As both hyper- and hypo-activation of the insulin pathway are known to contribute to increased genesis of the amyloid-β₄₂ protein (hallmark of dementia-associated neurodegeneration)(16,54,55) and hyperphosphorylation of the microtubule associated protein Tau^18,44,45, we assessed treatment effects on hemibrain Aβ₄₂ and Tau levels. Mice in all treatment groups displayed an increasing effect on Aβ₄₂ production compared to control (P/D_LPS = 0.077/0.776; P/D_hS = 0.059/0.783; P/D_hSL = 0.185/0.601, Fig. 4A, top). Only a mild effect on Aβ₄₀ levels was observed in the combined group (P/D_hSL = 0.177/0.560, Fig. 4A, middle). While both LPS (P/D_LPS = 0.030/0.946) and hS (P/D_hS = 0.085/0.701; Fig. 4A, bottom) treatment increased the ratio of Aβ₄₂ to Aβ₄₀ proteins individually, this effect was lost in combination, supporting an antagonistic interaction (interaction P = 0.013, Fig. 4A, bottom). Only a mild decrease of total Tau expression was observed in the combined group (P/D_hSL = 0.357/0.520). Treatment with LPS suppressed Tau phosphorylation, regardless of the presence or absence of high sucrose (LPS effect P = 0.006; P/D_LPS = 0.022/1.14; P/D_hSL = 0.015/1.18, Fig. 4B middle), though this effect was lost when normalized to total Tau protein (Fig. 3B bottom).

Figure 4: Effects of a high-sucrose diet and/or lipopolysaccharide on β-amyloidogenesis and Tau phosphorylation in reproductively normal female mice.

A. Aβ42 expression demonstrated an elevation in response to both LPS treatment and high-sucrose diet. Aβ40 was only affected in the combined treatment group. B. Although only the combined treatment affected total tau (top), LPS had a significant effect on phosphotau (middle). High sucrose in the water had a small effect (Cohen’s D = 0.39) on the ratio of phosphotau over total tau. Two-way ANOVA with Fisher’s LSD post-hoc test. *P < 0.05 vs control. # Cohen’s D > 0.5. n = 8-10 for all groups.

4.4 A high sugar diet alters behavior differently in the presence/absence of LPS

AD-like neurodegeneration often manifests phenotypically as memory impairments and behavioral abnormalities. Thus, we employed simple Barnes maze and Open Field protocols to assess spatial memory and anxiety-like behavior, respectively. Behavioral trials performed during the 6^th month of treatment demonstrated differing high-sucrose-associated behavioral phenotypes in the presence and absence of lipopolysaccharide. Animals sustained on hS alone displayed the largest impairment to spatial learning performance in the Barnes maze, characterized by increased latency to escape and frequency of errors (P/D_hS = 0.040/1.01; Fig. 5A, left). Such results are consistent with the effects of chronic corticosterone on spatial learning observed elsewhere [10, 56, 57], suggesting a glucocorticoid-mediated impairment in our hS mice. In contradiction to this notion, hSL mice demonstrated a mild impairment in spatial learning (P = 0.127; D_hSL = 0.842, Fig. 5A, left) that did not coincide with increased fecal corticosterone levels. Interestingly, although LPS or hS treatment had no effect on long-term spatial recall alone, when combined we observed an improvement in these reproductively normal female mice (Fig. 5A, right). During the Open Field test, there was an LPS-mediated effect that was only observed as a decrease of time spent in the center in the combined group (LPS effect P = 0.006, P/D_hSL = 0.021/1.85; Fig. 5B, left). Although no sucrose effect was observed, the hS group showed a mild increase in time spent in the center (P/D_hS = 0.063/0.854). Avoidance of the center of the field is often associated with anxiety-like behavior, suggesting that an anxiogenic interaction (interaction P = 0.003, Fig. 5B, left) between hS and LPS exists that is absent following the individual treatments. While hS-treatment reduced locomotion (sucrose effect P = 0.007) in the Open Field, both LPS (P/D_LPS = 0.138/0.591) and hS (P/D_hS = 0.056/0.891) had mild effects on locomotion that were lost in the combined group (Fig. 5B right). The lack of effect in the combined group suggests effects on locomotion do not account for the increased anxiety-like behavior observed in this group.

Figure 5: Differential alteration of Barnes maze-associated spatial learning performance and Open Field degree of thigmotaxis in response to high-sucrose feeding and/or systemic LPS injection.

