Fluorescent but Flawed: Fluorescent Labelling Alters Brain Distribution of Intranasally Administered Insulin
Description
Intranasal insulin and its therapeutic potential in Alzheimer’s disease have already been the focus of clinical trials, generally demonstrating cognitive improvement depending on the disease stage, dose and type of insulin. However, the distribution pattern of insulin in the brain following intranasal administration remains unclear. The aim of this study was to evaluate and compare the absorption and distribution profiles of intranasally administered regular or FITC-labelled insulin in a time-dependent manner. Male Wistar rats were administered 2 IU of FITC-labelled or regular insulin intranasally and sacrificed 3, 7.5, 15, 30, 60 and 120 min after. Control animals were sacrificed without intranasal administration. FITC fluorescence was measured in stomach. Insulin concentrations were measured in plasma, cerebrospinal fluid, nasal epithelia, and brain regions. Distribution patterns observed at 3 minutes post-administration differ significantly between native insulin and FITC-insulin, indicating they may follow distinct transport pathways. Brain levels of FITC-insulin were 10–260% lower across most regions within 3 minutes post-application, indicating uneven and less efficient distribution. Area under the curve analyses confirms slower metabolism and/or elimination of FITC-insulin from the central nervous system in comparison to regular insulin. Our findings demonstrate that the di- and tri-conjugated FITC-insulin mixture does not follow the same absorption timeline or regional distribution as native insulin. These discrepancies raise concerns about relying on FITC-labelled compounds for pharmacokinetic research. These findings emphasize the need for caution when interpreting distribution data based on fluorescently labelled insulin, as the observed patterns may reflect the label’s influence rather than insulin’s native physiology.