Intranasal Insulin Transport is Preserved in Aged SAMP8 Mice and is Altered by Albumin and Insulin Receptor Inhibition

Abstract

Insulin delivered to the level of the cribriform plate (intranasal insulin) is being investigated for its ability to enhance memory in people with Alzheimer’s disease (AD). Recent work has shown intranasal insulin can be detected in young CD-1 mice within 5 min and is still present 60 min after injection. The current study determined whether intranasal insulin transport and the subsequent brain distribution of insulin varies in young, healthy mice (CD-1) compared to those with an AD-like phenotype (aged SAMP8) or those pre-disposed to develop such a phenotype (young SAMP8). We showed transport does not vary among these three mouse cohorts, suggesting that intranasal uptake and brain pharmacokinetics do not differ with AD-like signs or the genetic predisposition to developing them. We found that co-administration with bovine serum albumin increased levels of insulin in most brain regions. In addition, the insulin receptor inhibitor, S961, decreases the amount of insulin transported throughout the brain after intranasal injection. These results show insulin delivery to the brain by intranasal administration can be modified with agents such as albumin, may be dependent on the insulin receptor, and is not affected by an AD-like phenotype as presented by the SAMP8 mouse.

Keywords

Alzheimer’s disease insulin intranasal administration pharmacokinetics

INTRODUCTION

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease. Alterations in brain insulin metabolism are suggested to be an underlying factor to this disease [1 –3]. Finding ways to increase brain insulin has been explored as a way to ameliorate cognitive deficits. Indeed, intracerebroventricular delivery of insulin in young rats has been shown to improve memory along with enhancing insulin signaling [4]. Another, more therapeutic way to increase brain insulin is through intranasal insulin. Delivery of insulin via the nares to the level of the cribriform plate results in its uptake into the central nervous system, negating the need to cross the blood-brain barrier (BBB) [5] and avoiding the peripheral side effects of insulin, including hypoglycemia [6]. Within the last decade, intranasal insulin has been shown to improve memory not only in young, healthy subjects, but also in older, demented adults with mild cognitive impairment or AD [7 –9]. In addition, Anderson et al. has shown that in aged rats intranasal Apidra (a zinc-free form of insulin) can increase insulin signaling in ventral brain areas 2 hours after intranasal delivery [10] and other insulin analogs, including Humalog and Levimir, can improve memory as well as reduce the Ca^2 +-dependent hippocampal after hyperpolarization [11]. However, these studies have not established the transport kinetics of insulin or the regional distribution of this peptide after intranasal delivery.

Intranasal delivery of peptides directly to the brain is increasingly gaining interest due to the translatable nature of delivery. Brain delivery from the nasal mucosa can occur along two principal cranial nerve routes, the olfactory and trigeminal nerve pathways, after crossing the olfactory epithelium [12]. Transport along these pathways can occur either via intracellular (endocytosis) or extracellular (perivascular or perineuronal) pathways. Transport along the perivascular space is involved in the rapid transport of intranasally administered tracers to widespread brain areas [13]. In addition, the perineuronal space can interact with the cerebrospinal fluid (CSF), also allowing for intranasally delivered substrates to distribute throughout the brain [12].

The blood-to-brain transport of insulin is not altered in a mouse model of AD, the SAMP8 mouse [14]. The SAMP8 is a strain used as a model of sporadic AD that with aging develops features similar to those observed in AD, including cortical atrophy, neuronal cell loss, gliosis, oxidative stress, increased amyloid-β peptide levels, tau hyperphosphorylation, decreased brain-to blood efflux of amyloid-β peptide, impaired bulk flow/glymphatic drainage, and deficits in learning and memory by 8 months of age [15]. However, it is unclear whether the transport of intranasal insulin to various brain regions is altered in AD. As current studies are focusing on intranasal insulin as a way to treat AD, we sought to investigate the transport of insulin throughout the brain after intranasal delivery in aged SAMP8 mice. We compared these values to young SAMP8 mice as well as across strains using CD-1 mice. We also determined whether the carrier protein, albumin, or the insulin receptor inhibitor, S961, alter transport of insulin after intranasal delivery.

MATERIALS AND METHODS

Animal use

Male CD-1 mice (2 mo) were purchased from Charles River Laboratories (Wilmington, MA). SAMP8 mice (young: 2 mo and aged: 12 mo) were kindly provided by Dr. Sue Farr (St. Louis, MO). Mice had ad libitum access to food and water and were kept on a 12/12 h light/dark cycle until study. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Veterans Affairs Puget Sound Health Care System and performed at an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) approved facility.

