulates the expression of nestin and glial fibrillary acidic protein in rats with cerebral ischemia-reperfusion injury. Biomed Rep 1(6): 895–900. https://doi.org/10.3892/br.2013.164
  45. Yamada M, Ohnishi H, Sano SA, Nakatani A, Ikeuchi T, Hatanaka H (1997) Insulin receptor substrate IRS-1 and IRS-2 are tyrosine-phosphorylated and associated with phosphatidylinositol 3-kinase in response to brain-derived neurotrophic factor in cultured cerebral cortical neurons. J Biol Chem 272(48):30334–30339. https://doi.org/10.1074/jbc.272.48.30334
  46. Grote CW, Morris JK, Ryals JM, Geiger PC, Wright DE (2011). Insulin receptor substrate 2 expression and involvement in neuronal insulin resistance in diabetic neuropathy. Exp Diabetes Res 2011: 212571. https://doi. org/10.1155/2011/21257
  47. Boura-Halfon S, Zick T (2009) Phosphorylation of IRS proteins, insulin action, and insulin resistance. Am J Physiol Endocrinol Metab 296(4): E581–591. https://doi.org/10.1152/ajpendo.90437.2008
  48. Li Y, Chen Y (2019) AMPK and autophagy. Adv Exp Med Biol 1206: 85–108. https://doi.org/10.1007/978-981-15-0602-4_4
  49. Kim J, Kundu M, Viollet B, Guan K-L (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13 (2):132–141. https://doi.org/10.1038/ncb2152
  50. Parzych KR, Klionsky DJ (2014) An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signals 20(3):460–473. https://doi. org/:10.1089/ars.2013.5371
  51. Yoon MS (2017) The role of mammalian target of rapamycin (mTOR) in insulin signaling, nutrients. 9 (11): 1176. https://doi. org/ 10.3390/nu9111176
  52. Sharma A, Anand SK, Singh N, Dwivedi UN, Kakkar P (2023) AMP-activated protein kinase: An energy sensor and survival mechanism in the reinstatement of metabolic homeostasis. Exp Cell Res 428(1):113614. https://doi.org/10.1016/j.yexcr.2023.113614
  53. Yang K, Chen Z, Gao J, Shi W, Li L, Jiang S, Hu H, Liu Z, Xu D, Wu L (2017) The key roles of GSK-3beta in regulating mitochondrial activity. Cell Physiol Biochem 44:1445–1459. https://doi.org/10.1159/000485580

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2. Fig. 1. The effect of insulin administration and inhibitors of autophagy and apoptosis (3-methyladenine and Ac-DEVD-CHO, respectively) on the level of the autophagy marker LC3B-II in the hippocampus and frontal cortex during ischemia and reperfusion of the forebrain of rats. (a), (c) and (e) - the level of LC3B-II in the hippocampus, (b), (d) and (f) –the level of LC3B-II in the frontal cortex. (a) and (b) – image blocks, (c), (d), (e) and (f) - densitometry results based on LC3B–I (c) and (d) or on GAPDH (e) and (f) are shown as an average ± SEM of 7-8 conducted experiments. Abbreviations in the figure: Sh-O – falsely operated rats, I/R – rats subjected to ischemia and reperfusion, 3-MA – 3-methyladenine. Rats were subjected to two-vessel forebrain ischemia in combination with hypotension and subsequent reperfusion for 3 days. Inhibitors of autophagy and apoptosis were administered rats were given intracerebroventricular (ICV) before ischemic exposure, and 0.5 IU of insulin was administered intranasally before ischemic exposure and then daily during 3 days of reperfusion. Differences before

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3. Fig. 2. The effect of insulin administration on the activity of caspase‑3 in the hippocampus and frontal cortex in ischemia and reperfusion of the forebrain of rats. The data are shown as an average ±SEM of 7-8 experiments. (a) – caspase‑3 activity in the hippocampus, (b) – caspase‑3 activity in the frontal cortex of the brain. The explanation of the abbreviations in the figure and other information are given in the legend to Fig. 1. The differences are significant according to the Student's t criterion: * – compared with data obtained in the same brain region of falsely operated rats, p<0.02, x and xx - compared with data obtained in the same brain region rats with ischemia and reperfusion of the forebrain, x – p<0.05, xx – p<0.02.

