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Semaglutide restores astrocyte–vascular interactions and blood–brain barrier integrity in a model of diet-induced metabolic syndrome

Diabetology & Metabolic Syndrome volume 17, Article number: 2 (2025) Cite this article

A Correction to this article was published on 23 April 2025

This article has been updated

Abstract

Introduction

Metabolic syndrome (MetS) is a metabolic disorder related to obesity and insulin resistance and is the primary determinant of the development of low-intensity chronic inflammation. This continuous inflammatory response culminates in neuroimmune-endocrine dysregulation responsible for the metabolic abnormalities and morbidities observed in individuals with MetS. Events such as the accumulation of visceral adipose tissue, increased plasma concentrations of free fatty acids, tissue hypoxia, and sympathetic hyperactivity in individuals with MetS may contribute to the activation of the innate immune response, which compromises cerebral microcirculation and the neurovascular unit, leading to the onset or progression of neurodegenerative diseases.

Objective

This study aimed to evaluate the effects of chronic treatment with a GLP-1 receptor agonist (semaglutide) on cerebral microcirculation and neurovascular unit (NVU) integrity.

Methods

C57BL/6 mice were fed a standard normolipidic diet or a high-fat diet (HFD) for 24 weeks and then treated for 4 weeks with semaglutide (HFD SEMA) or saline solution (HFD SAL). At the end of pharmacological treatment, biochemical analyses, immunohistochemistry analysis, and intravital microscopy of the brain microcirculation were carried out to quantify leukocyte–endothelium interactions and to assess structural capillary density, astrocyte coverage on cerebral vessels and microglial activation.

Results

We observed that SEMA attenuates high-fat diet-induced metabolic alterations in mice fed with HFD for 24 weeks. SEMA also reversed cerebral microcirculation effects of HFD by reducing capillary rarefaction and the interaction of leukocytes in postcapillary brain venules. The HFD-SEMA group exhibited improved astrocyte coverage on vessels. However, SEMA did not reverse microglial activation.

Conclusions

Semaglutide can reverse microvascular rarefaction in metabolic syndrome by restoring the integrity of the neurovascular unit. Adverse dietary stimuli can compromise microglial homeostasis that is not reversed by semaglutide.

Background

Metabolic syndrome (MetS) is defined as a low-intensity chronic proinflammatory condition in which abnormal metabolic factors increase the risk of developing diabetes, cardiovascular disease, and neurodegenerative disease, with diverse harmful effects on an individual's health and quality of life [1, 2]. The consumption of a high-fat diet (HFD) is strongly associated with this condition, leading to visceral obesity and insulin resistance, which can cause neurological issues such as anxiety and depression [3, 4] and may even contribute to the progression of Alzheimer's and Parkinson’s disease [5, 6].

In MetS, events such as increased oxidative stress and inflammatory cytokines generated by hyperglycemia and obesity can cause vascular endothelial dysfunction, compromising the vasomotor balance and increasing the adhesion of leukocytes to the endothelium in various organs and tissues, including the central nervous system (CNS) [7,8,9]. MetS damages small blood vessels, allowing inflammatory cells and cytokines to worsen brain tissue injuries. This can cause endothelial dysfunction, disturb the brain microvasculature and activate microglia [9, 10].

The primary goal of treating metabolic syndrome is to decrease the risk of developing atherosclerotic disease and diabetes. Currently, no medication has been proven effective in reducing neuroinflammatory and cerebral microvascular effects resulting from MetS. Despite extensive research into drug development for obesity and metabolic syndrome, success to date has primarily been limited to surgical interventions or treatment of associated risk factors. Lifestyle adjustments such as exercise, calorie ingestion control, and weight loss can improve health but are difficult to maintain if metabolic syndrome is present due to years of unhealthy habits.

