Abstract
Increasing data suggest a crucial relationship between glycolipid metabolic disorder and neuropsychiatric injury. The aim of this study is to investigate the behavioral performance changes and neuropathological injuries in mice challenged with high-fat diet (HFD) and streptozotocin (STZ). The glucose metabolism indicators and behavioral performance were detected. The mRNA expression of IL-1β, IL-6, TNF-α, ocln, zo-1, and clnds and protein expression of APP, p-Tau, p-IRS1, p-AKT, p-ERK, and TREM1/2 were measured. The fluorescence intensities of MAP-2, NeuN, APP, p-Tau, GFAP, and IBA-1 were observed. The results showed that combination of HFD and STZ/I.P. could induce glucose metabolic turmoil and Alzheimer's disease (AD)-like neuropsychiatric dysfunction in mice, as indicated by the increased concentrations of fasting blood glucose and impaired learning and memory ability. Moreover, the model mice presented increased levels of APP, p-Tau, p-IRS1, TREM2, IL-1β, IL-6, TNF-α, ocln, zo-1, and clnds; decreased levels of p-AKT, p-ERK, and TREM1; and neuron damage and the hyperactivation of astrocytes and microglia in the hippocampus as compared with control mice. Only male mice were used in this study. Although AD and type 2 diabetes mellitus (T2DM) are distinct pathologies, our results suggested that combination of HFD and STZ/I.P., a widely used T2DM modeling method, could successfully induce AD-like behavioral impairments and neuropathological injuries in mice; the mechanism might be involved with neuroinflammation and its associated dysfunction of IRS1/AKT/ERK signaling pathway. Our findings further support the potential overlap between T2DM and AD pathophysiology, providing insight into the mechanisms underlying the comorbidity of these diseases.
Significance Statement
Alzheimer's disease (AD) is a progressive neurodegenerative disorder whose prevalence is increasing with the rapidly growing global elderly population, but the accurate disease mechanisms and underlying therapeutic targets remain unclear. The aim of the present study is to investigate the behavioral performance changes and neuropathological injuries in mice challenged with the combination of a high-fat diet (HFD) and intraperitoneal injection of streptozotocin (STZ/I.P.). Our study suggested that combination of HFD and STZ/I.P. could successfully induce AD-like behavioral impairments and neuropathological injuries in mice, whose mechanism might be involved with neuroinflammation and its associated dysfunction of the IRS1/AKT/ERK signaling pathway. Our findings further support the potential overlap between T2DM and AD pathophysiology, providing insight into the mechanisms underlying the comorbidity of these diseases.
Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting elders globally with an increasing prevalence (Scheltens et al., 2016). According to the report from World Health Organization (WHO), approximately 5% of individuals aged 65 and older worldwide are affected by AD, and it has been estimated up to 115 million individuals worldwide living with AD by 2050 (Sengoku, 2020). However, only five treatment options are currently approved in the United States to address the cognitive symptoms of AD, with the most recent approval being memantine over a decade ago (Cummings et al., 2014). Regrettably, in the last 15 years, the majority of treatments under development have proven unsuccessful, with notable examples like aducanumab, which despite FDA approval, exhibited a striking 99.6% failure rate (Briggs et al., 2016). This highlights the critical necessity for advancements in comprehending disease mechanisms and pinpointing therapeutic targets.
Although the mechanism underlying the onset and progression of AD is not fully clear, mutations of amyloid precursor protein that result in abnormal production of Aβ peptides (Ghiso et al., 2004), and the intracellular formation of neurofibrillary tangles (NFTs) in the brain (J. Z. Wang and Liu, 2008), have been suggested to play a dominant role in the pathogenesis of AD (Maldonado-Díaz et al., 2024). Besides, recent studies have suggested a potential link between AD and brain insulin resistance (IR; Craft et al., 1998; Arnold et al., 2018; Kshirsagar et al., 2021). Moreover, increasing data suggested a close relation between metabolic disorders, especially diabetes mellitus, and the development of AD (Chakrabarti et al., 2015; Ashton et al., 2024). It has been reported that patients with type 2 diabetes mellitus (T2DM) have more than twice the risk of developing late-onset AD compared with nondiabetic individuals (Zhou et al., 2014) and that 80% of AD patients also exhibit symptoms of T2DM or abnormal glucose and insulin levels (S. Gao et al., 2019). It has been reported that the decreased insulin levels and impaired insulin signaling in the brain of AD patients could lead to the dysfunctions of glucose metabolism and synaptic plasticity, and the formation of NFTs and amyloid plaques, which are the critical processes involved in AD progression (Rad et al., 2018; Chen et al., 2023). Interestingly, accumulated Aβ and hyperphosphorylated tau protein have also been observed in the pancreatic tissue of T2DM patients and brain tissue of T2DM mice (Baglietto-Vargas et al., 2016; Marciniak et al., 2017). Therefore, understanding the intricate interplay between Aβ plaques, NFTs, and IR in the AD process may provide valuable insights into novel treatment strategies and diagnostic approaches for this neurodegenerative condition.
