A Mediterranean-style diet protects against cognitive and behavioral deficits,adiposity,and Alzheimer's disease-related markers,compared to a macronutrient-matched typical American diet in C57BL/6J mice

Abstract

Background

Research suggests that modifying risk factors may prevent or delay up to 40% of dementia cases, including Alzheimer's disease (AD). Thus, understanding the potential of healthful dietary patterns, like the Mediterranean diet (MD), in AD prevention is crucial. While supplementation of individual Mediterranean foods has demonstrated efficacy in reducing AD biomarkers and cognitive impairment in rodents, the effects of a comprehensive MD warrant further investigation. Additionally, while rodent studies often use a “Western diet” as a model for the typical American diet (TAD), these diets generally exceed the macronutrient densities of typical American consumption, particularly in fats and carbohydrates.

Objective

To better reflect human diets, we developed two diets for mice that more closely mirrored the macronutrient composition of the traditional MD or the TAD, each with matched macronutrient profiles (50% kcal from carbohydrates, 35% kcal from fat, 15% kcal from protein), and distinct food sources from Mediterranean regions or the U.S., respectively.

Methods

Male C57BL/6J mice were randomly assigned to one diet (MD or TAD) at weaning (21 days of age), which they consumed for six months.

Results

Compared to the TAD, MD animals had lower body weight, abdominal and hepatic fat, serum TNF-α, and central Aβ_1–42, while also exhibiting enhanced exploratory behavior, reduced anxiety-like behavior, and preserved spatial memory. The MD also protected against LPS-induced central inflammation and BDNF loss.

Conclusions

These findings suggest that a comprehensive MD provides protection against metabolic and AD-related markers in wildtype mice, despite matched caloric availability to the TAD.

Keywords

Alzheimer's disease amyloid-beta anxiety-like behavior cytokines fatty liver macronutrient-matched Mediterranean diet plant-based spatial memory Western diet

Introduction

Historically, Alzheimer's disease (AD) prevalence in the United States has consistently outstripped that of countries bordering the Mediterranean Sea in Southern Europe. For example, in 2018, approximately 10% of Americans over the age of 65 had AD,¹ whereas their Southern European counterparts had a comparatively lower incidence, at 6.88%.² Research on human subjects has identified several risk factors linked to an increased risk of AD, such as sedentary lifestyle,³ obesity,^4–6 and poor diet.^7–10 Based on these findings, the observed lower rate of AD prevalence in Southern Europe may be linked to the traditional Mediterranean diet (MD). For example, strict adherence to the MD has been associated with reduced weight gain and obesity,^11–14 along with diminished pro-inflammatory mediators,^15–17 amyloid-beta (Aβ),^9,18 brain atrophy,^10,19–21 cognitive decline,^22–28 and risk of AD and related dementias^28,29 in middle-aged and elderly adults. This largely plant-based diet offers longevity benefits, due to key dietary factors such as high fiber content, low glycemic load, high monounsaturated fatty acid (MUFA) intake, and abundant plant polyphenols.^30–32

In contrast, the typical American diet (TAD), a largely animal-based diet, is suffused with high levels of saturated fatty acids (SFA) and simple carbohydrates, while simultaneously devoid of many essential nutrients.³³ For example, recent evidence highlights a significant nutrient deficiency among Americans: merely 10% meet daily fruit and vegetable recommendations,³⁴ and only 7.4% achieve the recommended fiber goal.³⁵ Both obesity^36–38 and AD^38–40 prevalence are rapidly increasing in the U.S. Numerous studies using a “Western diet” that incorporate exaggerated levels of SFA, and about 42–60% kilocalories (kcal) from total fat,^41–48 provoke obesity-related biomarkers,^41,42 inflammation,^44,45 Aβ and beta-secretase 1 (BACE1),^44,46,49 along with spatial memory impairment^42,50–54 in C57BL/6J mice. However, the effects of a TAD reflecting the average macronutrient profile of Americans have not been thoroughly studied in rodent research.⁵⁵ Given the extensive existing studies on “Western diets,” further research is needed to understand the TAD's impact on metabolic markers and AD susceptibility. Additionally, while prior animal studies have demonstrated that individual MD components, like olive oil, prevent or mitigate AD pathologies, such as hippocampal and cortical Aβ_1–42 in wildtype mice⁵⁶ and transgenic models of AD,^57,58 few have aimed to replicate human Mediterranean dietary patterns in the context of AD research.

To address these crucial gaps in understanding diet-related AD risks, we designed two experimental diets to mimic either the MD or TAD, by providing the average macronutrient densities and diverse food sources typical of Mediterranean regions, including Greece and Italy,^26,30,32 or the United States.^59,60 Additionally, we designed the diets to reflect the typical percentage of kilocalories from different fat sources, including SFAs, MUFAs, and polyunsaturated fatty acids (PUFAs).^32,61,62 Further, we mimicked the typical omega-6 to omega-3 fatty acid ratio in the MD and the TAD,^61,63,64 while also accounting for typical insoluble and soluble fiber content, as the MD is fiber-rich compared to the TAD.⁶¹ Finally, we matched the macronutrient composition of both diets to control for energy availability. Through this distinctive approach that better reflects reality for human populations, we investigated the effects of early-life implementation and long-term consumption of the MD or TAD on AD-related markers and comorbidities in wildtype mice. In Experiment 1, we hypothesized that the MD would protect mice against (1) excessive weight gain, (2) increased abdominal and hepatic fat, (3) peripheral and central inflammation, (4) Aβ_1–42 in the cortex and hippocampus, and (5) behavioral and cognitive deficits, compared to the TAD.

In Experiment 2, an additional group of mice was utilized to examine the effects of lipopolysaccharide (LPS) treatment on the central gene expression of AD biomarkers. As wildtype mice do not typically produce high levels of pro-inflammatory cytokines without an inflammatory stimulus, we utilized LPS, a toll-like receptor 4 (TLR4) agonist, to induce an innate immune response, as established by prior research.^65–70 Our laboratory has repeatedly demonstrated that peripheral LPS injection induces mRNA expression of pro-inflammatory cytokines and reduces expression of brain-derived neurotrophic factor (BDNF) in the dorsal hippocampus of C57BL/6J mice.⁶⁷ Prior research has also established that LPS treatment rapidly induces a more robust inflammatory response in the dorsal hippocampus compared to the ventral hippocampus.^71,72 Therefore, we hypothesized that the MD would protect mice against elevated levels of LPS-induced gene expression of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, in addition to the amyloid-β protein precursor (AβPP) cleavage enzyme beta-secretase 1 (BACE1). Additionally, we hypothesized that the MD would protect against IL-10 and BDNF loss following LPS administration, compared to the TAD.

General methods for experiments 1–2

Subjects

The current study utilized male wildtype C57BL/6J mice bred at Texas Christian University's (TCU) vivarium (from stock animals initially obtained from The Jackson Laboratory, Bar Harbor, ME). All procedures adhered to the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and were approved by the TCU Institutional Animal Care and Use Committee (IACUC Protocol #2021-14). Mice were housed in standard polycarbonate cages with pelleted, paper-chip bedding and compressed cotton square nestlets (LabSupply, Fort Worth, TX) in groups of three to four, under a 12-h light/dark schedule.

Experimental design and timeline

At postnatal day (PND) 21, mice were weaned and randomly assigned to one of two experimental diets: a Mediterranean diet (MD) or typical American diet (TAD) (ns = 23) for six months, with access to food and water ad libitum. At six months of age, mice underwent behavioral testing, and at six months and 21 days of age, tissue and serum were collected.

Experimental diet formulations

The MD and the TAD were created in pelleted form, and purchased from Research Diets, Incorporated (New Brunswick, NJ). See Table 1 for experimental diet formulations and Supplemental Material for a complete list of diet ingredients.

Table 1.

Experimental diet formulations.

	MD Formula				TAD Formula
	g%	kcal%	gm	Key Ingredients	g%	kcal%	gm	Key Ingredients
Protein	14.3	15.0	147.2	Egg white, fish, soy	16.0	15.0	147.3	Casein
Carb	48.0	50.0	495.3	Brown rice, wheat starch	53.7	50.0	495.5	Corn starch
Fat	14.8	35.0	153.2	Olive, fish & flaxseed oil	16.6	35.0	153.1	Safflower oil, beef fat, butter
SFA		6.7	29.5			17.0	74.4
MUFA		19.6	86.2			12.3	54.0
PUFA		5.6	24.4			3.2	14.1
Ratio n-6/n-3			2.0				15.6
Insoluble Fiber	8.3		85.4	Cellulose	5.4		50.0	Cellulose
Soluble Fiber	5.8		59.4	Psyllium, inulin	0.0		0.0

Diets were purchased from Research Diets (New Brunswick, NJ).

Methods for experiment 1

Measures of food consumption and body weight

Food consumption was measured weekly by weighing and recording the total amount of food in each cage's hopper every Friday morning. On Monday mornings, the remaining food in each hopper was weighed and recorded, and average consumption was calculated. Additionally, body weight was measured and recorded once per week.

Open field testing

At six months of age, mice underwent open field testing in enclosed chambers (27 × 27 cm) equipped with infrared beams to measure horizontal and vertical movement (Med Associates Incorporated, St Albans, VT). The open field chamber lights were maintained at 5.5 lux. Prior to testing, two zones were defined: the center zone (304.85 cm²) and the outer zone (423.39 cm²). Time spent in these zones was utilized to assess anxiety-like behavior, with more time in the center zone indicating reduced anxiety-like behavior.⁷³ Mice were placed in the chamber for 10 min, and vertical counts (rearing behavior), ambulatory distance (cm), time in the center zone (seconds), and average speed of ambulatory episodes (cm per second) were measured.

Elevated zero maze

Mice were tested in the elevated zero maze to further measure anxiety-like behavior, as validated by previous research. The elevated zero maze (platform 50 cm tall, circular diameter 60 cm) consisted of four alternating quadrants: two open and two enclosed by walls. The testing room lights were maintained at 45–48 lux. At the beginning of the test, each mouse was placed in an open quadrant, facing a closed quadrant, and given five minutes of uninterrupted time to move. Time spent in each quadrant was used to measure anxiety-like behavior, with more time in the open quadrants indicating reduced anxiety-like behavior.⁷⁴ A video camera was mounted on the ceiling to record the total time spent in the open quadrants versus the enclosed quadrants using EthoVision XT software (Noldus Information Technology, Leesburg, VA).

