SB216763

Hyper-insulinemia increases the glutamate- excitotoxicity in cortical neurons: a mechanistic study

Ashok Kumar Datusalia, Piyush Agarwal, Jitendra Narain Singh, Shyam Sunder Sharma

www.elsevier.com/locate/ejphar

PII: S0014-2999(18)30368-6
DOI: https://doi.org/10.1016/j.ejphar.2018.07.001
Reference: EJP71866

To appear in: European Journal of Pharmacology Received date: 8 May 2018
Revised date: 20 June 2018 Accepted date: 2 July 2018

Cite this article as: Ashok Kumar Datusalia, Piyush Agarwal, Jitendra Narain Singh and Shyam Sunder Sharma, Hyper-insulinemia increases the glutamate- excitotoxicity in cortical neurons: a mechanistic study, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.07.001

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Hyper-insulinemia increases the glutamate-excitotoxicity in cortical neurons: a mechanistic study

Ashok Kumar Datusalia, Piyush Agarwal, Jitendra Narain Singh, Shyam Sunder Sharma*

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar-160 062 Punjab, India

*Address for correspondence: Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar-160 062, Punjab, India. [email protected]

ABSTRACT

Insulin resistance in type-2 diabetic condition increases the risk of stroke and cognitive deficits in which involvement of glutamate has been postulated. It has been hypothesized that hyper-insulinemia in cortical neurons increases the vulnerability towards glutamate-induced excitotoxicity. To mimic insulin resistance, cortical neurons were incubated with high insulin (1 µM) and high glucose (50 mM final concentration) in in-vitro condition for 24 h. Pre- treatment of cortical neurons with high insulin blocked acute insulin-induced activation of Akt and GSK-3 but not in the case of high glucose. Our results demonstrate that chronic high insulin exposure increases glutamate-induced excitotoxity, which was blocked by
insulin receptor antagonist (S961) and GSK-3 inhibitor (SB216763). These inhibitors also ameliorated pAkt (Ser473) and pGSK-3(Ser9) levels after chronic insulin exposure. Increase in glutamate-excitotoxicity in insulin resistant cortical neurons was found to be associated with increased expression of PICK1. However, GluR2 did not get altered in hyper- insulinemia condition. This study demonstrates that hyper-insulinemia increases glutamate excitotoxicity which could be attributed to activation of GSK-3β and increased expression of PICK1.

Keywords: Cortical neuron, Excitotoxicity, GSK-3 β inhibitor, Insulin resistance, S961

1.Introduction

Metabolism in the neurons is independent to the insulin but neurons have been shown to have insulin receptor and associated downstream insulin signalling which facilitate glucose metabolism during the growth period acting as a growth factor (Kim and Feldman, 2012). Insulin resistance, the state of hypo-responsiveness towards the insulin, in addition to peripheral system it is also phenomenally present in the brain (Kullmann et al., 2016). Recent epidemiological evidences suggested that insulin resistance condition (type-2 diabetes, obesity etc.) is associated with increased risk of stroke, Alzheimer’s disease, neurovascular dementia, depression etc. (Kullmann et al., 2016). Multiple factors, including insulin resistance itself and associated hyperglycaemic neuronal injury may contribute to perturbation of neuronal function and neuronal mortality in type-2 diabetes (Duarte et al., 2012). The mechanism of insulin resistance induced neuronal injury has not been identified yet and there is growing interest in metabolic disorder and their impacts on brain functions particularly ischemia reperfusion injury and cognitive deficits.

Under physiological conditions glutamate an excitatory transmitter; exerts neuronal excitation through glutamate receptors (GluRs) including ionotropic and metabotropic receptors. Inotropic GluRs exist (NMDA, AMPA and kainate) as heteromers of different subunit types and isoforms and combinatorial association of individual subunits diversifying their functional and signalling properties (Malinow and Malenka, 2002). AMPA receptor GluR2 subunits are differentially regulated by the interaction with PDZ domain-containing proteins GRIP1 (Glutamate receptor-interacting protein 1) and protein interacting with C kinase 1 (PICK1). The GluR2 interaction with GRIP1 and PICK1 has been shown to regulate AMPA receptor subunit composition and profound importance in both plasticity and pathological mechanisms (Isaac et al., 2007; Kim et al., 2001). Several studies have shown that AMPAR interacting protein PICK1 regulates the surface expression of glutamate