New antidiabetic drugs, such as glucagon-like peptide-1 (GLP-1) receptor agonists, are drugs licensed for weight management in individuals with obesity. GLP-1 is an intestinal hormone secreted by endocrine L cells after food intake. It reduces glucagon production, stimulates pancreatic insulin secretion, prolongs gastric emptying, and promotes satiety [11, 12]. Treatment with GLP-1 receptor agonists, such as liraglutide and semaglutide, can induce satiation in the central nervous system, achieve significant long-term weight loss and improve insulin sensitivity [13]. Although most studies suggest that GLP-1 receptor agonists promote weight loss primarily due to their inhibitory effect on food intake, other central impacts that have been described for native GLP-1 and some GLP-1 receptor agonists in rodents and humans inspire future clinical trials to explore additional mechanisms in the CNS. Semaglutide has already been proven to be effective in reducing weight and blood glucose levels in both clinical and experimental studies [14]. However, there is no evidence yet regarding the effects of semaglutide on long-term high-fat diet intake induced cerebral microcirculation changes and neuroinflammation. This study aimed to investigate the therapeutic potential of semaglutide, a MetS agent, for improving cerebral microcirculation and neuroinflammation.

Methods

Animals and ethics statement

Eight-week-old male C57BL/6 mice were obtained from the Oswaldo Cruz Foundation’s Animal Breeding Center. The animals were kept at a controlled temperature (23 ± 1 °C) in a room under a 12-h light/dark cycle with ad libitum access to food and water. The Animal Welfare Committee of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ) approved all animal procedures under license number L-012/2021.

Induction of metabolic syndrome and semaglutide treatment

We used a well-characterized experimental murine model of MetS. MetS was induced in a group of 10 animals by feeding them a high-fat diet (HFD) for 24 weeks, while a control group of 10 animals received a normalipid/regular fat diet (ND) (AIN93M) during the same period. Both chow diets were manipulated by Pragsoluções (Jau, São Paulo—Brazil).

The ND used was composed of corn starch, casein (14%), dextrinized starch, sucrose, soybean oil (4%), microcrystalline cellulose, mineral mix AIN 93M (sodium, iron, manganese, zinc, iodine, copper, selenium, cobalt and fluoride), mix Vit AIN 93 (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, choline bitartrate, vitamin D3, vitamin E, vitamin KE, niacin, biotin and folic acid), lysine, methionine, l-cystine, choline bitartrate and BHT (tertibutylhydroquinone). For the HFD, the same components as the control diet were used, but with changes in the amounts of casein (19%), increased porcine fat (32%), and sodium chloride (0.5%). In summary, the percentages of calories in each diet are as follows: carbohydrates: ND 75.81% vs. HFD 25.69%; lipids: ND 9.47% vs. HFD 60.19%; proteins: ND 14.73% vs. 14.12%.

For 24 weeks, C57BL/6 mice were first fed either a standard ND or a HFD. After this period, the mice on the HFD were randomly divided into two groups for an additional 4 weeks: one group received semaglutide treatment (HFD SEMA) and the other group received a saline solution (HFD SAL). The animals were administered a fixed daily dose of 0.2 mg/kg of semaglutide in 0.5 mL (Ozempic®, Novo Nordisk, Bagsværd, Denmark) subcutaneously on the back. The ND group also received the same semaglutide treatment (ND SEMA; n = 5) or saline solution (ND SAL; n = 5). The effects of semaglutide doses on mice with diabetes have been extensively studied to compare them to human doses. The dosage of semaglutide used in this study (0.2 mg/kg/day) is based on extrapolations from human dosages. In clinical settings, semaglutide is prescribed at doses of 0.5 mg to 1 mg per week for type 2 diabetes and obesity management, translating to approximately 0.01 to 0.02 mg/kg/day in humans. The higher dose used in mice accounts for differences in metabolism and body surface area scaling [15].