Radioactive labeling

Ten micrograms of human recombinant insulin (Sigma-Aldrich, St. Louis, MO) was radioactively labeled with 0.5 mCi Na ¹²⁵I (Perkin Elmer, Waltham, MA) by the chloramine-T (Sigma-Aldrich, St. Louis, MO) method. Addition of 10 μg of chloramine-T in 0.25 M chloride-free sodium phosphate buffer, pH 7.5, began the reaction. After 1 min, the reaction was terminated by adding 100 μg of sodium metabisulfite. Radioactively labeled insulin (¹²⁵I-insulin) was purified on a column of Sephadex G-10 (Sigma-Aldrich, St. Louis, MO) and collected in 100 μL lactated Ringer’s (LR) solution in glass tubes coated with Sigmacote® (Sigma-Aldrich, St. Louis, MO) to prevent sticking. Protein labeling by iodine was characterized by 30% trichloroacetic acid (TCA) precipitation. Greater than 90% radioactivity in the precipitated fraction was consistently observed.

Intranasal administration

Mice were anesthetized with an intraperitoneal injection of 40% urethane solution (Sigma-Aldrich, St. Louis, MO). With each mouse in the supine position, a single 1 μL injection of 5×10⁵–1×10⁶ CPM ¹²⁵I-insulin in LR was administered in the left nares to the level of the cribriform plate (4 mm depth) using a 10 μL Multi-flex tip (Thermo Fisher Scientific, Waltham, MA). There was no force applied during the administration to avoid puncturing the cribriform plate. In addition, after administration, the tip was investigated to confirm there was no blood present which could suggest puncturing of the cribriform plate. The mouse remained in the supine position for 30 s before being rolled to the right side. For the studies investigating the free ¹²⁵I transport, stock ¹²⁵I was diluted to 8×10⁵ CPM/μL. For the studies with co-administration of albumin, 1 μL injection was given in each naris to inject a total of 5×10⁵ CPM ¹²⁵I-insulin mixed with bovine serum albumin dissolved in LR for a final concentration of 0.5% albumin per mouse. For studies with co-administration of S961 [16], S961 was dissolved in ¹²⁵I-insulin, for a final concentration of 1 μg S961 per mouse.

Sample collection

Blood was collected from the right carotid artery and the whole brain and olfactory bulbs (Olf) were removed at times 2.5–60 min after intranasal administration. The brain was dissected into regions, including cortex (Cortex), striatum (Str), hypothalamus (Hy), hippocampus (Hpc), thalamus (Th), cerebellum (Cb), and midbrain (MBr) on ice by the method of Glowinski and Iversen [17]. Whole brain levels were calculated by combining the radioactivity for each brain region and dividing by the combined weights. Whole blood was centrifuged at 3200 xg for 10 min and a 50 μL aliquot of the serum was collected for radioactivity measurement. The amount of radioactivity in each of the brain regions, olfactory bulb, and serum was measured in a Wizard2 Automatic Gamma Counter (PerkinElmer, Waltham, MA) for 30 min. The percentage of the injected dose in one mL of serum (% Inj/mL) was calculated by:

% Inj/mL = 100(CPM/mL)/Inj

where Inj is the CPM administered and CPM/mL is the level of radioactivity in one mL of serum. The percent of injected dose taken up per gram of brain region (% Inj/g) was calculated at each time point using the following equation:

% Inj/g = 100(CPM)/[(Inj)W]

where Inj is the CPM administered, W is the weight of the given brain region in grams, and CPM is the level of radioactivity in each brain region.

To determine levels of insulin reaching the brain regions at 30 min, ¹²⁵I-insulin was co-injected with 1 μg of unlabeled insulin. The percentage of the injected dose taken up by brain was calculated as described above. Values were then multiplied by 1 μg and divided by 100 in order to determine the ng insulin/g of brain region.

Statistics

Data were analyzed using Prism 6.0 (GraphPad Software Inc., San Diego, CA). Means are reported with the standard error terms and compared by two-way ANOVA when multiple groups were compared or one-way ANOVA when only one group was compared for differences in time, followed by Bonferroni’s multiple comparisons test.