A. Rate of spatial memory acquisition was impaired in all animals sustained on a high-sucrose diet, though the aaddition of LPS attenuated - but did not abolish - these deficits. Only the combined treatment group showed an improvement in 48 hour recall of the escape location. B. The addition of systemic LPS to high-sucrose fed mice appeared to promote anxiety-like behavior (increased thigmotaxis) in the open field. Animals fed a high sugar diet demonstrated a trend toward increased duration in center. Locomotion did not differ significantly between groups, though a trend toward increased and reduced distance traveled was noted for LPS and hS animals, respectively. Two-way ANOVA with Fisher’s LSD post-hoc test. *P < 0.05 vs control. # Cohen’s D > 0.5. n = 10 for all groups.

5. Discussion

Given that trends in AD incidence coincide with similar trends in obesity, diabetes, and chronic inflammation - all known risk factors for AD-like neurodegeneration^7,50,51 - the present study combined a high-sucrose drinking water regimen with repeated monthly low-dose intraperitoneal LPS injections to accelerate neurodegenerative pathology in reproductively normal female wild-type mice. We proposed that elements of the high-sugar diet would enable ordinarily benign low-dose inflammatory stimuli to precipitate a chronic inflammatory state culminating in accelerated neurodegeneration. Female mice were used as the bulk of AD literature concerns male animal models despite nearly two-thirds of all AD patients being women [2]. We demonstrate that high sugar consumption promotes upregulation of early-stage neurodegenerative processes after just 6-7 months, as evidenced by elevated fecal corticosterone (Fig. 1C), increased liver sphingomyelinase activity (Fig. 1D), brain insulin pathway dysregulation (Fig. 2), increased β-amyloidogenesis (Fig. 4A), an altered brain inflammation state (Fig. 3 and Table 1), and worsened spatial learning in the Barnes maze (Fig. 5). Counter to our expectations, the addition of LPS prevented/lessened many of these effects; fecal corticosterone (Fig. 1C) was normal, tau phosphorylation (Fig. 4B) was suppressed, and spatial acquisition deficits were curtailed.

Previous studies have shown that caloric excess promotes lipogenesis and triglyceride storage in both the liver and adipose tissue [58]. In cases of chronic excess, exacerbated intake leads to liver insulin resistance and steatohepatitis (inflammation of fatty liver) [13, 14, 59]. This inflammatory state promotes lipolysis and degeneration of the liver, culminating in mitochondrial- and/or apoptotic-mediated cell-death and ceramide synthesis [60, 61]. The resultant free fatty acids, proinflammatory cytokines, and ceramides can induce both systemic and central dysregulation of the insulin pathway, leading to eventual neurotoxicity [14, 16]. In this study, animals sustained on 20% sucrose in their drinking water displayed weight gain, hepatic steatosis, elevated fecal corticosterone expression, and elevated liver neutral sphingomyelinase activity (ceramides) after 7 months. As noted prior, these conditions have been linked with insulin resistance, neurodegeneration and cognitive impairment [14, 17, 62, 63]. A recent study by Chen, et al (2017) demonstrated that liver ceramide synthesis is upregulated by glucocorticoid signaling [63], thus opening the possibility that glucocorticoids are partly responsible for the high-sucrose-induced liver pathologies in this study. While we did not assess systemic insulin sensitivity directly, observed increases in sphingomyelinase activity and liver steatosis suggest that our high-sucrose animals were likely insulin resistant. Ceramides have been shown to inhibit both peripheral and central insulin signaling [16, 62, 63], and to promote neuroinflammation [64] and oxidative stress in the CNS [14, 17, 62]. Total IRS1 proteins were found to be increased in whole hemi-brain homogenates of high sucrose fed mice without concomitant phosphorylation of downstream targets such as Akt and mTOR, suggesting a potential compensatory increase in IRS1 proteins in response to reduced insulin signaling. Although glucocorticoids are known to induce brain insulin resistance [12], observed irregularities within the brain insulin pathway appear to be attributable to ceramide-mediated mechanisms. Irregularities were displayed even in high sucrose fed mice with normal glucocorticoid activity (hSL group), implicating ceramides as the likely driving force behind the observed dysregulation. Furthermore, elevations in total IRS1 proteins without concurrent increase in Akt phosphorylation are consistent with results observed following injection with ceramides, as demonstrated by de la Monte, et al (2010) [62]. It should be noted that the second messengers discussed are not exclusive to the insulin pathway. Both insulin-like growth factor 1 (IGF1) [65]- and estrogen-receptor (ER) [66-68]-mediated signaling incorporate elements of the PI3K-Akt-mTOR pathway, and thus present potential confounds. As the mice used in this study are female, the interpretations regarding ceramides and insulin pathway dysfunction should be viewed within the context of these limitations: any differences in mTOR or Akt phosphorylation could have been influenced by estrogen, and therefore may not represent insulin resistance.

