Intranasal insulin activates Akt2 signaling pathway in the hippocampus of wild-type but not in APP/PS1 Alzheimer model mice

Type 2 diabetes mellitus (T2DM) increases the risk for Alzheimer's disease (AD). Human AD brains show reduced glucose metabolism as measured by [18F]fluoro-2-deoxy-2-D-glucose positron emission tomography (FDG-PET). Here, we used 14-month-old wild-type (WT) and APPSwe/PS1dE9 (APP/PS1) transgenic mice to investigate how a single dose of intranasal insulin modulates brain glucose metabolism using FDG-PET and affects spatial learning and memory. We also assessed how insulin influences the activity of Akt1 and Akt2 kinases, the expression of glial and neuronal markers, and autophagy in the hippocampus. Intranasal insulin moderately increased glucose metabolism and specifically activated Akt2 and its downstream signaling in the hippocampus of WT, but not APP/PS1 mice. Furthermore, insulin differentially affected the expression of homeostatic microglia markers P2ry12 and Cx3cr1 and autophagy in the hippocampus of WT and APP/PS1 mice. We found no evidence that a single dose of intranasal insulin improves overnight memory. Our results suggest that intranasal insulin exerts diverse effects on Akt2 signaling, autophagy, and the homeostatic status of microglia depending on the degree of AD-related pathology.


Introduction
It is well established that type 2 diabetes mellitus (T2DM) increases the risk for Alzheimer's disease (AD) (Irie et al., 2008;Liu et al., 2011;Vanhanen and Soininen, 1998;Xu et al., 2009). Several postmortem studies have revealed insulin resistance in the AD brain (Liu et al., 2011;Steen et al., 2005) and led to the suggestion that AD represents a "type 3" diabetes mellitus (Steen et al., 2005). T2DM has been associated with brain atrophy in the regions strongly affected in AD, including hippocampus (Moran et al., 2013). Furthermore, based on longitudinal studies, it has been estimated that the rate of global brain atrophy is even 3 times faster in patients with T2DM than in healthy elderly individuals (Kooistra et al., 2013;van Elderen et al., 2010), suggesting that impaired insulin signaling is adversely linked to neurodegeneration. Apart from regulating glucose metabolism in the brain, insulin acts as a neurotrophic factor and thereby may contribute to neuronal development and plasticity (Chiu et al., 2008). One of the key players in insulin signaling is the Akt family of serine/threonine kinases, whose downstream signaling has been associated with crucial physiological functions of the brain, such as promotion of dendritic spine and synapse formation (Lee et al., 2011). Furthermore, insulin/ Akt signaling is linked to the regulation of hyperphosphorylation of tau via glycogen synthase kinase 3b (GSK3b) (Hooper et al., 2008). The Akt family comprises Akt1, Akt2, and Akt3, of which the last is the least well characterized and mainly involved in brain development (Alcantara et al., 2017). The downstream signaling of Akt1, Akt2, and Akt3 is activated upon phosphorylation by mechanistic target of rapamycin complex 2 (mTORC2) at the serine 474, 473, and 472 sites, and by 2-phosphoinositide-dependent kinase 1 (PDK1) at the threonine 309, 308, and 305 sites, respectively (Noguchi and Suizu, 2012). Phosphorylation of both serine and threonine residues is required to fully activate each Akt isoform. However, the distinctive roles of each phosphorylation site remains to be elucidated (Noguchi and Suizu, 2012).
Reduced glucose metabolism in the parietal cortex as measured by fluorodeoxyglucose positron emission tomography (FDG-PET) is 1 of the key brain imaging findings in patients with AD (Cohen and Klunk, 2014). Reduced glucose metabolism in the posterior cingulate cortex and hippocampus can be observed years before the onset of dementia and is associated with increased risk of AD (Mosconi et al., 2006). Intriguingly, similar impairment in regional glucose metabolism to that in the AD brain has been reported in subjects with T2DM (Baker et al., 2011). Although most of the brain glucose uptake is based on insulin-independent glucose transporter subtypes 1 and 3 (Shah et al., 2012), the insulin-dependent glucose transporter 4 is present in certain brain regions, most notably in the hippocampus (Vannucci et al., 1998). Based on these findings, increasing brain insulin levels appears as a rational potential treatment for AD. However, to allow successful utilization of this treatment approach, 2 major challenges need to be overcome. First, the blood-brain barrier limits the passage of systemically administered insulin into the forebrain (Dhuria et al., 2010), although it can relatively freely access the hypothalamus (Ganong, 2000). Second, peripheral administration of insulin automatically leads to hypoglycemia. As demonstrated by animal studies, intranasally administered insulin has access deep into the brain along the olfactory and trigeminal pathways and reaches the brain at therapeutic concentrations (Chapman et al., 2017). Although intranasal insulin transiently raises serum insulin levels and lowers plasma glucose levels within the normal physiological range, these transient effects are considerably less problematic than the sustained increases caused by peripheral delivery of insulin (Chapman et al., 