RESULTS

¹²⁵I-Insulin transport after intranasal delivery

To measure the brain distribution of ¹²⁵I-insulin after intranasal delivery, brain regions were examined 5, 15, 30, and 60 min after intranasal administration. Figure 1 shows the percent of the administered intranasal ¹²⁵I-insulin taken up per gram of brain region (% Inj/g) or per mL of serum (% Inj/mL). Each region had ¹²⁵I-insulin present by 5 min and remaining at 60 min. There was no group effect as assessed by two-way ANOVA on ¹²⁵I-insulin transport. There was a significant effect of time in the cortex, thalamus, cerebellum, and whole brain. ¹²⁵I-insulin was detected in the serum within 5 min after intranasal delivery and was still present up to 60 min after administration (Fig. 1J). Similar to previous studies [6], 1.11% Inj/mL ¹²⁵I-insulin was present in the serum of 2 mo CD-1 mice at 30 min. No difference in the serum level was observed when comparing 2 and 12 mo SAMP8 mice 30 min after administration (1.86% Inj/mL and 1.23% Inj/mL, respectively).

As transport was not influenced between the three mouse cohorts, we combined the data across these groups for each time point to determine which brain regions contained the highest and lowest ¹²⁵I-insulin throughout the time course. As shown in Fig. 2, ¹²⁵I-insulin primarily collected in the olfactory bulb during the early time points (5, 15, and 30 min; Fig. 2A-C), while the hypothalamus contained the greatest amount of ¹²⁵I-insulin at 60 min (Fig. 2D). Table 1 shows the levels of ¹²⁵I-insulin present in each brain region (% Inj/g ± SEM) at each time point.

To determine the amount of insulin delivered to each brain region, 1 μg unlabeled insulin was co-injected with ¹²⁵I-insulin and tissues were collected after 30 min. The amount of ¹²⁵I-insulin (% Inj/g) was multiplied by the amount of unlabeled insulin injected, 1 μg, to yield the amount of insulin delivered (ng insulin per gram brain) (Table 2). Similar to the pharmacokinetics study, there was no group difference in intranasal transport of insulin to the brain regions. Therefore, the data were collapsed across the mouse cohorts to determine the transport of insulin to each brain region (Fig. 3). Thirty minutes after intranasal delivery of 1 μg, the olfactory bulb contains the most insulin with average levels of 6.03 ng insulin per gram of tissue.

In addition, to determine the stability of the insulin between the groups after intranasal delivery, an acid precipitation was performed on the serum and whole brain 30 min after intranasal ¹²⁵I-insulin injection. There was no difference in insulin stability between the three mouse cohorts (data not shown).

Free ¹²⁵I transport after intranasal delivery

Insulin is susceptible to degradation by multiple enzymes, including insulin degrading enzyme. Free ¹²⁵I, a product of ¹²⁵I-insulin degradation, may confound radioactive measurement of ¹²⁵I-insulin transport. We therefore sought to characterize distribution of free ¹²⁵I after intranasal delivery in young CD-1 mice. The distribution pattern of free ¹²⁵I, Olf>Cortex>Cb>Hy>Hpc (Fig. 4), differs from ¹²⁵I-Ins, Ob>Hy>Hpc>CB>Cortex (Fig. 1). Free ¹²⁵I levels throughout the brain decrease significantly with time. When measured at 5 min, serum ¹²⁵I levels were 12 times higher after the administration of free ¹²⁵I (3.53% Inj/mL, Fig. 4J) than after the administration of ¹²⁵I-insulin (0.29% Inj/mL,Fig. 1J).

Effect of albumin on ¹²⁵I-insulin transport after intranasal delivery

As there was no difference in intranasal insulin delivery between young CD-1 and young and aged SAMP8 mice, we carried out remaining experiments in young CD-1 mice to better define transport of intranasal insulin.

Transport to the cortex and midbrain was enhanced 2.5, 5, 20, and 30 min after delivery with albumin (Fig. 5B,H). In addition, albumin enhanced ¹²⁵I-insulin transport to the olfactory bulb 5 min after delivery (Fig. 5A), thalamus 20 min after delivery (Fig. 5F), and cerebellum 5 and 20 min after delivery (Fig. 5G). Co-administration of albumin significantly increased transport of ¹²⁵I-insulin into serum 30 min after delivery (2.53% Inj/g compared to 1.44% Inj/g without albumin) (Fig. 5J). Co-injection of albumin did not affect the saturable transport of ¹²⁵I-insulin nor the level of degradation in the serum or brain (data not shown).

