Detection of Brain‐derived Neurotrophic Factor Expression in Insulin‐Resistant SH‐SY5Y Neuroblastoma Cells

Abstract

AbstractBackgroundMore researchers are coining Alzheimer’s disease (AD) “type 3 diabetes” due to its significant comorbidity and shared pathology with type 2 diabetes mellitus (T2DM). A bi‐directional relationship between T2DM and AD exists, by which 50‐75% of individuals with T2DM have a greater risk of developing AD compared to non‐diabetes and that AD symptoms progress with impaired glucose utilization. Interestingly, brain‐derived neurotrophic factor (BDNF), a small protein that maintains neuron health and glucose homeostasis, has been implicated in both AD and IR. As such, this experiment investigates if insulin resistance (IR) alters BDNF expression in SH‐SY5Y neuroblastoma cells.MethodObjective 1: create a model of insulin resistance at 24, 48, 72 and 96 hours (hr) and determine expression of pAkt (S473), tAkt, insulin receptor b (INSRB) and pINSR (T1362) on Western blots. (n = 5)Objective 2: Detect mRNA and protein BDNF via Rt‐PCR and Western blotting. (n = 5)Objectives 3: Determine alterations to BDNF expression via Western blotting and qPCR. (n = 5)ResultLevels of pAkt expression were significantly decreased in insulin‐pretreated and activated samples at each timepoint of 24, 48, 72 and 96 hr; although, with each timepoint, pAkt does appear to steadily increase. pro‐BDNF and mature BDNF were detected at their respective sizes, 27kDa and 19kDa, on a Western blot and BDNF mRNA was detected on an agarose gel after PCR.ConclusionIt appears that insulin resistance is occurring at 24, 48, 72 and 96 hr solely based off Akt expression. This result will be further confirmed with the the completion of INSR‐B and p‐INSR blots. Ongoing experiments include western blots and qPCRs of BDNF to determine if there is any alteration to expression.