Object-location memory task

The object-location memory task (OLM) was utilized to assess spatial learning and memory. Arena settings were created with EthoVision XT software (Noldus Information Technology) and recorded with a ceiling-mounted video camera. OLM was comprised of habituation (2 sessions, 4 h apart, no objects), training (2 sessions, 4 h apart, two identical objects), and testing, involving the relocation of one object to the opposite corner (new spatial location). The testing room lights were maintained at 45–48 lux. During each session, mice had ten minutes to freely explore. See Figure 1 for OLM timeline. Exploration time was recorded when the mouse's nose entered the predetermined zone (three cm radius surrounding the object). Post-testing, the percentage of time spent exploring the object in the new location was calculated utilizing a previously-used formula.⁴² Object location exploration was also assessed during training to ensure unbiased location preference.

Figure 1.

Object-Location memory task. During each session, mice were placed in the OLM arena and provided ten minutes of uninterrupted exploration. The arena walls provided two distinct visual cues (one wall had a blue triangle, and one wall had three green stripes). During the training session, experimenters recorded the percentage of time that mice spent exploring two identical objects. During testing, one object was moved to the opposite corner of the chamber, and the percentage of time exploring the object in the new location was measured. This image was created using BioRender.

Tissue and serum collection

After six months consuming the prescribed diets, mice were fasted for six hours prior to euthanasia, and blood was immediately collected. Following blood collection, liver, spleen, and white adipose tissue were removed, weighed, and stored in neutral buffered formalin. Additionally, both hemispheres of the frontal cortex and hippocampus were collected and lysed in a protease inhibitor-containing cocktail (PRO-PREP, Bulldog Bio, Portsmouth, NH).

Peripheral cytokine analysis

A VPLEX Custom Mouse Cytokine Pro-Inflammatory Panel 1 multiplexing kit and QuickPlex SQ 120 instrument (Meso Scale Diagnostics, Rockville, MD) were utilized to measure the following analytes in serum: mouse TNF-α, IL-1β, IL-6, IL-10, and IFN-γ. Sample replicates with an intra-assay coefficient of variation of over 25% were excluded from the analysis, as advised by MSD technical support. See Supplemental Material for complete assay methodology.

Liver histology

Livers were collected from each mouse and divided for hematoxylin and eosin (H&E) and Oil Red O staining. Paraffin embedded sections of liver were collected on Millenia Command Adhesion Slides (StatLab, McKinney, TX) and stained with H&E.⁷⁵ Frozen sections of livers were also collected on Millenia Command Adhesion Slides (StatLab) and stained with Oil Red O, per manufacturer's instructions (StatLab). All images were collected on a Nikon Eclipse 90i microscope (Nikon Instruments, Melville, NY) and analyzed using NIS-Elements AR 4.60.00 software (Nikon Instruments).

Aβ_1–42 ELISA

Protein assays (DC Protein Assay; Bio-Rad Laboratories, Hercules, CA) were conducted to calculate the total amount of protein in each sample, and protein concentrations were standardized. A mouse Aβ_1–42 ELISA (Invitrogen, ThermoFisher Scientific, Waltham, MA) was conducted, per manufacturer's instructions, and read on a plate reader (BMG LabTech FLUOstar Omega, Cary, NC). Sample replicates with an intra-assay coefficient of variation over 25% were excluded from the analysis.

Statistical analyses

All data were analyzed with Statistical Package for Social Sciences (SPSS; Version 29.0, IBM, Armonk, NY). Normality was assessed prior to running statistical analyses, and transformations were performed to correct for violations of homogeneity of variance. Two-tailed independent samples t-tests and mixed-design analyses of variance (ANOVAs) were conducted to analyze behavioral and biological dependent variables. All outliers were detected utilizing SPSS's interquartile range, and for all analyses, an alpha level of p ≤ 0.05 was considered significant.

Methods for experiment 2

Subjects and experimental design

Following 6 months of diet administration (MD or TAD), male C57BL/6J mice were randomly assigned to one of two treatment conditions: one intraperitoneal (i.p.) injection of LPS (E. coli serotype O26:B6; Sigma-Aldrich, St Louis, MO) at a dose of 250 μg/kg of body weight, or an equivalent volume of sterile saline four hours prior to tissue collection. Injections were performed between 0800 h and 1100 h. This LPS model has previously been utilized in our laboratory.⁶⁷ Four hours after injections, both hemispheres of the dorsal hippocampus were collected under RNase-free conditions utilizing RNaseZap™ (Invitrogen, ThermoFisher Scientific, Wilmington, DE), and immediately placed in RNAlater™ Stabilization Solution (Invitrogen, ThermoFisher Scientific, Wilmington, DE). Tissue was stored at −80°C prior to RNA isolation.

Quantitative reverse-transcription polymerase chain reaction (qRTPCR)

Central gene expression for TNF-α, IL-1β, IL-10, IL-6, BDNF, BACE-1, and β-actin (internal control gene) was measured in the dorsal hippocampus, utilizing qRTPCR. First, RNA was isolated from hippocampal tissue utilizing a Maxwell LEV simplyRNA Purification kit (Promega Corporation, Madison, WI), and measured for quantity and purity with a NanoDrop™ OneC Microvolume Spectrophotometer (ThermoFisher Scientific, Wilmington, DE). Subsequently, reverse transcription was performed utilizing Script Reverse Transcriptase Supermix (Bio-Rad, Hercules, CA) and a 7500 Real-Time PCR Thermal Cycling System (Applied Biosystems, Foster City, CA). Expression levels of mRNA for the target genes were then measured using iTaq Universal Probes Supermix (Bio-Rad) and PrimePCR Probe Assay probes (Bio-Rad, Hercules, CA). Each gene sample was run in triplicate, using a control sample from mice on the MD that had been treated with saline. The relative normalized expression of cDNA from the fluorescence data was normalized to β-actin, relative to the control sample, utilizing the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA).

Statistical analyses

Analyses of variance (ANOVAs) were conducted utilizing Prism (GraphPad Software, Inc., San Diego, CA), to examine the effects of diet condition (MD or TAD) and treatment (LPS or saline) on gene expression. An alpha level of p ≤ 0.05 was considered significant. If a sample had a quantitation cycle (Cq) standard deviation of 0.25 or higher, the outlier of the triplicates was removed from the dataset. If the Cq standard deviation was still more than 0.25 following this correction, the sample was excluded from data analysis.

Experiment 1 results

Average food and kilocalorie consumption

Two mixed-design ANOVAs revealed a significant main effect of diet duration (number of months on diet) on daily food consumption (grams), F(5,60) = 9.988, p ≤ 0.001, $η_{p}^{2}$ = 0.454, and kilocalorie intake (% kcal), F(5,60) = 9.988, p ≤ 0.001, $η_{p}^{2}$ = 0.454, throughout six months of diet administration. Additionally, there was a significant main effect of diet condition (MD vs. TAD) on daily food consumption, F(1,12) = 6.276, p = 0.028, $η_{p}^{2} = 0.343$ , and kilocalorie intake, F(1,12) = 6.276, p = 0.028, $η_{p}^{2}$ = 0.343, such that mice assigned to the MD consumed more food and kilocalories than those assigned to the TAD, throughout six months of diet administration. These results were both supported by significant interactions between diet duration X food intake, F(5,60) = 2.673, p = 0.030, $η_{p}^{2}$ = 0.182, and diet duration X kilocalorie intake, F(5,60) = 2.673, p = 0.030, $η_{p}^{2}$ = 0.182. Further analyses utilizing Tukey's LSD revealed a significant difference in daily food consumption and kilocalorie intake between diet conditions following 3 months (p = 0.028) and 6 months (p = 0.025) of diet administration. See Figure 2.

Figure 2.

Mice on the MD consumed more daily kilocalories, but had lower body weight than mice on the TAD. (A) A mixed-design ANOVA revealed a significant main effect of diet condition (MD vs. TAD) on food consumption, such that mice on the MD consumed more food than those on the TAD, p = 0.028. (B) Mice assigned to the MD consumed more kilocalories per day than those on the TAD, p = 0.028. (C) There was a significant main effect of diet condition on body weight, p ≤ 0.001, in which males on the MD weighed less than those on the TAD. Bars represent ± SEM. Significant differences (p ≤ 0.05) are designated by *.

Body weight

A mixed-design ANOVA revealed a significant main effect of diet duration (number of months on diet) on body weight (grams) over the course of six months of diet administration, F(6, 240) = 2013.004, p ≤ 0.001, $η_{p}^{2}$ = 0.981. Additionally, there was a significant main effect of diet condition (MD vs. TAD) on body weight, F(1,40) = 16.365, p ≤ 0.001, $η_{p}^{2}$ = 0.290, such that mice on the MD, despite consuming more food and calories, weighed less than those on the TAD. These results were further supported by a significant interaction, F(6, 240) = 21.843, p ≤ 0.001, $η_{p}^{2}$ = 0.353. Further analyses utilizing Tukey's LSD revealed that there was a significant difference in body weight between diet conditions following 3, 4, 5, and 6 months of diet (ps < 0.016), such that males on the MD weighed less than those on the TAD. Four outliers were removed from the analysis because they fell outside of the interquartile range determined by SPSS. See Figure 2.

Organ and white adipose tissue weight

An independent samples t-test revealed a significant difference in white adipose tissue weight, t(27) = −9.194, p ≤ 0.001, and liver weight, t(27) = −4.355, p ≤ 0.001, in which mice on the TAD had heavier white adipose tissue and livers than those on the MD. The magnitude of the difference in the means of white adipose tissue weights (mean difference = -1.397, 95% CI [−1.085, −1.709]) was large (η2 = 0.758), and the magnitude of the difference in the means of liver weights (mean difference = -0.523, 95% CI [−0.277, −0.769]) was large (η2 = 0.413). However, there was no difference in spleen weight, t(33) = -1.583, p = 0.123. See Supplemental Material.

Liver health following diet administration

We imaged four, randomly-selected liver sections from three mice in each diet condition (MD or TAD). An independent samples t-test revealed a significant difference in the percentage of pixelated area that was unstained following H&E staining, in which mice on the MD had less percentage of pixelated area unstained compared to those on the TAD, t(21) = 39.173, p ≤ 0.001. The data violated homogeneity of variance, and a reflect and square root transformation was performed to correct this. The magnitude of the difference in the means of unstained area in the liver (mean difference = -22.486, 95% CI [−23.605, -21.368]) was large (η2 = 0.986). See Figure 3.

Figure 3.

The MD prevented hepatic fat compared to the TAD. (A) H & E-stained liver from a mouse on the MD. (B) H & E-stained liver from a mouse on the TAD. (C) An independent samples t-test revealed a significant difference in the percentage of pixelated area that was unstained following H & E staining, p ≤ 0.001. (D) Oil-Red O-stained liver from a mouse on the MD. (E) Oil-Red O-stained liver from a mouse on the TAD; lipids are stained red. Bars represent ± SEM. Significant differences (p ≤ 0.05) are designated by *.

