Age- and Sex-Associated Glucose Metabolism Decline in a Mouse Model of Alzheimer’s Disease

Abstract

Background:

Alzheimer’s disease (AD) is characterized by a high etiological and clinical heterogeneity, which has obscured the diagnostic and treatment efficacy, as well as limited the development of potential drugs. Sex differences are among the risk factors that contribute to the variability of disease manifestation. Unlike men, women are at greater risk of developing AD and suffer from higher cognitive deterioration, together with important changes in pathological features. Alterations in glucose metabolism are emerging as a key player in the pathogenesis of AD, which appear even decades before the presence of clinical symptoms.

Objective:

We aimed to study whether AD-related sex differences influence glucose metabolism.

Methods:

We used male and female APPswe/PS1dE9 (APP/PS1) transgenic mice of different ages to examine glucose metabolism effects on AD development.

Results:

Our analysis suggests an age-dependent decline of metabolic responses, cognitive functions, and brain energy homeostasis, together with an increase of Aβ levels in both males and females APP/PS1 mice. The administration of Andrographolide (Andro), an anti-inflammatory and anti-diabetic compound, was able to restore several metabolic disturbances, including the glycolytic and the pentose phosphate pathway fluxes, ATP levels, AMPKα activity, and Glut3 expression in 8-month-old mice, independent of the sex, while rescuing these abnormalities only in older females. Similarly, Andro also prevented Aβ accumulation and cognitive decline in all but old males.

Conclusion:

Our study provides insight into the heterogeneity of the disease and supports the use of Andro as a potential drug to promote personalized medicine in AD.

Keywords

Alzheimer’s disease andrographolide glucose metabolism neuroprotection sex differences

INTRODUCTION

Alzheimer disease (AD) is the most common age-related neurodegenerative disorder and is characterized by a progressive and gradual decline in cognitive functions, mainly memory performance [1, 2]. Neuropathologically, AD is defined by the extracellular accumulation of amyloid plaques, mainly composed of the amyloid-β (Aβ), as well as by intracellular tau aggregates [2, 3]. In addition, one of the common pathological features in AD patients is a severe decline in cerebral glucose metabolism, which correlates with disease progression and precedes clinical symptoms even decades before cognitive failure [4 –6]. While the causes of glucose hypometabolism in brains of AD patients are not fully understood, numerous studies have shown that promoting the uptake of glucose in neurons could halt the neurotoxic effects associated with this disease, improves memory and cognitive function [7 –10]. Indeed, previous results from our group have shown that Andro, a labdane diterpene obtained from Andrographis paniculate, increases the glucose uptake both in vivo and in vitro in an AD mouse model while also showing neuroprotective roles [9, 11].

According to Alzheimer’s Association, the prevalence of AD is expected to rise dramatically, affecting approximately 90 million people globally by 2050, which increases the health costs related to this disorder [12]. Although major advances have been made in understanding the disease pathogenesis, clinical trials have failed to find effective drugs, partly because of the high heterogeneity of the disease [13, 14]. One of the factors known to impact the variability in AD is differences in sex, affecting both clinical and pathological outcomes in patients [15 –17]. Interestingly, studies have reported females to be at greater risk of developing AD, which correlates with the fact that nearly two-thirds of Americans with AD are women [18 –20]. The higher incidence in women is not only attributed to their longer life expectancy, as emerging evidence has suggested gender-specific risk factors, together with higher tau and Aβ deposits in women than men [21 –23]. Similar observations have been described in transgenic AD mice, where females present higher levels of amyloid plaques, together with neuroinflammation and worse cognitive behavior compared to males [24, 25]. However, despite the importance of sex differences in the development, progression, and pathogenesis of AD, its effect on glucose metabolism remains largely understudied. In the present work, we treated APP/PS1 mice with Andro to evaluate the influence of sex differences on glucose metabolism. Collectively, our findings showed significant metabolic differences between males and females, together with a higher Aβ burden in females. Furthermore, Andro treatment was able to recover the metabolic functions and Aβ levels in younger animals, and reverted metabolic deficits in older females, but not in males, suggesting important differences among sexes. Together, our results emphasize the need of considering biological sex and age-specific differences to find more effective therapies for AD.

MATERIALS AND METHODS

Experimental overview

All the cognitive tests (training and experiments) and slice experiments were performed in a randomized double-blind manner. Animal experiments were usually performed in the beginning of the day and no sample calculation was performed.