In recent years, growing evidence highlighted the role of microglia and its related neuroinflammation in the pathogenesis and progression of AD (S. Hong et al., 2016; Hansen et al., 2018). As the innate immune cells of the central nervous system (CNS), microglia could play a crucial role in nervous system injury response and pathogen defense, as well as the developmental sculpting of neural circuits by engulfment and removal of unwanted neurons and synapses (Ransohoff and Cardona, 2010; Tay et al., 2017; Li and Barres, 2018). It has been reported that activated microglia (De Strooper and Karran, 2016) and aberrant proinflammatory phenotype contribute to neuronal damage and synaptic dysfunction by releasing cytokines, reactive oxygen species, and other inflammatory mediators, which could perpetuate neurodegeneration and exacerbate the progression of AD (Tang and Le, 2016). Moreover, microglial responses to plaque formation have also been considered an important factor in AD pathogenesis (Condello et al., 2015). It has been reported that the impairment of microglial function to reduce Aβ burden is a causal factor in AD. Specifically, microglial activation attempts to eliminate Aβ accumulation by phagocytosis and clearance (Yin et al., 2023); however, the generation and accumulation of Aβ could cause microglial dysfunction by releasing the inflammatory mediators (Sosna et al., 2018).
Triggering receptor expressed in myeloid cells 2 (TREM2) is a cell surface protein selectively and highly expressed by microglia in the brain, which has been proven to maintain the phagocytic ability of microglia toward Aβ deposits (Y. Wang et al., 2015; S. Wang et al., 2022). The results of our previous studies showed that the imbalance of TREM2 expression was not only involved in the process of neuronal injury induced by high cholesterol but also related to LPS-induced hyperactivation of microglia and increased secretion of inflammatory factors (X. R. Gao et al., 2021; Liu et al., 2022; Zheng et al., 2023). Moreover, both T2DM and AD mice models showed a neuroinflammation-related imbalance of TREM1/2 expression in the hippocampus, with a close relation with the impaired performance in behavioral tasks (Fan et al., 2022). Hence, it should be rational to hypothesize the potential role of TREM2 in linking AD and TREM2.
Focusing on the intersection of T2DM and AD, we aim to elucidate the mechanisms by which metabolic dysfunction may contribute to neurodegenerative changes. The T2DM mice model was established by the combination of high-fat diet (HFD) and STZ/I.P. Apart from the metabolic parameters, the behavioral performance was observed by open-field test (OFT), novel object recognition test (NOR), Y-maze test (Y-maze), and Morris water maze test (MWM). The neuron damage (mark proteins with Map-2 and NeuN), the pathological index of AD (mark proteins with APP and p-Tau), and the activation of astrocytes and microglia (mark proteins with GFAP and IBA-1) in the hippocampus were observed by immunofluorescence. The protein expression levels of the insulin signaling pathway including IRS1, Akt, and ERK and AD-related biomarkers including APP, p-Tau, and TREM1/2 in the hippocampus and PFC were detected via Western blot, and the mRNA expression levels of IL-1β, IL-6, TNF-α, ocln, zo-1, and clnds were measured by qPCR technique.
Materials and Methods
Animal experimental design and drug treatment
Sixteen male C57BL/6 mice, aged 6 weeks, were obtained from the Experimental Animal Center of Anhui Medical University. The mice were housed in cages with four mice per cage and subjected to alternating light and dark conditions for 12 h. They had access to adequate food and water, and the ambient temperature was maintained at 20 ± 2°C with a humidity of 50 ± 5%. The study protocol was approved by the Ethics Committee of Experimental Animals of Anhui Medical University and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Measures were implemented to minimize pain and discomfort for the mice throughout the entire experimental process. After 1 week of acclimatization, the mice were randomly divided into two groups, including the control group (Con) fed a standard chow diet (3.766 kcal/g) and the model group (Mod) fed a high-fat diet (60% of calories from fat, 5 kcal/g). After 8 weeks of feeding, the model mice were injected intraperitoneally with STZ (100 mg/kg), while the control mice were injected with a buffer solution. The experimental procedure is depicted in Figure 1A.