Peripheral cytokine production in serum

An independent samples t-test revealed that mice on the TAD produced significantly more TNF-α (pg/mL) compared to those on the MD, t(32) = -3.790, p ≤ 0.001. One sample was removed from the analysis due to an intra-assay coefficient of variation (CV) value over 25%, as recommended by MSD's technical team. The magnitude of the difference in the means (mean difference = -2.12, 95% CI [−3.253, −0.979]) was large (η2 = 0.31). Additionally, the difference in IL-1β (pg/mL) production between diet conditions was not significant, t(23) = -1.809, p = 0.084. Six samples were removed from the analysis due to CV values over 25%. Additionally, three outliers were removed from the analysis because they fell outside of the interquartile range determined by SPSS. The magnitude of the difference in the means (mean difference = -0.07967, 95% CI [0.0115, −0.171) was moderate (η2 = 0.125). Further, there was no significant difference in IL-10 (pg/mL) production between diet conditions, t(29) = -0.818, p = 0.420. Three outliers were removed from the analysis because they fell outside of the interquartile range determined by SPSS. Finally, an independent samples t-test failed to reveal a significant difference in IFN-γ (pg/mL) production between diet conditions, t(30) = 0.040, p = 0.968. Two samples were removed from the analysis due to CV values over 25%. See Figure 4. Although we also aimed to measure IL-6, numerous samples fell below the lowest detectable limit of the assay's standard curve, thus precluding analysis.

Figure 4.

The MD protected against serum TNF-α, but not IL-1β, IL-10, or IFN- γ compared to the TAD. Independent samples t-tests were performed to compare the quantity of peripheral cytokines produced in serum following six months of diet. (A) Mice on the MD produced significantly less serum TNF-α compared to mice on the TAD, p ≤ 0.001. (B–D) There was no significant difference in IL-1β, IL-10, or IFN- γ production between diet conditions, ps > 0.05. (A–D) Bars represent mean ± SEM. Significant differences (p ≤ 0.05) are designated by *.

Aβ_1–42 production in the cortex and hippocampus

An independent samples t-test revealed a significant difference in Aβ_1–42 (pg/mg) in the cortex, t(22) = -3.067, p = 0.006, in which mice on the MD had less Aβ_1–42 in the cortex, in comparison to those on the TAD. The data violated homogeneity of variance, and a log transformation was performed to correct this. The magnitude of the difference in the means (mean difference = -9.63, 95% CI [−3.10, −16.16,]) was very large (η2 = 0.299). Approximately 30% of variance was explained by diet. Two outliers were outside of the interquartile range determined by SPSS and removed from the analysis.

An independent samples t-test also revealed a significant difference in Aβ_1–42 (pg/mg) in the hippocampus, such that mice on the MD had less hippocampal Aβ_1–42 compared to those on the TAD, t(29) = -2.103, p = 0.044. The magnitude of the difference in the means (mean difference = -4.522, 95% CI [−0.124, −8.920]) was moderate (η2 = 0.132). Three outliers were removed from the analysis. Additionally, two samples were removed from the analysis due to intra-assay CV values over 25%. See Figure 5.

Figure 5.

The MD protected against increased Aβ_1–42 in the cortex and hippocampus compared to the TAD. Mice on the TAD had significantly more Aβ_{1– 42} (pg/mg) in the cortex, p ≤ 0.01, and hippocampus, p ≤ 0.05, compared to mice on the MD. Bars represent mean ± SEM. Significant differences (p ≤ 0.05) are designated by *.

Open field behavior

Vertical counts

An independent samples t-test revealed a significant difference in vertical counts t(27) = 2.767, p ≤ 0.01, such that mice on the MD exhibited significantly more vertical counts than mice on the TAD. The magnitude of the difference in the means during the total testing time (mean difference = 14.111, 95% CI [24.573, 3.648]) was very large (η2 = 0.221). Three outliers were detected utilizing SPSS and were removed from the analysis. See Figure 6.

Figure 6.

Locomotor activity, exploratory and anxiety-like behavior. (A–D) Multiple independent samples t-tests were performed to examine exploratory behavior and locomotor activity during the open field test. (A) Mice on the MD exhibited more vertical counts compared to mice on the TAD, p ≤ 0.01. (B) There was no significant difference in the total ambulatory distance traveled (cm/s) between the two diet conditions, p = 0.072. (C) A paired-samples t-test revealed that mice on the MD traveled significantly more (cm) in the center zone compared to the outer zone of the open field, p ≤ 0.05. (D) There was no significant difference in time (s) spent in the center zone of the open field between the two diet conditions, p ≥ 0.05. (E) Mice on the MD spent significantly more time in the open quadrants of the elevated zero plus maze in comparison to mice on the TAD, p ≤ 0.01. Bars represent mean ± SEM. Significant differences (p ≤ 0.05) are designated by *.

Distance traveled

An independent samples t-test did not reveal a significant difference in the total ambulatory distance traveled t(30) = 1.867, p = 0.072. The magnitude of the difference in the means during the total testing time (mean difference = 213.878, 95% CI [447.887, − 20.130]) was moderate (η2 = 0.104). However, when analyzing the total ambulatory distance traveled in the center zone, the difference in the distance traveled approached significance, t(30) = 1.961, p = 0.059. There was no difference in the total ambulatory distance traveled in the outer zone, t(30) = 0.702, p = 0.488. Additionally, a paired-samples t-test revealed that mice on the MD traveled a significantly farther distance in the center zone in comparison to the outer zone, t(16) = -2.777, p = 0.013, whereas there were no differences in the amount of distance traveled between the center and outer zone in mice on the TAD, t(14) = -0.954, p = 0.356. See Figure 6.

Time spent in the center zone of open field

An independent samples t-test did not reveal a significant difference in the amount of time spent in the center zone during testing, t(27) = 1.748, p = 0.092. See Figure 6.

Average speed

An independent samples t-test did not reveal a significant difference in the average speed (cm/s) during the total duration of testing, t(28) = 1.347, p = 0.189. See Supplemental Figure 2.

Elevated zero maze behavior

An independent samples t-test revealed that mice on the MD spent significantly more time in the open quadrants of the elevated zero maze in comparison to those on the TAD, t(31) = 3.121, p = 0.004. The data violated homogeneity of variance, and a log transformation was conducted to correct this. The magnitude of the difference in the means (mean difference = 0.214, 95% CI [0.354, 0.743]) was large (η2 = 0.239). One outlier was detected by SPSS and excluded from the analysis. See Figure 6.

Spatial learning and memory in OLM

Post-testing, the percentage of time spent exploring the object in the new location was calculated using the formula: new object location time / [new object location time + old object location time] x 100, as adopted from Heyward and colleagues.⁴² As expected, an independent samples t-test revealed that there was no difference in the percentage of time spent exploring the new object location during training, t(18) = -0.787, p = 0.441. However, an independent samples t-test revealed that mice on the MD spent significantly more time exploring the object moved to the new location, compared to mice on the TAD, during testing, t(18) = 2.259, p = 0.037. The magnitude of the difference in the means (mean difference = 21.919, 95% CI [42.305, 1.532]) was large (η2 = 0.221). See Figure 7.

Figure 7.

The MD preserved spatial learning and memory in the OLM test in comparison to the TAD. Mice on the MD spent significantly more time exploring the object moved to the new location, compared to mice on the TAD, during testing, p = 0.037. Bars represent ± SEM. Significant differences (p ≤ 0.05) are designated by *.

Experiment 2 results

Gene Expression of BDNF and BACE1 in the Dorsal Hippocampus

A two-way ANOVA revealed a significant main effect of diet condition (MD or TAD) on hippocampal BDNF mRNA, F(1, 38) = 9.263, p = 0.004, in which mice on the MD had increased BDNF gene expression compared to those on the TAD. This effect was largely driven by LPS-treated mice, and post-hoc analyses revealed that mice on the MD had increased BDNF gene expression compared to those on the TAD following LPS treatment, p = 0.006. There was no significant main effect of injection (LPS or saline), F(1, 38) = 0.069, p = 0.794, on BDNF gene expression. In contrast to our hypothesis, a two-way ANOVA did not reveal a significant main effect of injection, F(1,39) = 3.485, p = 0.070, or diet condition, F(1,39) = 2.606, p = 0.115, on BACE1 gene expression. See Figure 8.

Figure 8.

The MD protected against both elevated LPS-induced TNF-α and IL-1β gene expression, and LPS-induced BDNF loss compared to the TAD. (A) Mice on the TAD had significantly decreased gene expression of BDNF compared to male mice on the MD, p ≤ 0.05. (B) There was no significant main effect of diet on relative normalized gene expression of BACE1, p > 0.05. (C) Mice on the TAD had increased TNF-α gene expression compared to mice on the MD, p ≤ 0.05. (D) Mice on the TAD had increased IL-1β gene expression compared to MD fed counterparts, p ≤ 0.05. (E) There was no significant main effect of diet on relative normalized gene expression of IL-6, p > 0.05. (F) There was no significant main effect of diet on relative normalized gene expression of IL-10, p > 0.05. (A–F) Bars represent ± SEM. Significant differences (p ≤ 0.05) are designated by *.

Gene Expression of TNF-a, IL-1β, IL-6, and IL-10 in the Dorsal Hippocampus

A two-way ANOVA revealed a significant main effect of injection on TNF-α gene expression, F(1,38) = 73.55, p ≤ 0.001, such that LPS-injected mice had increased TNF-α gene expression, compared to saline-treated mice. Additionally, there was a significant main effect of diet condition, F(1,38) = 4.320, p = 0.045, in which mice on the TAD had increased TNF-α gene expression compared to MD-fed counterparts. This was further supported by a significant interaction between injection (LPS or saline) X diet condition (MD or TAD), F(1,38) = 4.242, p = 0.046, in which the combined effects of the TAD and LPS treatment significantly elevated TNF-α gene expression compared to the TAD and saline treatment. Additionally, post-hoc analyses revealed that the TAD and LPS treatment significantly increased TNF-α gene expression compared to the MD and LPS treatment, p = 0.019.

A two-way ANOVA also revealed a significant main effect of injection on IL-1β gene expression, F(1,35) = 113.4, p ≤ 0.001, such that LPS-treated mice had increased IL-1β gene expression compared to saline-treated mice. Additionally, there was a significant main effect of diet condition, F(1,35) = 5.484, p = 0.025, in which mice on the TAD had increased IL-1β gene expression compared to MD fed counterparts. Finally, the interaction between injection (LPS or saline) X diet condition (MD or saline) was significant, F(1,35) = 4.106, p = 0.050. Post-hoc analyses revealed that the TAD and LPS treatment significantly increased IL-1β gene expression compared to the MD and LPS treatment, p = 0.011.