We previously demonstrated that a 20% sucrose diet increases Tau phosphorylation after just 4-months in male mice [10]. Similar - though lesser - observations were noted in the present work, confirming for the first time that the high-sucrose diet contributes to neurodegenerative processes in female wild-type mice. Both total Aβ₄₂ and the ratio of Aβ₄₂ to Aβ₄₀ were increased by the high sugar diet. Furthermore, high-sucrose animals displayed increased nitrate + nitrite expression, suggesting enhanced NO production (perhaps through upregulated iNOS). Exaggerated NO has been linked to neuroinflammation and nitrosative activity, both of which are known to enhance the processing of APP to Aβ [26, 27]. Increased β-amyloidogenesis and NO expression were associated with worsened spatial learning performance, highlighting a potential decline in cognition. Given the data presented, it seems possible that a high-sugar diet upregulated neurodegenerative processes (i.e. β-amyloidogenesis, nitrosative activity, etc.) through glucocorticoid [12] - and hepatic ceramide-mediated mechanisms [14, 17, 17] to influence spatial memory.

It should be noted that the mild phenotype observed may be related to the sex of the animals. Estrogen has been shown to exert neuroprotection in a variety of models (cell culture as well as animal) and can diminish many pathological processes associated with AD, such as β-amyloidopathy, glucocorticoid over-expression, mitochondrial dysfunction, and oxidative stress [3, 6]. Also, the mild elevation in Aβ₄₂ coupled with impaired spatial memory acquisition but intact long-term recall could represent an early time-point in late-onset AD pathogenesis [69-72] (Fig. 6), at least in females. Given the proposed neuroprotective effects of estrogen against the AD-related pathological processes associated with insulin dysregulation - such as inhibition of GSK3 [66], suppression of tau phosphorylation [66, 73], and attenuation of Aβ production [74] - it is possible that male and/or post-menopausal female mice would display more severe neurodegenerative phenotypes under the experimental conditions employed herein; i.e. employing similar treatment parameters, we previously demonstrated a more robust phenotype after just 4 months in male mice. Regardless, the results presented in this study using reproductively normal females assist in the establishment of an important baseline for future exploration of the role of estrogen in dietary-stressor-induced neurodegeneration. The modest effects of hS treatment on Tau phosphorylation and β-amyloidogenesis could also have been due to the use of hemibrain homogenates, which may have diluted region-specific differences. In addition, it has been proposed that while diabetes alone may be insufficient to generate a robust AD phenotype, it could serve as a cofactor in the etiology and progression of the disease [59]. Nonetheless, when paired with corticosterone, sphingomyelinase, Aβ, and pTau data, the results obtained in a non-transgenic wild-type model at this early time-point support a high-sucrose diet-mediated upregulation of neurodegenerative processes.

Figure 6: Seven months on a high-sucrose diet induces behavioral abnormalities and upregulation of processes suggestive of early AD-like neurodegeneration.

Early AD is characterized, in part, by impairments in short-term memory formation. As the disease progresses, long-term recall begins to diminish. This coincides with increased Aβ and pTau burden. Our mice demonstrate a mild though present Aβ and pTau phenotype that coincides with worsened spatial acquisition (delayed learning of a visual escape location in the Barnes maze) and spared long-term recall. This may suggest that our mice display an AD-like phenotype representative of an early time-point within late-onset pathogenesis.

Counter to our prediction that a high-sugar diet would sensitize the CNS to inflammation and spur transient inflammatory events to precipitate a chronic inflammatory state and accelerated neurodegeneration, the addition of low-dose LPS to high-sucrose fed mice proved to be protective, with antagonistic interactions noted at several levels. First and perhaps most notably, animals on the combined high-sucrose and LPS regimen did not display an elevation in fecal corticosterone. Given the proposed central nature of glucocorticoids to high-sucrose diet-mediated pathology [12], attenuation of corticosterone expression may have proved to be quite beneficial. In fact, acute LPS-mediated inflammation was found to suppress Tau phosphorylation (with or without sucrose) in animals sustained on a high sugar diet. Thus, the high-sugar diet did not appear to sensitize the CNS to induction of chronic inflammation in response to transient inflammatory events. Interestingly, the only pathology that arose due to the combination was observed in the Open Field, where the addition of LPS to high-sucrose animals enhanced thigmotaxis. Avoidance of the center has been linked to anxiety-like behavior, suggesting the combination treatment to be anxiogenic.

