Long-term intranasal insulin aspart: a profile of gene expression, memory, and insulin receptors in aged F344 rats

Intranasal insulin is a safe and effective method for ameliorating memory deficits associated with pathological brain aging. However, the impact of different formulations and the duration of treatment on insulin’s efficacy and the cellular processes targeted by the treatment remain unclear. Here, we tested whether intranasal insulin aspart, a short-acting insulin formulation, could alleviate memory decline associated with aging and whether long-term treatment affected regulation of insulin receptors and other potential targets. Outcome variables included measures of spatial learning and memory, autoradiography and immunohistochemistry of the insulin receptor, and hippocampal microarray analyses. Aged Fischer 344 rats receiving long-term (3 months) intranasal insulin did not show significant memory enhancement on the Morris water maze task. Autoradiography results showed that long-term treatment reduced insulin binding in the thalamus but not the hippocampus. Results from hippocampal immunofluorescence revealed age-related decreases in insulin immunoreactivity that were partially offset by intranasal administration. Microarray analyses highlighted numerous insulin-sensitive genes, suggesting insulin aspart was able to enter the brain and alter hippocampal RNA expression patterns including those associated with tumor suppression. Our work provides insights into potential mechanisms of intranasal insulin and insulin resistance, and highlights the importance of treatment duration and the brain regions targeted. All subjects were male F344 rats. Subjects were young (2 months old) or aged (18 months old) at arrival, and were treated daily for 12 weeks with intranasal insulin aspart or saline (control). On the last week of treatment, all animals underwent Morris water maze protocol. Right hippocampi were dissected from N = 26 animals (young saline n = 6, young aspart n = 5, aged saline n = 8, aged aspart n = 7) were isolated over ice and placed in a -80 °C freezer until processed for RNA extraction. To extract RNA, hippocampi were thawed on ice and homogenized in RiboZol™ Extraction Reagent (#97064-948; VWR®, Radnor, PA). RNA was precipitated with chloroform and isopropanol, then resuspended in a 75% ethanol solution. Following extraction, RNA integrity numbers (RIN) were obtained for each sample using standard protocols (University of Kentucky Genomics Core, Lexington, KY). Mean values for each group were as follows: young saline = 7.32 ± 0.14, young aspart = 7.34 ± 0.15, aged saline = 7.33 ± 0.12, aged aspart = 7.29 ± 0.13. No significant difference in RIN was noted between the groups (2-way ANOVA; p > 0.5). Samples were stored in a -80 °C freezer until thawed for microarray analysis (Affymetrix™ rat Clariom™ S Assay; Thermo Fisher Scientific, Waltham, MA). Gene signal intensities were calculated using the Robust Multiarray Average algorithm at the transcript level and data were associated with vendor-provided annotation information.