Novolin

At 6 weeks of age mice were randomly separated into T_S or T_N housing facilities. After 2 weeks of acclimation, mice were fed either an irradiated high fat diet (HFD; Research Diets #D12492; 60% of calories from fat) or a chow diet (LAB Diet #5010; calories provided by carbohydrates [58%], fat [13%] and protein [29%])). All food was replaced weekly to avoid contamination. All mice were fasted overnight prior to, glucose metabolism testing, insulin tolerance testing, or terminal harvest (completed between 7–10am). Glucose and insulin tolerance tests were done as previously described^{11 (link)}. Briefly, following an overnight fast, glucose tolerance levels were determined by injecting mice with 10μL of a 10% dextrose solution per gram of body weight and glucose levels were quantified kinetically at the times shown. For insulin tolerance testing, mice were given 10μL of a 0.15 units/mL solution of insulin (Novolin) per gram of body weight. Hepatic triglyceride deposition, and serum alanine transaminase levels were quantified as previously described^{11 (link)}. NAFLD activity score (NAS) was determined from H&E staining by a certified pathologist according to standard practice^{37 (link)}. Total body fat, lean and water mass were determined by nuclear magnetic resonance (Whole Body Composition Analyzer; Echo MRI)^{68 (link)}.

A nurse unaffiliated with the trial used a table of random numbers to randomly assign participants to receive a daily dosage of 20 IU of insulin (ie, 36 participants received 10 IU of insulin twice a day), 40 IU of insulin (ie, 38 participants received 20 IU of insulin twice a day), or placebo (ie, 30 participants received saline twice a day) for 4 months. Participants were stratified by whether or not they were carriers of the APOE ε4 allele. Saline or insulin (Novolin R; Novo Nordisk, Princeton, New Jersey) was administered after breakfast and dinner with a ViaNase nasal drug delivery device (Kurve Technology, Bothell, Washington) designed to deliver drugs to the olfactory region to maximize transport to the central nervous system. This device released a metered dose of insulin into a chamber covering the participant’s nose, which was then inhaled by breathing regularly for 2 minutes until the prescribed dose was delivered.
Parallel versions of the cognitive and functional protocol were administered at baseline, months 2 and 4 of treatment, and 2 months after treatment. Testing occurred in the morning after a standard meal. Participants were instructed to skip their morning dose on the day of testing and thus had received their last dose more than 12 hours prior to cognitive testing. The coprimary outcome measures were the delayed story recall score and the Dementia Severity Rating Scale (DSRS) score, both of which had previously demonstrated the beneficial effects of insulin.^{13 (link)} The protocol consisted of the following measures: (1) The delayed story recall score^{13 (link)} was determined after a story containing 44 informational bits was read a single time to participants who were then asked to recall the story immediately and after a 20-minute delay. (2) The DSRS score^{15 (link)} was determined after a questionnaire was completed by the study partner; this questionnaire was used to rate the change in the participant’s cognitive, social, and functional status over a specified period of time, with higher scores indicating greater impairment. (3) The Alzheimer Disease’s Assessment Scale–cognitive subscale (ADAS-cog)^{16 (link)} includes measures of memory, praxis, orientation, and language, with higher scores indicating greater impairment. (4) The Alzheimer’s Disease Cooperative Study–activities of daily living (ADCS-ADL) scale^{17 (link)} was completed by the study partner and used to rate the participant’s ability to perform daily activities within the past month, with lower scores indicating greater impairment.