Behavioral tests
All behavioral tests were conducted in a quiet laboratory, with testing times scheduled between 08:00 and 12:30 to ensure consistent conditions across all groups. Prior to each experiment, the mice were acclimated to the behavioral laboratory environment to minimize anxiety and hyperactivity. After each experiment, all equipment and utensils were thoroughly cleaned and wiped with 75% alcohol to prevent any residual odors or excrement that could potentially impact the behavior of subsequent mice. The ANY-maze video imaging software (Stoelting) was utilized to record and analyze the performance of the mice in the behavioral tests.
Open-field test (OFT)
The OFT was conducted to assess the spontaneous exploratory activity of mice, whose apparatus consisted of a white rectangular box measuring 50 × 50× 45 cm. Each mouse was placed in one corner of the box and allowed to freely explore the field for 5 min. The total distance, average speed, and line crossing of mice traveled in the OFT were recorded and analyzed.
Novel object recognition test (NOR)
The NOR was used to assess the cognitive function of mice, whose apparatus is similar to the OFT with some modifications. The experiment consists of two stages: the adaptation period and the experimental period. During the adaptation period, the mouse was placed into the apparatus for 10 min to familiarize with the environment. The experimental period is divided into two stages: during the first stage, two identical objects were placed on the floor, and the mouse was allowed to freely explore the objects for 10 min; after 1 h, during the second stage, one of the familiar objects was replaced with a novel object, which had a different color and shape but was placed in the same position as the familiar object, and the mouse was then reintroduced into the apparatus and allowed to explore the novel and familiar objects for 5 min. The novel object recognition index was calculated as the ratio of the exploration time the mouse spent on the novel object and the old object.
Y-maze test (Y-maze)
The Y-maze was conducted to evaluate the spatial memory of mice, whose apparatus consisted of three interconnected arms (40 × 10 × 20 cm) arranged radially at 120° angles from one another. The arms were randomly designated as the start, familiar, or novel arm. During the adaptation period, the novel arm was blocked off, and the mouse was placed from the start arm and allowed to explore the apparatus for 10 min. One hour later, with the novel arm open, the mouse was placed back and allowed to explore the three arms for 5 min freely. The novel arm preference index was calculated as the ratio of duration the mouse spent in the novel arm and the familiar arm.
Morris water maze test (MWM)
The MWM was utilized to assess the spatial memory of mice, whose apparatus consisted of a black circular pool with a diameter of 120 cm and a height of 80 cm. The water temperature was maintained at 21–23°C and colored white using food coloring. The maze was divided into four equal quadrants: the first, second, third, and fourth quadrants. A circular platform, positioned 1 cm below the water surface, was placed in one of the quadrants and designated as the target quadrant. The test comprised two stages: the acquisition phase and the probe test. During the acquisition phase, the mouse was initially allowed to adapt to the platform’s location by spending 30 s on it. Subsequently, the mouse was placed in the water, facing the wall, in each quadrant and trained to locate the hidden platform within 60 s. This training was repeated for 3 consecutive days, and the average time the mouse spent in four trials per day was recorded as the escape latency. During the probe test, the submerged escape platform was removed, and the mouse was placed into the pool opposite to the platform and allowed to swim freely for 60 s. The time mice spent in the target quadrant and the latency to the target quadrant were recorded and analyzed.
Measurement of serum samples
The blood glucose levels were measured using a Roche glycemic meter. Twenty-four hours after the last behavioral test, the mice were deeply anesthetized after an overnight fast. Blood was collected and placed at room temperature for 2 h. The serum was isolated by centrifugation at 3,000 rpm for 15 min, and the upper layer was collected. The serum levels of insulin were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Wuhan ColorfulGene Biological Technology). The HOMA-IR index was calculated using the following formula: HOMA-IR = (fasting glucose (mmol/L) × fasting insulin (mU/L)) / 22.5.
