Effects of Menopause and High Fat Diet on Metabolic Outcomes in a Mouse Model of Alzheimer’s Disease

Abstract

Background:

About two-thirds of those with Alzheimer’s disease (AD) are women, most of whom are post-menopausal. Menopause accelerates dementia risk by increasing the risk for metabolic, cardiovascular, and cerebrovascular diseases. Mid-life metabolic disease (obesity, diabetes/prediabetes) is a well-known risk factor for dementia. A high fat diet can lead to poor metabolic health in both humans and rodents.

Objective:

Our goal was to determine the effects of a high fat diet on metabolic outcomes in the App^NL-F knock-in mouse model of AD and assess the effects of menopause.

Methods:

First, 3-month-old App^NL-F and WT female mice were placed on either a control or a high fat diet until 10 months of age then assessed for metabolic outcomes. Next, we did a more extensive assessment in App^NL-F mice that were administered VCD (4-vinylcyclohexene diepoxide) or vehicle (oil) and placed on a control or high fat diet for 7 months. VCD was used to model menopause by causing accelerated ovarian failure.

Results:

Compared to WT controls, AD female mice had worse glucose intolerance. Menopause led to metabolic impairment (weight gain and glucose intolerance) and further exacerbated obesity in response to a high fat diet. There were interactions between diet and menopause on some metabolic health serum biomarkers and the expression of hypothalamic markers related to energy balance.

Conclusions:

This work highlights the need to model endocrine aging in animal models of dementia and will contribute to further understanding the interaction between menopause and metabolic health in the context of AD.

Keywords

Alzheimer’s disease glucose metabolism hypothalamus menopause metabolic disease obesity

INTRODUCTION

Worldwide, it is estimated that about 22% of people over 50 years of age suffer from Alzheimer’s disease (AD) at various clinical stages. 1 Currently, approximately 6 million Americans are living with AD. This number is rapidly increasing, and it is estimated that in 2060, about 14 million Americans will have AD. 2 AD neuropathology, such as amyloid-β plaques, neurofibrillary tangles, and neuroinflammation, begins accumulating in mid-life, decades before cognitive impairment and diagnosis. Since some of the most striking symptoms associated with AD are related to cognitive decline, the most commonly studied brain regions are those that are implicated in cognitive function. However, metabolic dysfunction is another symptom of AD that has recently been gaining attention. The hypothalamus, responsible for the regulation of several homeostatic functions, is affected by AD pathology, resulting in the dysregulation of metabolism, sleep, and neuroendocrine alterations in AD patients.3,4, 3,4 Previously, we and others have reported that metabolic disturbances are associated with pathological changes to the hypothalamus in rodent models of AD.3,5–8 , 3,5–8

Not only does AD contribute to hypothalamic and metabolic disturbances, but conversely, metabolic disturbances are a driver of AD pathology. 3 Metabolic disease (including obesity and diabetes or prediabetes), especially in mid-life, is a major risk factor for developing AD.4,9–11 , 4,9–11 A recent study showed that in individuals older than 60 years of age, metabolic disease was associated with a greater than 11-fold higher risk of AD compared to metabolically healthy individuals. 11 Additionally, up to 80% of AD patients suffer from impaired glucose metabolism and insulin resistance has been reported in the brain of AD patients.12 –14 Animal studies, including our own, are in line with these clinical observations. Inducing metabolic disease via consumption of a high fat (HF) diet has been shown to worsen AD pathology and cognitive deficits in animal models.5,15–19 , 5,15–19 Previously, we reported that consumption of a HF diet from ∼3– 7 months of age in 3xTg-AD mice resulted in a wider array of cognitive deficits as well as more severe weight gain, glucose intolerance, and hypothalamic inflammation in females compared to males.5,20, 5,20 These results not only highlight the association between metabolic impairment and AD, but also shed light on underlying sex differences, with a greater vulnerability of females. Female sex is a major risk factor for AD, with women constituting 2/3 of all those suffering from the disease.21,22, 21,22 Since the greatest risk factor for AD is advanced age, most women with AD are post-menopausal. Menopause is a mid-life endocrine transition during which women experience a loss of ovarian hormones, leading to increased risk for cognitive decline and dementia.23 –27 This is in part due to the loss of the neuroprotective effects of estrogens, as well as the exacerbation of dementia risk factors, as menopause has been associated with increased weight gain and visceral fat accumulation, increased rates of type 2 diabetes, and altered brain glucose metabolism.23–25,27–31 , 23–25,27–31

In this study we sought to determine the effect of menopause on metabolic disease in the context of AD. Using a knock-in mouse model of AD, App^NL-F mice, we induced metabolic disease using a HF diet and modeled menopause using an accelerated ovarian failure model. 32 This ovary-intact accelerated ovarian failure model of menopause is more clinically relevant than an ovariectomy model, as the mice go through a perimenopausal period. 33 We found that both menopause and HF diet had negative effects on several metabolic outcomes, potentially through modulation of hypothalamic gene expression.