The protective effects of LPS can likely be attributed to the low dose employed and the transient nature of the inflammation induced. Glucocorticoids engage in a form of negative feedback through a hippocampal-HPA axis circuit. Once a temporal and spatial threshold of interaction between glucocorticoids and their receptors in the hippocampus is reached, activation of the HPA is suppressed. This process allows for robust yet transient corticosterone/cortisol responses to stressful stimuli [75]. As transient inflammatory events have been shown to promote a robust increase in circulating glucocorticoids [76] capable of triggering negative feedback, it is possible that this spike in corticosterone activity may have ‘reset’ the high-sucrose diet-induced chronic elevation in glucocorticoids through activation of the aforementioned feedback loop (Fig. 7). Furthermore, LPS demonstrates dose-specific effects, with high doses leading to sepsis [77]; however, the effects at low concentrations are far more controversial. While it has been reported that sustained treatment with low-dose LPS promotes low-grade inflammation [78], transient exposure has been found to result in suppressed inflammatory responses to future challenge [28-30]. Short-term exposure to low-dose LPS has even been shown to suppress inflammation and prevent neurodegeneration following cerebral ischemia-reperfusion injury [31-33]. Regarding the present study, it is important to note that the final injection of LPS occurred four months prior to sacrifice, making it difficult to compare the neuroinflammation results herein with the literature. The addition of low-dose LPS to our hS-fed mice suppressed proinflammatory cytokine expression (IL-5, IL-6 and IL-1β), suggesting a possible state of inflammatory hypo-responsiveness caused by low-dose LPS-induced tolerance to TLR4 ligands [28-30]. Alternatively, Everhardt and associates (2016) observed that female mice injected with LPS display subdued proinflammatory responses relative to males [4], which raises the possibility that the lack of LPS-induced neuroinflammation observed in this study could be attributable to estrogen interference. Furthermore, gonadectomy leads to an increase in mortality in female rats exposed to LPS that is attenuated by estrogen replacement therapy [79]. Taking potential estrogen interference into account, it is possible that male and/or post-menopausal females would demonstrate more robust neuroinflammatory phenotypes in response to the dose of LPS administered in this study. Regardless, the high-sucrose diet failed to potentiate the inflammation induced by the low-dose LPS stimuli in female wild-type model employed. If transient low-dose LPS-induced inflammation did protect against select elements of the deleterious effects of a high-sucrose diet by ‘re-setting’ the corticosterone response in these reproductively normal female mice, it would reinforce the notion that high sugar diet-induced pathology is mediated primarily by glucocorticoids, and that transient inflammatory events may be beneficial in the long-term management of chronic stress.

Figure 7: LPS may have conferred protection via attenuation of chronic glucocorticoid signaling.

LPS is known to induce a robust inflammatory response associated with elevated glucocorticoid release via activation of the HPA axis. Glucocorticoids self-regulate their activity through induction of negative feedback via a hippocampal-HPA axis-dependent loop. High-sucrose diets could potentially promote chronic increases in glucocorticoid expression without triggering this negative feedback (sub-threshold elevations). Transient and robust glucocorticoid responses following LPS administration may have ‘re-set’ this chronic increase.

6. Conclusion

This study supports for the first time the efficacy of high sugar diets in the induction of pathologic processes involved in neurodegeneration in reproductively normal female wild-type mice, and demonstrates that transient low-grade inflammatory events may antagonize pathologies associated with said diets, at least in females. While it remains possible that modern lifestyle changes and inflammatory medical complications can intersect to accelerate development of an AD phenotype, much work is to be done to untangle the relationships between diet, stress and neurodegeneration. Furthermore, this study may highlight a potential reason as to why AD occurrence varies among individuals. If transient stress/inflammation is protective in females, then perhaps differences in lifestyles, diet, exercise and work/home environments contribute to long-term cognitive health; i.e. stress is needed to ‘re-set’ cortisol homeostasis. It also remains likely that males, females and post-menopausal females respond differently to inflammatory events on account of differing sex hormone profiles, thus raising the possibility that the combination of a high sugar diet and low-grade LPS injection would be neurotoxic in males and/or post-menopausal females.

Author Contributions Statement

All authors contributed equally to this work.

Funding

This work was supported by the Saskatchewan Health Research Foundation (grant # 3075) and University of Saskatchewan (College of Medicine Research Award).

Conflict of interest:

The authors declare that there are no conflicts of interest.

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