Immunofluorescence staining (IF)
Three mice were randomly selected and treated with a perfusion of PBS and 4% paraformaldehyde until systemic spasm occurred. Then the whole brains were fixed overnight in a 4% paraformaldehyde solution and dehydrated in a 30% sucrose solution for 48 h. Subsequently, the brains were embedded in an OCT embedding agent (4583, Solarbio) and frozen at −80°C. Brain slices were cut for 30 µm using a cryostat. After antigen retrieval with EDTA in a 95°C water bath, the sections were blocked with 5% BSA and 0.3% Triton X-100 at room temperature for 1 h. Following the blocking step, the sections were incubated overnight at 4°C with primary antibodies: anti-APP (1:100; 25524-1-AP, Proteintech), anti-p-Tau (Ser396; 1:100; sc-32275, Santa Cruz Biotechnology), anti-GFAP (1:100; 16825-1-AP, Proteintech), anti-IBA1 (1:100; DF6442, Affinity Biosciences), anti-CD68 (1:100, 28058-1-AP, Proteintech), anti-NeuN (1:100; ET1602-12, Huabio), and anti-Map-2 (1:100; EM1709-48, Huabio). Then the sections were incubated with either goat anti-rabbit Alexa Fluor 488–conjugated IgG (1:100; ZF-0511, ZSGB-BIO) or goat anti-mouse Alexa Fluor 647–conjugated IgG (1:100; A0473, Time) at room temperature for 2 h. Finally, nuclei were counterstained with DAPI. The stained slices were observed under a fluorescence microscope (Olympus Life Science VS120), and fluorescence intensity was quantified using Image-Pro software (Media Cybernetics).
Western blotting
Three mice were randomly selected from each group, and the hippocampus and the PFC were carefully separated, rapidly frozen in liquid nitrogen, and stored at −80°C. Then, the hippocampus and PFC tissues were cleaved using RIPA buffer (P0013B, Beyotime) containing protease inhibitors (ST507-10, Beyotime) and phosphatase inhibitors (ST019-10, Beyotime). Equal amounts of protein were separated on 10% SDS–PAGE gels, transferred onto polyvinylidene difluoride (PVDF) membranes, and incubated overnight at 4°C with the relevant primary antibodies: anti-phosphorylated AKT (p-AKT) and total AKT (1:1,000; Zen-Bio), anti-phosphorylated ERK (p-ERK) and total ERK (1:1,000; Santa Cruz Biotechnology), anti-phosphorylated IRS1 (p-IRS1) and total IRS1 (1:1,000; Abcam), anti-APP(1:1,000; Proteintech), anti-phosphorylated Tau (p-Tau) and total tau (1:1,000; Santa Cruz Biotechnology), anti-TREM1 and TREM2 (1:1,000; Abcam), and anti-β-actin (1:1,000; Zhongshan Biotechnology). After washing with TBST for 30 min, the membranes were incubated with corresponding secondary antibodies (HRP-conjugated anti-rabbit or anti-mouse antibodies) at room temperature for 1 h. In addition, in order to detect multiple targets, the Western blot fast stripping buffer (PS107, Epizyme) was used due to the similar molecular weight of the target bands. Protein images were captured using a gel imaging system, and the images were processed and analyzed using ImageJ software (National Institutes of Health) and normalized relative to that of the internal control β-actin.
Quantitative real-time PCR
Equal quantities of total RNA from hippocampal and PFC tissues were used for qPCR analysis. Briefly, the total RNA was extracted using the TRIzol reagents and transcribed into cDNA using commercial kits according to the instructions. The reverse transcription reaction was performed at 37°C for 15 min and 85°C for 5 s. The mRNA expression levels of ocln, zo1, clnds, IL-1β, LI-6, and TNF-α in the hippocampus and PFC of mice were detected using the SYBR Green PCR kit on an ABI Prism Sequence Detector System in a 20 µl volume for 40 cycles (10 s at 95°C and 30 s at 55°C). Three replicates were conducted for each qPCR analysis, and the data were analyzed using the 2−ΔΔCt method. The primer sequences used in the PCR are provided in Table 1.
Statistical analysis
All experimental data were analyzed and visualized using SPSS Statistics 17.0 software, and the charts were drawn using GraphPad Prism 8.0 software. The differences between the two groups were assessed using the Student’s t test. Although the differences between the two groups were initially assessed using the Student's t test, we also conducted the Wilcoxon rank-sum test to account for potential deviations from normality and small sample size. For comparisons of more than two groups, two-way (repeated measures) ANOVA was used to assess statistical significance, followed by multiple comparisons between groups using Bonferroni’s post hoc test. Data are presented as mean ± standard error of the mean (SEM), and p < 0.05 was considered statistically significant. All data points were included in the plots to maintain transparency and facilitate a comprehensive analysis of the results.