receptor 2 (GluR2) and promotes internalization or inhibits recycling of GluR2 or retains GluR2 intracellularly (Lin and Huganir, 2007). Incorporation of GluR2-lacking AMPARs at membrane and increased association of GluR2 with PICK1 results in increased Ca2+ permeability a condition that might place neurons at a risk for subsequent excitotoxic deterioration if the process is unregulated (Balkhi et al., 2014; Iihara et al., 2001). It has been demonstrated that glutamate is massively released in ischemic brain tissue and many neurons actively respond to this neurotransmitter through the ionotropic receptors (Kostandy, 2012). Experimental studies on ischemic stroke have shown IRS1/Akt/GSK-3 signalling as a key regulator of neuronal survival (Chuang et al., 2011; Gao et al., 2010; Lai et al., 2014). However, it is yet to be investigated whether insulin resistance alter glutamate excitotoxicity. Here, we hypothesized that hyper-insulinemia within the central nervous system, contributes to enhanced glutamate mediated excitotoxic neuronal injury. We demonstrate the increased effect of hyper-insulinemia on sensitivity of glutamate mediated excitotoxicity in cortical neurons in-vitro. The effects of the insulin receptor antagonist (S961) and GSK-3 inhibitor (SB216763) were investigated on the glutamate-induced neurotoxicity under insulin resistance/ hyperinsulinemia conditions. In addition, the effects of S961 and SB216763 on alteration of downstream protein were also examined.

2.Materials and methods

2.1Primary culture of rat cortical neurons culture and treatment

All studies were performed according to the Guideline by CPCSEA, Ministry of Environment and Forest, Govt. of India and were approved by the Institutional Animal Ethics Committee, NIPER, S.A.S. Nagar. Primary cortical neuronal cultures were prepared from postnatal (P0) Sprague Dawley rat pups as described previously (Kaech and Banker, 2006). Briefly, the cortical regions of the pups brains were aseptically dissected and all the meninges

were removed in Ca2+ and Mg2+-free Hanks balanced salt solution (HBSS). Cortical tissues were digested in papain (0.20% in HBSS) for 15 min at 37°C with occasionally swirling. At the end of digestion, papain solution was decanted and tissue was washed twice with F-12 Ham media (containing 10% FBS, 1% antibiotic mixture, and 25 mM glucose). The digested tissues were subjected to trituration in F12 Ham media using fire polished Pasteur’s pipette. The suspensions of dissociated cells were centrifuged at 200 g for 5 min. The supernatant was discarded and cells pellet was suspended in F12 Ham media. Then the suspension was
filtered through cell strainer (70 µm) to obtain uniform size of cells. Neurons were plated to a density of 0.2 – 1.0 X 106 cells/ml previously coated with 0.1 mg/ml sterile poly-D-lysine in H2O. The cultures were maintained at 37 °C in 5% CO2 and 95% air in a humidified incubator. After 4 h, the initial medium was replaced with Neurobasal-A medium containing 2% B-27, 1% Glutamax-I and 1 % penicillin and streptomycin. Half of the medium was replaced with fresh medium at every third day (at 3rd, 6th, 8th and 10th days). The DIV3 (Day in-vitro 3) feeding included cytosine arabinoside (AraC) at final concentration of 1µM to inhibit non-neuronal cells proliferation. Insulin receptor antagonist (S961) was used at equimolar concentration to insulin because its affinity for the insulin receptor is comparable to that of insulin on the basis of previous studies (Knudsen et al., 2012; Schaffer et al., 2008). SB216763 (a GSK-3 inhibitor) was used at 30 µM concentration (IC50=34 µM) (Li et al., 2015). Cortical neurons were also exposed to high glucose (50 mM final concentration) and high insulin (1 μM) to mimic experimental condition. Drug treatment schedules and experimental design are described in Fig. 1 and with the respective results.

2.2Immunocytochemistry

To verify the predominance of neurons in this cell culture, microtubule-associated protein 2 (MAP2) immunostaining was performed. Neurons were fixed using 4%

paraformaldehyde/sucrose mixture for 10 min at 37 °C. The paraformaldehyde/sucrose mixture was aspirated and 0.1% (vol/vol) of Triton X-100 solution was added in Dulbecco’s Phosphate-Buffered Saline (DPBS) and incubated for 10 min at room temperature. Then, cells were washed gently with DPBS and blocked with 5% bovine serum albumin (BSA; wt/vol) for one hour (h) at room temperature. After blocking with BSA, fixed cells were incubated with primary antibody anti-MAP2 (1:100) (overnight at 4 °C). Then cells were
washed three times with DPBS and were incubated with FITC conjugated secondary antibody (Santa Cruz Biotechnologies, USA) for 1 h in the dark at room temperature. After washing three times with DPBS cover slips were mounted on slide. Image was captured on Confocal Laser Scanning Microscopes (Olympus).