Animals and ethical standards

Four-, eight-, and eleven-month-old male and female APPswe/PS1dE9 (APP/PS1) [resource identification initiative (RRID): 34832-JAX] and C57BL/6J (RRID: 000664) mice were used in this study (a total of 160 mice; 8 per group). APP/PS1 animals co-express the Swedish (K594M/N595L) mutation of a chimeric mouse/human APP (Mo/HuAPP695swe) together with the human exon-9-deleted variant of PS1 (PS1-dE9), and secrete elevated levels of human Aβ peptide [26, 27]. Both strains were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Animals were maintained at the animal facility of Pontificia Universidad Católica de Chile under sanity barrier in ventilated racks and in closed colonies. Mice age was selected based on previously described appearance of neuropathological and cognitive hallmarks; 4, 8, and 11 months old are considered as pre-symptomatic stage of AD, early disease stage and advanced/stablished pathological stage, respectively [26, 27]. The animals were kept under standard cage density conditions and had access to food and water ad libitum. To avoid animal suffering, the animals were reviewed by technical personal every day to look evidence of distress (National Institutes of Health tables of supervision). Animals were allocated randomly to experimental groups. All animal work was approved by the bioethical and biosafety committee of the Faculty of Biological Sciences from Pontificia Universidad Católica de Chile (ethical approval CBB-158/2014). The inclusion/exclusion criteria for this study were the health and body weight of the animals. No animals had to be excluded in this study. Our analysis showed no significant differences between 8- and 11-month-old wild-type (WT) mice, unless otherwise stated, and thus only 11-month-old data is shown (Supplementary Table 1). Intraperitoneal (i.p.) injections of Andrographolide (Andro, Sigma-Aldrich, 5508-58-7) (two groups, 2 mg/kg) or saline solution as vehicle (two groups) were carried out three times per week for the four months before euthanasia, as described previously [28] (Fig. 1A).

Fig. 1

Improvement of cognitive performance caused by Andro depends on sex and age. A) Schematic of the study design and timeline. Male and female APP/PS1 mice received Andro (2 mg/kg) or vehicle injections 3 times/week for 4 weeks before euthanasia at 8 or 11 months. 4-, 8-, and 11-month-old male or female mice, treated with or without Andro, were subjected to cognitive tests, including (B) open field, (C) novel object localization (NOL), (D) novel object recognition (NOR) and memory flexibility for (E) males and (F) females. Means±SEM are shown (n = 8 independent experiments). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, compared with 4 months old group or specified group; ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001, compared with APP/PS1 mice of the corresponding sex within experimental group.

Hippocampal slices preparation

Hippocampal slices were prepared as previously described [29]. Briefly, transverse slices (350 μm) from the dorsal hippocampus were sectioned in cold artificial cerebrospinal fluid (119 mM NaCl, 26.2 mM NaHCO₃, 2.5 mM KCl, 1 mM NaH₂PO₄, 1.3 mM MgCl₂, 10 mM glucose, 2.5 mM CaCl₂) and incubated in artificial cerebrospinal fluid for 1 h at 22°C before using.

Large open-field test

A 120×120 cm transparent Plexiglas platform with 35 cm-high transparent walls was used to study locomotor and stress behavior in our mouse model. The open field, which measured 40×40 cm, was defined as the center area of the field. Data were collected using an automatic tracking system (HVS Imagen, Buckingham, Buckingham, UK). Each mouse was placed alone in the center of the open field, and its behavior was tracked for 20 min. At the end of the session, the mouse was returned to its home cage. The parameters measured included total time moving and number of times the mouse crossed the center area [30, 31].

Novel object recognition and novel object localization

The novel object recognition (NOR) and novel object localization (NOL) tasks were performed as previously described [32, 33]. Mice were habituated to the experimental room in the experimental cages for three consecutive days for 30 min per day (three consecutive days) and for 1 h on the testing day. The task occurred in a 120×120 cm transparent Plexiglas platform with 35 cm-high transparent walls containing two identical objects places at specific locations. For object familiarization, mice were allowed to explore the platform for 10 min. The animals were subsequently returned to their home cages for 1 h, followed by a 5 min exposure to a novel localization of one of the familiar objects (NOL). The mice were again returned to their home cages for 1 h and were subsequently exposed to a NOR for 5 min. The mice had no observed baseline preference for the different objects. An object preference index was determined by calculating the time spent near the relocated/novel object divided by the cumulative time spent with both the familiar and relocated/novel objects. The cages were routinely cleaned with ethanol following mouse testing/habituation of the mice.

Memory flexibility test

This test was performed as previously described [34, 35], and the pool conditions of the pool were the same as those of a Morris Water Maze [9]. Each animal was trained for one pseudo-random location of the platform per day, for 5 days, with a new platform location each day. Training was conducted for up to 10 trials per day, until the criterion of three successive trials with an escape latency of 20 s was achieved. When testing was completed, the mouse was removed from the maze, dried, and returned to its cage. Animals were tested for the next location on the following day. Data were collected using a video tracking system (HVS Imagen).

D-[1-¹⁴C] glucose biodistribution

Upon completing the cognitive tests, three mice from each group were injected with D-[1-¹⁴C] glucose (PerkinElmer, NEC043X) via the tail vein. Briefly, mice were anesthetized with isoflurane, a widely used volatile anesthetic agent, and injected intravenously via the tail with 50μCi of tracer diluted to a final volume of 20μL in isotonic saline. Following a 15 min uptake period, the animals were killed by cervical dislocation, and tissues were collected. Tissue radioactivity was quantified by liquid scintillation. D-[1-¹⁴C] glucose levels were normalized to the weight of resected tissue and expressed as the percent of injected dose [9 , 37].