Effect of insulin receptor inhibition on ¹²⁵I-insulin transport after intranasal delivery

Co-administration of 1 μg of the selective insulin receptor antagonist, S961, significantly impaired transport of ¹²⁵I-insulin into cortex, striatum, hypothalamus, hippocampus, thalamus, cerebellum, midbrain, and whole brain at 60 min (Fig. 6B-I). The decrease in ¹²⁵I-insulin levels in the olfactory bulb with inhibition of the insulin receptor was not statistically significant (Fig. 6A). Similarly, levels of ¹²⁵I-insulin present in the serum at 60 min were significantly decreased with S961 co-administration (Fig. 6J). Levels in the serum without insulin receptor inhibition at 60 min were 2.42% Inj/mL compared to 0.43% Inj/mL with inhibition.

DISCUSSION

Although success of clinical trials investigating intranasal insulin as a way to improve memory and cognition have proved promising, transport after intranasal delivery has not been well defined. Here, we examined transport of intranasal insulin in various contexts, including: different mouse strains, mice exhibiting dementia of the Alzheimer’s type, co-injection with the carrier protein albumin, and inhibition of the insulin receptor. We found insulin is transported rapidly throughout the entire brain by 5 min and remains present after 60 min.

Intranasal insulin transport did not vary among healthy, young CD-1 mice compared to young, pre-disposed SAMP8 mice or aged, AD-like SAMP8 mice. These results suggest the transport of insulin itself is not altered due to strain or AD-like phenotype. These results are consistent with the clinical studies which have shown beneficial effects of intranasal insulin on memory in young, healthy cohorts [7] as well as aged, demented subjects [8, 9]. Similar levels of intranasal insulin delivery to the brain have been observed in young CD-1 mice [6]. Whole brain levels were 0.112±0.0117% Inj/g 30 min after intranasal insulin administration previously compared to 0.133±0.0227% Inj/g in the currentstudy.

The data described here can help begin to understand the transport process of insulin after intranasal delivery. As mentioned before, brain distribution of peptides after intranasal delivery can occur via the CSF. However, not all proteins delivered by intranasal administration reach the CSF. For example, ¹²⁵I-insulin-like growth factor-1 (IGF-1) is not detected in CSF after intranasal administration despite significant brain distribution [18]. As transport did not vary between young or aged SAMP8 mice, we believe that CSF bulk flow did not play a significant role in insulin uptake as measured in these studies. Aged SAMP8 mice have an approximate 50% reduction in the rate of CSF reabsorption [19]. Decreased bulk flow is suggested to contribute to AD development due to reduced clearance of toxic materials such as amyloid-β peptide [20, 21]. The results presented here suggest insulin is transported by a mechanism that does not involve CSF bulk flow but might involve perivascular transport based on the rapid distribution.

Due to lack of varying distribution patterns discerned among our three mouse cohorts, we combined data to determine which brain region insulin was targeting at each time point. Intranasal insulin concentrated in the olfactory bulb early on as expected due to its proximity to the site of delivery. However, at 60 min, the highest concentration of insulin was in the hypothalamus (0.3918±0.071% Inj/g), a region containing a high number of insulin receptors [22, 23], and lowest in the pons (0.0907±0.017% Inj/g), an area containing many fewer insulin receptors [24]. It is still unclear how insulin might be concentrated in specific brain regions like the hypothalamus, but the importance of insulin in the hypothalamus is clear due to the role of this peptide in the regulation of peripheral energy metabolism.

In order to determine the dose of insulin reaching the different brain regions, we co-injected the radiolabeled insulin with 1 μg unlabeled insulin, a dose previously shown to be therapeutic in the SAMP8 AD mouse model [6]. This calculation assumes that the distinction the transporter makes between radiolabeled and unlabeled insulin is negligible. Knowing the concentration of insulin reaching each brain region will aid in interpreting levels of insulin necessary to elicit changes in behavior or signaling. As there were no differences in levels between the three mouse cohorts, we combined the data. In this study, the amount of insulin reaching the hippocampus, an area important in cognition, 30 min after intranasal delivery is 3.01 ng insulin/g brain region. This is in contrast to the olfactory bulb, located near the site of delivery, which has double the level at 6.03 ng insulin/g brain region.

The transport of ¹²⁵I-insulin across the BBB is unaffected by excess addition of tyrosine, consistent with the transport of ¹²⁵I-insulin and ¹²⁵I-tyrosine across the BBB being unrelated [25]. Generation of free iodine by brain iodinases could confound these results, although efflux of free ¹²⁵I out of the brain occurs, and so should prevent ¹²⁵I accumulation in the brain. In order to determine the transport of free ¹²⁵I after intranasal delivery, we measured levels in brain and serum at different time points. We show after intranasal delivery of free ¹²⁵I, serum levels are immediately saturated within 5 min. Free ¹²⁵I levels in brain regions drop significantly with time, further supporting the efflux mechanism. Therefore, we can confidently conclude our ¹²⁵I-insulin studies are not measuring free ¹²⁵I.