2017). Studies in cognitively healthy humans have shown encouraging results, which indicate that intranasal insulin may improve cognition in a dose-dependent and memory taskedependent manner without major adverse effects (Benedict et al., 2004(Benedict et al., , 2007(Benedict et al., , 2008Brünner et al., 2015;Chapman et al., 2017;Novak et al., 2014). Also clinical trials in individuals with mild cognitive impairment and patients with AD suggest that both acute and chronic administration of intranasal insulin ameliorate multiple aspects of cognition, including verbal memory, memory storage, and selective attention (Craft et al., 2012;Reger et al., 2006Reger et al., , 2008. Cognitively impaired patients also exhibit functional improvement after treatment with intranasal insulin (Craft et al., 2012;Reger et al., 2008). However, although patients not carrying the apolipoprotein E ε4 (APOE ε4) allele mostly benefitted from intranasal insulin treatment (Craft et al., 2012;Reger et al., 2008Reger et al., , 2006, the treatment of APOE ε4 allele carriers has yielded mixed responses (Chapman et al., 2017). Intranasal insulin treatment has also been shown to alter the levels of AD biomarkers in the blood and cerebrospinal fluid (Chapman et al., 2017). A number of animal studies on the effects of intranasal insulin on the hallmark AD brain pathologies and cognitive impairment have generally demonstrated promising results (Chapman et al., 2017). Both acute and repetitive intranasal insulin administration have been reported to improve spatial and object recognition memory, motor learning, and decision-making of wild-type (WT), senescence-accelerated SAMP8, and AD model mice (3xTG) (Apostolatos et al., 2012;Chapman et al., 2017;Mao et al., 2016;Salameh et al., 2015). There are also some studies that do not indicate improvements in memory tests after intranasal insulin treatment in mice (Bell and Fadool, 2017), but such studies are in the minority. Apart from these aforementioned effects, it is expected that intranasal insulin may modulate the function of glia because insulin deficit has been linked to AD-associated neuroinflammation (Zhao and Townsend, 2009). This is a particularly relevant and timely issue given the recent characterization of a unique type of microglia associated with neurodegenerative diseases (disease-associated microglia [DAM]) and characterized by specific RNA profiles (Keren-Shaul et al., 2017). Importantly, DAM have the potential to restrict neurodegeneration, which emphasizes the need for assessing the RNA signature of DAM targets on different treatment procedures, which are aimed to modify AD-associated outcome measures.
Here, we have addressed the effect and mechanisms of intranasal insulin treatment in the well-characterized APP Swe /PS1 dE9 (APP/PS1) transgenic AD mouse model (Borchelt et al., 1997). APP/ PS1 mice develop peripheral glucose intolerance, but not insulin resistance, nor hyperinsulinemia (Hiltunen et al., 2012;Stanley et al., 2016;Takalo et al., 2014). Previous studies in APP/PS1 and APP23 mice, which carry the same APP Swe mutation, have shown that this AD genotype predisposes to insulin resistance but requires another triggering factor, such as the overexpression of insulin-like growth factor 2 (Hiltunen et al., 2012;Takalo et al., 2014), knockdown of leptin (Takeda et al., 2010), or high-fat diet (Hiltunen et al., 2012) to develop insulin resistance. Although the role of insulin signaling along the Akt pathway in the brain has previously been investigated, no studies have yet elucidated the distinctive responses of the Akt1 and Akt2 signaling pathways in different brain areas with respect to insulin treatment. Here, we demonstrate that intranasal insulin treatment increases glucose metabolism in the ventral brain areas and hippocampus of WT mice, but a similar increase is not detected in APP/PS1 mice. We also show that intranasal insulin specifically activates Akt2 and its downstream signaling and differentially affects the expression of homeostatic microglia and autophagy markers in the hippocampus of WT and APP/PS1 mice.

Animals
Fourteen-month-old male APP Swe /PS1 dE9 (APP/PS1) mice (n ¼ 17) and their age-matched WT littermates were used in the study (n ¼ 15). In total, these 32 mice were used during the study. However, a different number of these mice were used in each analysis as indicated in their corresponding methods section. The APP/PS1 colony founders were obtained from D. Borchelt and J. Jankowsky (Johns Hopkins University, Baltimore, MD, USA), while the mice were raised locally at the Laboratory Animal Center in Kuopio, Finland. Mice were created by co-injection of chimeric mouse/human APP Swe and human PS1-dE9 (deletion of exon 9) vectors controlled by independent mouse prion protein promoter elements. The 2 transgenes co-integrated and co-segregate as a single locus (Jankowsky et al., 2004). Mice were backcrossed to C57BL/6J for 21 generations. The mice were kept in a controlled environment (constant temperature, 22 AE 1 C, humidity 50%e 60%, lights on 07:00e19:00) and had food and water available ad libitum. All animal procedures were carried out in accordance with the guidelines of the European Community Council Directives 86/609/EEC and approved by the Animal Experiment Board of Finland.