Measurement of hexokinase and glucose-6-phosphate dehydrogenase activity

Quantification of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) activity was performed as previously described [9]. Briefly, hippocampal slices were washed with PBS, treated with trypsin/EDTA, and centrifuged at 500 g for 5 min at 4°C. The tissue was then resuspended in isolation medium (250 mM sucrose [Sigma-Aldrich, S9378], 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA [Sigma-Aldrich, E6758], 1 mM DTT, 2 mg/mL aprotinin [Sigma-Aldrich, A1153], 1 mg/mL pepstatin A [Sigma-Aldrich, 77170], and 2 mg/mL leupeptin [Sigma-Aldrich, L8511]) at a 1 : 3 dilution, sonicated at 4°C, and centrifuged at 1500 g for 5 min at 4°C. For HK activity determination, supernatant was collected and mixed with the reaction medium (25 mM Tris-HCl, 1 mM DTT, 0.5 mM NADP/Na⁺, 2 mM MgCl₂, 1 mM ATP, 2 U/mL glucose-6-phosphate dehydrogenase [Sigma-Aldrich, G6378] and 10 mM glucose [Sigma-Aldrich, G8270]) and the mixture was incubated at 37°C for 30 min. For G6PDH determination, the pellet was discarded, and the supernatant was further separated by centrifugation at 13,000 g for 30 min at 4°C. The supernatant was then used to quantify the G6PDH activity in a reaction buffer containing 1 mM ATP and 10 mM glucose-6-phosphate (G6P) for 30 min at 37°C. For both HK and G6PDH, the reactions were stopped by adding 10% trichloroacetic acid (Sigma-Aldrich, T6399) and the generation of NADPH was measured at 340 nm [38, 39].

Quantification of ATP, ADP, and ATP/ADP ratio

ATP and ADP levels were quantified from hippocampal lysates using ATP determination kit (Invitrogen, A22066) or ADP assay kit (Abcam, ab83359), respectively, according to the manufacturer’s instructions and as described previously [9, 28]. The ATP/ADP ratio was calculated accordingly.

ELISA quantification

Total Aβ₄₂, total Aβ₄₀ levels, and active (phospho-T172) AMPK from RIPA homogenates were measured using the human amyloid Aβ₄₂ ELISA kit (Sigma-Aldrich, EZBRAIN42), human amyloid Aβ₄₀ ELISA kit (Sigma-Aldrich, EZBRAIN40), or AMPK alpha phospho human ELISA kit (Thermo Fisher Scientific Inc., KHO0651) respectively, according to the manufacturer’s instructions and as described previously [40, 41].

Pentose phosphate pathway measurements

The pentose phosphate pathway (PPP) flux was determined as previously [39]. Briefly, hippocampal slices were washed once with ice-cold PBS and collected. Tissue was then incubated in the presence of either 0.5μCi D-[1-¹⁴C] glucose or 2μCi D-[6-¹⁴C] glucose in O₂ saturated Krebs-Henseleit buffer (11 mM Na₂HPO₄, 122 mM NaCl, 3.1 mM KCl, 0.4 mM KH₂PO₄, 1.2 mM MgSO₄ and 1.3 mM CaCl₂ pH 7.4) supplemented with 5.5 mM D-glucose (final concentration) in a flask containing 500μl benzethonium hydroxide in a fixed Eppendorf tube. Flasks were then flushed with O₂ for 20 s, sealed with a rubber cap and incubated for 60 min at 37°C with shaking. The reactions were stopped by adding 0.2 ml of 1.75 M HClO₄ and further incubated for 20 min to facilitate ¹⁴CO₂ trapping by benzethonium hydroxide. Radioactivity was measured by liquid scintillation spectrometry. PPP flux was determined by calculating the difference between D-[1-¹⁴C]glucose and D-[6-¹⁴C]glucose incorporated into ¹⁴CO₂.

Glycolytic rate determination

The glycolytic rate was measured as detailed previously [39]. Shortly, hippocampal slices were placed in tubes containing glucose (5μM) and washed twice with Krebs-Henseleit buffer (11 mM Na₂HPO₄, 122 mM NaCl, 3.1 mM KCl, 0.4 mM KH₂PO₄, 1.2 mM MgSO₄, and 1.3 mM CaCl₂ pH 7.4) supplemented with appropriate glucose concentration. After equilibration in 0.5 ml of Hank’s balanced salt solution/glucose at 37°C for 10 min, 0.5 mL of Hank’s balanced salt solution containing different D-[3-³H]-glucose concentrations was added, with a final specific activity of 1–3 disintegrations/min/pmol (approximately 1 mCi/mmol). Aliquots of 100μl were then transferred to another tube, placed inside a capped scintillation vial containing 0.5 mL of water, and incubated at 45°C for 48 h. After this vapor-phase equilibration step, the tube was removed from the vial, a scintillation mixture was added, and the ³H₂O content was determined by counting over a 5 min period. Glycolytic rates were determined by measuring the rate of ³H₂O production from D-[3-³H]-glucose.