Additionally, a two-way ANOVA revealed a significant main effect of injection on IL-6 gene expression, F(1,37) = 21.22, p ≤ 0.001, in which LPS-treated mice had increased IL-6 gene expression compared to vehicle-treated mice. However, contrary to our hypothesis, there was no significant main effect of diet condition on IL-6 gene expression, F(1,37) = 0.003, p = 0.958. Finally, a two-way ANOVA did not reveal a significant main effect of injection, F(1,22) = 0.001, p = 0.980, or diet condition, F(1,22) = 3.624, p = 0.070, on IL-10 gene expression. See Figure 8.

Discussion

To fill key gaps in the current understanding of diet-related AD risks, as well as explore potential, nutritional AD prevention strategies in animal research, we designed two macronutrient-matched diets (MD and TAD) reflecting comprehensive dietary patterns and average macronutrient intake in Mediterranean regions and the U.S. Although there is evidence that the MD is associated with a reduction in AD-related markers in human subjects research, the current study aimed to further explore the underlying biological mechanisms responsible for the potential, protective effects of the MD in mice. The current findings align closely with those observed in human subjects.

For example, research utilizing magnetic resonance imaging (MRI) has shown that close adherence to the MD is associated with protection from AD pathologies, including Aβ,^9,18 brain atrophy,^10,19–21 and cognitive decline^22,23,25,26 in middle-aged and elderly adults. Specifically, high adherence to the MD has been associated with reduced ventricular enlargement and atrophy in the hippocampus and temporal cortex of middle-aged adults,^10,19 while low adherence has been shown to increase atrophy and induce cortical thinning.¹⁰ In further support of the current research, a recent study reported that long-term consumption of the MD was associated with reduced global AD pathology and Aβ in postmortem brains of elderly adults.⁷⁶

In further support of the current findings, research in elderly adults has shown that excessive consumption of refined grains and sugars is associated with increased Aβ production in the cortex and lower test scores on cognitive tests in cognitively healthy, elderly adults.⁷⁷ Further, human subjects research has demonstrated that a MD (with olive oil as the main fat source) has been associated with reduced peripheral pro-inflammatory mediators,^16,17 while high quantities of SFA^78–80 and red meat⁸¹ have been associated with increased pro-inflammatory mediators in adults. For example, a Western-style diet, rich in SFA, was associated with increased pro-inflammatory gene expression in the adipose tissue of adults at risk of developing metabolic syndrome.⁸⁰

Although the previously discussed research has provided evidence that there is a connection between diet and AD susceptibility, there is limited research on the underlying biological and behavioral mechanisms of a long-term, comprehensive MD, and how it potentially diminishes AD susceptibly in rodents. Further, potential health consequences stemming from long-term adherence to the TAD remain inadequately explored in rodent studies. The overarching term “Western diet” lacks a precise definition, as prior studies have utilized numerous variations of the Western diet, including “high-fat”, “high-sugar”, or cafeteria diets, with exaggerated macronutrient densities that are not representative of human diets.^82,83 Therefore, the current experiments addressed these limitations by exploring the impact of early-life implementation and long-term adherence to the more realistic MD or TAD on AD vulnerability in adulthood.

Prior to testing, we conducted a comprehensive literature review to ensure that the experimental diets accurately modeled dietary patterns observed in both Mediterranean and U.S. populations. Prior research indicates that the MD provides approximately 35–40% kcal from fat.^30,32,84 Specifically, the MD provides about 19% kcal from monounsaturated fatty acids (MUFAs) and 5% kcal from polyunsaturated fatty acids (PUFAs), with a typical omega-6 to omega-3 ratio of 1:1 or 2:1.^61,64,85 Additionally, the MD generally provides 15–20% kcal from protein, with its primary macronutrient source being complex carbohydrates, constituting approximately 40–48% of daily kcal intake.^61,85 In contrast, the average American adult consumes about 34% kcal from fat, and the majority of macronutrients are provided by refined carbohydrates and sugars.^33,59

We specifically designed this experiment to compare the MD and TAD without an additional group consuming a low-fat, “standard” diet. All diets were designed with the same percentage of kcals from macronutrients to account for macronutrient availability; thus, we did not add a low-fat, “standard,” diet. The “standard” diet typically provided to rodents in laboratory settings does not mimic a human diet, as they are grain-based and low in fat.⁸⁶ Moreover, standard laboratory diets for rodents are inconsistent in both macronutrient composition and typically only provide about 10% kcal from fat.⁸⁶ Numerous “standard” grain-based diets are available for rodents, and typically provide a wide variety of macronutrient sources. However, the improper use of “standard” diets in rodent research has recently become an important topic of discussion within the scientific community. A recently published article highlighted potential problems with widely-used rodent diets: “they [standard diets for rodents] are generally considered to maintain a healthy phenotype in the animal (though it can be argued ‘compared to what?’).”⁸⁶

The formulas for standard chow diets tend to fluctuate, and the ingredients are not strictly regulated.⁸⁶ For example, a previous experiment tested over thirteen different laboratory diets globally, and found contaminants such as glyphosate and heavy metals.⁸⁷ Thus, this large variation of macronutrient density ranges, ingredients, pesticides, and heavy metals in “standard” diets has considerable variability that could affect scientific data. In contrast, purified ingredient diets use highly refined ingredients, such as casein, fewer chemicals, and more consistent product testing.⁸⁶ Therefore, the current study compared two macronutrient-matched, purified diets to control for variability and prevent confounding variables, aiming to clearly demonstrate that the two experimental diets (MD and TAD) were driving differences in the physiological and behavioral dependent variables. Likewise, prior experiments examining the effects of diet on rodent physiology and behavior have also only utilized two experimental diets without the use of a “standard” rodent diet.^45,88

Through this approach, we found that the MD protected mice from adverse alterations in: (1) body weight, (2) abdominal and hepatic fat, (3) serum TNF-α, (4) Aβ_1–42 in both the cortex and hippocampus, (5) exploratory behavior, (6) anxiety-like behavior, and (7) spatial memory, compared to the TAD. Although our hypotheses were largely supported, we only saw a moderate, but not significant, increase in serum IL-1β in mice on the TAD, and did not find any differences in serum IL-10, or IFN-γ between diet conditions. Interestingly, although the diets were macronutrient-matched, mice on the MD consumed more food per day compared to mice on the TAD, indicating a potential appetitive preference. However, despite consuming more kilocalories, mice assigned to the MD had reduced body weight, white adipose fat, and hepatic fat compared to mice on the TAD. A previous study investigating the effects of the “Western” diet found that male C57BL/6J mice consumed less food when placed on a “Western” diet, in comparison to those fed a low-fat chow diet, most likely due to higher kilocalorie content in the “Western” diet.⁸⁹ However, contrary to most prior research, the two diets utilized in the current study were macronutrient-matched, and thus provided the same amount of energy availability.

Although prior research has established that high-fat “Western” diets (providing about 40–60% kcal from fat) induce excess weight gain compared to low-fat diets,^42,55 the current study is one of the first to illustrate that the TAD induces weight gain in male C57BL/6J mice.

We infer that fatty acid ratios in the MD protected against increased body weight. The TAD was formulated to mimic the fatty acid ratios consumed by the typical American, and thus provided more SFA compared to the MUFA-rich MD utilized in the study. Although there is limited research on the effects of a comprehensive MD in rodents, some studies have found that olive oil supplementation protects rodents against excess body weight compared to SFA rich diets.^57,90 When examining the effects of Mediterranean dietary factors on body weight, there are conflicting results in the scientific literature, as other studies have not found significant effects of MD factors on body weight in wildtype mice and transgenic mouse models of AD.^91–93

However, despite these contradictory findings, we hypothesize that fatty acids in the MD contributed to protection against weight gain. For example, olive oil has been associated with reduced postprandial glucose and insulin, and increased glucagon-like peptide-1 (GLP-1) in insulin resistant patients.⁹⁴ GLP-1 plays an important role in the regulation of blood sugar and appetite, and GLP-1 agonists are commonly utilized to improve blood sugar and insulin levels, as well as reduce body weight, in diabetic patients.⁹⁵ Interestingly, the MD, rich in MUFAS, has been shown to increase basal GLP-1 in patients with diabetes mellitus type II, a risk factor and comorbidity of AD.⁹⁵

Mediterranean fatty acids have also been shown to decrease adipocyte formation in mice that were previously fed a “Western diet.”⁹⁶ For example, omega-3 is an important fatty acid in the MD that has been shown to increase GLP-1 in obese mice.⁹⁷ The ratio of fatty acids, such as the two PUFA subtypes, omega-6 and omega-3, has been associated with adipocyte formation and obesity.^98,99 Prior research has found that the ratio of omega-6:omega-3 affects the production of prostaglandins, which are derived from omega-6-AA (arachidonic acid), and some prostaglandins have been shown to impact the ability for white fat cells to brown.⁹⁹ Brown fat is important for energy storage and usage, which contributes to a healthy body weight.⁹⁹ Thus, high levels of dietary omega-6 could potentially increase prostaglandins and prevent white fat cells from being converted into brown fat.⁹⁹ The recommended ratio of omega-6 to omega-3 is 1:1 or 2:1, which is typical of the MD.⁶⁴ In contrast, the average American typically consumes an omega-6 to omega-3 ratio of 15:1.^33,64,98 This evidence provides support for the current findings, demonstrating that fatty acid ratios in the MD formula potentially contributed to the prevention of weight gain compared to the TAD.

We further explored the therapeutic capacity of the MD against the inflammatory trigger LPS, as prior studies have established that peripheral LPS administration provokes neuroinflammation that is associated with cognitive impairment.^65,67–69 The results revealed that the MD was more effective at preventing an increase in LPS-induced gene expression of TNF-α and IL-1β, and loss of BDNF expression, in the dorsal hippocampus compared to the TAD. However, our hypotheses were only partially supported, as the MD was not effective at reducing LPS-induced BACE1 or IL-6 expression, and did not affect IL-10 mRNA, compared to the TAD.

To our knowledge, this is the first study to directly compare the long-term effects of a macronutrient-matched MD and TAD on obesity-related variables, AD biomarkers, behavior, and cognition in wildtype mice, modeling sporadic AD. Although the TAD has not been thoroughly explored, a few researchers have aimed to create a diet that models the average American like the current study. For example, despite their use of the term “Western diet,” two separate research teams employed diets that attempted to mirror the average American dietary pattern, similar to our TAD. For example, Johnson, Shively and colleagues recently utilized two macronutrient-matched diets for nonhuman primates that closely resemble those used in our experiments.^100–104 Experimenters found that long-term consumption of their “Western diet” induced excess body fat, insulin resistance, hepatosteatosis,¹⁰³ anxiety,^100,101 and the expression of pro-inflammatory genes,¹⁰¹ compared to the MD in nonhuman primates.