MATERIALS AND METHODS

Animals and experimental design

All experiments were approved by the Albany Medical College Animal Care and Use Committee and in compliance with the ARRIVE guidelines. App^NL-F (App^tm2.1Tcs/App^tm2.1Tcs) mice were acquired congenic on a C57BL/6J background from Dr. John Cirrito at Washington University following MTA from Riken, Japan. 32 These mice were bred in-house and were fed a standard chow diet (Purina Lab Diet 5P76) until the start of this study. Female wild type mice (WT; C57BL/6J; #000664) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) at ∼2.75 months of age and left to acclimate for at least a week before enrolling in the study. All mice were housed (3– 5 per cage) at ∼21°C, 30– 70% humidity, with a 12-h light/dark cycle. At three months of age, cages of mice were randomized to treatment groups and placed on either a high fat (HF) diet (60 kcal% fat, D12492, Research Diets, New Brunswick, NJ, USA) or a low fat (LF) diet (10 kcal% fat, D12450J, Research Diets, New Brunswick, NJ, USA). The chosen control diet has only 7% sucrose thus avoiding negative effects of a high-sucrose diet. This study includes two independent experiments (see Figs. 1A and 2A for timelines). The first experiment compared WT and App^NL-F female mice on both diets and evaluated weight gain and glucose tolerance. The second study compared the effects of menopause and diet within App^NL-F mice. To model menopause, we induced accelerated ovarian failure as described previously. 34 Starting at diet onset (3 months of age), mice received once daily i.p. injections of 4-vinylcyclohexene diepoxide (VCD, 160 mg/kg) for 20 consecutive days. Control mice on each diet received vehicle (sesame oil) injections. For this study, the mice were divided into 4 cohorts with each cohort including mice from all experimental groups. Cyclicity was assessed using vaginal cytology starting 2 months after the 1st injection (mice ∼5.5 months old), and mice that remained in diestrus for 10 consecutive days were declared acyclic/menopausal. Average cycle length was tracked for oil injected mice and is shown in Supplementary Figure 1A. Six months after diet onset, mice received a glucose tolerance test (GTT) to evaluate their diabetic status, as described below. One month later (at 10 months old), unfasted mice were euthanized by deep anesthesia followed by cardiac puncture to collect blood, then perfused with ice cold heparinized saline. Brains were collected and bisected, with one hemisphere randomly selected to undergo post-fixation for use with immunofluorescent labeling, and the other hemisphere regionally dissected, and flash frozen for qPCR. Visceral, subcutaneous, and brown fat weights were collected at endpoint. Additionally, mice underwent vaginal cytology on the day they were euthanized, and ovaries were collected to confirm menopause.

Fig. 1

Metabolic effects of HF diet in WT versus APP^NL-F female mice. An experimental timeline is shown in A. Metabolic status was assessed using body weight, adiposity, and glucose intolerance as outcome measures. Body weight change at the end of the experiment as a percentage of the start weight is shown in B. Adiposity was evaluated by measuring the percentage of the wet weights of several fat tissue types at endpoint, including visceral fat (C) and subcutaneous fat (D). Glucose tolerance test (GTT) was used for pre-diabetic status evaluation. Blood glucose levels (mg/dL) before (fasting) and up to 2 h after injection of a glucose bolus (2 g/kg in saline i.p.) E) Area under the curve for blood glucose during GTT (F). A 2-way ANOVA was used to compare the effect of diet and menopause on each variable. For the GTT (E), a 3-way repeated measures ANOVA was used. ^*p < 0.05, ^****p < 0.0001. n = 8– 22 mice/group. WT, wild type; HF, high fat; LF, low fat; VCD, 4-vinylcyclohexene diepoxide (menopause model). Timeline figure created with BioRender.

Fig. 2

Metabolic effects of HF diet and menopause in female APP^NL-F mice. A timeline of the experiment is shown in A. Metabolic status was assessed using body weight, adiposity, and glucose intolerance as outcome measures. Body weight was measured monthly from the beginning of the study (B). Blue arrow indicates the time of the GTT. Body weight change as a percentage of the start weight is shown in C at the end of the experiment and in D at the time of the GTT. Weight gain between GTT and the endpoint is shown in E. Adiposity was evaluated by measuring the percentage relative to body weight of wet weights of several fat tissue types at endpoint, including visceral fat (F), subcutaneous fat (G), and brown fat (H). Prediabetic status was evaluated using GTT. Blood glucose levels (mg/dL) before (fasting) and up to 2 h after injection of a glucose bolus (2 g/kg in saline i.p.) (I). Area under the curve for blood glucose during GTT (J). A 2-way ANOVA was used to compare the effect of diet and menopause on each variable. For the GTT (E), a 3-way repeated measures ANOVA was used. # is used for main effects of menopause. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^****p < 0.0001. n = 14– 19 mice/group. HF, high fat; LF, low fat; VCD, 4-vinylcyclohexene diepoxide (menopause model); GTT, glucose tolerance test. Timeline figure created with BioRender.

Glucose tolerance test

Glucose tolerance test was performed as previously described.20,35,36 , 20,35,36 Mice were fasted for 16 h overnight. Baseline blood glucose levels in saphenous vein blood were measured by glucometer (Verio IQ, OneTouch, Sunnyvale CA, USA). Mice received 2 g/kg of glucose via i.p. injection and blood glucose levels were re-measured at 15-, 30-, 60-, 90-, and 120-min post-injection.

Estrous cycle and ovarian cytology

Vaginal cytology was performed daily starting 2 months after VCD injections. For the first cohort, cytology was performed daily for all mice. For the rest of the cohorts, vaginal cytology was performed on Mondays and continued up to 10 days if the mouse was in diestrus. If mice exited diestrus (were still cycling), then cytology was stopped and resumed the following week to minimize stress. Oil injected mice were paired with a VCD mouse and treated similarly in parallel to make sure both groups received similar amounts of handling related stress. All mice received vaginal cytology on the day of euthanasia.

Vaginal cytology took place between 9:00 AM and 11:00 AM by performing a vaginal lavage with 10μL of PBS. Contents were examined under a microscope at 20× (Zeiss PrimoVert), and estrus cycle phase was determined by analyzing the dominant composition of cells as described previously. 35 A mouse was considered menopausal if it was stuck in diestrus for 10 consecutive days. Vaginal cytology at euthanasia was used to confirm that menopausal mice were no longer cycling. Only one mouse in the LF VCD group was excluded. The cycle distribution can be seen in Supplementary Figure 1C. Vaginal cytology was scored independently by 2 different experimenters blinded to the groups.

Ovaries were dissected and postfixed in a 4% paraformaldehyde (PFA) solution overnight then transferred and stored in 70% ethanol. Ovaries were dehydrated, paraffin embedded and sectioned at 5μm thickness before being stained with hematoxylin and eosin by the Albany Medical College research pathology core. Images were taken at 10× on a Zeiss PrimoVert microscope and analyzed for the presence or absence of ovarian follicles. Representative images can be found in Supplementary Figure 1B.