Results
Combination of HFD and STZ/I.P. induced hyperglycemia, hyperinsulinemia, and insulin resistance in mice
According to the longitudinal epidemiological studies, there have been elevated plasma insulin levels (Craft et al., 1998) in AD patients, with a close association between cognitive disorder and insulin resistance in T2DM patients (Arnold et al., 2018; Kshirsagar et al., 2021). Similarly, in the present study, the mice in the Mod group exhibited a significant increased blood glucose levels and serum levels of insulin when compared with the Con group, as shown in Figure 1B,C. Furthermore, the results showed that the HOMA-IR index was significantly increased in the Mod mice as compared with the Con mice, as shown in Figure 1D. The tendency of the bodyweight in the Mod group was increased after HFD induction and began to decline upon STZ treatment after 8 weeks when compared with the Con group in Figure 1E.
Combination of HFD and STZ/I.P. induced an AD-like behavioral dysfunction in mice
The general behavior of mice is shown in Figure 2. As shown in Figure 2A,D, there was no significant difference between the two groups in the total moving distance, average speed, and line crossing in the OFT. The behavioral performance of mice in the NOR and Y-maze test are shown in Figure 2E–H. Compared to the Con mice, the Mod mice exhibited a significant decrease in the novel object recognition index (Fig. 2E,F), together with a decreased novel arm preference index (Fig. 2G,H). Moreover, in the MWM test, the Mod mice showed a decreased distance traveled in the target quadrant and an increased latency found in the target quadrant in the probe trial (Fig. 2I–K). The typical swimming orbits of the two groups in the probe trial are shown in Figure 2L.
Combination of HFD and STZ/I.P. induced neuron injuries and blood–brain barrier damage in the hippocampus in mice
Map-2 and NeuN are the neuronal marker proteins used to assess the morphology and function of neurons, and detecting the expression of Map-2 and NeuN could help know the extent of neuronal damage and degeneration in the present study (Götz and Ittner, 2008; Ghatak et al., 2019; Sattarov et al., 2023). As shown in Figure 3A,B, the Mod group exhibited significantly lower fluorescence intensity for Map-2 in the hippocampal region, suggesting a reduction in the expression of this neuronal marker compared with the control group. Similarly, the immunofluorescent staining of NeuN was also reduced in the hippocampus of Mod mice as compared with the Con mice (Fig. 3C,D), indicating that combination of HFD and STZ/I.P. could induce neuron damage in the hippocampus of mice. Besides, the mRNA expression levels of ocln, zo-1, and clnds in the hippocampus and PFC of Mod mice were all remarkably reduced compared with the Con ones, suggesting potential BBB damage induced by the combination of HFD and STZ/I.P. in mice (Fig. 3E,F).
Combination of HFD and STZ/I.P. induced Aβ accumulation and tau hyperphosphorylation in mice
As shown in Figure 4A,B, compared with the Con mice, there has been a significant increase in APP immunofluorescence intensity in the hippocampus of Mod mice. Furthermore, the immunofluorescent staining of p-Tau was also increased in the hippocampus of Mod mice as compared with the Con mice (Fig. 4C,D). We also conducted a colocalization of APP and neuronal marker (Fig. 4E). Similarly, as shown in Figure 4F,H, the protein expression levels of APP, p-Tau, and Tau in the hippocampal and PFC tissues of mice in the Mod group were significantly increased compared with the Con ones, suggesting that combination of HFD and STZ/I.P. could induce Aβ accumulation and tau hyperphosphorylation in the hippocampus and PFC of mice.
Combination of HFD and STZ/I.P. promoted the activation of microglia and astrocytes in mice
As shown in Figure 5A–D, compared with the Con mice, the number of IBA-1–positive cells (Fig. 5B) and the ratio of activated microglia cells to the total number of microglia cells (Fig. 5C) were both significantly increased in the hippocampus of Mod mice. Additionally, the Mod group demonstrated a higher prevalence of CD68+ microglia than the Con group (Fig. 5D). Representative images of different states of microglial cells are shown in Figure 5A. Furthermore, the immunofluorescent staining of GFAP-positive cells was also increased in the hippocampus of Mod mice as compared with the Con mice (Fig. 5E,F), suggesting that combination of HFD and STZ/I.P. could promote the activation of microglia and astrocytes in the hippocampus of mice.