Further, scientists focused on AD,⁵⁵ cancer,⁸³ and obesity¹⁰⁵ research have sought to develop diets for rodents that better reflect the average American diet. Like the present study, Graham and colleagues (2016) formulated an animal fat and protein-based diet for mice that mirrors the typical diet of Western societies. However, they compared this diet to a lower-fat, plant-based diet that was neither modeled after an MD, nor macronutrient-matched.⁵⁵ They also addressed an additional research gap by placing mice on the experimental diets at weaning, thereby enabling the observation of long-term effects of diet administration into adulthood, in contrast to the prevalent focus on short-term dietary interventions in prior studies.⁵⁵ The results of the current study support and extend these findings, demonstrating that long-term consumption of a TAD increases body weight and fat compared to a plant-based diet in C57BL/6J mice. Like the previously described experiment,⁵⁵ the TAD in the current study provided approximately 5–25% fewer kilocalories from fat compared to commonly used “Western diets” in research with C57BL/6J mice,^{41,42,44,45,47,48} yet it still increased body weight and adiposity compared to the MD. As obesity arguably represents the top modifiable risk factor for AD,⁴ these findings in non-human primates¹⁰³ and C57BL/6J mice⁵⁵ highlight the risk of long-term TAD consumption on body weight, whereas the MD could be utilized as a potential strategy for weight gain prevention.

Additionally, our results demonstrate that the TAD, as opposed to the more exaggerated “Western diets”^88,106,107 employed in the majority of scientific literature, is sufficient to increase fat deposition in the liver. Notably, metabolic dysfunction-associated steatotic liver disease (MASLD) has been associated with increased risk of AD¹⁰⁸ and cognitive decline in human subjects.¹⁰⁹ Although the underlying biological interplay between the liver and brain during AD development is still being explored, a recent study reported that partial blockage of blood to the liver hinders clearance of peripheral Aβ_1–40 and Aβ_1–42 in the body, and thus exacerbates central Aβ_1–40 and Aβ_1–42 in APP/PS1 mice.¹¹⁰ Interestingly, recent studies have reported that the MD is associated with a reduced risk of MASLD,¹¹¹ and improved liver health in patients with MASLD.¹¹² Likewise, animal research has demonstrated that the MD protects against hepatosteatosis in non-human primates,¹⁰³ whereas plant-based diets have been shown to inhibit MASLD compared to a “Western diet” in C57BL/6J mice.⁸⁸ Collectively, our results, in conjunction with prior evidence, indicate that the MD reduces markers of MASLD, which is potentially linked to the MD-associated Aβ reduction.

Accordingly, we measured Aβ_1–42, a hydrophobic amino acid consisting of 42 residues, due to its higher susceptibility to forming insoluble Aβ plaques compared to Aβ_1–40.¹¹³ Previous studies utilizing C57BL/6J mice have also utilized similar methodology, and only measured Aβ_1–42.^49,56,114 The current data revealed that the MD prevented elevated levels of Aβ_1–42 in the cortex and hippocampus compared to the TAD. This finding aligns with previous studies demonstrating that high-fat “Western diets” induce Aβ_1–42 in C57BL/6J mice,⁴⁴ and exacerbate amyloid plaque pathology in transgenic AD models.^43,115–118

Similarly, a moderate “Western diet” exacerbated Aβ_1–42 plaque burden in the hippocampus of APP/PS1 mice; however, the experimenters only measured Aβ_1–42 in hippocampal lysates in APP/PS1 mice, and not in their wildtype counterparts.⁵⁵ Thus, we addressed this research gap in the current study, and demonstrated that the TAD induces Aβ_1–42 production in wildtype mice without genetic predisposition to AD. In further support, prior studies have revealed the amyloid-reducing capacity of individual MD components, like olive oil, in transgenic models of AD^57,58 and wildtype mice.⁵⁶ In addition, both fish oil and DHA supplementation have been shown to mitigate hippocampal and cortical Aβ production in transgenic mice.^{116,119–122} As the current study utilized a whole MD approach, it is one of the first to demonstrate that a comprehensive MD effectively protects against Aβ_1–42 production in wildtype mice.

To further examine the relationship between peripheral inflammation and Aβ_1–42, we measured cytokine production in serum, and cytokine gene expression in the dorsal hippocampus. The current findings underscored the anti-inflammatory effects of the MD against elevated peripheral TNF-α, in addition to LPS-induced TNF-α and IL-1β gene expression in the dorsal hippocampus. Prior studies have shown that long-term consumption of a high-fat diet exacerbates gene expression of central pro-inflammatory cytokines in wildtype mice^45,46 and transgenic models of AD.¹¹⁸ In contrast, research on MD components have shown that hydroxytyrosol effectively reduces peripheral pro-inflammatory mediators in C57BL/6J mice,¹²³ and olive oil supplementation attenuates LPS-induced inflammation in BALB/c mice,^124,125 and C57BL/6J mice.¹²⁶

Although our hypotheses were largely supported, our hypotheses that the TAD would increase gene expression of BACE1 and IL-6, and decrease gene expression of IL-10 in the dorsal hippocampus were not supported. In contrast to the current results, prior evidence has shown that a “Western diet,” providing an exaggerated 60% kilocalories from fat, increases BACE1 in male C57BL/6J mice.⁴⁴ However, our TAD only provided 35% kilocalories from fat, making direct comparison between these studies challenging. Our null findings align with a previous study, in which a “Western diet”, providing 40% kilocalories from fat, did not affect pro-inflammatory cytokine and chemokine production compared to a low-fat diet in C57BL/6J mice.⁴⁵ However, when the experimenters compared an extremely high-fat (60% kcal from total fat) and high-SFA diet to a low-fat diet, they observed significant differences in TNF-α, IL-6, MCP-1, and BDNF in the cortex,⁴⁵ suggesting that higher SFA content engenders greater inflammation in the brain. Notably, more evidence has revealed that SFA provokes inflammation because it functions like a TLR4 agonist.¹²⁷ Indeed, treatment with a TLR4 inhibitor has been shown to mitigate inflammation and other harmful effects induced by a high-SFA diet in C57BL/6J mice.⁴⁴ While our diet mimicked the average SFA content in the American diet, rather than exaggerated amounts of SFA in commonly utilized high-fat “Western diets”, these results demonstrate that the TAD is also a pro-inflammatory diet.⁴⁵ While the inflammation induced by our TAD may be lower than that caused by commonly used “Western diets”, we demonstrate that the average American diet is sufficient to engender obesity and AD-related deleterious effects.

“Western diets” have also been shown to reduce proteins related to long-term potentiation and neuronal plasticity, such as synaptophysin, PSD95, and BDNF,^{45,46,128,129} whereas plant polyphenols and probiotics, commonly found in the MD, have been shown to protect against high-fat diet-induced BDNF loss^46,129 in the cortex and hippocampus of mice. The current results align with prior findings, and demonstrate that a comprehensive MD effectively sustains BDNF gene expression after LPS treatment, compared to the TAD. Given the reduced serum BDNF levels and increased pro-inflammatory cytokines in AD patients,^130,131 our results collectively suggest that the MD may prove protective against BDNF loss in an inflammatory state.

The results of behavioral testing revealed that mice on the TAD exhibited reduced exploratory behavior and locomotor activity compared to mice on the MD. This aligns with findings demonstrating that olive oil or fish oil supplementation increases locomotor activity in rats⁹⁰ and mice. Moreover, the MD brought about protective effects against spatial memory impairment in the OLM test. These results are consistent with prior studies highlighting the negative impact of “Western diets” on spatial learning and memory, specifically in the OLM task,⁴² novel object recognition task,⁴⁸ T-maze,^45,50 and Morris water maze,^48,51 in mice. In contrast, Mediterranean dietary factors, like plant polyphenols, fatty acids, or butyrate, have been shown to improve learning and memory in transgenic models of AD^93,132 and wildtype mice.⁸⁸ We also found that higher levels of cortical Aβ_1–42 were moderately associated with spatial memory impairment, suggesting that the induction of Aβ_1–42 by the TAD disrupts spatial learning and memory compared to the MD. These findings underscore our hypothesis that a comprehensive MD helps prevent the emergence of AD biomarkers associated with cognitive impairment, and thus, should be further explored as an AD prevention strategy, along with the mechanisms involved in its efficacy.

In addition to the current study, our laboratory is presently exploring the effects of the MD and TAD in female C57BL/6J mice; however, our preliminary results indicate that the effects are biologically and behaviorally sexually dimorphic. Similarly, recent studies have also reported that female C57BL/6J mice are less susceptible to the harmful effects of the “Western diet” compared to their male counterparts. For example, female C57BL/6J mice reportedly do not develop increased adiposity, obesity, glucose intolerance, insulin resistance, or behavioral and cognitive alterations following a “Western diet” compared to their male counterparts.¹³³ We intend to continue the investigation of the MD or TAD in female C57BL/6J mice, as well as potential protective mechanisms, such as estrogen and progesterone, against AD-related markers.

There were limitations in our current experiments that should be addressed in future research. First, we aimed to design two novel rodent diets that mimicked human diets in pellet form. Whole fruits, vegetables, and other components of the MD were not included in our diet formulation, although we tried to represent them as best as possible. For example, although we did not include whole fruits and vegetables, we included complex carbohydrates from brown rice, and dietary fiber from inulin, psyllium, and cellulose, to mimic nutrients that would be attained from fruits and vegetables. In the future, it would be important to incorporate fruits and vegetables in a comprehensive MD, like a formulation designed for non-human primates.^100–104 An additional limitation is that we did not examine the effects of lifelong adherence to the MD or TAD in aged mice, or the ability of MD consumption later in life to counteract the effects of the early life TAD consumption. Therefore, we aim to address these limitations in our future experiments.

With estimates projecting a nearly two-fold increase in AD cases in the U.S. over the coming decades, the urgency of preventive health strategies is crucial. Better efforts are needed to develop rodent diets that are more translatable to humans, as this would enhance experimental designs in biomedical research and improve our understanding of potential, nutritional strategies for AD prevention. In this context, the current set of experiments stands out as one of the first investigations to establish that the TAD, designed to mimic average human dietary patterns in the U.S., nonetheless provokes AD-related pathology, even in the absence of human transgenes or exaggerated “Western diets” most often employed in the scientific literature. Collectively, these results generally support our hypothesis that long-term TAD consumption is linked to heightened sporadic AD susceptibility, whereas the MD protects against it.