Serum diabetes markers

During euthanasia, blood was collected via cardiac puncture and left to coagulate then spun at 1500×g for 10 min at 4°C to collect serum. Serum samples were aliquoted and stored at – 80°C until assayed. Diabetes-associated markers were assessed using the Bio-Plex Pro Mouse Diabetes 8-Plex Assay (171F7001M, Bio-Rad, Carlsbad, CA, USA) according to the manufacturer’s instructions. Glucose levels were also measured in frozen serum samples taken during euthanasia using the same glucometer as for the GTT (OneTouch Verio IQ). Mice were not fasted before tissue collection.

Quantitative polymerase chain reaction (qPCR)

RNA was extracted from flash frozen hypothalamic tissue using Qiagen RNeasy plus mini kit according to the manufacturer’s instructions (74131, Qiagen, USA). Next, 240 ng of RNA were converted to cDNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (4374967, Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative PCR was performed in triplicates on 10 ng of cDNA in a 10μl reaction using TaqMan probe technology on a Bio-Rad CFX-384 real time system. Taqman assays (Thermo Fisher Scientific) used were: Agrp (Mm00475829_g1), Npy (Mm01410146_m1), Pomc (Mm00435874_m1), Lepr (Mm00440181_m1), Mc4r (Mm00457483_s1). The housekeeping genes used were Rpl13a (Mm01612986_gH) and Rps17 (Mm01314921_g1). Bio-Rad CFX Maestro 1.1 software was used to analyze the data. The relative expression levels of genes of interest were calculated using the ΔΔCq method relative to either of the housekeeping genes using the females on a LF diet with oil injections as reference group.

Immunofluorescent labeling of hypothalamus

Hemispheres used for immunofluorescent labeling were fixed overnight in a 4% paraformaldehyde (PFA) solution, cryoprotected in 30% sucrose, then frozen in optimal cutting temperature (O.C.T.) solution (23-730-571, Thermo Fisher Scientific) and stored at – 80°C until further processing. Tissue sections (35μm thickness) were obtained using a cryostat (Cm1950, Leica) and stored at 4°C in cryopreserve. A series of sections containing the hypothalamus (4– 6 sections per animal) was divided into 2 alternating sets: one processed for labeling AgRP and the other for MC4R. Slices were permeabilized and blocked for one hour at room temperature using 0.2% Triton X-100 in PBS (TPBS) with 5% donkey serum solution. Primary antibody of rabbit anti-AgRP (1:1000; H-003-57 Phoenix Pharmaceuticals, lot #01826-6) was applied for 36 h at 4°C in blocking solution. Rhodamine Red-X donkey anti-rabbit (1:500; 711-295-152, Jackson ImmunoResearch) was added in 0.2% TPBS for 1 h at room temperature. Primary antibody of rabbit anti-MC4R (1:1000; ab24233 AbCam, lot # 1073198-1) was applied overnight at 4°C in blocking solution. Alexa Fluor 647 donkey anti-rabbit (1:1000; 711-295-152, Jackson ImmunoResearch) was added in 0.2% TPBS for 1 h at room temperature. Slices were counterstained with DAPI and washed 3 times before mounting between slides and coverslips using ProLong^™ Gold Antifade Mountant (P36930 Thermo Fisher Scientific). Images of brain slices were obtained using the Axio Observer Fluorescent Microscope (Carl Zeiss Microscopy, Oberkochen, Germany). Regions of interest (ROIs) were drawn around the arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), lateral hypothalamus area (LH), and paraventricular nucleus (PVN) using ImageJ software (NIH, Bethesda, MD, USA) to quantify the average area covered (% ) by each stain after thresholding of each image. All measurements were made by an experimenter who was blinded to the treatment group.

Microglia analysis

Immunofluorescent labeling for microglia was performed as previously described on a different set of sections than above.5,20,37 , 5,20,37 Briefly, slices were permeabilized and blocked for one hour at room temperature using 0.3% Triton X-100 in PBS (TPBS) with 5% donkey serum solution. Primary antibodies: goat anti-Iba-1 (1:1000; PA5-18039, lot #TI2638761, Thermo Fisher Scientific), rat anti-CD68 (1:1000; MCA1957, lot #1708, Bio-Rad) and rabbit anti-Amyloid-β (1:500; 71-5800, lot# SH257822, Thermo Fisher Scientific) were applied overnight at 4°C in blocking solution. Alexa Fluor 647 donkey anti-goat (705-605-147, Jackson ImmunoResearch), Alexa Fluor 488 Donkey anti-rabbit (ab150073, Jackson ImmunoResearch), and Rhodamine Red-X donkey anti-rat (712-295-150 Jackson ImmunoResearch) were added in blocking buffer (1:500) for 2 h at room temperature. Slices were counterstained with DAPI and washed 3 times before mounting between slides and coverslips using ProLong^™ Gold Antifade (P36930 Thermo Fisher Scientific). Images of brain slices were obtained at 10× magnification using the Axio Observer Fluorescent Microscope (Carl Zeiss Microscopy, Oberkochen, Germany). Regions of interest (ROIs) were drawn around the ARC, DMH, VMH, LH, and PVN of every 8th section using ImageJ. ROIs were drawn for each hypothalamic nucleus. Microglia were hand counted and classified according to their morphology (ramified or ameboid) and presence of CD68 labelling as described before. 38 All measurements were made by an experimenter who was blinded to the treatment group.

Open field testing

To evaluate locomotor activity, mice were placed in a square arena (45×45 cm) and allowed to explore freely for 10 min, then removed and placed in a “recovery cage” so as not to expose them to naïve cage mates. Videos were analyzed using ANY-maze software (Stoelting, Wood Dale, IL, USA). Total distance traveled (m) was measured as a proxy for locomotor activity.