Biochemical analyses

Blood glucose was measured using an automatic glucometer (One Touch Ultra 2, Johnson & Johnson Medical SA, Argentina), and triglyceride levels were measured by Accutrend Plus (Roche). At the end of the experiments, blood samples were collected from all animals via intracardiac puncture, and serum was collected by centrifugation at 1500×g for 10 min at 4 °C, aliquoted, and stored at −80 °C for analysis. A biometric immunoassay, which uses fluorescent-dyed microspheres conjugated with a monoclonal antibody specific for a target protein such as insulin, leptin, or resistin, was conducted using a multiplex array reader from the Luminex™ Instrumentation System (Bio-Plex Workstation from Bio-Rad Laboratories). The concentration of the analyte was calculated using the software provided by the manufacturer (Bio-Plex Manager Software).

Histological analyses of brain and adipose tissues

To assess changes in adipose tissue, the epididymal fat contents of the animals were harvested immediately following the termination of the experiments. The tissues were fixed in 4% paraformaldehyde for 48 h at 4 °C before sectioning and H&E staining. Twelve-micron-thick adipose tissue sections were stained with hematoxylin and eosin (H&E) and imaged at 40X magnification using a Zeiss Primo Star light microscope (Oberkochen, Germany). The area of white adipocytes was determined using ImageJ software (NIH, Bethesda, MD, USA) by outlining white adipocytes with ellipses. For each animal, two to three images, each representing a distinct sample area, were obtained. The adipocyte size was quantified in 10–20 adipocytes per image and reported as the area of adipocyte size.

Twelve-micron-thick hippocampal sections stained with hematoxylin and eosin (H&E) were imaged using a Zeiss Primo Star light microscope at 40X magnification (Oberkochen, Germany). Analysis was conducted using ZenBlue software (Zeiss), through which the thickness of the hippocampus was measured. By counting the number of cells across a transverse line from the inner to the outer layers of the hippocampus, the cellular density in the hippocampus was calculated and is expressed as the ratio of cells per hippocampal thickness.

Brain intravital microscopy

The animals were randomly assigned to microcirculatory analysis at the end of the experiment. Mice were anesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg, i.p.) (Cristália, SP, Brazil). The tail vein was catheterized to allow injection of the fluorescent dye. To assess cerebral microcirculation, the mice were fixed in a stereotaxic apparatus, and a cranial window over the left parietal bone (1–5 mm lateral, between the coronal and lambdoid sutures) was created using a high-speed drill to expose the brain’s microvascular surface, as previously described [16]. The animals were placed under the light beam of a fluorescence microscope (Zeiss model AXIO SCOPE A1, Obercochen, Germany), and microcirculation images were acquired using Zen Blue software (Zeiss, Obercochen, Germany).

Leukocyte–endothelium interactions were assessed as previously described [17]. Rhodamine 6G (0.3 mg/kg) was injected into the tail vein to label circulating leukocytes. The interaction of leukocytes with the endothelial wall was evaluated by determining the number of adhered leukocytes that remained static along 100 µm of the venular wall for 30 s, and the number of rolling leukocytes that moved within the vessel at a slower speed than did the number of circulating erythrocytes showing slow contact with the inner venular walls. The values are expressed as the number of cells/min/100 μm. We determined these parameters in brain surface venules with diameters ranging from 50 to 100 µm, and images were acquired with a 10× ocular and 10× objective microscope (Zeiss—AXIO SCOPE A1, Obercochen, Germany), producing a final magnification of 100× on the monitor.