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4. Fig. 3. The effect of insulin administration and inhibitors of autophagy and apoptosis (3-MA and ac-DEVD-CHO, respectively) on the level of glial acid fibrillar protein (GFAP) in the hippocampus and cerebral cortex during ischemia and reperfusion of the forebrain of rats. The data are shown as an average ± SEM of 6-8 experiments. (a) and (c) – GFAP level in the hippocampus, (b) and (d) – GFAP level in the frontal cortex, (a) and (b) – immunoblotts, (c) and (d) – densitometry results are presented as GFAP/GAPDH. The explanation of the abbreviations in the figure and other information are given in the legend to Fig. 1. The differences are significant according to the Student's t criterion compared with the data obtained in the same brain region of falsely operated rats: * – p <0.05, ** – p<0.02.

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5. Fig. 4. The effect of insulin administration on the activity of protein kinase B (Akt) in the hippocampus and frontal cortex during ischemia and reperfusion of the forebrain of rats. The data are shown as an average ± SEM of 5-7 experiments. (a) and (c) – Akt protein kinase activity in the hippocampus, (b) and (d) – Akt protein kinase activity in the frontal cortex, (a) and (b) – immunoblotts, (c) and (d) – densitometry results are presented as pAkt (Ser473)/Akt. Abbreviations in the figure: Sh-O – false- operated rats, I/R – rats with ischemia and reperfusion of the brain. Rats were subjected to two-vessel forebrain ischemia in combination with hypotension and subsequent reperfusion for 2 hours. 0.5 IU of insulin was administered intranasally 1 hour before ischemic exposure. The differences are significant according to the Student's t criterion: * and ** – compared with data obtained in the same brain region of falsely operated rats, * - p<0.05, ** - p<0.02, x and xx – compared with data obtained in the same brain region of rats with ischemia and reperfusion of the forebrain, x – p<0.05,

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6. Fig. 5. The effect of insulin administration and inhibitors of autophagy and apoptosis (3-MA and Ac-DEVD-CHO, respectively) on AMPK activity in the hippocampus and cerebral cortex during ischemia and reperfusion of the forebrain of rats. The data are shown as an average ± SEM of 7-8 experiments. (a) and (c) – AMPK protein kinase activity in the hippocampus, (b) and (d) – AMPK protein kinase activity in the frontal cortex, (a) and (b) - immunoblotts, (c) and (d) – densitometry results are presented as pAMPK‑alpha (Thr172)/AMPK‑alpha. The explanation of the abbreviations in the figure and other information are given in the legend to Fig. 1. The differences are significant according to the Student's t criterion: * – compared with data obtained in the same brain region of falsely operated rats, p <0.05, x and xx - compared with data obtained in the same brain region of rats with ischemia and reperfusion of the forebrain, x – p<0.05, xx – p<0.02.

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7. Fig. 6. The effect of insulin administration and inhibitors of autophagy and apoptosis (3-MA and ac-DEVD-CHO, respectively) on the level of pIRS‑2 (Ser731) in the hippocampus and cerebral cortex in rat forebrain ischemia and reperfusion. The data is shown as the average ± SEM of 7-8 experiments. (a) and (c) – the level of pIRS‑2 (Ser731) in the hippocampus, (b) and (d) – the level of pIRS‑2 (Ser731) in the frontal cortex, (a) and (b) – immunoblotts, (c) and (d) – densitometry results are presented as the pIRS‑2 ratio (Ser731)/pIRS‑2. The explanation of the abbreviations in the figure and other information are given in the legend to Fig. 1.

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