2.3Neuronal viability assay by Lactate Dehydrogenase ( LDH) release and calcein-AM assay

To check neuronal viability LDH release and calcein-AM based fluorescent assay were used. For calcein-AM assay, cells were washed with Ca2+ and Mg2+ -free Dulbecco`s Phosphate Buffered Saline (DPBS) pre-warmed at 37 °C. Cells were incubated with 3 μM calcein-AM diluted in 1X DPBS (pre-warmed at 37 °C). After 30 min incubation, fluorescence was measured for calcein (excitation wavelength ~495 nm; emission wavelength ~515 nm) from the top of the plate using a plate reader (Cary Eclipse, Agilent Technologies, USA). Cell viability was calculated as percentage of control. For LDH release assay, 50 μl of supernatant from each well was collected. The samples were incubated with reduced form of nicotinamide-adenine dinucleotide (NADH) and pyruvate for 15 min at 37 °C and the
reaction was stopped by adding 0.4 mol/l NaOH. The activity of LDH was calculated by measuring the absorbance at 440 nm using Infinite® M1000 PRO Multiplate reader (Tecan Group Ltd., Männedorf, Switzerland). Percentage of LDH release in relation to control was calculated.

2.4Preparation of cell lysate for immunoblotting

Cultured cortical neurons were harvested in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, 50 mM NaF, 200 mM Na3VO4, and a protease inhibitor mixture). Neurons were lysed on ice for 30 min by vortexing for 1 min at 10-min intervals. Cell lysates were centrifuged at 13,000 g for 15 min at 4°C to remove debris. Equal amount of proteins was separated by SDS–PAGE and transferred to a nitrocellulose (Pall Life Sciences, USA)/PVDF membrane (Millipore, USA). The blots were blocked by nonfat dried milk (Sigma Aldrich, USA)/BSA (Sigma Aldrich, USA) for 1 h and were probed overnight at 4C with primary antibodies for Akt (1:500), pAkt(Ser473) (1:250) GSK-3β (1:500), pGSK-3β(Ser9) (1:500), PICK1 (1:250), GluR2 (1:250) and β-actin (1:1000) obtained from Santa Cruz Biotechnologies, USA or Sigma Aldrich, USA. After, washing with TBST blots were incubated with respective HRP- conjugated secondary antibody (1:10000) for 1 h at 37 C and developed with the ECL Western blotting detection system (Santa Cruz Biotechnology, Inc. USA). Densitometric analysis was carried out using NIH software ImageJ and results are expressed as percentage of control (Datusalia and Sharma, 2014).