Immunohistochemical analysis and confocal microscopy

At the end of the intravital microscopy analyses, the animals were euthanized by an overdose of anesthetic drugs. Specifically, we used an overdose of pentobarbital sodium at a dose of 150 mg/kg, administered intraperitoneally. The mice were perfused with a saline solution through the heart, followed by 4% paraformaldehyde. Subsequently, the brains were collected and postfixed in paraformaldehyde for 48 h at 4 °C and sectioned to obtain 12-μm slices, which were permeabilized with 0.05% Triton X-100 (Vetec, Speyer am Rhein, Germany) solution in PBS for 30 min and subsequently incubated with blocking solution containing 5% bovine serum albumin (Sigma‒Aldrich), 2.5% normal goat serum (Thermo Fisher Scientific, Waltham, MA), and 0.02% Triton X-100 diluted in PBS for 1 h. Then, the tissues were incubated with primary antibodies and diluted in a blocking solution overnight at 4 °C. The sections were incubated with Biotinylated isolectin B4 (IB4) (Vector; 1:100) and streptavidin-Cy3 (1:400) to label cerebral microcirculation vessels. We used primary antibodies against astrocytes (anti-GFAP, Dako, 1:400 dilution), microglia (anti-Iba-1, Wako, 1:200 dilution), and ICAM-1 (Monoclonal Antibody for CD54, ICAM1, eBioKAT-1, eBioscience). Then, we used secondary antibodies and photographed the slides using a LSM 510 META confocal microscope (Zeiss). The BBB integrity was considered by measuring the parenchymal abundance of IgG using immunomicroscopy. Briefly, after blocking with 10% goat serum, 20 μm cryosections were incubated with goat anti-mouse IgG conjugated with Alexa488 (1:50, Life Technologies) for 20 h at 4 °C. The sections were counterstained with DAPI.

Structural capillary density analysis

To analyze the cerebral microvascular network, we used the AngioTool program (available in the public domain at https://ccrod.cancer.gov/confluence/display/ROB2/Downloads), a validated source for measuring vascular networks. We calculated the total length of IB4-labeled brain capillaries, which represented the brain capillary density. Lacunarity describes the distribution of spaces between vessels.

Analysis of microglial activation

To quantify morphological changes in Ionized calcium‑binding adaptor molecule 1 (Iba-1+) cells, consecutive Z-stack images were converted to a maximum intensity projection image by ImageJ software (NIH Bethesda, MD, USA). Using the Sholl analysis plugin, concentric circles were created centered on the soma, beginning at 4 μm radii and increasing by 2 μm with every circle. We determined the number of intersections made by microglial branching processes with each successive increasing circle, the maximum number of intersections for the cell (Nm), the critical value at which Nm occurred, and the maximum length at which a branch intersection was observed. We used the image tool “Sholl analysis” from the ImageJ Program to analyze microglial morphology. It was possible to analyze the number of processes of each microglia separately through concentric and consecutive circles every 2 µm around the cell and to compute the number of intersections of microglial processes with each circle, representing the degree of branching of the cell. The less branched the cell was, the more activated the cell was. At least 30 microglia per field were analyzed in 3 sections/4 slides per animal.

Analysis of vessel coverage by astrocytes

The degree of colocalization between the astrocyte marker GFAP and the blood vessel marker IB4 was analyzed using the ImageJ program. The tool "colocalization", which uses Mander's M1 coefficients, was used. These coefficients indicate the percentage of pixels in the green channel (GFAP+) intersecting with a red channel signal (IB4). In other words, the M1 coefficients show the fraction of intensity in each channel that coincides with some intensity in the other channel.

Cognition tests

Fear conditioning memory test

Mice were placed into a specific chamber individually. After 3 min of habituation, mice were subjected to an electric foot shock (0.75 mA, 3 s) simultaneously to a tone (2.5 kHz, 85 dB, 3 s) and returned to their home cages. The next day, mice were placed in the same chamber for 3 min, and the tone was applied without a foot-shock. A digital video camera recorded the behavior of the mice and was manually analyzed. Continuous immobility for 1 s was defined as freezing behavior, and the total freezing time (i.e., length of immobility) was measured. Throughout the experiment and analysis of results, the group of animals was not revealed to the researcher. The procedures followed the described by Zhang et al. [18] and Granja et al. [19].

Light/dark box test

The light/dark box test reliably predicts anxiolytic and anxiogenic-like effects in rodents and offers quick, easy performance without prior animal training or food/water deprivation. Transitions in this test indicate activity and exploration, while the time spent in each compartment reflects aversion (light) and attraction (dark). The apparatus consists of a wooden box (48 cm × 24 cm × 27 cm) divided into two compartments by a barrier with a doorway (10 cm × 10 cm). One compartment is black and covered, while the other is white and illuminated by a 60-W light bulb positioned 40 cm above the box. The test procedure involves transporting the mice to a darkened room, allowing them to acclimate in their home cages for 2 h, then placing them in the lit compartment to explore freely for 5 min. We recorded the time spent in the lit compartment, the number of entries into the lit compartment, and the latency to enter the dark compartment [20]. Throughout the experiment and analysis, the group of animals remained blinded to the researcher.