Treatment
Mice were treated intranasally with natural insulin (Humulin, 100 U/mL; Eli Lilly, Indianapolis, IN, USA) according to the protocol described by Hanson et al. (2013) at a total dose of 30 mL (3.0 U) per animal (Hanson et al., 2013). First, the mice were handled daily for 5 days. During the next week, the mice were familiarized with intranasal application of saline for another 5 days. The first intranasal application for a PET imaging session took place on the following day. The second PET session a week later was also preceded by a practice day with intranasal saline. The mouse was gently held supine on a towel, and a droplet of 3 mL insulin was administered using a pipette into each nostril at a time. The mouse was kept supine for 30 seconds after each administration and then let to walk for 1 minute before the following administration. The same cycle was repeated until the desired total dose was reached. Alternatively, mice were administered 0.9% saline according to the same protocol.

PET imaging
In total, 11 APP/PS1 and 11 WT littermate mice were used in [ 18 F] fluoro-2-deoxy-2-D-glucose (FDG) PET study to assess potential genotype differences and insulin effects on brain glucose uptake. The animals were imaged using a dedicated PET scanner (Inveon DPET; Siemens Healthcare, Erlangen, Germany), followed immediately by computed tomography analysis (Flex SPECT/CT; Northridge Trimodality Imaging, Chatsworth, CA, USA) for anatomical reference images using the same animal holder. The mice were placed on a heated animal holder on the scanner bed in a prone position and secured with adhesive tape to prevent movement during scanning. Each animal underwent PET imaging twice with 1 week between the sessions. Mice were fasted 5e7 hours before the imaging. The mice received either insulin or saline intranasally in a counterbalanced order 4 hours before the imaging session. For anesthesia during imaging, 2% isoflurane was used with N 2 /O 2 flow (70%/30%) through a facemask. Dynamic imaging of 110 minutes was started at the time of the administration of (10.49 AE 0.4) MBq of FDG that was administered through the tail vein. Data were gathered in list-mode form; corrected for dead-time, randoms, scatter, and attenuation; and reconstructed with 2D-OSEM. Regions of interest were drawn for olfactory bulb, whole ventral brain, hippocampus and cerebellum, and heart as a reference using Carimas 2.9 software (Turku PET Centre, Finland). The accumulated activity data were normalized to the accumulated activity in the heart. The time window from 40 to 110 minutes after injection was chosen for further analysis.

Behavioral testing
Some mice (7 APP/PS1 and 4 WT) could not be accepted to the swim test because of wounds in the tail after repeated injections. They were replaced by available male mice of the corresponding genotype and age (6 APP/PS1 and 4 WT). Thus, 10 APP/PS1 and 11 WT mice were included in the behavioral testing. Spatial learning and memory were assessed in the Morris swim task. The test was conducted in a white circular wading pool (diameter 120 cm) with a transparent submerged platform (diameter 14 Â 14 cm) 1.0 cm below the surface serving for escape from the water. The pool was open to landmarks in the room (white screen blocking the view to the computer and the experimenter, green water hose, door, 1-m high black pattern on the wall). Temperature of the water was kept at 20 AE 0.5 C. The acquisition phase was preceded by 2 practice days with a guiding alley to the platform (day-4 and day-3, not shown). During the acquisition phase (days 1e5), the location of the hidden platform was kept constant (SE quadrant) and the starting position varied between 4 different locations at the pool edge, with all mice starting from the same position in a given trial. Each mouse was placed in the water with its nose pointing toward the pool wall. If the mouse failed to find the escape platform within 60 seconds, it was placed on the platform for 10 seconds by the experimenter (the same time was allowed for mice that found the platform). The acquisition phase consisted of 5 daily trials with a 10-minute intertrial interval. The mice received no treatment during the acquisition phase. On day 8, the platform was placed in a new location (NW, different distance from the pool wall), and 5 trials were run as before. After the last trial, the mice received intranasally either insulin or saline and were returned to their home cage. On day 9 (24 hours after treatment), a probe trial of 60 seconds was run without the platform to determine the search bias as an index of spatial memory. On day 13, the platform was placed in a new location (NE), and 5 acquisition trials were run as before. After the last trial, the mice that previously received insulin were given saline intranasally and vice versa. On day 14, the search bias was tested in a 60-second probe trial without the platform. The experimenter was blind to the genotype and treatment of the mice. The mouse was video-tracked, and the video analysis program calculated the escape latency, swimming speed, path length and time in the pool periphery (10 cm from the wall), and the platform zone (diameter 30 cm).

Sample preparation
At the end of the study, all but 1 mouse from those that underwent PET imaging (with or without behavioral testing, 10 APP/ PS1 and 11 WT mice) were treated once more with either intranasal insulin or saline. Two hours later, the mice were deeply anesthetized with pentobarbiturate-chloralhydrate cocktail and transcardially perfused with ice-cold saline for 3 minutes to rinse blood from the brain. The brains were removed, and hippocampi and olfactory bulb were dissected on ice and snap-frozen in liquid nitrogen. The samples were stored at À70 C. Olfactory bulb and hippocampus tissue samples were collected into microcentrifuge tubes and weighed. Samples were homogenized in 250 mL of phosphate-buffered saline (DPBS; Lonza) using a stirrer. Fractions of homogenates were taken to RNA isolation (50 mL of homogenate and 500 mL Trizol) and Western blot analysis (100 mL of homogenate was supplemented with protease inhibitors and phosphatase inhibitors 1:100; Thermo Scientific). The remaining 100 mL of homogenate was left unprocessed and stored at À80 C.