2.5Statistical analysis

Quantitative data are presented as mean ± S.E.M. For comparing the difference between two groups Students t-test was used. Comparison between multiple groups was carried out using one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test (GraphPad Prism 5). P<0.05 was considered statistically significant. 3.Results 3.1Induction of hyper-insulinemia in cortical neurons Most of cell population in the culture at day 10 (DIV10) contained MAP2 immunoreactivity which indicates pure neuronal culture (Fig. 2A). Cortical neurons were treated with high glucose (50 mM final concentration) or high insulin (1 μM) for 24 h for development of insulin resistance. In one way ANOVA of western blot, acute insulin (20 nM for 15 min) show significant effect (F (5, 12) = 36.33; P<0.001) on Akt activation. Under untreated condition Akt activation (increased pAkt) was observed after 15 minute of insulin treatment (20 nM). 24 h high insulin exposure resulted in significant (P<0.001) decrease in insulin stimulated Akt phosphorylation (Ser473) (Fig. 2). In contrast, high glucose did not show any effect on the activation of Akt in response to short term insulin treatment. Furthermore, significant (one way ANOVA; F (5, 12) = 27.69; post-hoc Tukey’s test P<0.001) down regulation of pGSK-3 were observed with 24 h insulin treatment (Fig. 2B and C). Insulin stimulated phosphorylation of GSK-3(Ser9) was also found to significantly decrease on long exposure of high insulin but found to unaffected on long exposure with high glucose. Both high insulin and high glucose did not alter basal Akt and GSK-3 levels after 24 h treatment (Fig. 2B). During 24 h high insulin treatment, cortical neurons cultures were incubated with or without insulin receptor antagonist (S961) or inhibitor of GSK-3 (SB216763). Co-exposure of insulin with equimolar concentration of S961 attenuated the high insulin-induced decrease in phosphorylation of pAkt (Ser473) (P<0.05) and pGSK-3(Ser9) (P<0.01) (Fig. 3). SB216763 had not shown significant increase in pAkt levels but significantly (P<0.001) restored the levels of pGSK-3 on co-incubation with chronic high insulin. 3.2Effect of hyper-insulinemia on glutamate-induced toxicity Cortical neurons were exposed to glutamate (100 μM for 15 min) and showed significant reduction in neuronal viability measured after 24 hrs by calcein AM assay (one way ANOVA; F (5, 18) = 84.39; P<0.001) and LDH release assay (one way ANOVA; F (5, 18) = 43.87; P<0.001) (Fig. 4A and C respectively). Neurons pre-treated with insulin (1μM) for 24 h significantly enhanced (P<0.001; post-hoc Tukey’s test) the cell loss induced by the glutamate exposure in calcein AM assay compared glutamate-induced cell loss in normal control and pre-treated with high glucose (Fig. 4A). In contrast, glutamate-induced cells death on high glucose incubation did not differ significantly compared to glutamate-induced cell death in normal medium. These finding were consistent with cell cytotoxicity as determined by % LDH release (Fig. 4C). Co-treatment of cortical neurons with S961 and high insulin showed significant effect ( F(5, 18) = 115.9) in the neuronal death induced by the glutamate in calcein AM assay (Fig. 4B). In post-hoc analysis (Tukey’s test), SB216763 a GSK-3 inhibitor, significantly reduced the glutamate-induced neuronal death compared to cells incubated with high insulin alone and glutamate. SB216763 showed significantly higher (P<0.05) protection compared to S961 in calcein AM assay, however similar trend was observed in LDH assay but it was not significant (Fig. 4B-C). Moreover, S961 did not show any effect on normal and high glucose condition (Data not shown). 3.3Involvement of PICK1 and GluR2 in glutamate-induced neuronal death under hyper- insulinemia condition We next evaluate the possible effect of high insulin exposure in PICK1 and GluR2 levels (Fig. 5A). 24 h high exposure of high insulin led to significant (t(4)=5.42; P< 0.01) increase in the expression of PICK1 protein in cortical neurons but did not alter the total GluR2 levels (t(4)=0.89; P= 0.42). S961 and SB216763 showed significant reduction (one way ANOVA; F (5, 12) = 48.04 ; P < 0.001) in PICK1 expression under high insulin condition. Moreover, S961 showed significant (post-hoc Tukey’s test; P<0.05) reduction in the elevated PICK1 levels (Fig. 5B). SB216763 also decrease the elevated PICK1 levels significantly (P<0.001) under high insulin condition. However, both S961 and SB216763 did not alter the GluR2 levels (one way ANOVA; F (5, 12) = 0.5047; P = 0.77) in normal or high insulin condition (Fig. 5B). 4.Discussion Systemic insulin-resistance and disturbed glucose metabolism are the hallmark characteristic of type-2 diabetes and also part of the constellation of the symptoms associated with metabolic disorders. In the present study, chronic exposure (24h) of high glucose (50 mM) did not affect the cortical neurons sensitivity towards the acute insulin. Acute insulin (20 nM for 15 min) treatment showed significantly increase in pAkt and pGSK-3 levels in normal culture as well as high glucose incubation. Interestingly, both glucose deprivation and exposure to elevated glucose levels have been shown to be toxic to cells (Semra et al., 2004; Suh et al., 2003). Primary neuronal cultures were observed healthy with longer survival at 25 mM glucose (Russell et al., 1999; Shi and Liu, 2006). Addition of 25 mM more glucose for 24 h did not alter primary cortical neuronal viability; this is consistent with the literature (Gaspar et al., 2010; Noh et al., 1999). In the present study, downstream effector of insulin, pAkt was diminished after 24 h of high insulin treatment. However, the basal Akt level was unaffected by the high insulin treatment. There are several studies on Neuro2A, PC12, SH- SY5Y neuronal cells and primary cortical neurons, reporting altered insulin downstream signalling after 24 h high insulin incubation (Gupta and Dey, 2012; Rhee et al., 2013). In addition, the phosphorylation of downstream effectors, GSK-3β was also found to be downregulated after chronic insulin treatment. It has been demonstrated that decrease in pAkt and pGSK-3 levels are specific for insulin signalling via decreased IRS-1 activation, as acute IGF-I activation of Akt was not affected by chronic insulin treatment (Kim et al., 2011). Cortical neurons were exposed to high insulin resulted in Akt down-regulation and exhibited increased sensitivity toward the glutamate-induced neuronal death. Chronic high insulin produces 1.5 times more death of cortical neurons compared to control. Inhibition of the effect of high insulin with insulin receptor antagonist (S961) significantly reversed pAkt, and GSK-3 with increase in the neuronal survival against glutamate-induced excitotoxicity in- vitro. Downstream to Akt, inhibition of GSK-3 by