Statistical analysis

The results are expressed as the mean ± standard error of the mean (SEM). All graphs and statistics were generated using GraphPad Prism (GraphPad Software, Inc.) (San Diego, CA, USA). Comparisons between all groups were made using analysis of variance (one-way ANOVA) with Tukey’s post hoc test to determine differences for p < 0.05. The normality of data distribution was verified using the Shapiro–Wilk test. For data that were normally distributed, we used parametric tests and presented the results as mean ± SEM. For data that did not follow a normal distribution, non-parametric tests were applied. Normally Distributed Data: Parametric tests such as the Student's t-test or ANOVA were used. Non-Normally Distributed Data: Non-parametric tests such as the Mann–Whitney U test or Kruskal–Wallis test were applied.

Results

Semaglutide restores the metabolic profile of animals with metabolic syndrome

Compared with those in the ND SAL control group, the body weight (p < 0.001), blood glucose (p < 0.05), insulin (p < 0.001), leptin (p < 0.001), triglycerides (p < 0.01) and resistin (p < 0.05) in the HFD SAL-treated group increased. The 4-week treatment with semaglutide significantly reduced all the abovementioned parameters in HFD-treated SEMA animals (p < 0.05) compared to those in HFD-treated SAL animals (Fig. 1A–F).

Fig. 1
figure 1

Effect of semaglutide treatment (4 weeks) on the metabolic parameters of body weight (A), fasting glucose (B), insulin (C), leptin (D), triglycerides (E), and resistin (F) in mice fed a high-fat (HFD) or normolipid (ND) diet and treated subcutaneously with semaglutide 0.2 mg/kg/day (SEMA) or saline solution (SAL). The values represent the means ± SEMs of 5 animals per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. ND SAL, and #p < 0.05 and ##p < 0.01 vs. HFD SAL

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Semaglutide treatment reduces adipocyte size in animals with metabolic syndrome

We performed histological analysis on HE-stained slices of visceral adipose tissue to evaluate the association between weight loss and the reduction in white adipose tissue after treatment with semaglutide. Compared with those in the ND SAL group, the visceral adipocytes in the HFD SAL group were significantly hypertrophic (p < 0.05). Compared with those in the HFD SAL group, the size of these cells in the HFD SEMA groups significantly decreased (p < 0.05), indicating a reduction in adipocyte content (Fig. 2). In the control groups, no significant differences were detected in the adipocyte areas between the ND SEMA and ND SAL groups (Fig. 2).

Fig. 2
figure 2

Representative photomicrographs (A) and quantitative analysis of the size (B) of epididymal adipocytes from mice fed a high-fat (HFD) or normolipid (ND) diet and treated subcutaneously with 0.2 mg/kg/day semaglutide (SEMA) or saline solution (SAL). The values represent the means ± SEMs of 5 animals per group. Magnification: 400x, scale bar = 30 µm. *p < 0.05 vs. ND SAL, and #p < 0.05 vs. HFD SAL

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Semaglutide reduced leukocyte–endothelium interactions in cerebral microvascular inflammation in metabolic syndrome animals

An increase in leukocyte rolling and adhesion in cortical microvessels was observed in animals in the HFD SAL group (p < 0.001) compared to those in the control ND SAL group. In the HFD SEMA group, there was a significant decrease in the interaction of cells within the endothelial walls of the microcirculation. Semaglutide successfully reduced the number of leukocytes that rolled and adhered to the endothelial cells. However, the rolling of leukocytes in the HFD SEMA group did not completely return to the basal control values similar to the ND SAL group (*p < 0.05) (Fig. 3A, B and C).

