Influence of Insulin Resistance and Metabolic Dysregulation on Synaptic Protein Profile in SH‐SY5Y Neuroblastoma Cells

Abstract Background Brain insulin resistance and metabolic dysregulation are significantly associated with Alzheimer’s disease development[1,2]. Metformin is the most prescribed type‐2‐diabetic drug and has been used to treat peripheral insulin resistance (IR) driven metabolic dysfunction through the activation of AMP‐kinase (AMPK)[3,4], but whether metformin is effective in improving brain metabolism is unknown. We have previously shown that chronic AMPK activation negatively impacts neuronal morphology and impairs synaptic protein content in healthy neurons[5]. However, whether AMPK activation under an IR state will exacerbate neuronal impairments is not known. Thus, we examined the effect of metformin‐driven AMPK activation on SH‐SY5Y cells with and without high‐insulin induced IR. Methods Retinoic acid‐differentiated SH‐SY5Y neuroblastoma cells were treated for 48h with either 1) vehicle (Ctl), 2) 100 M insulin (INS, mimicking hyperinsulinemia), 3) 2 mM metformin (MET), or 4) a combination of INS/MET. Results INS‐treated cells demonstrated higher Akt S473, mTOR S2448, and p70s6k T389 phosphorylation, indicative of hyperactive mTORC1 signaling associated with brain IR. INS cells had lower insulin receptor‐ (IR‐) content and higher IRS‐1 S636 phosphorylation, further demonstrating insulin signaling abnormalities. MET increased AMPK T172 phosphorylation, and increased phosphorylation of its downstream effectors ULK S555 and raptor S792, which, collectively, is indicative of mTORC1 inhibition. These phosphorylation increases occurred alongside higher IRS‐1 and insulin receptor‐ content and reduced IR‐ content. INS/MET‐treated cells produced no change in AMPK T172 or raptor S792 phosphorylation but produced increased ULK S555 phosphorylation. INS/MET did not recover increased Akt S473, mTOR S2448, p70s6k T389, or IRS‐1 S636 phosphorlyation nor did it recover IR‐ content. No differences were seen between groups for pre‐/post‐synaptic protein content. INS and MET treatments showed reduced NeuN and synaptophysin content. INS/MET treatment showed reductions in synaptophysin. Morphologically, cell size & area, and neurite length & area all increased with INS and MET treatments, however, these increases were absent with the INS/MET treatment. Conclusion These findings indicate that IR and MET impact neuronal morphology and reduce the development of mature neurons. These impairments are likely driven through Akt and AMPK signaling seperately and suggest that MET is not effective in treating IR‐driven impairments in the brian.

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