Supplemental Material

sj-docx-1-alz-10.1177_13872877251319467 - Supplemental material for A Mediterranean-style diet protects against cognitive and behavioral deficits, adiposity, and Alzheimer's disease-related markers, compared to a macronutrient-matched typical American diet in C57BL/6J mice

Supplemental material, sj-docx-1-alz-10.1177_13872877251319467 for A Mediterranean-style diet protects against cognitive and behavioral deficits, adiposity, and Alzheimer's disease-related markers, compared to a macronutrient-matched typical American diet in C57BL/6J mice by Paige N Braden-Kuhle, Vivienne A Lacy, Kelly N Brice, Morgan E Bertrand, Hatice Buse Uras, Catherine Shoffner, Bridgette E Fischer, Ashish Rana, Jada L Willis, Gary W Boehm and Michael J Chumley in Journal of Alzheimer's Disease

Footnotes

Acknowledgments

Thank you to all the incredible undergraduate research assistants in the Neurobiology of Aging Laboratory at TCU.

ORCID iDs

Paige N Braden-Kuhle

Vivienne A Lacy

Kelly N Brice

Morgan E Bertrand

Catherine Shoffner

Jada L Willis

Gary W Boehm

Michael J Chumley

Author contributions

Paige Nicole Braden-Kuhle (Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Project administration; Writing – original draft; Writing – review & editing); Vivienne A Lacy (Conceptualization; Data curation; Investigation; Methodology; Project administration; Writing – original draft; Writing – review & editing); Kelly N Brice (Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Visualization; Writing – original draft; Writing – review & editing); Morgan E Bertrand (Data curation; Investigation; Methodology; Writing – review & editing); Hatice Buse Uras (Investigation; Methodology); Catherine Shoffner (Investigation; Methodology); Bridgette E Fischer (Investigation; Methodology); Ashish Rana (Methodology); Jada L Willis (Conceptualization; Formal analysis; Funding acquisition; Supervision; Writing – review & editing); Gary W Boehm (Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Supervision; Visualization; Writing – original draft; Writing – review & editing); Michael J Chumley (Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Supervision; Visualization; Writing – original draft; Writing – review & editing).

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: TCU Invests in Scholarship Grant, The transgenerational effects of diet on gene expression, microbiome, and prevention of Alzheimer's disease, 06/2022– 06/2023, $20,000, PIs: Dr Michael Chumley (TCU), Dr Gary Boehm (TCU), Dr Jada Willis (TCU), Dr Matthew Hale (TCU), and Dr Michael Allen (UNTHSC). Graduate and Undergraduate Science and Engineering Research Center Grants, Texas Christian University (2021–2023).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

Data are publicly available via Open Science Framework (OSF):

Supplemental material

Supplemental material for this article is available online.

References

Alzheimer's Association. 2018 Alzheimer's disease facts and figures. Alzheimers Dement 2018; 14: 367–429.

Niu

Álvarez-Álvarez

Guillén-Grima

, et al. Prevalence and incidence of Alzheimer's disease in Europe: a meta-analysis. Neurología 2017; 32: 523–532.

Raichlen

Aslan

Sayre

, et al. Sedentary behavior and incident dementia among older adults. JAMA 2023; 330: 934–940.

Livingston

Huntley

Sommerlad

, et al. Dementia prevention, intervention, and care: 2020 report of the lancet commission. Lancet 2020; 396: 413–446.

Naderali

Ratcliffe

Dale

. Obesity and Alzheimer's disease: a link between body weight and cognitive function in old age. Am J Alzheimers Dis Other Demen 2009; 24: 445–449.

Baranowski

Hayward

Fajardo

, et al. Increased prevalence of obesity/type 2 diabetes and lower levels of lithium in rural Texas counties may explain greater Alzheimer's disease risk. J Alzheimers Dis 2018; 64: 303–308.

Grant

. Trends in diet and Alzheimer's disease during the nutrition transition in Japan and developing countries. J Alzheimers Dis 2014; 38: 611–620.

Grant

Blake

. Diet’s role in modifying risk of Alzheimer’s disease: history and present understanding. J Alzheimers Dis 2023; 96: 1353–1382.

Berti

Walters

Sterling

, et al. Mediterranean Diet and 3-year Alzheimer brain biomarker changes in middle-aged adults. Neurology 2018; 90: e1789–e1798.

10.

Mosconi

Murray

Tsui

, et al. Mediterranean Diet and magnetic resonance imaging-assessed brain atrophy in cognitively normal individuals at risk for Alzheimer's disease. J Prev Alzheimers Dis 2014; 1: 23–32.

11.

Poulimeneas

Anastasiou

Santos

, et al. Exploring the relationship between the Mediterranean diet and weight loss maintenance: the MedWeight study. Br J Nutr 2020; 124: 874–880.

12.

Sánchez-Villegas

Bes-Rastrollo

Martínez-González

, et al. Adherence to a Mediterranean dietary pattern and weight gain in a follow-up study: the SUN cohort. Int J Obes (Lond) 2006; 30: 350–358.

13.

Schröder

Marrugat

Vila

, et al. Adherence to the traditional Mediterranean diet is inversely associated with body mass index and obesity in a Spanish population. J Nutr 2004; 134: 3355–3361.

14.

Razquin

Martinez

Martinez-Gonzalez

, et al. A 3 years follow-up of a Mediterranean diet rich in virgin olive oil is associated with high plasma antioxidant capacity and reduced body weight gain. Eur J Clin Nutr 2009; 63: 1387–1393.

15.

Casas

Sacanella

Estruch

. The immune protective effect of the Mediterranean diet against chronic low-grade inflammatory diseases. Endocr Metab Immune Disord Drug Targets 2014; 14: 245–254.

16.

Casas

Sacanella

Urpí-Sardà

, et al. Long-term immunomodulatory effects of a Mediterranean diet in adults at high risk of cardiovascular disease in the PREvención con DIeta MEDiterránea (PREDIMED) randomized controlled trial. J Nutr 2016; 146: 1684–1693.

17.

Casas

Urpi-Sardà

Sacanella

, et al. Anti-inflammatory effects of the Mediterranean diet in the early and late stages of atheroma plaque development. Mediators Inflamm 2017; 2017: 3674390.

18.

Rainey-Smith

Gardener

, et al. Mediterranean Diet adherence and rate of cerebral Aβ-amyloid accumulation: data from the Australian imaging, biomarkers and lifestyle study of ageing. Transl Psychiatry 2018; 8: 238.

19.

Brickman

Stern

, et al. Mediterranean Diet and brain structure in a multiethnic elderly cohort. Neurology 2015; 85: 1744–1751.

20.

Kaplan

Zelicha

Yaskolka Meir

, et al. The effect of a high-polyphenol Mediterranean diet (Green-MED) combined with physical activity on age-related brain atrophy: the dietary intervention randomized controlled trial polyphenols unprocessed study (DIRECT PLUS). Am J Clin Nutr 2022; 115: 1270–1281.

21.

Staubo

Aakre

Vemuri

, et al. Mediterranean Diet, micronutrients and macronutrients, and MRI measures of cortical thickness. Alzheimers Dement 2017; 13: 168–177.

22.

Féart

Samieri

Rondeau

, et al. Adherence to a Mediterranean diet, cognitive decline, and risk of dementia. JAMA 2009; 302: 638–648.

23.

Qin

Adair

Plassman

, et al. Dietary patterns and cognitive decline among Chinese older adults. Epidemiology 2015; 26: 758–768.

24.

Scarmeas

Stern

Mayeux

, et al. Mediterranean Diet and mild cognitive impairment. Arch Neurol 2009; 66: 216–225.

25.

Tangney

Kwasny

, et al. Adherence to a Mediterranean-type dietary pattern and cognitive decline in a community population. Am J Clin Nutr 2011; 93: 601–607.

26.

Trichopoulou

Kyrozis

Rossi

, et al. Mediterranean Diet and cognitive decline over time in an elderly Mediterranean population. Eur J Nutr 2015; 54: 1311–1321.

27.

Tsivgoulis

Judd

Letter

, et al. Adherence to a Mediterranean diet and risk of incident cognitive impairment. Neurology 2013; 80: 1684–1692.

28.

Charisis

Ntanasi

Yannakoulia

, et al. Mediterranean Diet and risk for dementia and cognitive decline in a Mediterranean population. J Am Geriatr Soc 2021; 69: 1548–1559.

29.

Luchsinger

Stern

, et al. Mediterranean Diet, inflammatory and metabolic biomarkers, and risk of Alzheimer's disease. J Alzheimers Dis 2010; 22: 483–492.

30.

Trichopoulou

Martínez-González

Tong

, et al. Definitions and potential health benefits of the Mediterranean diet: views from experts around the world. BMC Med 2014; 12: 112.

31.

Bach-Faig

Berry

Lairon

, et al. Mediterranean Diet pyramid today. Science and cultural updates. Public Health Nutr 2011; 14: 2274–2284.

32.

Trichopoulou

Costacou

Bamia

, et al. Adherence to a Mediterranean diet and survival in a Greek population. N Engl J Med 2003; 348: 2599–2608.

33.

Cordain

Eaton

Sebastian

, et al. Origins and evolution of the Western diet: health implications for the 21st century. Am J Clin Nutr 2005; 81: 341–354.

34.

Lee

Moore

Park

, et al. Adults meeting fruit and vegetable intake recommendations - United States, 2019. MMWR Morb Mortal Wkly Rep 2022; 71: 1–9.

35.

Miketinas

Tucker

Patterson

, et al. Usual dietary fiber intake in US adults with diabetes: NHANES 2013–2018. Curr Dev Nutr 2021; 5: 1061.

36.

Hales

Fryar

Carroll

, et al. Trends in obesity and severe obesity prevalence in US youth and adults by sex and age, 2007-2008 to 2015-2016. JAMA 2018; 319: 1723–1725.

37.

Fryar

Carroll

Ogden

. Prevalence of overweight, obesity, and severe obesity among adults aged 20 and over: United States, 1960–1962 through 2017–2018, https://www.cdc.gov/nchs/data/hestat/obesity_adult_15_16/obesity_adult_15_16.pdf (2020).

38.

Nianogo

Rosenwohl-Mack

Yaffe

, et al. Risk factors associated with Alzheimer disease and related dementias by sex and race and ethnicity in the US. JAMA Neurol 2022; 79: 584–591.

39.

Alzheimer's Association. Alzheimer's disease facts and figures. Alzheimers Dement 2023; 2023: 1598–1695.

40.

Matthews

Gaglioti

, et al. Racial and ethnic estimates of Alzheimer's disease and related dementias in the United States (2015-2060) in adults aged ≥65 years. Alzheimers Dement 2019; 15: 17–24.

41.

Guillemot-Legris

Masquelier

Everard

, et al. High-fat diet feeding differentially affects the development of inflammation in the central nervous system. J Neuroinflammation 2016; 13: 206.

42.

Heyward

Walton

Carle

, et al. Adult mice maintained on a high-fat diet exhibit object location memory deficits and reduced hippocampal SIRT1 gene expression. Neurobiol Learn Mem 2012; 98: 25–32.