Fig. 3
figure 3

Representative images (A) of postcapillary venules in the brain microcirculation of mice fed a high-fat diet (HFD) and treated subcutaneously with 0.2 mg/kg/day semaglutide (SEMA) or saline solution (SAL). Quantitative analysis of leukocyte–endothelial interactions in the brain microcirculation. Rolling (B) and adhesion (C) of rhodamine 6G‑labeled leukocytes in the cerebral postcapillary venules were evaluated by intravital fluorescence microscopy. The values represent the means ± SEMs of 5 animals per group. Magnification: 400×. *p < 0.05, ***p < 0.001 vs. ND SAL, and #p < 0.05 vs. HFD SAL

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Semaglutide treatment does not reverse microglial activation in the hippocampus of animals with metabolic syndrome

Using the Sholl Analysis tool, we evaluated changes in microglial morphology to determine alterations in function. The number of intersections and the area under the curve (AUC) were used to assess the impact of semaglutide treatment on microglial activation.

As shown in Fig. 4A, microglial cell bodies in the hippocampus were enlarged in the HFD SAL group, and their branches were significantly shortened and decreased compared to those in the ND SAL control group (p < 0.05) (Fig. 4A, depicted images). These alterations are compatible with an activation response of microglia. Sholl analysis (Fig. 4B) and the area under the curve of microglia (Fig. 4C) did not detect statistically significant alterations in microglial morphology in animals with MetS treated with semaglutide (HFD SEMA), indicating that semaglutide cannot reverse the microglial activation profile in the MeS.

Fig. 4
figure 4

Confocal images (A) of microglia in hippocampal sections from mice fed a high-fat (HFD) or normolipid (ND) diet and treated subcutaneously with 0.2 mg/kg/day semaglutide (SEMA) or saline solution (SAL). A single cell from each image is cropped to show the details of the Sholl analysis. Sholl analysis graphs (B) and area under the curve (C). The data represent the mean ± SEM of up to 20 microglia per group of 5 animals. Magnification 400×, scale bars 50 µm for the full-sized figures and 10 µm for the depicted images in the images. **p < 0.01 vs. ND SAL

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Semaglutide treatment reverses structural capillary rarefaction in the cerebral microcirculation of animals with metabolic syndrome

We assessed the density of capillaries in the cortex and hippocampus, which are important regions for memory storage and consolidation (Fig. 5). The AngioToll program obtained the following parameters: average total vessel length, which is the average of the total length of vessels in the image, and lacunarity, which is the number of spaces between vessels.

Fig. 5
figure 5

Graphical representation of the AngioTool analysis of cerebral cortex sections (A) and hippocampal sections (D) from mice fed a high-fat (HFD) or normolipid (ND) diet and treated subcutaneously with 0.2 mg/kg/day semaglutide (SEMA) or saline solution (SAL). Average vessel length in the cortical area and hippocampus (B and E) and lacunarity in the cortical area and hippocampus (C and F). The skeleton is shown in red, and the branching points are shown in blue. Vessels labeled with IB4. Magnification: 400x; scale bar, 50 µm for all images. n = 6. *p < 0.05 versus the ND SAL group; #p < 0.05 versus the HFD SAL group

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Figure 5A, B shows a significant reduction in the average total vessel length in the parietal cortex of the HFD SAL group. Additionally, there was greater lacunarity (Fig. 5C) in the parietal cortex area in the HFD SAL group than in the ND SAL group (p < 0.05). This effect is called capillary rarefaction, which suggests a possible decrease in blood flow in the affected area. In the HFD SEMA group, there was a significant reversion of microvascular rarefaction characterized by an increased total length and reduced lacunarity (Fig. 5A, B and C). No significant differences were detected in the hippocampus among the analyzed groups. Despite data indicating an increase in both capillarity and lacunarity in the angiotool analysis of HFD SEMA group, these were not statistically significant (Fig. 5D, E and F).