mRNA extraction, RT-PCR, and qPCR

Total RNA was extracted from hippocampus using TRIzol, following the manufacturer’s protocol. RNA sample concentrations were determined at 260 nm absorbance using a spectrophotometer. RNA integrity was verified in denaturing agarose gel. 500 ng of total RNA was used for cDNA synthesis using Superscript IV random primers, according to the manufacturer’s instructions. The cDNA was analyzed by qPCR using the SYBR^™ green PCR master Mix (Life Technologies, 4385612) using the following program: 20 s at 95°C, 40 cycles of 5 s at 95°C and 30 s at 60°C, with a subsequent termination step between 55°C and 95°C (to verify the specificity of the amplification). Primer used for qPCR were the following, Glut1-S: 5^′-ATGGATCCCAGCAGCAAGAAG-3^′, Glut1-AS: 5^′-AGAGACCAAAGCGTGGTGAG-3^′, Glut3-S: 5^′-GGATCCCTTGTCCTTCTGCTT-3^′, Glut3-AS: 5^′-ACCAGTTCCCAATGCACACA-3^′, Cyclin D1-S: 5^′-AAAATGCCAGAGGCGGATGA-3^′, Cyclin D1-AS: 5^′-GCAGTCCGGGTCACACTT G-3^′, Hexokinase-S: 5^′-GGATGGGAACTCTCCCCTG-3^′, Hexokinase-AS: 5^′-GCATACGTGCTGGACCGATA-3^′, CamKVI-S: 5^′-TTATGCAACTCCAGCCCCTG-3^′, CamKVI-AS: 5^′-AGC CTC GGA GAA TCT CAG GT-3^′,

Phosphofructokinase-1-S: 5^′-AGGGCCTTGTCATCATTGGG-3^′ and Phosphofructokinase-1-AS: 5^′-ACTGCTTCCTGCCTTCCATC-3^′, housekeeping cyclophilin-S: 5^′-TGGAGATGAATCTGTAGGAGGAG-3^′ and cyclophilin-AS: 5^′-TACCACATCCATGCCCTCTAGAA-3. Data were analyzed using the comparative ΔCT method, as previously described [42].

Statistical analysis

All experiments were performed at least 3 times and no sample size calculation was performed. Data were analyzed by two-way ANOVA, followed by a posterior Bonferroni’s test for multiple comparisons. Statistical analysis was performed using the software Prism8 version 9.1. All data are presented as mean±standard error of the mean (SEM). Assessment of data normality and test for determining outliers were not performed for the datasets. Differences were considered significant when ^* or ⁺p < 0.05, ^** or ⁺⁺p < 0.01 and ^*** or ⁺⁺⁺p < 0.001.

RESULTS

Andro administration prevents cognitive decline in a sex and age dependent manner

With the aim to determine the effect of Andro on behavioral performance, we subjected 4- (pre-symptomatic stage), 8- (early disease stage), and 11- (advanced disease stage) month-old male and female APP/PS1 mice to an open field test to assess for anxiety and general behavioral activity. Additionally, and to rule out the possibility that Andro effects are caused by overall improvement and not by a specific rescue of AD-related abnormalities, we also treated female and male WT mice (Supplementary Table 1). Our results show no significant differences in open field performance between male or female sexes in any group of any mice tested (Fig. 1B, Supplementary Table 1).

Next, to evaluate spatial recognition, we performed the NOL test. As expected, we observed an overall tendency to decrease in the preference index with age in both sexes of APP/PS1 mice but not in WT mice (Fig. 1C, Supplementary Table 1). However, both 8- and 11-month-old females, but not males, displayed a significant recovery in spatial memory after Andro treatment, relative to untreated controls of same age. To further examine the impact of short-term memory we also performed the NOR. In the NOR behavioral test, both male and female APP/PS1 mice of different ages spent a similar amount of time exploring the novel object, which decreased with age (Fig. 1D). However, similar to NOL, females treated with Andro at 8 and 11 months of age significantly increased their preference for novel object exploration as compared with age-matched controls, suggesting a rescue of short-term memory. Males, on the other hand, show only significant differences at 8 months after Andro treatment.

Finally, we subjected mice to the memory flexibility test to study learning and memory performance. On average, the control group of 4-month-old males took an average of 10 trials to reach criterion, whereas 8- and 11-month-old male mice needed 12.3 and 13.4 trials, respectively (Fig. 1E, left panel). Interestingly, Andro treatment resulted in a significant decrease in the number of trials in 8-month-old male mice at day 3, as compared to untreated animals, but not in 11-month-old males at any experimental day. After 5 days of testing, we observed a significant drop in number of trials after Andro administration only in 8-month-old animals, indicating that only young male mice are able to recover learning and memory functions (Fig. 1E, right panel). In contrast to males, the 4-month-old female group needed an average of 10.5 trials to reach criterion. Females of 8 and 11 months, on the other hand, needed an average of 13.6 and 13.9 trials, respectively, showing a decrease in learning and memory performance, which was recovered after Andro treatment (Fig. 1F, left panel). However, this enhancement was not maintained and by day 5 of testing, only the younger group treated with Andro showed a recovery of functions (Fig. 1F, right panel).