Statistics
IBM SPSS, versions 21 and 23, were used to analyze the data. The PET data were analyzed using the paired-sample t-test. The acquisition phase of the Morris swim task was analyzed with the analysis of variance (ANOVA) for repeated measures (ANOVA-RM) using day as the within-subject factor and genotype as the between-subjects factor. Spatial search bias during the probe trials was analyzed with ANOVA-RM using treatment as the within-subject factor and genotype as the between-subjects factor. Statistical comparisons of biochemical analysis results were performed using two-way ANOVA followed by Fisher's least significant difference post hoc test. Statistical comparisons of correlations were performed using Spearman's rho test. Results are expressed as mean AE standard error of mean of control samples; p-values <0.05 were considered statistically significant.

Results
To study the effects of intranasal insulin in the AD brain, we used the APP/PS1 mouse model and age-matched WT littermates. After intranasal insulin or saline treatment, the mice underwent FDG-PET imaging to assess glucose metabolism in different brain regions. Subsequently, the spatial memory of the mice was analyzed using the Morris swim task after intranasal insulin or saline administration. With some exceptions, the same animals underwent FDG-PET and spatial memory testing twice, with intranasal insulin or saline treatment, and were then divided into intranasal insulin or saline groups before sacrificing. The timeline of the FDG-PET imaging and spatial memory testing is illustrated in Figure 1. To study the biochemical effect of intranasal insulin, the brain regions of interest were collected and homogenized.

Moderately increased glucose uptake in FDG-PET in ventral brain regions after intranasal insulin in WT mice as compared with APP/PS1 mice
The effect of intranasal insulin treatment on glucose uptake was determined separately for the olfactory bulb, cerebellum, and ventral half of the remaining brain using anatomical boundaries by FDG-PET imaging ( Fig. 2A). In addition, the hippocampal voxels were extracted using an Atlas-based template for quantification of hippocampal FDG uptake. The main insulin effect on FDG uptake was nonsignificant (F 1,13 ¼ 2.2, p ¼ 0.17). The effect approached significance in the ventral brain (F 1,13 ¼ 4.2, p ¼ 0.06; Fig. 2B) but was nonsignificant in other brain regions (hippocampus, p ¼ 0.08; cerebellum, p ¼ 0.11; olfactory bulb, p ¼ 0.54). There was no statistically significant main effect of genotype (p > 0.26 in all brain regions).  The sequence of procedures during the study is shown above the timeline with indicated duration in days (d) for each procedure. The numbers below the timeline denote the start days of each procedure. (B) Detailed description of the Morris swim task. The task began with 5 days of acquisition of the first platform position (SE). A new platform position (NW) was introduced on day 8, and the memory was tested with a probe without platform on day 9. Similarly, another new platform position was introduced on day 13 (NE), followed by a probe test on day 14. The arrows denote administration of intranasal saline or insulin 4 hours before PET imaging on days 14 and 21 (A) as well as after 5 trials of task acquisition on days 8 and 13 (B). Indices: i.n. ¼ intranasal, Sal ¼ saline, Ins ¼ insulin, Acq ¼ acquisition. Abbreviations: PET, positron emission tomography; WT, wild type.

Postacquisition intranasal insulin impairs spatial memory retention overnight in both WT mice and APP/PS1 mice
Here, our aim was to test whether a single intranasal dose of insulin given after the acquisition period affects encoding and consolidation of a discrete memory (unique platform location) in mice. At all stages of task acquisition (learning the initial platform location on days 1e5, as well as learning a new location on day 8 and day 13), it took longer for APP/PS1 mice to find the escape platform than for WT mice (F 1,19 ¼ 21.2, p < 0.001; Fig. 3A). On the other hand, the genotypes did not differ in their swimming speed (F 1,19 ¼ 0.5, p ¼ 0.47). After learning a new platform location on day 8 or day 13, the mice were given intranasal insulin or saline, and their memory retention for the newly learned versus original (days 1e5) platform location was tested in a 60-second probe trial without the platform on days 9 and 14. There was no main effect of postacquisition insulin on remembering the newly learned platform location 24 hours later (F 1,19 ¼ 0.0, p ¼ 1.0; Fig. 3B). In contrast, the genotype main effect was statistically significant, so that APP/PS1 mice spent less time in the new platform zone (F 1,19 ¼ 6.0, p ¼ 0.02; Fig. 3B). On the other hand, WT mice spent also more time in the original platform zone than APP/PS1 mice (F 1,16 ¼ 7.5, p ¼ 0.01; Fig. 3B). Intranasal insulin further increased the time in the original platform zone, more clearly for APP/PS1 mice, but also for WT mice, so that the main treatment effect became significant (F 1,16 ¼ 6.6, p ¼ 0.02; Fig. 3B). Importantly, intranasal insulin had no effect on the swimming speed (F 1,19 ¼ 0.1, p ¼ 0.73), speaking against any systemic hypoglycemic effect of the treatment. These findings suggest that a single intranasal dose of insulin given right after the learning session impairs memory retention at least when assessed 24 hours later.