Semaglutide treatment improves coverage of astrocytes in the cortical and hippocampal capillaries of animals with metabolic syndrome

Immunohistochemical analysis allowed the evaluation of vessel coverage by astrocytes on the cerebral microcirculation, which is an essential feature of the neurovascular unit and blood brain barrier (BBB) integrity.

Confocal microscopy revealed a significant reduction in the colocalization of astrocytes with the cerebral cortex and hippocampal vessels in the HFD SAL group. As shown in Fig. 6A and B, the astrocytes are marked with an anti-GFAP antibody in green, the cerebral vessels are marked with IB4 in red in the center, and the image on the right is the “merge” overlay tool showing the astrocytes covering the vessels. The HFD SEMA group presented a significant increase in the number of IB4+ vessels surrounded by GFAP+ astrocytes in the cortex (Fig. 6A) and hippocampus (Fig. 6B), indicating the recovery of astrocyte coverage in the vessels. To evaluate the impact of semaglutide treatment on the blood–brain barrier of animals with metabolic syndrome, we used the “colocalization” tool from the ImageJ program to analyze the coverage of astrocytes in the cerebral vessels of the cortex (Fig. 6C) and hippocampus (Fig. 6D) of the animals studied. The HFD SAL group presented significantly reduced values of colocalization of GFAP+ on IB4+ pixels, compared to the ND SAL group in both cortex (p < 0.001) and hippocampus (p < 0.05), and the treatment with semaglutide restored these effects in the cortex (p < 0.001) and hippocampus (p < 0.05).

Fig. 6
figure 6

Colocalization of astrocytes with cortical and hippocampal vessels from mice fed a high-fat diet (HFD) and treated subcutaneously with 0.2 mg/kg/day semaglutide (SEMA) or saline solution (SAL). Representative confocal images of parietal cortex (A) and hippocampus (B) GFAF+ (green), IB4+ (red) and merged immunostaining (scale bar 50 µm). The merged images show examples of contact between the immunoreactive astrocytic processes and IB4 of the vessel. Magnification: 400×, scale bar 50 µm for all images. Graphical representation of cortical (C) and hippocampal (D)  colocalization of GFAP/IB4 in all groups; *p < 0.05, ***p < 0.001 versus the ND SAL group; ##p < 0.01, ###p < 0.001. HFD-fed SAL, n = 5

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Semaglutide protects against metabolic syndrome-induced reductions in hippocampal volume and neuronal density

We used light microscopy to observe changes in the hippocampus of the animals studied. We found significant structural disorganization in H&E-stained hippocampal samples from the HFD SAL group compared to those from the HFD SEMA group. Specifically, the hippocampal CA1 region of HFD fed animals showed noticeable damage, thinning of the pyramidal cell layers, disorderly and sparse cell arrangement, and clear signs of neuronal degeneration and necrosis; all these parameters were qualitatively analyzed (Fig. 7A). However, the thickness of the hippocampus and the number of cells were quantified and are expressed as the ratio of the number of cells to the thickness of the hippocampus (Fig. 7B). The differences in hippocampal thickness between the HFD SAL group and the ND SAL group were not statistically significant. However, the ratio of hippocampal cell number to hippocampal thickness was significantly reduced, indicating a deterioration in cell density in HFD SAL group. Conversely, animals treated with semaglutide in the HFD SEMA group showed an increased number of cells per hippocampal thickness, suggesting the treatment's efficacy in these animals. This pathological state suggested a disruption in the architectural integrity of the hippocampus and potential apoptotic effects induced by the HFD regimen.

Fig. 7
figure 7

H&E staining photomicrographs (A) of the hippocampus of the brains of mice fed a high-fat (HFD) or normolipid (ND) diet and treated subcutaneously with 0.2 mg/kg/day semaglutide (SEMA) or saline solution (SAL). Magnification: 400x, scale bar 50 µm. Graphical representation (B) of hippocampal thickness (upper graph) and hippocampal cellular density (lower graph), represented by the ratio of the number of cells to the hippocampal thickness

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Evaluation of the anxiolytic and cognitive effects of semaglutide treatment through behavioral tests.