Combination of HFD and STZ/I.P. induced the dysregulation of the IRS1/AKT/ERK signaling pathway in the hippocampus and the PFC in mice
Figure 6 shows the expression of proteins in the IRS1/AKT/ERK signaling pathway in the hippocampus and PFC of the mice. Compared with that in the Con group, the relative protein expression of p-IRS1/IRS1 was increased in the hippocampus and PFC of Mod mice, while the relative protein expression of p-AKT/AKT and p-ERK/ERK were both decreased in the hippocampus and PFC of Mod mice when compared with the Con mice (Fig. 6A–C), suggesting that combination of HFD and STZ/I.P. could induce the dysregulation of the IRS1/AKT/ERK signaling pathway in the hippocampus and the PFC of mice.
Combination of HFD and STZ/I.P. induced the imbalance of TREM1/2 and increased inflammatory factors in the hippocampus and the PFC in mice
Figures 7, A, D, and E, and 8B show the expression of TREM1 and TREM2 in the hippocampus and PFC of the mice. Compared to the Con mice, the Mod mice exhibited a significant decrease in the protein expression of TREM1 and an obvious increase in the protein expression of TREM2 in both the hippocampus (Fig. 7A,B) and PFC (Fig. 7D,E), indicating an imbalanced expression of TREM1/2 induced by the combination of HFD and STZ/I.P. in mice. Additionally, as depicted in Figure 7C,F, the mRNA expression levels of the inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus and PFC of Mod mice were all remarkably increased as compared with the Con ones, suggesting an increased inflammatory response induced by the combination of HFD and STZ/I.P. in mice.
Discussion
In the present study, the AD-like neuropsychiatric dysfunction, both the behavioral performance and the neuropathological changes, was investigated in a mice model induced by the combination of HFD and STZ/I.P. The results showed that combination of HFD and STZ/I.P. could induce AD-like metabolic disorders and behavioral impairments in mice, including increased blood glucose levels and serum insulin levels and and impaired cognitive ability in NOR, Y-maze, and MWM tests. Moreover, compared with the Con group, combination of HFD and STZ/I.P. could also induce neuron damage and activation of microglia and astrocytes in the hippocampus of mice, as well as the APP accumulation, tau hyperphosphorylation, imbalanced protein expression of the IRS1/AKT/ERK signaling pathway and TREM1/2, and increased inflammatory factors in the hippocampus and PFC of mice.
Hyperglycemia and insulin resistance, which are typical clinical features of T2DM, have also been increasingly recognized as an important metabolic index in the pathogenesis of AD (Grodstein et al., 2001; Luchsinger et al., 2004). It has been reported that chronic hyperglycemia could promote the formation of advanced glycation end products and oxidative stress (Goh and Cooper, 2008; Joubert et al., 2019), and insulin resistance could affect neuronal function and Aβ accumulation in the brain, which contribute to neuroinflammation and synaptic dysfunction and finally lead to cognitive decline (Arnold et al., 2018). Given the killing effect of STZ on the pancreatic β-cell, it has been widely used in establishing T2DM animal models (Ganda et al., 1976; Grieb, 2016). Moreover, besides its efficiency in inducing T2DM-like metabolic disorders including hyperglycemia and hyperlipidemia, the results of our previous studies suggested that STZ could induce behavioral impairments in rodents (Qi et al., 2021; Fan et al., 2022; X. Gao et al., 2023b). Consistently, in the present study, our results showed that combination of HFD and STZ/I.P. could induce a significant increase in blood glucose level, serum insulin, and the HOMA-IR index in mice. Moreover, the model mice showed decreased learning and memory ability in the NOR, Y-maze, and MWM tasks, although there was no significant difference between groups in the behavioral performance in the OFT. These results suggested that the combined use of HFD and STZ/I.P. could successfully induce AD-like metabolic and behavioral injuries in mice.
The hippocampus is a vital brain structure that serves as a critical hub for cognitive processes, playing a central role in learning function, memory consolidation, and spatial navigation (Preston and Eichenbaum, 2013). Research studies have shown that hippocampal structural abnormalities and neuronal loss are the key neuropathological hallmarks of AD, contributing to the cognitive decline observed in the disease (Venkateshappa et al., 2012; Scheltens et al., 2021). The microtubule-associated protein 2 (Map-2) and neuronal nuclei (NeuN) are widely used in marking the mature neurons and neuronal nuclei in the brain (Götz and Ittner, 2008). In this study, compared with the control group, immunofluorescence staining of Map-2 and NeuN in the hippocampal tissue of Mod mice exhibited a significant reduction, indicating that the combined effect of HFD and STZ/I.P. can induce hippocampal neuronal damage in mice, which is consistent with our previous findings (X. Gao et al., 2023b).