Insulin treatment activates Akt2 in the WT, but not in the APP/ PS1 mouse hippocampus
Because intranasal insulin treatment showed the most prominent effect on glucose uptake in FDG-PET in the ventral brain regions, including the hippocampus, we next focused on the hippocampus to elucidate the role of Akt kinases, central mediators in the insulin signaling pathway, in this process. The olfactory bulb was used as an insulin nonresponsive control region. To investigate the activation of Akt kinases, we assessed the level of the phosphorylation of Akt1 and Akt2 at the serine 473 and serine 474 residues, respectively, in hippocampal lysates of WT and APP/PS1 mice treated with intranasal insulin or saline. Phosphorylation at these sites leads to activation of the Akt kinase activity. Insulin treatment did not affect the phosphorylation level of Akt1 at serine 473 residue in either the olfactory bulb (F 1,14 ¼ 1.7, p ¼ 0.21; Fig. 4A) or the hippocampus (F 1,14 ¼ 0.1, p ¼ 0.79; Fig. 4A). However, increased total levels of Akt1 protein (phosphorylated þ nonphosphorylated) were observed in the olfactory bulb of both saline- and insulin-treated APP/PS1 mice as compared with WT mice (F 1,14 ¼ 10.6, p ¼ 0.006; Fig. 4A). Interestingly, insulin treatment increased the phosphorylation of Akt2 at serine 474 residue by w70% in the WT mouse hippocampus, but not in the APP/PS1 mouse hippocampus (Fig. 4B). No treatment effect was seen (F 1,14 ¼ 2.1, p ¼ 0.17). However, when 2 obvious outliers (1 in saline and 1 in insulin group) were removed in the APP/PS1 group of mice, the treatment main effect became statistically significant (F 1,12 ¼ 11.1, p ¼ 0.006). We also analyzed the phosphorylation status of Akt1 and Akt2 at other phosphorylation sites regulating Akt kinase activity at threonine 308 and 309 residues, respectively (this antibody does not differentiate between the Akt isoforms). However, insulin treatment did not affect the phosphorylation status of Akt1 or Akt2 at threonine 308 or 309 in either the olfactory bulb (F 1,14 ¼ 2.9, p ¼ 0.11; Fig. 4C) or the hippocampus (F 1,14 ¼ 0.04, p ¼ 0.85; Fig. 4C).
3.4. The levels of autophagosomal markers p62 and LC3-I are altered on insulin treatment in the WT, but not in the APP/PS1 mouse hippocampus After the observation that insulin treatment specifically activated Akt2 in the hippocampus of WT mice (Fig. 4B), we wanted to study the downstream effects of Akt2 activation. We analyzed the phosphorylation levels of GSK3b at the inhibitory serine 9 residue and tau protein at the serine 202 and threonine 205 and serine 208 residues in the hippocampal lysates of WT and APP/PS1 mice treated with intranasal saline or insulin. Insulin treatment did not affect the phosphorylation status of GSK3b at Ser9 inhibitory residue in the hippocampus of WT or APP/PS1 mice (F 1,15 ¼ 2.2, p ¼ 0.16; Fig. 5A). However, we observed a significant genotype effect (F 1,15 ¼ 8.4, p ¼ 0.01), such that the phosphorylation levels of GSK3b at Ser9 were 36% lower in the insulin-treated APP/PS1 hippocampus than in the insulin-treated WT hippocampus (Fig. 5A). Total GSK3b protein levels were significantly elevated by w30% in the hippocampus of APP/PS1 as compared to WT mice in both salineand insulin-treated groups (F 1,15 ¼ 22.7, p < 0.001; Fig. 5A). Insulin treatment had no significant main effect on phosphorylation of tau protein in the hippocampus (F 1,15 ¼ 0.1, p ¼ 0.72; Fig. 5B) when using an antibody detecting phosphorylated Ser202, Thr205, and Ser208 residues in tau. However, there was a significant genotype Â treatment interaction, such that insulin decreased phosphorylated tau levels in WT mice but increased those in APP/PS1 mice (F 1,15 ¼ 5.0, p ¼ 0.04; Fig. 5B). In addition, owing to the intimate link between Akt and mammalian target of rapamycin complex 1   hippocampus of WT and APP/PS1 mice treated with saline (Sal) or insulin (Ins) showed that insulin had no effect on the phosphorylation level of Akt1 at serine 473 (Ser473). Total Akt1 levels were increased in the olfactory bulb of both saline-and insulin-treated APP/PS1 mice as compared to WT mice. (B) Insulin treatment moderately increased the phosphorylation of Akt2 at serine 474 (Ser474) in the WT hippocampus, but not in the APP/PS1 hippocampus. (C) Phosphorylation levels of the threonine 308 and 309 (Thr308/309/ 305) sites in Akt1, Akt2, and Akt3, respectively, were not altered after insulin treatment in either brain area. Phosphorylated protein levels were normalized to their respective total protein levels in cell lysates, and total protein levels were normalized to those of GAPDH in each sample. All results are shown as mean % þ SEM of WT OB Sal. n ¼ 4e5, two-way ANOVA, post hoc LSD. *p < 0.05, **p < 0.01. Abbreviations: ANOVA, analysis of variance; LSD, least significant difference; WT, wild type.