Statistical analysis

Statistical analyses were completed using GraphPad Prism (GraphPad Software v10, San Diego, CA, USA). Statistical outliers were removed following identification using Grubbs’ test with alpha set at 0.05. Data were analyzed using 2-way ANOVAs followed by Fisher’s LSD post hoc test, except for measures tracked over time (GTT) that used 3-way repeated measures ANOVAs. Only main effects are presented on graphs unless an interaction effect is observed or to make results clearer and easier to interpret. Correlations were run for all animals and separately for each group using Pearson’s correlations. Statistical significance was set at p < 0.05. Data are expressed as mean+SEM.

RESULTS

HF diet causes more severe glucose intolerance in App^NL-F versus WT female mice

Metabolic deficits have been observed in AD patients.3,24, 3,24 These are also found in several AD mouse models.5 –7 We have shown when comparing 3xTg-AD and WT mice, that on a HF diet 3xTg-AD females gain more weight and have worse glucose tolerance that WT females or males. 32 In the current study, we compared the effect of HF diet between WT and App^NL-F female mice by measuring several metabolic outcomes including body weight, adiposity, and glucose tolerance (timeline in Fig. 1A). We found that App^NL-F mice gained less weight than WT controls (Fig. 1B, main effect of dementia, p = 0.0367) but both strains gained significant weight on the HF diet (Fig. 1B, main effect of diet p < 0.0001). When comparing adipose tissue accumulation, both WT and App^NL-F female mice on a high fat diet accumulated similar percentages of visceral (Fig. 1C, main effect of diet p < 0.0001), and subcutaneous fat (Fig. 1D, main effect of diet p < 0.0001). No difference between strains was observed for fat accumulation. When examining glucose tolerance, unsurprisingly we found a main effect of diet in increasing values of glucose over time (Fig. 1E, main effect of diet p < 0.0001) resulting in a greater area under the curve (AUC, worse glucose intolerance, Fig. 1F, main effect of diet p < 0.0001) in mice on a HF diet compared to those on a LF diet. Additionally, we observed a diet by dementia interaction, where App^NL-F mice were more impaired by a HF diet than WT controls (Fig. 1E, diet×dementia p = 0.0429, Fig. 1F, diet×dementia p = 0.0424) as evident by a higher AUC in App^NL-F versus WT mice on a HF diet (Fig. 1F, HF WT versus HF App^NL-F, p = 0.0157). These results show that HF diet causes more severe glucose intolerance in App^NL-F female mice compared to WT controls.

Menopause and HF diet cause weight gain and glucose intolerance in App^NL-F mice

Menopause is known to be associated with increased metabolic deficits in women.3,24, 3,24 In a mouse model of AD, we assessed differences in the effect of HF diet and menopause as indicated in the experimental design outlined in Fig. 2A. We tracked body weight over time (Fig. 2B). As expected, females on a HF diet gained significantly more weight than those on the LF diet (Fig. 2B, main effect of diet p < 0.0001). Additionally, we found a main effect of menopause in increasing body weight over time (Fig. 2B, main effect of menopause p = 0.0011). When evaluating weight gain at the end of study, unsurprisingly, females on a HF diet weighed significantly more than those on the LF diet (Fig. 2C, main effect of diet p < 0.0001), however no main effect of menopause was found. Conversely, when evaluating weight gain at the time of the GTT (before behavior testing), in addition to the diet effect (Fig. 2D, main effect of diet p < 0.0001), we found a main effect of menopause in increasing body weight (Fig. 2D, main effect of menopause p = 0.0018). We observed that menopausal mice gained less weight than oil injected controls during the period of potentially stressful handling after GTT and behavior testing (Fig. 2E, main effect of menopause p = 0.0006). We sought to determine whether there were any differences in adiposity. HF diet increased visceral fat (Fig. 2F, main effect of diet p < 0.0001), subcutaneous fat (Fig. 2G, main effect of diet p < 0.0001), but not brown fat weights (Fig. 2H). No effect of menopause was observed for fat accumulation.

Additionally, we evaluated the prediabetic status of mice by performing a GTT 6 months after diet onset (∼9 months of age). When examining glucose clearance over time, there was a main effect of HF diet to impair glucose clearance (Fig. 2I, main effect of diet p < 0.0001). This diet effect translated into a larger area under the curve (AUC, worse glucose intolerance) in mice on a HF diet compared to those on a LF diet (Fig. 2J, main effect of diet p < 0.0001). Additionally, we observed a main effect of menopause in worsening glucose intolerance, independent of diet (Fig. 2I, main effect of menopause p = 0.0286). This was supported by a larger AUC for VCD injected mice (Fig. 2J, main effect of menopause p = 0.0346). Taken together, these results show that menopause, on its own or in addition to HF diet, leads to metabolic impairment in App^NL–F mice.