The most significant pathological changes in Alzheimer's disease are the accumulation of abnormal protein aggregates in the brain, primarily including Aβ plaques and tau neurofibrillary tangles. These abnormal aggregates lead to neuronal dysfunction, synaptic damage, and neurodegeneration in Alzheimer's disease patients (Scheltens et al., 2016, 2021). Studies have shown that Aβ aggregation and excessive phosphorylation of tau can impair microtubule stability, thereby affecting axonal transport, leading to neuronal degeneration and synaptic loss, and exacerbating the progression of cognitive impairment in AD (Selkoe and Hardy, 2016; Jack et al., 2018). In the present study, the results of Western blot and immunofluorescence staining both indicated that the expression levels of Aβ and p-Tau were significantly increased in the hippocampus and PFC of Mod mice as compared with the Con mice. These results indicated that combination of HFD and STZ/I.P. could induce an AD-related pathology in mice, which was in line with the findings in our previous study.
In addition to Aβ and NFTs, growing evidence has highlighted the role of neuroinflammation in the pathogenesis and progression of AD (Medeiros et al., 2010). Astrocytes and microglia are the two important regulatory factors in neuroinflammatory responses. Microglia, accounting for ∼20% of the brain's glial cells, are the immune cells of the CNS and play a critical role in monitoring the brain and maintaining homeostasis by clearing damaged neurons, plaques, and pathogens (Prinz et al., 2019). However, excessive activation or dysfunction of microglia may lead to neurotoxicity, which also could be found around amyloid plaques in the brains of AD patients (Tang and Le, 2016). In addition, astrocytes also play an important role in maintaining brain structure and function, which could undergo morphological and functional changes and transform into reactive astrocytes under pathological conditions. It has been reported that reactive astrocytes are commonly found in postmortem brain tissues of AD patients in areas with high Aβ or tau pathology (Bussian et al., 2018). Consistently, the results of our present study showed that the number of activated microglia cells and the number of astrocyte cells were both significantly increased in the hippocampus of Mod mice as compared with the Con mice, indicating that combination of HFD and STZ/I.P. could promote the activation of microglia and astrocytes in the hippocampus of mice.
Reactive microglia and astrocytes could promote neuroinflammation by releasing cytokines and inflammatory factors such as IL-1β and IL-6 in AD (H. S. Hong et al., 2003). It has been reported that combined stimulation of IL-1β, IL-6, and IFN-γ in U373 glioblastoma cell lines and primary human astrocytes could induce APP production and lead to increased Aβ levels (Blasko et al., 2000). In addition, IL-1β is the key proinflammatory cytokine associated with age-related cognitive decline, and growing evidence has suggested that synaptic plasticity, learning, and memory are more susceptible to IL-1β–induced impairment, particularly in the aging brain (Prieto et al., 2015). Consistently, the results of our previous study also showed increased mRNA and protein expression levels of IL-1β, IL-6, and TNF-α in AD or T2DM mice and oleic acid or palmitic acid (PA) induced BV2 cells (Fan et al., 2022; X. Gao et al., 2023a). Similarly, in the present study, the expression levels of the inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus and PFC of Mod mice were all remarkably increased, suggesting an increased inflammatory response induced by the combination of HFD and STZ/I.P. in mice.
Recent studies have suggested a potential link between AD and insulin resistance, which exhibits a decreased level of key insulin signaling proteins and an increased level of insulin resistance markers in the brains of AD mice (Craft et al., 1998; Arnold et al., 2018; Kshirsagar et al., 2021). Insulin receptor substrate 1 (IRS1), protein kinase B (AKT), and ERK are the key components of the insulin signaling pathway; impaired IRS1 signaling leads to decreased activation of AKT and subsequent dysregulation of ERK involved in insulin resistance and metabolic dysfunction (Ding et al., 2019). Of note, activated AKT and ERK were also suggested to be the major kinase for tau phosphorylation and APP deposition in AD, and the brain insulin resistance emerging in AD further induces the decreased phosphorylation levels of AKT and ERK (Oliveira et al., 2021). Consistent with this, the results of our previous study demonstrated a decreased expression level of phosphorylated Akt and ERK in PA-induced HT-22 cells, and an imbalanced expression of PI3K/Akt and ERK pathways was also presented in the hippocampus of T2DM mice through DESeq screening (X. Gao et al., 2023a). In line with these findings, the results of the present study showed that the protein expression levels of p-IRS1/IRS1 were increased, while the levels of p-AKT/AKT and p-ERK/ERK were decreased in the hippocampus and PFC of mice induced by the combination of HFD and STZ/I.P., which provides additional evidence linking insulin resistance and AD.