43.

Julien

Tremblay

Phivilay

, et al. High-fat diet aggravates amyloid-beta and tau pathologies in the 3xTg-AD mouse model. Neurobiol Aging 2010; 31: 1516–1531.

44.

Moser

Uchoa

Pike

. TLR4 Inhibitor TAK-242 attenuates the adverse neural effects of diet-induced obesity. J Neuroinflammation 2018; 15: 306.

45.

Pistell

Morrison

Gupta

, et al. Cognitive impairment following high fat diet consumption is associated with brain inflammation. J Neuroimmunol 2010; 219: 25–32.

46.

Liu

Lan

, et al. Luteolin protects against high fat diet-induced cognitive deficits in obesity mice. Behav Brain Res 2014; 267: 178–188.

47.

Rutkowsky

Lee

Puchowicz

, et al. Reduced cognitive function, increased blood-brain-barrier transport and inflammatory responses, and altered brain metabolites in LDLr -/-and C57BL/6 mice fed a western diet. PLoS One 2018; 13: e0191909.

48.

Denver

Gault

McClean

. Sustained high-fat diet modulates inflammation, insulin signalling and cognition in mice and a modified xenin peptide ameliorates neuropathology in a chronic high-fat model. Diabetes Obes Metab 2018; 20: 1166–1175.

49.

Busquets

Ettcheto

Pallàs

, et al. Long-term exposition to a high fat diet favors the appearance of β-amyloid depositions in the brain of C57BL/6J mice. A potential model of sporadic Alzheimer's disease. Mech Ageing Dev 2017; 162: 38–45.

50.

Arnold

Lucki

Brookshire

, et al. High fat diet produces brain insulin resistance, synaptodendritic abnormalities and altered behavior in mice. Neurobiol Dis 2014; 67: 79–87.

51.

Meng

Liu

, et al. Sterol O-acyltransferase 1 deficiency improves defective insulin signaling in the brains of mice fed a high-fat diet. Biochem Biophys Res Commun 2018; 499: 105–111.

52.

Spinelli

Fusco

Mainardi

, et al. Brain insulin resistance impairs hippocampal synaptic plasticity and memory by increasing GluA1 palmitoylation through FoxO3a. Nat Commun 2017; 8: 2009.

53.

Thirumangalakudi

Prakasam

Zhang

, et al. High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice. J Neurochem 2008; 106: 475–485.

54.

Wei

Yao

Zhao

, et al. High-fat diet aggravates postoperative cognitive dysfunction in aged mice. BMC Anesthesiology 2018; 18: 20.

55.

Graham

Harder

Soto

, et al. Chronic consumption of a western diet induces robust glial activation in aging mice and in a mouse model of Alzheimer's disease. Sci Rep 2016; 6: 21568.

56.

Abuznait

Qosa

Busnena

, et al. Olive-oil-derived oleocanthal enhances β-amyloid clearance as a potential neuroprotective mechanism against Alzheimer's disease: in vitro and in vivo studies. ACS Chem Neurosci 2013; 4: 973–982.

57.

Lauretti

Iuliano

Praticò

. Extra-virgin olive oil ameliorates cognition and neuropathology of the 3xTg mice: role of autophagy. Ann Clin Transl Neurol 2017; 4: 564–574.

58.

Qosa

Mohamed

Batarseh

, et al. Extra-virgin olive oil attenuates amyloid-β and tau pathologies in the brains of TgSwDI mice. J Nutr Biochem 2015; 26: 1479–1490.

59.

Wright

Wang

. Trends in intake of energy and macronutrients in adults from 1999-2000 through 2007-2008. NCHS Data Brief 2010; 49: 1–8.

60.

Nutrient intakes from food and beverages: mean amounts consumed per individual, by gender and age, What We Eat in America, NHANES 2013–2014. https://www.ars.usda.gov/arsuserfiles/80400530/pdf/1314/table_1_nin_gen_13.pdf (2016).

61.

Davis

Bryan

Hodgson

, et al. Definition of the Mediterranean diet; A literature review. Nutrients 2015; 7: 9139–9153.

62.

Willett

Sacks

Trichopoulou

, et al. Mediterranean Diet pyramid: a cultural model for healthy eating. Am J Clin Nutr 1995; 61: 1402S–1406S.

63.

Simopoulos

. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: nutritional implications for chronic diseases. Biomed Pharmacother 2006; 60: 502–507.

64.

Simopoulos

. The Mediterranean diets: what is so special about the diet of Greece? The scientific evidence. J Nutr 2001; 131: 3065S–3073S.

65.

Biesmans

Meert

Bouwknecht

, et al. Systemic immune activation leads to neuroinflammation and sickness behavior in mice. Mediators Inflamm 2013; 2013: 271359.

66.

Kahn

Kranjac

Alonzo

, et al. Prolonged elevation in hippocampal Aβ and cognitive deficits following repeated endotoxin exposure in the mouse. Behav Brain Res 2012; 229: 176–184.

67.

Kranjac

McLinden

Deodati

, et al. Peripheral bacterial endotoxin administration triggers both memory consolidation and reconsolidation deficits in mice. Brain Behav Immun 2012; 26: 109–121.

68.

Lee

Yuk

, et al. Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation. J Neuroinflammation 2008; 5: 37.

69.

Zhao

Xiao

, et al. Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice. Sci Rep 2019; 9: 5790.

70.

Weintraub

Bisson

Nouri

, et al. Imatinib methanesulfonate reduces hippocampal amyloid-beta and restores cognitive function following repeated endotoxin exposure. Brain Behav Immun 2013; 33: 24–28.

71.

Onufriev

Uzakov

Freiman

, et al. The dorsal and ventral hippocampus have different reactivities to proinflammatory stress: corticosterone levels, cytokine expression, and synaptic plasticity. Neurosci Behav Physiol 2018; 48: 1024–1031.

72.

Parnet

Kelley

Bluthé

R-M

, et al. Expression and regulation of interleukin-1 receptors in the brain. Role in cytokines-induced sickness behavior. J Neuroimmunol 2002; 125: 5–14.

73.

Seibenhener

Wooten

. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp 2015; 96: e52434.

74.

Shepherd

Grewal

Fletcher

, et al. Behavioural and pharmacological characterisation of the elevated “zero-maze” as an animal model of anxiety. Psychopharmacology (Berl) 1994; 116: 56–64.

75.

Cui

Han

, et al. Optimized analysis of in vivo and in vitro hepatic steatosis. J Vis Exp 2017; 121: 55178.

76.

Agarwal

Leurgans

Agrawal

, et al. Association of Mediterranean-DASH intervention for neurodegenerative delay and Mediterranean diets with Alzheimer disease pathology. Neurology 2023; 100: e2259–e2268.

77.

Taylor

Sullivan

Swerdlow

, et al. A high-glycemic diet is associated with cerebral amyloid burden in cognitively normal older adults. Am J Clin Nutr 2017; 106: 1463–1470.

78.

Kien

Bunn

Fukagawa

, et al. Lipidomic evidence that lowering the typical dietary palmitate to oleate ratio in humans decreases the leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes. J Nutr Biochem 2015; 26: 1599–1606.

79.

Poppitt

Keogh

Lithander

, et al. Postprandial response of adiponectin, interleukin-6, tumor necrosis factor-alpha, and C-reactive protein to a high-fat dietary load. Nutrition 2008; 24: 322–329.

80.

van Dijk

Feskens

Bos

, et al. A saturated fatty acid-rich diet induces an obesity-linked proinflammatory gene expression profile in adipose tissue of subjects at risk of metabolic syndrome. Am J Clin Nutr 2009; 90: 1656–1664.

81.

Montonen

Boeing

Fritsche

, et al. Consumption of red meat and whole-grain bread in relation to biomarkers of obesity, inflammation, glucose metabolism and oxidative stress. Eur J Nutr 2013; 52: 337–345.

82.

Więckowska-Gacek

Mietelska-Porowska

Wydrych

, et al. Western diet as a trigger of Alzheimer's disease: from metabolic syndrome and systemic inflammation to neuroinflammation and neurodegeneration. Ageing Res Rev 2021; 70: 101397.

83.

Hintze

Benninghoff

Cho

, et al. Modeling the Western diet for preclinical investigations. Adv Nutr 2018; 9: 263–271.

84.

Trichopoulou

. Traditional Mediterranean diet and longevity in the elderly: a review. Public Health Nutr 2004; 7: 943–947.

85.

Trichopoulou

Orfanos

Norat

, et al. Modified Mediterranean diet and survival: EPIC-elderly prospective cohort study. BMJ 2005; 330: 991.

86.

Pellizzon

Ricci

. The common use of improper control diets in diet-induced metabolic disease research confounds data interpretation: the fiber factor. Nutr Metab 2018; 15: 3.

87.

Mesnage

Defarge

Rocque

L-M

, et al. Laboratory rodent diets contain toxic levels of environmental contaminants: implications for regulatory tests. PLoS One 2015; 10: e0128429.

88.

Liu

Wang

, et al. Identification of a sustainable two-plant diet that effectively prevents age-related metabolic syndrome and extends lifespan in aged mice. J Nutr Biochem 2018; 51: 16–26.

89.

Casimiro

Stull

Tersey

, et al. Phenotypic sexual dimorphism in response to dietary fat manipulation in C57BL/6J mice. J Diabetes Complications 2021; 35: 107795.

90.

Nakajima

Fukasawa

Gotoh

, et al. Saturated fatty acid is a principal cause of anxiety-like behavior in diet-induced obese rats in relation to serum lysophosphatidyl choline level. Int J Obes (Lond) 2020; 44: 727–738.

91.

Marchlewicz

McCabe

Djuric

, et al. Gestational exposure to high fat diets and bisphenol A alters metabolic outcomes in dams and offspring, but produces hepatic steatosis only in dams. Chemosphere 2022; 286: 131645.

92.

Nardiello

Pantano

Lapucci

, et al. Diet supplementation with hydroxytyrosol ameliorates brain pathology and restores cognitive functions in a mouse model of amyloid-β deposition. J Alzheimers Dis 2018; 63: 1161–1172.

93.

Sharman

Gyengesi

Liang

, et al.

Assessment of diets containing curcumin, epigallocatechin-3-gallate, docosahexaenoic acid and α-lipoic acid on amyloid load and inflammation in a male transgenic mouse model of Alzheimer's disease: are combinations more effective?

Neurobiol Dis 2019; 124: 505–519.

94.

Paniagua

de la Sacristana

Sánchez

, et al. A MUFA-rich diet improves posprandial glucose, lipid and GLP-1 responses in insulin-resistant subjects. J Am Coll Nutr 2007; 26: 434–444.