Effects of menopause and HF diet on serum levels of diabetes-associated markers

Since HF diet and menopause had effects on metabolic outcomes, we sought to assess serum levels of several diabetes-associated markers. Leptin is a hormone that is released by adipose tissue to limit food intake. 39 As expected, HF diet resulted in hyperleptinemia, with leptin levels in HF fed mice reaching ∼3-fold higher than those on a LF diet (Fig. 3A, main effect of diet p < 0.0001), but menopause had no effect on leptin levels. No group differences were observed in ghrelin levels, a hormone that stimulates feeding behavior (Fig. 3B). As expected, HF diet also increased levels of plasminogen activator Inhibitor-1 (PAI-1, a biomarker of fibrinolysis that has been shown to increase with age, obesity, and insulin resistance) (Fig. 3C, main effect of diet p < 0.0001).39,40, 39,40 Menopause did not alter PAI-1 levels. Next, we examined hormones that control blood glucose levels, including insulin (decreases blood glucose) and glucagon (increases blood glucose). As expected, HF diet increased insulin levels (Fig. 3D, main effect of diet p = 0.0361). Additionally, we observed a trend in menopausal mice for lower insulin (p = 0.077). HF diet led to decreased glucagon levels (Fig. 3E, main effect of diet p = 0.0025), with no effect of menopause. As these hormones control serum glucose levels, we measured glycemia in the same serum sample and its ratios to both insulin and glucagon. We found a diet by menopause interaction in glucose levels (Fig. 3F, p = 0.0365) but no post-hoc significance using Fisher’s LSD multiple comparisons test. We noted that mice on a HF diet had a lower glucose/insulin ratio (Fig. 3G, main effect of diet p = 0.0473) and a diet×menopause interaction (p = 0.0060) driven by VCD injected mice on LF diet having a lower ratio than LF oil injected mice (Fisher’s LSD p = 0.0203) or HF VCD injected mice (Fisher’s LSD p = 0.0067). Conversely, mice on a HF diet had a lower glucose/glucagon ratio (Fig. 3H, main effect of diet p = 0.0027) but no effect of menopause was observed. HF diet also increased levels of resistin (a hormone that suppresses insulin function) (Fig. 3I, main effect of diet p < 0.0001). 40 No differences were observed between groups for levels of gastric inhibitory polypeptide (GIP; Fig. 3J) and glucagon-like-peptide 1 (GLP-1, Fig. 3K); these hormones have opposing actions on glucagon secretion, respectively enhancing, and repressing it. 41 Further, we looked for associations between these serum markers and metabolic outcomes measured above (Supplementary Figure 2). As expected, we found a positive association between leptin levels and both weight gain and visceral fat percentages when pooling all animals together or when stratifying by menopausal status. However, some associations were found in VCD injected menopausal mice but not in oil injected controls. For example, high glucagon levels were associated with lower fat accumulation in VCD injected but not oil injected mice. Taken together, these results show that metabolic health (weight gain, visceral fat, etc.) is most strongly associated with serum levels of metabolic hormones in App^NL-F female mice however, menopause can alter some of these effects.

Fig. 3

Effects of HF diet and menopause on serum levels of diabetes-associated markers. At endpoint (∼7 months after diet onset, mice ∼10 months), blood was collected via cardiac puncture and serum was extracted and stored at – 80°C. Diabetes associated markers were later measured using the Bio-Plex Pro Mouse Diabetes 8-Plex Assay. Graphs represent serum levels (ng/mL) of leptin (A), ghrelin (B), plasminogen activator inhibitor-1 (PAI-1; C), insulin (D), glucagon (E), resistin (I), gastric inhibitory polypeptide (GIP; J) and glucagon-like-peptide 1 (GLP-1; K). We measured glucose levels in the serum (F) and calculated ratios of glucose to insulin (G) and to glucagon (H). A 2-way ANOVA was used to compare the effect of diet and menopause on each marker. Main effects are indicated above the graphs in case of a trend or an interaction at which point posthoc differences are indicated on the graph. ^*p < 0.05, ^**p < 0.01, ^****p < 0.000.1. n = 8-9 mice/group. HF, high fat; LF, low fat; VCD, 4-vinylcyclohexene diepoxide (menopause model).

Interaction between menopause and HF diet on hypothalamic markers of energy balance

Hypothalamic dysfunction, which can lead to disturbances in feeding behavior and metabolic function, is well known to occur in AD. 3 Therefore, we measured the expression of several hypothalamic genes involved in regulating metabolic function and feeding behavior. We did not observe any changes in the expression of the fast-acting orexigenic neuropeptide Y (Npy, Fig. 4A). However, menopause altered levels of the delayed, longer-acting orexigenic agouti-related peptide (AgRP, Fig. 4B) (main effect of menopause p = 0.037), with a decrease in Agrp mRNA expression specifically in menopausal females on a LF diet (LF oil versus LF VCD p = 0.0054). This resulted in a menopause by diet interaction (p = 0.040). Next, we examined the expression of melanocortin 4 receptor (Mc4r), because MC4R deficiency is linked to hyperphagia.42,43, 42,43 HF diet trended towards decreasing Mc4r (main effect of diet p = 0.062); conversely, menopause trended towards increasing Mc4r (Fig. 4C, main effect of menopause p = 0.053). No differences were observed between groups in expression levels of leptin receptor (Lepr, Fig. 4D) or pro-opiomelanocortin (Pomc, Fig. 4E), which play roles in energy balance. We sought to validate changes in mRNA levels for AgRP and MC4R using immunofluorescent labeling. We found a main effect of menopause in decreasing AgRP labeling density in the only the PVN of the hypothalamus (Fig. 4F and 4G, main effect of menopause p = 0.0068). For MC4R (Supplementary Figure 3A), we found a diet by menopause interaction in the VMH (Supplementary Figure 3B, p = 0.0379) driven by oil injected mice on HF diet having a higher positive area density than LF oil injected mice (Fisher’s LSD p = 0.0217) or HF VCD injected mice (Fisher’s LSD p = 0.0456). In the DMH, we noted a trend for menopause to increase the positive area density (Supplementary Figure 3C, p = 0.0679). No changes were observed in the other nuclei (Supplementary Figure 3D and 3E). We additionally sought to examine differences in amyloid-β plaques in the hypothalamus, however, we did not observe any plaques in most animals, despite plaques being visible in other brain regions such as the cortex and the hippocampus (Supplementary Figure 3F).

Fig. 4

Effects of menopause and HF diet on the expression of hypothalamic markers. Hypothalami were dissected, flash frozen and then RNA was extracted. qPCR was used to measure the expression levels of mRNA of genes coding for several markers related to food consumption and energy balance. Graphs represent relative expression levels (fold change compared to LF oil mice) of Npy (A), Agrp (B), Mc4r (C), Lepr (D), Pomc (E); n = 7– 9 mice/group. AgRP immunofluorescent labeling was performed (magenta in F, representative images of each group) and the percentage of the positive area was calculated in hypothalamic nuclei, such as the PVN (G); n = 5-6 mice/group. A 2-way ANOVA was used to compare the effect of diet and menopause on each marker. Main effects are indicated above the graphs in case of a trend or an interaction at which point posthoc differences are indicated on the graph. ^**p < 0.01, # is used for main effects of menopause; ^#p < 0.05. HF, high fat; LF, low fat; VCD, 4-vinylcyclohexene diepoxide (menopause model); PVN, paraventricular nucleus.