Collectively, our results suggest that age seems to cognitively affect females more strongly than males. Importantly, the administration of Andro led to an improvement of cognitive decline for the 8-month-old animals independently of sex; however, it was only able to rescue 11-month-old females but no males of same age.

Andro-mediated restoration of Aβ levels depends on sex and age

Next, to examine the impact of sex on Aβ levels, we measured Aβ₄₀ and Aβ₄₂, two of the most abundant Aβ species in amyloid plaques, with the latter being more hydrophobic, prone to aggregation and more toxic to neurons [43]. Similar to previous findings, we found a positive correlation between total Aβ₄₂ levels and age in both sexes of APP/PS1 mice (Fig. 2A) [44, 45]. However, we only observed significant differences in Aβ₄₂ levels between females and males in the 11-month-old group. Importantly, the administration of Andro led to a significant decrease of Aβ₄₂ at 8 months in both sexes but had a significant effect only in 11-month-old APP/PS1 females, but not males. In contrast to Aβ₄₂, we observed a significant decrease of total Aβ₄₀ for both APP/PS1 males and females at 8 and 11 months, as compared to young animals (Fig. 2B). Interestingly, males had significantly higher levels of Aβ₄₀ at months 8 (69%) and 11 (95%) relative to age-matched females, respectively. Moreover, and similarly as for Aβ₄₂, Andro treatment resulted in a significant change for 8-month-old groups, independently of the sex, but only had an effect for 11-month-old females and not males. As for the Aβ₄₂/Aβ₄₀ ratio, 11-month-old females showed significantly higher levels, relative to males, which decreased to levels similar to 4-month-old control group or WT female mice after Andro administration (Fig. 2C).

Fig. 2

Andro rescue of Aβ levels is influenced by sex and age. Hippocampal lysates from 4-, 8-, and 11-month-old male or female mice, treated with or without Andro were analyzed by ELISA for (A) Aβ₄₂ or (B) Aβ₄₀. C) The ratio of Aβ₄₂/Aβ₄₀ was calculated accordingly. Means±SEM are shown (n = 5 independent experiments). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, compared with 4 months old group or specified group/sex; ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001, compared with APP/PS1 mice of the corresponding sex within experimental group.

Sex and age influence Andro-mediated stimulation of hippocampal glucose consumption

Cerebral glucose hypometabolism has been reported as a common pathological hallmark in many neurodegenerative diseases, which has been associated with a strong decline in cognitive skills [7 , 46]. Importantly, several studies have shown a progressive decline in brain glucose consumption, which correlates with clinical impairment in AD [4, 47]. Interestingly, gender has also been associated with glucose metabolism alteration and decline of cognitive features [48, 49]. Thus, to examine whether sex has an effect on glucose metabolism, we followed several parameters of glucose utilization in male and female mice hippocampus. First, we measured glucose accumulation by intraperitoneal injection of radioactive D-[1-14C] glucose. Our results showed no significant differences in untreated APP/PS1 or WT animals when comparing sexes within each age group or between age groups for a given sex (Fig. 3A, Supplementary Table 1). As expected, Andro administration led to a strong 52% and 48% increase in hippocampal glucose accumulation in males and females at 8 months, respectively. Interestingly, we also observed a significant 1.2-fold increase in 11-month-old females, relative to untreated age- and sex-matched controls; however, males at this age did not show any differences after Andro treatment.

Fig. 3

Effect of age and sex in glucose metabolism. Hippocampal slices from 4-, 8-, and 11-months old male or female mice, treated with or without Andro were analyzed for (A) glucose uptake, (B) glycolytic flux, (C) hexokinase activity, (D) PPP flux, and (E) glucose-6-phosphate dehydrogenase (G6PDH). Means±SEM are shown (n = 5 independent experiments). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, compared with 4 months old group or specified group/sex; ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001, compared with APP/PS1 mice of the corresponding sex within experimental group.

After glucose is transported into the cell, it can be metabolized either by the glycolytic pathway or by the PPP [50]. Thus, and given the sex-related change in hippocampal glucose accumulation after Andro administration, first, we measured the glycolytic rate and the PPP fluxes in male and female mice. No significant changes were observed in the rate of glycolysis between untreated males of different ages; however, and as expected, Andro administration caused a strong increase in 8 (∼78%), but not in 11-month-old APP/PS1 or WT males, as compared to age-matched controls (Fig. 3B). On the other hand, untreated females showed a significant ∼36% decrease in the glycolytic rate at both 8 and 11 months, as compared to 4 months, an effect that was reverted after Andro treatment. Interestingly, when compared by sex within the same age group, we observed significant differences in the glycolytic rate in months 8 and 11 for both treated and untreated mice.