SB216763 also blocks the effect of high insulin on pGSK-3 levels and glutamate-induced excitotoxicity. In the present study, high insulin exposure showed a significant increase in the Protein Interacting with C kinase 1 (PICK1) expression in cortical neurons. However, total GluR2 protein levels were found unchanged after the 24 h of high insulin incubation. In the studies on hippocampal neuron, insulin showed distinct effect on GluR1/GluR2 receptor localization at neuronal surface (Ferrario and Reagan, 2017). In a model of neuronal insulin resistance (Insulin and palmitic acid-induced), presence on excess palmitic acid enhanced the GluR1 palmitoylation hindering its activity-dependent trafficking to the plasma membrane. However, insulin and palmitic acid-induced insulin residence did not show any change in total level of GluR1 and GluR2 (Spinelli et al., 2017). In different studies, insulin has been shown to induce reductions in AMPAR surface expression which was predominantly mediated by the removal of GluA2-containing AMPARs, whereas it has shown to increase in AMPAR surface expression which predominantly mediated by the addition of GluA1- containing AMPARs (Ferrario and Reagan, 2017). Thus, it is possible that insulin may have distinct effects on GluA1 vs GluA2-containing AMPA receptors, although this has not been directly tested. AMPA receptor GluR2 subunit differentially regulated by the interaction with the PDZ domain-containing proteins GRIP1 (Glutamate receptor-interacting protein 1) and PICK1. The modulation of GluR2 interaction with PICK1 has been shown to regulate AMPA receptor internalization (Chong et al., 2001; Isaac et al., 2007). Incorporation of GluR2-lacking AMPARs and increased association of GluR2 with PICK1 resulted in increased Ca2+ permeability, a condition that might place neurons at risk for subsequent excitotoxic deterioration if the process is unregulated (Iihara et al., 2001). We observed a significant neuroprotection offered by S961 and SB216763 against glutamate excitotoxicity with parallel decrease in the PICK1 levels. Here, both S961 and SB216763 also decrease the expression the PICK1 in the cortical neurons incubated with high insulin. In summary, our experiments reveal that chronic insulin stimulation blunted insulin stimulated Akt activation, a form of hyper-insulinemia in cortical neurons. Our results also provide primary evidences that hyper-insulinemia sensitize the cortical neurons towards glutamate excitotoxicity via upregulation of PICK1 (Fig 6). Further studies are required to understand the underlying mechanism involved in the degree of down-regulation of acute insulin action after chronic insulin exposure; as if exposure to high insulin leads to loss of insulin receptors, and if basal and insulin-stimulated activity of the insulin receptor, and/or IRS-1 are diminished by chronic insulin exposure. Insulin dose-responses are needed to be seen if there are spare receptors that allow higher doses of insulin to activate downstream elements. Conflict of interest The authors declare no conflicts of interest. Acknowledgements Ashok Kumar Datusalia was the recipient of fellowship grant from CSIR New Delhi (Grant no: 09/727(0108)/2.14-EMR-I). The authors are grateful to Dr. Lauge Schaffer, Novo Nordisk, Denmark for providing the insulin receptor antagonist S961 used in the present investigation. The authors also thank the Department of Pharmaceuticals, Ministry of Chemical and Fertilizers, Government of India for the financial support. Reference Balkhi, H.M., Gul, T., Banday, M.Z., Haq, E., 2014. Glutamate Excitotoxicity: An Insight into the Mechanism. Int. J. Adv. Res. 2, 361-373. Chong, H.K., Chung, H.J., Lee, H.K., Huganir, R.L., 2001. Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression. Proc. Natl. Acad. Sci. U. S. A. 98, 11725-11730. Chuang, D.M., Wang, Z., Chiu, C.T., 2011. GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke. Front. Mol. 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Brain insulin resistance impairs hippocampal synaptic plasticity and memory by increasing GluA1 palmitoylation through FoxO3a. Nature communications 8, 2009. Suh, S.W., Aoyama, K., Chen, Y., Garnier, P., Matsumori, Y., Gum, E., Liu, J., Swanson, R.A., 2003. Hypoglycemic neuronal death and cognitive impairment are prevented by poly(ADP-ribose) polymerase inhibitors administered after hypoglycemia. J. Neurosci. 23, 10681-10690. Fig. 1. Schematic representation of the experimental plan. Experimental design (I) Represents development of insulin resistance and effect of inhibitors (S961: an insulin receptor antagonist/SB216763: GSK-3β inhibitor) on different protein expression. Experimental design (II) represents effect of high insulin (1µM)/high glucose (50 mM final concentration) on glutamate-induced excitotoxicity and effect of inhibitors on glutamate-induced excitotoxicity. Fig. 2. Chronic insulin exposure mimicking hyperinsulinemia and downregulation of Akt and GSK-3 activation after acute insulin stimulation. (A) Representative image of 10 days neuronal culture with MAP2 immunostaning (FITC conjugated) and brightfield. (B) Representative immunoblot of cortical neurons with/without high glucose (HI; 50 mM)/high insulin (HI; 1 µM) exposure and acute insulin (20 nM) stimulation. (C) Densitometeric analysis of Akt(Ser(473) and GSK-3(Ser9) phosphorylation. Values are represented as mean ± S.E.M. (n=3). Data were analysed using one way ANOVA post hoc Tukey’s test. ***p<0.001. Fig. 3. Effect of insulin antagonist (S961) and GSK-3 inhibitor (SB216763) on insulin downstream proteins (A) Representative immunoblot Akt and GSK-3 after chronic high insulin exposure (24h) with/without S961/ SB216763) and acute insulin (20 nM) stimulation. (B) Densitometeric analysis of Akt(Ser(473) phosphorylation and (C) Densitometeric analysis of GSK-3(Ser9) phosphorylation. Values are presented as mean ± S.E.M. (n=3 independent experiments). One way ANOVA post hoc Tukey’s test were used to compare multiple groups. *p<0.05, **p<0.01, ***p<0.001. Fig. 4. Effect of 24 h exposure of high glucose and high insulin on glutamate-induced neuronal death measured (A and C). Effect of S961 and SB216763 on glutamate-induced neuronal death under high insulin condition (B and D). Neuronal viability was evaluated by calcein-AM (A and B) and LDH release assay (C and D). Values are presented as mean ± S.E.M (n=4). One way ANOVA post hoc Tukey’s test were used to compare multiple groups. *p<0.05, **p<0.01, ***p<0.001. Fig. 5: Effect of (A) chronic high insulin (24 h) on PICK1 and total GluR2 expression (B) Effect of S961 and SB216763 on PICK1 and GluR2 expression after chronic insulin exposure (Representative blot and densitometric analysis).Values are presented as mean ± S.E.M. (n=3 independent experiments). Student t-test was used to compare two groups.