(mTORC1) signaling (Caccamo et al., 2018), we next investigated whether insulin treatment affected the levels of autophagosomal markers p62 and LC3 in the hippocampus of WT and APP/PS1 mice treated with intranasal saline or insulin. There was a significant genotype Â treatment interaction in p62 levels (F 1,13 ¼ 6.6, p ¼ 0.02) such that saline-treated APP/PS1 mice showed lower p62 levels than WT mice, but no genotype difference was found among insulin-treated animals (Fig. 5C). Conversely, insulin treatment decreased p62 levels on average by 30% in WT mice but had no effect in APP/PS1 mice. LC3-I levels were lower in APP/PS1 mice than WT mice (F 1,12 ¼ 7.3, p ¼ 0.02), and insulin tended to decrease LC3-I levels in both genotypes (F 1,12 ¼ 4.4, p ¼ 0.06) (Fig. 5C).
3.5. FDG uptake correlates with the Akt2 activity in the WT, but not in the APP/PS1 mouse hippocampus Because intranasal insulin treatment increased both FDG uptake and the phosphorylation levels of Akt2 in serine 474 in the WT mouse hippocampus (Figs. 2B and 4B), we next investigated the possible correlation between these results. As expected, there was a significant positive correlation between FDG uptake and Akt2 phosphorylation levels in the hippocampus of saline-or insulintreated WT (n ¼ 6, r ¼ 0.94, p ¼ 0.005; Fig. 6A) but not in  Fig. 6. Akt2 phosphorylation correlates with glucose uptake in the WT, but not in APP/ PS1, mouse hippocampus. The correlation analysis of WT (A) or APP/PS1 (B) mice treated with saline or insulin revealed a significant correlation between FDG uptake and Akt2 phosphorylation levels in the WT hippocampus (n ¼ 6, r ¼ 0.94, p ¼ 0.005), but not in the APP/PS1 hippocampus (n ¼ 7, r ¼ À0.14, p ¼ 0.76). FDG uptake was assessed using PET imaging. Accumulated [18F] activity (Bq/mL) in the hippocampus was normalized to accumulated activity in the heart, and results are shown as relative activity. Akt2 phosphorylation levels were analyzed using Western blotting. Phosphorylated protein levels were normalized to total Akt2 protein levels and are shown as % of the levels in WT Sal samples. Abbreviations: FDG, [ 18 F]fluoro-2-deoxy-2-Dglucose; PET, positron emission tomography; WT, wild type.
3.6. Intranasal insulin differentially affects the expression of homeostatic microglia markers in the hippocampus of WT and APP/ PS1 mice Apart from regulating GSK3b by inhibitory phosphorylation, Akt2 inhibits FoxO1, which is a transcription factor known to be a key regulator of endogenous glucose production (Latva-Rasku et al., 2017). To investigate the transcriptional response of FoxO1 to insulin treatment, we analyzed the expression levels of 3 known target genes of FoxO1, namely, Rictor, Sestrin3 (Sesn3), and Pak1 (Chen et al., 2010;de la Torre-Ubieta et al., 2010), in the hippocampus of WT and APP/PS1 mice. The expression levels of Rictor (F 1,15 ¼ 15.6, p ¼ 0.001) and Sesn3 (F 1,16 ¼ 12.7, p ¼ 0.003) showed a significant genotype Â treatment interaction, such that their expression levels were reduced on insulin treatment in the WT hippocampus but increased in the APP/PS1 hippocampus (Fig. 7A).

Discussion
In this study, we have investigated how intranasal insulin treatment modulates glucose uptake, Akt signaling cascade, the phosphorylation status of Tau protein, autophagy, and the expression of glial and neuronal markers in WT and APP/PS1 mice. Furthermore, we assessed how this treatment affects spatial learning and memory in these mice. We also for the first time delineated the specific involvement of Akt1 and Akt2 in the hippocampus on intranasal insulin treatment. FDG-PET imaging showed that insulin treatment increased glucose uptake in the ventral brain and hippocampus of WT mice, but not APP/PS1 mice. In the Morris swim task, we observed that WT mice learned the first location of the platform and subsequently found the new platform locations significantly faster than APP/PS1 mice. However, intranasal insulin treatment delayed the learning of new platform locations of both WT and APP/PS1 mice. We also found out that intranasal insulin treatment specifically activated Akt2, but not Akt1, in the hippocampus of WT mice, whereas a similar effect was not observed in APP/PS1 mice. Consistent with the insulin effect on the activation of Akt2 signaling, insulin treatment decreased the transcriptional activity of FoxO1, a downstream target of Akt2, and a key regulator of endogenous glucose production (Latva-Rasku et al., 2017). This was shown by significantly lower expression levels of FoxO1-regulated target genes Rictor and Sesn3 in the hippocampus of WT mice, but not in APP/PS1 mice. Thus, our results suggest that on intranasal insulin treatment, Akt2 is specifically activated in the hippocampus of WT mice, which in turn coincides with the increased uptake of FDG and altered FoxO1-mediated gene expression in the hippocampus. Importantly, these changes did not take place in the hippocampus of APP/PS1 mice, suggesting a disadvantageous link between AD-associated genetic background and insulin-induced Akt2 signaling in the brain. Also, the observations that intranasal insulin differentially affected the expression of homeostatic microglia markers P2ry12 and Cx3cr1 and the levels of autophagosomal markers p62 and LC3-I between APP/PS1 and WT mice suggest diverse cellular responses for insulin in the brain tissue depending on the degree of AD-related pathology.