We examined the association of these hypothalamic genes/proteins with metabolic measures, and serum levels of diabetes-associated markers (Supplementary Figure 4). We observed different associations in oil-injected controls versus VCD-injected menopausal mice between hypothalamic gene expression and metabolic markers (Supplementary Figure 4A) and serum levels of diabetes associated hormones (Supplementary Figure 4B). For example, in menopausal mice, but not in controls, Mc4r expression was significantly associated with several metabolic measures, as well as insulin and leptin levels. MC4R positive area density in the LH is associated with higher visceral fat accumulation in oil injected but not VCD injected mice (Supplementary Figure 4A). On the other hand, ghrelin levels were negatively associated with LepR expression in control but not menopausal mice (Supplementary Figure 4B). These results indicate a potential shift in metabolic control post-menopause, and that some menopause-induced metabolic changes might in part result from central effects on hypothalamic gene expression.

Changes in microglia response in the hypothalamus are region specific

Microglia activation is a prominent feature of AD. 44 Additionally, in both humans and rodent models, obesity is associated with increased gliosis notably in the hypothalamus.45 –47 Immunolabeling was performed for Iba1 and CD68 to assess microgliosis in several hypothalamic nuclei (Fig. 5). In the LH (Fig. 5B, C), as well as the the DMH (Fig. 5D, E) we did not observe any changes in total microglia density (Iba1+ cells) however, mice on HF diet had a lower percentage of activated microglia (Iba1+CD68+ ameboid cells, main effect of diet p = 0.0113 LH and p = 0.0206 DMH). Conversely in the VMH, mice on a HF diet, regardless of menopause status, had a higher density of microglia (Fig. 5F, main effect of diet p = 0.0384) but similar percentage of activated cells compared to LF mice (Fig. 5G). No statistically significant changes were observed in the ARC or the PVN (Fig. 5H-K). These results show that menopause does not necessarily affect microglia response in the hypothalamus and that HF diet effects are different depending on the hypothalamic nucleus.

Fig. 5

Effects of menopause and HF diet on microglial response in the hypothalamus. Microglia density and activation was assessed by immunofluorescent labeling of Iba1 (cyan) and CD68 (magenta). DAPI (blue) was used to counterstain cell nuclei (A). Dotted lines represent different regions of interest indicating different nuclei. Quantification of the density (cells/mm²) of the Iba1+ cells and the percentage of Iba1+CD68+ ameboid cells respectively in the (B, C) lateral hypothalamus area (LH), (D, E) dorsomedial hypothalamus (DMH), (F, G) ventromedial hypothalamus (VMH), (H, I) the arcuate nucleus (ARC), and (J, K) the paraventricular nucleus (PVN) of the hypothalamus. A 2-way ANOVA was used to assess the effect of diet and menopause. Main effects are indicated above the graphs in case of a trend. ^*p < 0.05 n = 5– 7 mice/group. HF, high fat; LF, low fat; VCD, 4-vinylcyclohexene diepoxide (menopause model).

Menopause does not alter overall activity levels

General locomotor activity was assessed in the open field test. As expected, HF diet fed mice covered less distance; however, no effect of menopause was observed (Supplementary Figure 5A). Further, locomotor activity did not correlate with any of the metabolic measures (Supplementary Figure 5B), suggesting that changes in metabolic measures might not be mediated by physical activity.

DISCUSSION

AD patients often have metabolic dysfunction, which has been shown to be in part due to hypothalamic pathology.3,7,48 , 3,7,48 Female sex is also a risk factor for AD, such that ∼2/3 of AD patients are women.21,22, 21,22 Given that the majority of those who suffer from AD are elderly, most female AD patients are post-menopausal. On its own, menopause has been shown to increase metabolic risk factors for AD, such as obesity and diabetes.23,49–52 , 23,49–52 Here, we sought to determine the interactions between HF diet, menopause, and dementia on metabolic health. By comparing WT and App^NL-F female mice, on either a LF or HF diet, we show that even though AD mice gained less weight, they had worse glucose intolerance. Using an accelerated ovarian failure model in App^NL-F female mice, on either a LF or HF diet, menopausal mice exhibited worse outcomes on some metabolic measures. Our data further suggest that some of the effects of menopause may stem from central changes in the hypothalamus, such as altered expression of genes regulating energy balance without necessarily affecting microglial response.

Metabolic disease (including obesity and glucose intolerance) is a major dementia risk factor.4,9–11 , 4,9–11 Further, glucose intolerance (diabetes and prediabetes) confers an even greater risk for dementia in women compared to men.53,54, 53,54 Animal studies show that middle-aged female mice are more vulnerable to the negative metabolic effects of a high fat diet.35,36, 35,36 These findings also extend to mouse models of AD.5,6,55 , 5,6,55 For example, in both 3xTg-AD and TgAPP mice, females gain more weight, accumulate more visceral fat, and develop worse glucose intolerance than males or WT females.5,55, 5,55 Here we show that App^NL-F female mice gain less weight than WT controls, which can be due to differences in AD models or degree of pathology. When immunolabeling for amyloid-β plaques in App^NL-F female mice, we did not observe plaques in the hypothalamus at this stage, even with HF diet or menopause. Conversely, in accordance with previous studies in AD models, App^NL-F female mice on a HF diet had worse glucose intolerance than HF diet fed WT female mice. Impaired glucose tolerance seems to be a consistent pathological feature in female mice across different models of AD.5,20,55 , 5,20,55