In order to determine whether uptake of intranasal insulin can be increased or targeted to different brain regions, we co-injected insulin with albumin. Previously, we showed that albumin is itself slowly taken up after intranasal administration and can to some degree redirect the brain distribution of leptin [26]. As albumin binds insulin readily and has been used clinically to enhance transport and stability of various other proteins and peptides [27, 28], we considered that albumin might affect the uptake or distribution of intranasal insulin [26]. Here, our studies showed albumin increased the level of insulin in each brain region, but did not alter its relative distribution. This differential effect of albumin on insulin versus leptin could be due to the differences in the leptin and insulin transport systems [29]. Since albumin does not influence the saturation of insulin after its intranasal delivery and has no effect on degradation, the overall increase in insulin concentration indicates a role for albumin in insulin retention. The prolonged half-life of insulin detemir is attributed to enhanced proteinbinding capabilities [30]. Whereas insulin only binds albumin transiently, the presence of albumin may still extend insulin bioavailability. However, whether a long-acting insulin is used such as Levimir versus a short-acting insulin such as Humalog, memory improvements are still observed after intranasal administration [11], suggesting the effects due to insulin occur quickly.

To determine the role of the insulin receptor in insulin brain transport, we used S961, a selective receptor antagonist [16]. With exception of the olfactory bulb, the decrease in insulin transport resulting from co-injection of the insulin receptor inhibitor reached statistical significance in all brain regions.It has been suggested insulin transport across endothelial cell membranes occurs via the insulin receptor by receptor mediated transcytosis [31, 32], although the exact mechanism as to how insulin crosses endothelial cell membranes or epithelial cell membranes present in the nares, has not been tested directly. Salameh et al. has shown protein kinase C (PKC) inhibition enhances intranasal transport of insulin [6]. PKC activation can decrease the tyrosine kinase activity of the insulin receptor, inhibiting the insulin signaling cascade [33, 34]. Our S961 inhibition studies build on the PKC inhibitory studies and suggest intranasal transport of insulin might be mediated by the insulin receptor.

In conclusion, using the SAMP8 model, we determined that transport of intranasal insulin into brain or brain regions is unaffected by strain or the AD-like phenotype. Therefore, further investigations on the effect of intranasal insulin in enhancing memory can continue to focus on molecular and biochemical differences after intranasal insulin delivery rather than differences in transport. Indeed, others have shown insulin levels in the brain whether after intracerebroventricular [4] or intranasal [35] delivery can impact insulin signaling and inflammation. Both of these mediators have been linked to improvements in memory. The results presented here also allow us to continue investigating the mechanism of insulin transport after intranasal delivery in an established model used in transport studies, CD-1 mice. Using this model, we were able to show increased levels of insulin throughout the brain when co-administered with albumin and inhibition of transport when the insulin receptor was inhibited. Since clear enhancements of memory occur in clinical studies after intranasal insulin delivery, investigating ways to improve the amount of insulin delivered to the brain will serve great importance.

Footnotes

ACKNOWLEDGMENTS

We thank Lauge Schäffer (Novo Nordisk) for providing the insulin receptor inhibitor, S961. EMR is supported by the Genetic Approaches to Aging Training Grant at the University of Washington (T32AG000057); SRH and SN were supported by the Medical Student Training in Aging Research Program (2T35AG026736-11); and WAB is supported by the NIA (R01AG046619).

Authors’ disclosures available online ().

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Intranasal Insulin Transport is Preserved in Aged SAMP8 Mice and is Altered by Albumin and Insulin Receptor Inhibition

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animal use

Radioactive labeling

Intranasal administration

Sample collection

Statistics

RESULTS

125I-Insulin transport after intranasal delivery

Free 125I transport after intranasal delivery

Effect of albumin on 125I-insulin transport after intranasal delivery

Effect of insulin receptor inhibition on 125I-insulin transport after intranasal delivery

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

¹²⁵I-Insulin transport after intranasal delivery

Free ¹²⁵I transport after intranasal delivery

Effect of albumin on ¹²⁵I-insulin transport after intranasal delivery

Effect of insulin receptor inhibition on ¹²⁵I-insulin transport after intranasal delivery