Previous studies have assessed changes in Akt levels in the context of brain insulin signaling without assessing the contribution of the individual Akt subtypes (Stanley et al., 2016;Takeda et al., 2010). Here, we show that Akt2 is the Akt family member, which is specifically activated on intranasal insulin treatment in the healthy mouse brain. This is an interesting finding as the key role of Akt2 in glucose metabolism is supported by population-based studies in humans demonstrating that individuals carrying the AKT2 P50T genetic variant have on average a 40% reduction in glucose uptake in the whole body (Latva-Rasku et al., 2017). We observed that the insulin-induced activation of Akt2 was associated with an increased phosphorylation status at serine 474 residue known to be phosphorylated by mTORC2, but not at threonine 308/ 309/305, which are phosphorylated by PDK1. It should be noted, however, that we cannot completely rule out the possibility that insulin might also activate Akt2 via threonine 309 as the antibody used detects phosphorylated threonine 308/309/305 residues in all Akt1, Akt2, and Akt3 isoforms, respectively, and thus the effect on Akt2 may be masked. Previous studies have demonstrated a more robust insulin effect on Akt phosphorylation in the mouse hypothalamus (Takeda et al., 2010) and hippocampus (Stanley et al., 2016). However, these studies have either used genetically obese leptin-deficient mice (Takeda et al., 2010), which have reduced Akt phosphorylation levels in the hippocampus and cortex (Clodfelder-Miller et al., 2005), or delivered insulin directly into the hippocampus via reverse microdialysis (Stanley et al., 2016). Although insulin receptors are found in the olfactory bulb (Duarte et al., 2012), we did not observe insulin-related effects in the olfactory bulb of WT or APP/PS1 mice. These findings are consistent with a recent study in aged rats, showing significant metabolic effect of acute intranasal insulin in the ventral brain, excluding the olfactory bulb (Anderson et al., 2017).
In addition to regulating glucose metabolism, insulin has been shown to play a key role in neuronal development, neurotransmission, neuroprotection, and learning and memory (Duarte et al., 2012). However, it should be emphasized that not all studies have reported positive effects of intranasal insulin treatment on memory (Chapman et al., 2017). This may derive from variations in the dose that reaches the critical brain region (which is difficult to estimate) and also from the timing of the administration. Our aim here was to test whether a single dose of intranasal insulin given after the acquisition period affects encoding and consolidation of a discrete memory (unique platform location) in mice. Testing 24 hours after the insulin treatment did not find evidence for enhanced spatial memory. Rather, mice in the insulin group remembered better the original platform location than the new platform locations during the task acquisition phase than the saline-treated mice. This suggests that insulin might have impaired memory related to the most recent platform location. Interestingly, a recent study in aged rats under similar test conditions revealed that a single intranasal dose of insulin given on the test day (24 hours since learning) did not improve memory recall (Anderson et al., 2017). In general, the most beneficial effects of intranasal insulin have been obtained with repetitive dosing (Chapman et al., 2017), suggesting involvement of slowly developing metabolic or biochemical processes in insulin effects. Recent studies have found that insulin promotes dendritic spine and synapse formation in rat hippocampal primary neuron cultures (Lee et al., 2011). However, we did not find changes in Bdnf expression levels in the hippocampus of insulin-or saline-treated middle-aged WT or APP/PS1 mice, suggesting that a single dose of intranasal insulin did not induce a marked neurotrophic response. This is consistent with the lack of positive insulin effect on spatial memory, but most likely not the only underlying mechanism that remains to be disclosed in future studies.
Downstream of Akt2 signaling, the FoxO-Pak1 transcriptional pathway regulates neuronal polarity (de la Torre-Ubieta et al., 2010). Akt2 inhibits the transcription factor FoxO1 by phosphorylating it, after which FoxO1 translocates from the nucleus to the cytosol (Dong, 2018). Here, we observed a moderate downregulation of Pak1, a target gene of FoxO1, in response to insulin treatment in the hippocampus of WT mice, but not in APP/PS1 mice, reinforcing the idea of impaired insulin signaling pathways in APP/PS1 mice. Insulin-induced PI3K-Akt-mTOR pathway is also closely linked to autophagosomal regulation (Caccamo et al., 2018), and the rapamycin-mediated mammalian target of rapamycin (mTOR) inhibition has been shown to decrease the levels of Ab 42 in the hippocampus and consequently ameliorate the memory deficits by increasing autophagy in AD mouse models (Caccamo et al., 2010;Spilman et al., 2010). In the present study, we observed a significant decrease in the levels of autophagosomal markers p62 and LC3-I in the hippocampus on intranasal insulin treatment in WT, but not in APP/PS1 mice. Also, these markers were significantly lower in saline-treated APP/PS1 mice than WT mice, suggesting that their autophagosomal activity was already altered in the basal condition. Because we were unable to detect the phosphatidylethanolamineconjugated form of LC3 (LC3-II), it is difficult to interpret whether decreased levels of LC3-I reflect increased autophagosomal activity. However, simultaneous reduction of p62 levels on intranasal insulin treatment in WT mice further suggests increased, rather than decreased, autophagosomal activity. This is contrary to what one would expect if Akt activation leads to mTORC1 activation and thereby inhibition of autophagy . On the other hand, some reports suggest that Akt phosphorylated at threonine 308, but not at serine 473, is required for mTORC1 activation (Guertin et al., 2006;Rodrik-Outmezguine et al., 2011). We observed here insulin-induced phosphorylation of Akt2 predominantly at serine 474 and not threonine 309 (corresponding to serine 473 and threonine 308 in Akt1).