TREM1/2 are the members of the immunoglobulin superfamily involved in the regulation of inflammation and immune responses. It has been reported that there has a close association between genetic polymorphisms of TREM1/2 and the pathogenesis of AD (Y. Wang et al., 2015). Knocking out TREM1 in the brains of APP/PSEN1 mice has been shown to increase Aβ1–42 levels and total amyloid plaque burden, and selective overexpression or activation of TREM1 on microglia cells could improve Aβ neuropathology and rescue AD-related spatial cognitive impairments (Jiang et al., 2016). TREM2-expressing microglia cells have been found in the peripheral regions of amyloid plaques in APP23 transgenic (Tg) mice, and increased TREM2 expression is consistent with the progression of amyloid deposition (Frank et al., 2008). Moreover, the knockdown of TREM2 could alleviate the neuroinflammation and prevent neurodegeneration of tau pathology in TREM2−/−PS mice compared with wild-type mice (Leyns et al., 2017). The results of our previous studies showed that the imbalance expression of TREM2 was not only involved in the neuronal injury induced by high cholesterol but also related to LPS-induced homeostatic imbalance of microglia and increased secretion of inflammatory factors (Liu et al., 2022; Zheng et al., 2023). Moreover, the neuroinflammation-related imbalance of TREM1/2 expression was also found in the hippocampus of AD and T2DM mice model, with a closely related to the glyeolipid metabolism disorder and cognitive impairment (Fan et al., 2022; X. Gao et al., 2023b). Similarly, the present study also demonstrates a significantly decreased TREM1 expression and increased TREM2 expression in the hippocampus and PFC of mice induced by the combination of HFD and STZ/I.P. Together with the increased activation of microglia and astrocytes, as well as the elevated expression of IL-1β, IL-6, and TNF-α, these results further support the crucial role of neuroinflammation in the pathogenesis of AD.
There are also some limitations in this study. Firstly, the mouse model used in this study may not fully represent the complexity of human AD. Animal models could only partially replicate the disease processes seen in humans, and the findings may not directly translate to human patients. Secondly, the present study identified several potential mechanisms, and further research is needed to elucidate the underlying pathways and their interactions. Additionally, the role of other factors such as genetic predisposition and environmental influences in the development of AD-like neurophysiological features in T2DM needs to be further explored. Thirdly, the sample size in the present study was relatively small, and larger studies with more animals are needed to confirm and validate the findings. Moreover, the present study only focused on the hippocampus and PFC of mice, and potential changes in other brain regions were not investigated. While mRNA expression data provide compelling evidence of potential BBB damage and increased inflammatory response in our model, it is important to note that mRNA expression does not directly equate to protein function or localization. Future studies are necessary to validate these inflammatory response findings at the protein level or through ELISA assays, while also employing more direct methods to evaluate functional blood–brain barrier permeability (Ben-Zvi et al., 2014; Wood et al., 2021). It is important to note that the findings may not be generalizable to female mice. Given that females are twice as likely as males to receive a diagnosis of AD (Rajan et al., 2021; Lopez-Lee et al., 2024), future studies should include female mice to provide a more comprehensive understanding of the disease across genders.
In summary, our results showed that combination of HFD and STZ/I.P. could induce not only T2DM-like metabolic disorders but also AD-like neuropsychiatric behavior in mice, as indicated by the increased blood glucose levels and serum insulin levels and the impaired cognitive ability in NOR, Y-maze, and MWM tests. Apart from the APP accumulation and tau hyperphosphorylation, the mechanism might be associated with neuron damage and activation of microglia and astrocytes in the hippocampus. Moreover, the imbalanced protein expression of the IRS1/AKT/ERK signaling pathway and TREM1/2, as well as the increased inflammatory factors, may also be involved in AD-like pathological process. These findings might provide new evidence for understanding the pathogenesis of AD-like neuropsychiatric injuries. Future research efforts should focus on further elucidating the underlying mechanisms and identifying potential therapeutic targets for the prevention and treatment of AD.
Data availability
The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.
Footnotes
The authors declare no competing financial interests.
We thank the Center for Scientific Research of Anhui Medical University for their valuable help in our immunofluorescence experiment. This work was supported by the National Natural Science Foundation of China (81870403), Anhui Province Postgraduate Education Quality Project (2023xscx050), and Scientific Research Promotion Plan of Anhui Medical University (2022xkjT009).
↵*H.S. and X.G. contributed equally to this work.
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