Weight gain and glucose intolerance are known metabolic changes that can occur in women after menopause.49 –52 These changes have also been described in the ovariectomy animal model of surgical menopause, and in the VCD induced menopause model.34,56–59 , 34,56–59 In agreement with these studies, our study shows that in the context of AD, menopausal female mice exhibit greater weight gain over time and worse glucose tolerance compared to LF diet fed controls. We did not observe effects of menopause on visceral or subcutaneous adiposity at the end of the study. This could be due to the absence of difference in body weight gain between oil and VCD injected mice at that time point. At the time of GTT (one month before the end of the study), we did observe a menopause effect on body weight gain wherein VCD injected mice weighed more than oil injected ones. However, we found that menopausal mice gained less weight than controls specifically between GTT and endpoint when mice were subjected to behavioral testing. This might indicate that menopause increases vulnerability to stress. We are currently investigating the interaction between stress and menopause in other studies. Of note, in young wild type (C57BL6/J) mice, without weight loss prior to endpoint analysis, we have previously found that the VCD menopause model does result in weight gain and increases in both visceral and subcutaneous fat. 34 However, in middle-aged mice (with either young or middle-aged onset of menopause), we only observe increased weight gain and glucose intolerance with menopause (and with peri-menopause), but no changes in adiposity. 60 Thus, across several studies the increase in weight and glucose intolerance is consistent, yet the age of the animals and stressors may influence the effects of menopause on adiposity.

To further characterize the metabolic state of these mice, we measured serum levels of diabetes-associated markers. Menopause had no main effect on any of the measured markers (only a trend p = 0.077 to lower insulin levels). However, we found a diet by menopause interaction on plasma levels of glucose at endpoint and in the Glucose/Insulin ratio where menopausal mice on a LF diet had a higher ratio than LF oil injected controls. This potentially indicated a deficit in insulin secretion in response to the same levels of glucose and might explain the higher glucose levels observed in these mice at the time of GTT. One caveat is that these glucose levels were taken in nonfasted mice on the day of euthanasia day which was after the GTT and behavior; this may create a confounding variable as glucose levels are affected by stress. Higher insulin levels were associated with increased body weight and worse glucose tolerance in VCD-injected menopausal mice. These findings indicated that the adverse metabolic effects observed in menopausal mice on a LF diet could be mediated by insulin dysregulation. Previous studies using this menopause model in WT mice do not report a difference in fasting insulin levels between control and VCD-injected mice on a control diet. 56 However, on a HF diet, menopausal females had significantly higher fasting insulin levels than cycling controls. 56 These differences could be due to the AD pathology in our mice that starts at around 6 months of age. Insulin resistance drastically increases in women after menopause.51,61–63 , 51,61–63 Studies have shown that resistin levels vary in parallel and in the same direction as insulin and glucose levels.64,65, 64,65 In our study, menopause did not affect resistin levels. This is in line with other studies showing no change in resistin production by adipose tissue in ovariectomized female rats. 66 This decoupling between insulin and resistin levels in LF-fed AD mice could be a potential mechanism behind the adverse effects of menopause on glucose tolerance. In this study, our samples were examined from non-fasting animals during their inactive period (light cycle); values for insulin and glucagon, among others, could be different if examined after a fasting period. Further studies, beyond the scope of this paper, are needed to elucidate the interaction between menopause and insulin signaling in the context of AD.

Balancing food consumption with energy expenditure is centrally regulated in the hypothalamus. This brain region is gaining interest in AD research as it has been shown to be affected by both amyloid plaques and tau neurofibrillary tangles, contributing to metabolic and non-cognitive deficits seen in AD patients that are observed prior to the onset of cognitive impairment.3,7,48 , 3,7,48 We therefore sought to explore how menopause and diet can influence changes in the hypothalamus in the context of AD. We found a decrease in Agrp expression in menopausal mice, and that VCD injected mice had lower labeling density for AgRP in the PVN. This effect could be due to the loss of estrogen. Estradiol is known to be anorexigenic and regulates food intake in part by inhibiting the excitability of the hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons. 67 Further, in ovariectomized female mice, treatment with estradiol restores the response of NPY/AgRP neurons to insulin. 67 Aside from NPY/AgRP, estrogen has also been shown to promote POMC excitability; however, we did not observe any group differences in Pomc mRNA levels. Some of estrogen’s effects may also be mediated through peripheral effects on other organs, such as the liver. 68 For example, conditional knock-out of estrogen receptor alpha in the liver leads to alterations of hepatic lipid metabolism and dysregulation of AgRP neurons. 69 These results provide a mechanistic insight into the effects of estrogen loss induced by menopause in a mouse model of AD and suggest that AgRP may be a promising target for future studies.

AgRP also acts as an MC4R antagonist, driving energy intake. 70 Menopausal mice tended to have higher Mc4r mRNA expression in the hypothalamus and higher labeling density for MC4R in the DMH which controls among other things food intake and locomotor activity. 71 MC4R is activated principally by α-melanocyte stimulating hormone (MSH, bioactive products of POMC processing), which promotes a cessation of feeding, increased energy expenditure and weight loss. Estrogen has been described to increase Mc4r gene expression via estrogen receptor alpha. 72 Surprisingly, VCD mice tended to have higher Mc4r mRNA expression and higher labeling density for MC4R in the DMH regardless of diet; however, studies have shown that androgens can also activate Mc4r gene in female rats.73,74, 73,74 Female rats treated with subcutaneous dihydrotestosterone pellets exhibit higher expression of Mc4r than controls. 73 In women, postmenopausal ovaries stop producing estrogens but continue to produce androgens, a hormonal change that is also present in the VCD menopause model but lacking in the ovariectomy model.75,76, 75,76 The increase in Mc4r following menopause could be a compensatory mechanism, potentially induced by androgens, to reduce food consumption and prevent excessive weight gain following menopause. Further studies are needed to elucidate the link between androgens and MC4R in postmenopausal mice. One limitation of the current study is the lack of non-AD mice; however, in a previous study, we thoroughly compared wild type and 3xTg-AD mice and did not observe any effects of AD background on any of these hypothalamic markers in females. 5