Next, and due to the observed changes in glucose utilization by glycolysis in Andro-treated mice, we also measured the hexokinase activity in hippocampal slices. In accordance with our findings, the administration of Andro resulted in a 40% increase in the hexokinase activity for both sexes at month 8; however, only 11-month-old females, and not males, responded to Andro to levels similar to WT (Fig. 3C). On the other hand, the rate of glucose oxidized through the PPP in hippocampal slices was ∼5.1 nmol/min/mg protein for untreated males at 4 months, which decreased ∼80% in both the 8- and 11-month-old APP/PS1 mice groups, but not in WT mice (Fig. 3D). Similar to males, we observed a significant reduction in the PPP activity in untreated APP/PS1 females at 8 and 11 months, relative to the 4-month-old group. Interestingly, the PPP flux in Andro-treated animals was only recovered in females, independent of the age. To further determine the effect of sex on PPP, we analyzed the activity of G6PDH, the first and rate-limiting enzyme of the PPP. Surprisingly, we only observed a significant ∼16.5-fold increase in 11-month-old males treated with Andro, relative to age-matched control (Fig. 3E).

Females displayed a stronger increase in ATP levels and AMPKα phosphorylation

Next, to investigate whether the sex-related metabolic shift has an effect on ATP synthesis, we measured ATP levels. Our results showed a significant negative correlation between age and ATP levels that was independent of sex only in APP/PS1 mice (Fig. 4A). Notably, Andro administration caused a significant ∼36% and ∼58% increase in ATP for 8-month-old male and female APP/PS1 mice, relative to untreated animals, respectively. However, at month 11, Andro only had an effect in females, which resulted in a ∼2.1-fold ATP increase, as compared to untreated age-matched control. To explore whether Andro increases ATP levels by promoting its release or by enhancing its production, we also measured ADP levels and ATP/ADP ratio. We did not observe differences in ADP levels between sexes or for any group of APP/PS1 mice tested, suggesting that ATP synthesis was not promoted and that the ATP rise was possibly due to an increase release (Fig. 4B). Furthermore, we found that Andro led to a significant ∼40% increase in ATP/ADP ratio for 8-month-old males, but not females, while only promoting it in 11-month-old females, as compared to age-matched untreated controls (Fig. 4C).

Fig. 4

Restoration of ATP and AMPKα activity by Andro in females is independent of age. Hippocampal lysates from 4-, 8-, and 11-month-old male or female mice, treated with or without Andro were analyzed for total (A) ATP, (B) ADP, or (D) AMPKα activity. C) The ratio of ATP/ADP was calculated accordingly. Means±SEM are shown (n≥4 independent experiments). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, compared with 4 months old group or specified group/sex; ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001, compared with APP/PS1 mice of the corresponding sex within experimental group.

AMP-activated protein kinase (AMPK) is one of the key metabolic kinases that regulates cellular ATP levels by stimulating catabolic processes and inhibiting anabolic pathways to restore ATP levels. Thus, to evaluate whether sex affects AMPK, we also measured the activation state of AMPK in hippocampus lysates. As described previously, the activation capacity of AMPK declined with aging, in a sex-independent manner only in APP/PS1 mice (Fig. 4D) [51]. Moreover, and as expected, Andro administration caused a significant rise in AMPK activation in both sexes at month 8, with a 0.83- and 1.2-fold increase for male and female APP/PS1 mice, compared to untreated controls, respectively. However, only females were affected by Andro in the older group, showing a 1-fold increase higher in AMPK activation, which correlated with ATP levels.

Sex and age impact the Andro-mediated transcriptional change of several metabolic genes

Finally, we examined the expression of several key glucose metabolic genes in mice of different sex. First, we quantified the mRNA expression of the glucose transporter 1 (Glut1) and Glut3, which transport glucose across the blood-brain barrier into the extracellular space of the brain and into neurons, respectively, and have been shown to be reduced in AD [7 , 53]. We found that the expression of Glut1 did not vary between sexes or for any group analyzed, whereas Glut3 expression decreased significantly with age only in APP/PS1 mice (Fig. 5A, B). Importantly, the administration of Andro led to a strong increase of Glut3 mRNA expression, which affected 8-month-old mice equally; however, it only had a significant effect on older APP/PS1 females, but not males. Furthermore, and given the observed change in hexokinase activity, we evaluated the mRNA expression of hexokinase and phosphofructokinase 1 (Pfk 1), two key enzymes of glycolysis. We found a significant drop in hexokinase and Pfk 1 expression in both 8- and 11-month-old APP/PS1 mice groups, relative to 4-month-old animals, which was independent of sex (Fig. 5C, D). As observed, the increase of hexokinase and Pfk 1 expression levels caused by Andro was independent of age in females but only generated an effect in younger males, indicating a more pronounced age-related effect in males. Andro is a known agonist of the Wnt signaling pathway, thus to confirm the effect of Andro, we also quantified the expression of CamKIV and Cyclin D1, two Wnt target genes [54, 55]. Our results showed a significant Andro-mediated increase in the expression of both CamKIV and Cyclin D1 in females, independent of age, relative to untreated animals (Fig. 5E, F). However, in males, the effect of Andro was only observed at 8 months and not in the older group. Taken together, these results demonstrate that Andro is able to restore the expression of several metabolic genes altered in APP/PS1 younger animals. However, and in contrast to females, older males become insensitive to Andro and are incapable of restoring gene expression possibly due to an inactivation of the Wnt pathway.