One way ANOVA post hoc Tukey’s test were used to compare multiple groups. **p<0.01. HI: High insulin. Fig. 6. Schematic summary of findings observed in current study. Insulin resistance increases (pAkt and GSK-3) the expression of PICK1. It has been demonstrated that PICK1 promotes GluR2 internalization. GluR2 internalization lead to a switch from Ca2+- impermeable AMPARs (GluR2-containing AMPARs) to Ca2+-permeable AMPARs (GluR2-lacking AMPARs) (Chong et al. 2001; Isaac et al. 2007). The increase in Ca2+- permeable AMPARs further enhances Ca2+-influx and this Ca2+-influx induces the activity of intracellular Ca2+-dependent kinases and excitotoxic death to the neuronal cells. Insulin receptor antagonist (S961) and GSK-3 inhibitor (SB 216763) ameliorates insulin resistance-induced excitotoxicity to the neuronal cells. Glu = glutamate; GluR1 and GluR2: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits GluR1 and GluR2; NR2: N-methyl-D-aspartic acid receptor subunit 2; PICK1 = protein interacting with C kinase 1. Fig 1 (A) Immuno staining with MAP2 Bright field (B) Control HG HI - + - + - + Insulin pAKT AKT pGSK-3GSK-3Actin Fig 2 Fig 3 Fig. 4 Fig. 5 Fig 6