A state of neuroinflammation, marked by microgliosis, has been described in both AD and metabolic disease.44 –47 Studies show that hypothalamic inflammation has been linked to energy balance disruptions.3,77–79 , 3,77–79 To assess whether this might contribute to the metabolic status of our mice, we assessed microglia density and activation in several hypothalamic nuclei involved in energy balance and glucose homeostasis. We did not find any main effect of menopause other than a trend for increasing Iba1 cell density in the ARC. These results indicated that menopause may not increase hypothalamic microglia response in AD mice, however this may change as at this timepoint we did not observe any amyloid plaques in the hypothalamus. These results are in line with a previous study showing no alteration of Iba1 immunoreactivity or morphology in the PVN or the hypothalamus of young WT mice treated with VCD. 80 Similar results were shown in the Tg-SwDI mouse model of AD, where no increased microglia activation was found in the hippocampus of VCD injected mice. 81 Our results are in agreement with these studies and show that menopause does not increase microglia activation in AD models even with comorbid metabolic disease. Conversely, we found that HF diet did not increase microglia density, except in the VMH. Moreover, HF diet led to a decreased percentage of ameboid phagocyting microglia (signs of microglia activation). Most prior studies on the effects of HF diet were conducted in young male rodents, which show that HF diet increases microglia activation. However, our studies and others show that HF diet increases microglia activation in male mice but has no effect or even leads to a decrease in female mice.5,38,82 , 5,38,82 This highlights the need for further studies on microglia response in females as increasing evidence suggests they are different than males.

Changes in hypothalamic gene expression observed above may result in changes in feeding behavior and/or energy expenditure. However, we do not have measurements of food intake, and/or indirect calorimetry to assess reasons underlying differences in metabolic outcomes. When testing general locomotor activity using the open field test, we did find that HF diet fed mice covered less distance, but no effect of menopause was observed. Further, locomotor activity did not correlate with any of the metabolic measures, suggesting that changes in metabolic measures might not be mediated by physical activity.

This is the first study to examine the interaction between metabolic disease, modeled by a HF diet, and menopause using an accelerated ovarian failure model in an AD mouse model. This is an important contribution because the accelerated ovarian failure model is a follicle depleted ovary-intact model in which mice go through a peri-menopausal phase before entering a menopausal phase in which the ovaries cease to produce estrogens but continue to produce androgens (similar to post-menopausal women). We found that in addition to HF diet, menopause caused metabolic impairment in AD mice. Some effects of menopause may be centrally regulated by affecting hypothalamic expression of orexigenic genes in certain hypothalamic nuclei. How these metabolic changes affect AD pathology in other brain regions and cognitive impairment is the subject of other current studies in our lab. The current data show menopause-specific changes in metabolic control, highlighting the need to model menopause in preclinical studies of dementia (AD) and co-morbid risk factors such as metabolic disease.

AUTHORS CONTRIBUTIONS

Charly Abi-Ghanem (Data curation; Formal analysis; Investigation; Methodology; Project administration; Supervision; Writing – original draft; Writing – review & editing); Abigail E. Salinero (Investigation; Methodology); Rachel M. Smith (Formal analysis; Investigation; Methodology); Richard D. Kelly (Formal analysis; Investigation; Methodology); Kasey M. Belanger (Formal analysis; Investigation; Methodology); Riane N. Richard (Formal analysis; Investigation; Methodology); Aaron S. Paul (Formal analysis); Ava A. Herzog (Formal analysis); Christina A. Thrasher (Formal analysis; Investigation; Methodology); Krystyna A. Rybka (Formal analysis); David Riccio (Investigation; Methodology); Olivia J. Gannon (Investigation; Methodology); David Kordit (Investigation; Methodology); Nyi-Rein Kyaw (Formal analysis); Febronia M. Mansour (Formal analysis); Emily A. Groom (Formal analysis); Heddwen L. Brooks (consultation, training in methodology); Lisa S. Robison, PhD (Writing – review & editing; consultation); Kevin Pumiglia (Funding acquisition; Supervision); Damian G. Zuloaga (Conceptualization; Funding acquisition; Supervision); Kristen L. Zuloaga (Conceptualization; Data curation; Formal analysis; Funding acquisition; Project administration; Supervision; Writing – review & editing).

Footnotes

ACKNOWLEDGMENTS

The authors would like to thank Dr. Takaomi Saido for providing the MTA for the App^tm2.1Tcs/App^tm2.1Tcs (App^NL-F) mice, Dr. Paul Feustel for help with statistical analysis, and Matthew K. Wang for technical assistance with experiments.

FUNDING

This work was funded by BrightFocus Foundation postdoctoral fellowship A2022001F (CAG); American Heart Association pre-doctoral award 908878 (AES); NINDS/NIA R01 NS110749 (KLZ); NIA U01 AG072464 (KLZ, KP); and Alzheimer’s Association AARG-21-849204 (KLZ); AARG-1024658 (DGZ, KLZ).

CONFLICT OF INTEREST

The authors have no conflicts to report.

DATA AVAILABILITY

The data supporting the findings of this study are available on request from the corresponding author.

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

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Effects of Menopause and High Fat Diet on Metabolic Outcomes in a Mouse Model of Alzheimer’s Disease

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animals and experimental design

Glucose tolerance test

Estrous cycle and ovarian cytology

Serum diabetes markers

Quantitative polymerase chain reaction (qPCR)

Immunofluorescent labeling of hypothalamus

Microglia analysis

Open field testing

Statistical analysis

RESULTS

HF diet causes more severe glucose intolerance in AppNL-F versus WT female mice

Menopause and HF diet cause weight gain and glucose intolerance in AppNL-F mice

Effects of menopause and HF diet on serum levels of diabetes-associated markers

Interaction between menopause and HF diet on hypothalamic markers of energy balance

Changes in microglia response in the hypothalamus are region specific

Menopause does not alter overall activity levels

DISCUSSION

AUTHORS CONTRIBUTIONS

Footnotes

ACKNOWLEDGMENTS

FUNDING

CONFLICT OF INTEREST

DATA AVAILABILITY

References

HF diet causes more severe glucose intolerance in App^NL-F versus WT female mice

Menopause and HF diet cause weight gain and glucose intolerance in App^NL-F mice