Fig. 5

Andro induces the expression of several metabolic genes in a sex and age-dependent manner. A-F) Quantitative RT-PCR of Glut1, Glut3, hexokinase, Pfk 1, CamKIV, and Cyclin D1 expression from 4-, 8-, and 11-month-old male or female mice hippocampal lysates, treated with or without Andro, normalized to the cyclophilin control gene. Means±SEM are shown (n≥4 independent experiments). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, compared with the 4-month-old group or specified group; ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001, compared with APP/PS1 mice of the corresponding sex within experimental group.

DISCUSSION

In the last decades, several epidemiological studies have suggested that women are more likely to develop AD than men [56, 57]. Although the causes that result in these differences are still unclear, growing evidence has suggested that sex can give rise to major differences in metabolic capabilities [22 , 58–60]. Moreover, several studies have shown that a decrease in glucose metabolic rates can disrupt energy homeostasis, which has been tightly associated with the appearance of AD [9 , 61]. In the present study, we examined the influence of sex on glucose metabolism during AD. Collectively, the data presented here shows an age-associated decline in metabolic and cognitive functions that appear to be independent of sex in younger (8-month-old) APP/PS1 animals. However, at older ages, males and females show marked metabolic, cognitive, and pathological restoration differences when treated with Andro. Importantly, Andro was able to restore several of the AD-related deficiencies observed in APP/PS1 mice.

Effect of age and sex on glucose metabolism and Aβ levels

Our results show a similar age-related reduction in the expression of Glut3 between males and females; however, females, but not males, show a decrease in the glycolytic rate. Interestingly, Glut3 protein levels have been found to be lower in AD brains, relative to healthy controls, while lower rates of glycolysis correlate with more severe deposition of amyloid plaques [62, 63]. Positron emission tomography studies have further validated these results, while also showing that females have higher Aβ deposition, together with a lower glucose metabolism [5, 22]. Similarly, our findings demonstrate that relative to males, APP/PS1 females present higher Aβ₄₂ levels_, which appears to correlate with a reduction in the glycolytic flux at older ages. However, it is worth noting that although alterations in glucose metabolism in AD have been widely documented, a reduction in the cerebral metabolic state in both males and females has also been shown to occur during normal aging [64 –66].

Moreover, and in addition to the alterations in glycolysis, we observed a strong sex-independent reduction in the PPP flux. The PPP pathway plays a critical role in the generation of NADPH, which helps to maintain redox homeostasis and prevent oxidative stress [67]. Importantly, our results are validated by several studies showing altered PPP, together with dysregulations in G6PDH in both AD patients and APP/PS1 mice [68, 69]. Furthermore, our results show an age-related and sex-independent decline in AMPKα activation. Although several reports have validated our findings, the decrease of AMPK responsiveness with aging in the brain has been contradictory and possibly depends on several stimuli in a context-dependent manner [51 , 71]. While the sex-associated effect on AMPK has been poorly demonstrated, its activation has been also shown to be tissue-specific. Indeed, previous studies have shown a sex-dependent AMPK activation in cardiac and skeletal muscle in mice, where males had higher AMPK activity relative to females [72, 73]. On the other hand, using a stroke model in mice, no differences in AMPK activation were observed between males and females [74]. As expected, the age-dependent drop in AMPK activity also correlated with a reduction in ATP levels [75, 76]. An age-related decline in ATP production, mostly due to mitochondrial impairment, has also been widely documented in AD [77 –79]. Interestingly, sex-associated differences in mitochondrial function and ATP levels have been also reported in the brain of healthy men and women, suggesting deeper differences between sexes [80, 81]. In addition, and in accordance with previous studies the observed drop in ATP levels can also be caused by the reduction of glycolysis. Additionally, our findings show an age-related decrease in the expression of the Wnt target genes, CamKIV and Cyclin D1. Interestingly, the reduction of the Wnt signaling pathway has also been associated with a decrease in AMPKα activity, and both pathways have been shown to be altered in AD [82 –85].

Amelioration of metabolic disturbances, cognitive impairment, and Aβ₄₂ levels after Andro treatment

We found that Andro administration ameliorated both metabolic and cognitive impairment, together with the recovery of Aβ levels in young (8-month-old) APP/PS1 animals, independent of the sex.