Apart from the differential activation of Akt2 in the hippocampus of WT and APP/PS1 mice on intranasal insulin treatment, the homeostatic microglia markers, P2ry12 and Cx3cr1, displayed dissimilar expression response between APP/PS1 and WT mice on insulin treatment. However, the expression analysis of the astrocytic marker Gfap did not reveal prominent insulin-dependent changes. These are important findings given the recent advances in the characterization of a unique microglia type associated with neurodegenerative diseases (DAM) (Keren-Shaul et al., 2017). DAM are regulated through a two-step activation mechanism in the mouse models of AD. In the first step, the increased expression of specific set of genes such as TREM2-signaling adaptor protein (Tyrobp) is observed, whereas the homeostatic microglial markers, such as P2ry12 and Cx3cr1, show decreased expression. The second step, which is TREM2-dependent, involves a switch toward increased expression of a specific set of genes, such as Trem2 and Cst7. Importantly, DAM have the potential to restrict neurodegeneration, which emphasizes the importance of assessing the RNA signature of DAM targets on different treatment procedures, which are aimed to modify AD-associated outcome measures. Although intranasal insulin did not affect the expression of DAM target genes, we detected the expected increase of Trem2, Tyrobp, and Cst7 expressions in the hippocampus of APP/PS1 mice as compared to WT mice (Ulrich et al., 2018). Importantly, the fact that intranasal insulin increased the expression of both P2ry12 and Cx3cr1 in the hippocampus of APP/PS1 mice, but tended to decrease it in WT mice, suggests that intranasal insulin treatment significantly promotes the prevailing homeostatic status of microglia in APP/PS1 mice as compared to WT mice. Thus, it is important in future studies to assess, in a similar experimental setting, the effects of intranasal insulin on the microglia status by, for example, analyzing their activity using PET radiotracers available for microglia. Collectively, these are crucial findings in the context of ongoing intranasal insulin trials targeted against AD and mild cognitive impairment because insulin deficit has been linked intimately to AD-associated neuroinflammation ("Nasal Insulin," n.d.).
Although previous studies with intranasal insulin in humans and rodents have shown generally encouraging results in improving cognition, they have not consistently demonstrated if intranasal insulin treatment ameliorates tau pathology in AD mouse models (Chapman et al., 2017). We did not observe significant changes in the phosphorylation at the inhibitory Ser9 site of the Akt2 downstream target GSK3b, the main kinase phosphorylating tau, in either WT or APP/PS1 mice. In line with this, no changes in tau phosphorylation in these mice were detected. Nevertheless, we found a significant positive correlation between Akt2 (Ser474) and GSK3b (Ser9) phosphorylation levels in the hippocampus of insulin-treated WT and APP/PS1 mice, confirming that increased activation of Akt2 results in inhibition of GSK3b activity. In addition, there was a significant negative correlation between Akt2 (Ser474) and tau (Ser202, Thr205 and Ser208) phosphorylation levels, suggesting that Akt2 activation leads to decreased tau phosphorylation likely through decreased activity of GSK3b (Hooper et al., 2008). These correlations are consistent with insulin-induced activation of Akt2. However, the timing and repetition of the insulin administration appear to crucially affect the results in behavioral testing, in vivo imaging, physiological analysis, and postmortem biochemical analysis (Chapman et al., 2017). We also suspect that while acute insulin treatment increases glucose uptake in the brain, chronic or repeated treatment may be required to achieve trophic effects, such as restoring synaptic plasticity and eventually ameliorating cognition in AD mouse models or patients with AD.
In conclusion, we demonstrate here that intranasal insulin treatment moderately increases glucose uptake in the WT mouse hippocampus via activating the Akt2 signaling pathway. We also suggest that peripheral glucose intolerance in APP/PS1 mice could associate with impaired insulin-induced Akt2 signaling in the brain. Our results highlight the therapeutic potential of intranasally administered insulin as a noninvasive delivery method, which overcomes the blood-brain barrier and reaches the brain at biochemically effective levels. Further studies with chronic intranasal insulin treatment are required to elucidate the downstream effects of Akt2 activation in the hippocampus as well as the potential trophic effects of insulin on recovering synaptic plasticity and enhancing cognition in the AD brain.

Disclosure
The authors have no actual or potential conflicts of interest.