95.

Di Mauro

Tuccinardi

Watanabe

, et al. The Mediterranean diet increases glucagon-like peptide 1 and oxyntomodulin compared with a vegetarian diet in patients with type 2 diabetes: a randomized controlled cross-over trial. Diabetes Metab Res Rev 2021; 37: e3406.

96.

Castellanos-Tapia

Tejero-Barrera

Salas-Silva

, et al. Mediterranean-like mix of fatty acids induces cellular protection on lipid-overloaded hepatocytes from western diet fed mice. Ann Hepatol 2020; 19: 489–496.

97.

Pavlisova

Horakova

Kalendova

, et al. Chronic n-3 fatty acid intake enhances insulin response to oral glucose and elevates GLP-1 in high-fat diet-fed obese mice. Food Function 2020; 11: 9764–9775.

98.

Simopoulos

. An increase in the omega-6/omega-3 fatty acid ratio increases the risk for obesity. Nutrients 2016; 8: 128.

99.

Pisani

Amri

E-Z

Ailhaud

. Disequilibrium of polyunsaturated fatty acids status and its dual effect in modulating adipose tissue development and functions. OCL 2015; 22: D405.

100.

Johnson

CSC

Frye

, et al. Mediterranean Diet reduces social isolation and anxiety in adult female nonhuman primates. Nutrients 2022; 14: 2852.

101.

Johnson

CSC

Shively

Michalson

, et al. Contrasting effects of Western vs Mediterranean diets on monocyte inflammatory gene expression and social behavior in a primate model. eLife 2021; 10: e68293.

102.

Shively

Appt

Chen

, et al. Mediterranean Diet, stress resilience, and aging in nonhuman primates. Neurobiol Stress 2020; 13: 100254.

103.

Shively

Appt

Vitolins

, et al. Mediterranean Versus Western diet effects on caloric intake, obesity, metabolism, and hepatosteatosis in nonhuman primates. Obesity 2019; 27: 777–784.

104.

Shively

Frye

, et al. Mediterranean Versus western diet effects on cerebral cortical thickness and volume in cynomolgus macaques. Alzheimers Dement 2020; 16: e044554.

105.

Speakman

. Use of high-fat diets to study rodent obesity as a model of human obesity. Int J Obes 2019; 43: 1491–1492.

106.

Kim

D-G

Krenz

Toussaint

, et al. Non-alcoholic fatty liver disease induces signs of Alzheimer’s disease (AD) in wild-type mice and accelerates pathological signs of AD in an AD model. J Neuroinflamm 2016; 13: 1.

107.

Recena Aydos

Aparecida do Amaral

Serafim de Souza

, et al. Nonalcoholic fatty liver disease induced by high-fat diet in C57bl/6 models. Nutrients 2019; 11: 3067.

108.

Jeong

Kim

Park

. Non-alcoholic fatty liver disease and risk of dementia: unmet need for a pooled analysis of cohort studies. Clin Mol Hepatol 2022; 28: 933–934.

109.

Seo

Gottesman

Clark

, et al. Nonalcoholic fatty liver disease is associated with cognitive function in adults. Neurology 2016; 86: 1136–1142.

110.

Cheng

C-Y

Tian

D-Y

, et al. Physiological β-amyloid clearance by the liver and its therapeutic potential for Alzheimer’s disease. Acta Neuropathol 2023; 145: 717–731.

111.

George

Georgousopoulou

Mellor

, et al. Exploring the path of Mediterranean diet, non-alcoholic fatty liver disease (NAFLD) and inflammation towards 10-year cardiovascular disease (CVD) risk: the ATTICA study 10-year follow-up (2002-2012). Nutrients 2022; 14: 2367.

112.

Ryan

Itsiopoulos

Thodis

, et al. The Mediterranean diet improves hepatic steatosis and insulin sensitivity in individuals with non-alcoholic fatty liver disease. J Hepatol 2013; 59: 138–143.

113.

LaFerla

Oddo

. Alzheimer's disease: abeta, tau and synaptic dysfunction. Trends Mol Med 2005; 11: 170–176.

114.

Liu

Hao

, et al. TLR2 Is a primary receptor for Alzheimer's amyloid β peptide to trigger neuroinflammatory activation. J Immunol 2012; 188: 1098–1107.

115.

Hooijmans

Rutters

Dederen

, et al. Changes in cerebral blood volume and amyloid pathology in aged Alzheimer APP/PS1 mice on a docosahexaenoic acid (DHA) diet or cholesterol enriched typical western diet (TWD). Neurobiol Dis 2007; 28: 16–29.

116.

Oksman

Iivonen

Hogyes

, et al. Impact of different saturated fatty acid, polyunsaturated fatty acid and cholesterol containing diets on beta-amyloid accumulation in APP/PS1 transgenic mice. Neurobiol Dis 2006; 23: 563–572.

117.

Vandal

White

Tremblay

, et al. Insulin reverses the high-fat diet-induced increase in brain Aβ and improves memory in an animal model of Alzheimer disease. Diabetes 2014; 63: 4291–4301.

118.

Walker

Dixit

Saulsberry

, et al. Reversal of high fat diet-induced obesity improves glucose tolerance, inflammatory response, β-amyloid accumulation and cognitive decline in the APP/PSEN1 mouse model of Alzheimer's disease. Neurobiol Dis 2017; 100: 87–98.

119.

Green

Martinez-Coria

Khashwji

, et al. Dietary docosahexaenoic acid and docosapentaenoic acid ameliorate amyloid-beta and tau pathology via a mechanism involving presenilin 1 levels. J Neurosci 2007; 27: 4385–4395.

120.

Lim

Calon

Morihara

, et al. A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. J Neurosci 2005; 25: 3032–3040.

121.

Perez

Berg

Moore

, et al. DHA Diet reduces AD pathology in young APPswe/PS1 Delta E9 transgenic mice: possible gender effects. J Neurosci Res 2010; 88: 1026–1040.

122.

Zhou

M-m

Ding

Wen

, et al. Mechanisms of DHA-enriched phospholipids in improving cognitive deficits in aged SAMP8 mice with high-fat diet. J Nutr Biochem 2018; 59: 64–75.

123.

Illesca

Valenzuela

Espinosa

, et al. Hydroxytyrosol supplementation ameliorates the metabolic disturbances in white adipose tissue from mice fed a high-fat diet through recovery of transcription factors Nrf2, SREBP-1c, PPAR-γ and NF-κB. Biomed Pharmacother 2019; 109: 2472–2481.

124.

Bitler

Viale

Damaj

, et al. Hydrolyzed olive vegetation water in mice has anti-inflammatory activity. J Nutr 2005; 135: 1475–1479.

125.

Fuccelli

Fabiani

Rosignoli

. Hydroxytyrosol exerts anti-inflammatory and anti-oxidant activities in a mouse model of systemic inflammation. Molecules 2018; 23: 3212.

126.

Rincón-Cervera

Valenzuela

Hernandez-Rodas

, et al. Supplementation with antioxidant-rich extra virgin olive oil prevents hepatic oxidative stress and reduction of desaturation capacity in mice fed a high-fat diet: effects on fatty acid composition in liver and extrahepatic tissues. Nutrition 2016; 32: 1254–1267.

127.

Huang

Rutkowsky

Snodgrass

, et al. Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways. J Lipid Res 2012; 53: 2002–2013.

128.

Jena

Sheng

Di Lucente

, et al. Dysregulated bile acid synthesis and dysbiosis are implicated in Western diet-induced systemic inflammation, microglial activation, and reduced neuroplasticity. FASEB J 2018; 32: 2866–2877.

129.

Jena

Setayesh

Sheng

, et al. Intestinal microbiota remodeling protects mice from Western diet-induced brain inflammation and cognitive decline. Cells 2022; 11: 504.

130.

Dursun

Gezen-Ak

Hanağası

, et al. The interleukin 1 alpha, interleukin 1 beta, interleukin 6 and alpha-2-macroglobulin serum levels in patients with early or late onset Alzheimer's disease, mild cognitive impairment or Parkinson's disease. J Neuroimmunol 2015; 283: 50–57.

131.

Gezen-Ak

Dursun

Hanağası

, et al. BDNF, TNFα, HSP90, CFH, and IL-10 serum levels in patients with early or late onset Alzheimer's disease or mild cognitive impairment. J Alzheimers Dis 2013; 37: 185–195.

132.

Fernando

Martins

Morici

, et al. Sodium butyrate reduces brain amyloid-β levels and improves cognitive memory performance in an Alzheimer's disease transgenic mouse model at an early disease stage. J Alzheimers Dis 2020; 74: 91–99.

133.

Pereira-Silva

Machado-Silva

Castro-Pinheiro

, et al.

Does gender influence cardiovascular remodeling in C57BL/6J mice fed a high-fat, high-sucrose and high-salt diet?

Int J Exp Pathol 2019; 100: 153–160.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.36 MB

A Mediterranean-style diet protects against cognitive and behavioral deficits,adiposity,and Alzheimer's disease-related markers,compared to a macronutrient-matched typical American diet in C57BL/6J mice

Abstract

Background

Objective

Methods

Results

Conclusions

Keywords

Introduction

General methods for experiments 1–2

Subjects

Experimental design and timeline

Experimental diet formulations

Methods for experiment 1

Measures of food consumption and body weight

Open field testing

Elevated zero maze

Object-location memory task

Tissue and serum collection

Peripheral cytokine analysis

Liver histology

Aβ1–42 ELISA

Statistical analyses

Methods for experiment 2

Subjects and experimental design

Quantitative reverse-transcription polymerase chain reaction (qRTPCR)

Statistical analyses

Experiment 1 results

Average food and kilocalorie consumption

Body weight

Organ and white adipose tissue weight

Liver health following diet administration

Peripheral cytokine production in serum

Aβ1–42 production in the cortex and hippocampus

Open field behavior

Vertical counts

Distance traveled

Time spent in the center zone of open field

Average speed

Elevated zero maze behavior

Spatial learning and memory in OLM

Experiment 2 results

Gene Expression of BDNF and BACE1 in the Dorsal Hippocampus

Gene Expression of TNF-a, IL-1β, IL-6, and IL-10 in the Dorsal Hippocampus

Discussion

Supplemental Material

sj-docx-1-alz-10.1177_13872877251319467 - Supplemental material for A Mediterranean-style diet protects against cognitive and behavioral deficits, adiposity, and Alzheimer's disease-related markers, compared to a macronutrient-matched typical American diet in C57BL/6J mice

Footnotes

Acknowledgments

ORCID iDs

Author contributions

Funding

Declaration of conflicting interests

Data availability

Supplemental material

References

Supplementary Material

Aβ_1–42 ELISA

Aβ_1–42 production in the cortex and hippocampus