Consistent with our previous results, we have shown an Andro-associated recovery of cognitive functions in J20 mice, without altering the cognitive behavior of WT mice [28, 86]. Interestingly, we observed a transient improvement in the memory flexibility test for both males and females (Fig. 1E, F). Previous pharmacokinetics studies of Andro, have shown rapid blood absorption, followed by a strong and fast reduction of Andro levels only 3-4 h after administration of the drug in both rats and humans [87]. Importantly, the elimination half-life of Andro is approximately 1.3 h in rats while ranging from 2–10 h in humans [87 –89]. Although we did not test for Andro bioavailability, we believe that the fast absorption and elimination could account for the transient effect observed in the memory flexibility assay.

In addition, several studies have reported similar reductions of AD pathological features in Andro-treated male mice of similar ages [28 , 90]. To our knowledge, no studies have been conducted to examine the effect of Andro on female mice. However, previous studies have reported no significant changes in several biochemical or hematological analyses between sexes in young, orally-treated mice with Andro or A. paniculata extracts [91, 92]. Interestingly, when compared by sex, we only observe a significant recovery in both metabolic effects and Aβ levels in old females (11-month-old) after Andro treatment, relative to old males, indicating that males become unresponsive to Andro with age. Among the possibilities that could be causing this discrepancy in older animals is a reduction in the activation of the canonical Wnt signaling pathway, a known Andro effector [11, 93]. Indeed, several studies have shown a significant decrease in the expression of numerous Wnt genes with aging in male mouse brain [94, 95]. However, it is important to note that the expression of Wnt genes has been reported to depend not only on age, but also on sex in humans [96]. Importantly, the inactivation of the Wnt pathway has been associated with the worsening of pathological hallmarks in AD, including increased levels of Aβ₄₂ and tau hyperphosphorylation [82 , 97]. Interestingly, the activation of the Wnt pathway by Andro or other Wnt agonists have been shown to decrease the amyloidogenic processing of AβPP by repressing β-secretase 1 (BACE1) expression and activity, whereas inhibition of the Wnt pathway results in higher AβPP and BACE1 expression [11 , 99].

However, our mRNA expression results showed a reduction in the expression of Cyclin D1 and CamKIV in 11-month-old males after Andro treatment, whose expression was comparable between 8- and 11-month-old untreated males. Moreover, untreated 11-month-old females had similar expression levels of Cyclin D1 and even lower levels of CamKIV mRNA, relative to age-matched males, suggesting that the observed metabolic effects and alterations in Aβ levels could be independent of the Wnt pathway. Interestingly, Serrano and coworkers showed that Andro was able to significantly activate the Wnt pathway in both young (7-month-old) and mature (12-month-old) APP/PS1 male mice; however, this activation did not cause changes in Aβ deposition [86]. Thus, further analyses have to be performed to determine what is causing the unresponsiveness to Andro in older male mice and whether this inactivation is involved in Aβ and metabolic alterations.

In summary, our study shows an age- and sex-associated decline of metabolic functions. While these metabolic alterations appear to correlate with an increase in Aβ₄₂ levels in both males and females, the latter were also more strongly affected at older ages. Interestingly, the administration of Andro was able to restore both metabolic changes and Aβ levels in 8-month-old mice, independently of the sex. However, at older ages, APP/PS1 female mice, but not males showed significant metabolic, cognitive, and pathological recovery after Andro treatment. Thus, even though no significant differences between the sexes were observed in younger animals, determining the underlying sex-specific molecular mechanisms that result in Andro differential responsiveness in later stages could be critical to develop appropriate therapeutic interventions in AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the Basal Center of Excellence in Aging and Regeneration (CONICYT-AFB 170005) to N.C.I., We also thank Sociedad Química y Minera de Chile (SQM) for the special grants “The role of K⁺ on Hypertension and Cognition” and “The role of Lithium in Human Health and Disease”. We also thank to FONIS-Miades T010132 and FONIS-FALZHEIMER T010131 to N.C.I.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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Age- and Sex-Associated Glucose Metabolism Decline in a Mouse Model of Alzheimer’s Disease

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Experimental overview

Animals and ethical standards

Hippocampal slices preparation

Large open-field test

Novel object recognition and novel object localization

Memory flexibility test

D-[1-14C] glucose biodistribution

Measurement of hexokinase and glucose-6-phosphate dehydrogenase activity

Quantification of ATP, ADP, and ATP/ADP ratio

ELISA quantification

Pentose phosphate pathway measurements

Glycolytic rate determination

mRNA extraction, RT-PCR, and qPCR

Statistical analysis

RESULTS

Andro administration prevents cognitive decline in a sex and age dependent manner

Andro-mediated restoration of Aβ levels depends on sex and age

Sex and age influence Andro-mediated stimulation of hippocampal glucose consumption

Females displayed a stronger increase in ATP levels and AMPKα phosphorylation

Sex and age impact the Andro-mediated transcriptional change of several metabolic genes

DISCUSSION

Effect of age and sex on glucose metabolism and Aβ levels

Amelioration of metabolic disturbances, cognitive impairment, and Aβ42 levels after Andro treatment

Footnotes

ACKNOWLEDGMENTS

References

D-[1-¹⁴C] glucose biodistribution

Amelioration of metabolic disturbances, cognitive impairment, and Aβ₄₂ levels after Andro treatment