We conducted both the Fear Conditioning Memory Test (freezing) and the Light/Dark Box Test to evaluate memory and anxiety-like behaviors in rodents. Our results indicated that the HFD SEMA group exhibited less anxious behavior, characterized by more frequent entries and exits in the lit compartment of the Light/Dark Box Test, although this finding was not statistically significant. This suggests a trend towards reduced anxiety, which warrants further investigation with a larger sample size to achieve statistical power (Supplementary Fig. 3A).

In the Fear Conditioning Memory Test, the HFD SEMA group showed a significantly better learning response than the untreated HFD group, characterized by a longer hesitation time before stepping onto the electrified grid. This behavior indicates that the animals remembered the foot shock, demonstrating enhanced memory retention. These findings, confirmed by the Student's t-test, highlight the potential cognitive benefits of SEMA treatment in this model (Supplementary Fig. 3A, B, and C).

Discussion

This study examined the ability of semaglutide to mitigate the harmful effects of metabolic syndrome on the central nervous system in mice fed a long-term high-fat diet. This study showed that semaglutide effectively reversed the metabolic changes caused by prolonged high-fat diet in animals, even after metabolic syndrome had already been established. At the end of the experimental protocol, the weight, blood glucose, insulin, triglyceride, and resistin levels of the HFD SEMA group were similar to those of the ND SAL control group. Semaglutide treatment did not affect the control ND SEMA group. This finding corroborates previous studies demonstrating that GLP-1 receptor agonists have no effect on normoglycemic animals, and in nonobese animals, the effect on body weight is minimal [21, 22]. In clinical trials testing exenatide in nonobese and nondiabetic Parkinson’s disease patients, no noticeable effects on body weight or blood glucose were detected [23].

The dysregulation of adipose tissue significantly influences the pathophysiology of brain disorders in individuals with MetS. Adipocyte hypertrophy is considered a key event in insulin resistance in individuals with obesity and MetS, as it increases the levels of proinflammatory cytokines and leads to leptin resistance. However, the total number of adipocytes is determined in childhood and adolescence, regardless of body mass [23]. This finding suggested that adipocyte hypertrophy represents the most critical mechanism for expanding tissue adiposity during weight gain. As we started feeding the animals a high-fat diet in adulthood, our data suggest that treatment with semaglutide reduced the size of the adipocytes.

We investigated the effect of semaglutide on leptin and resistin levels. Resistin, a protein that contributes to insulin resistance, is primarily produced by macrophages. In cases of obesity, macrophages infiltrate white adipose tissue, increasing resistin levels in the bloodstream explaning why increased levels of resistin are often observed in individuals with obesity and diabetes [24]. When high serum levels of resistin and leptin were combined, the reduction in food intake induced by leptin decreased in magnitude. Similarly, the decrease in insulin secretion induced by leptin was also reduced. This finding suggested that the interaction between these two hormones may not improve metabolic regulation in hyperleptinemia and hyperresistinemia. Our research showed that the HFD SAL group had increased leptin and resistin levels. However, semaglutide significantly reversed these changes, indirectly reducing the visceral adipocyte content in adipose tissue. Studies have shown that semaglutide can activate adipocyte browning, which leads to improved mitochondrial biogenesis and increased expression of thermogenic markers, ultimately resulting in weight loss [25].

The neuroprotective and anti-inflammatory effects of semaglutide were shown in experimental stroke models in which semaglutide was administered acutely and shortly after stroke induction in nondiabetic animals [26]. In this case, the effects were evident in reducing the stroke area, neuronal loss, and resulting microglial activation. Another study showed the effectiveness of semaglutide in improving episodes of severe epilepsy in nondiabetic animals and reducing cognitive dysfunction via the inhibition of NLRP3 inflammasome activation and inflammatory cytokine secretion in these animals [27]. It was also shown that semaglutide attenuates microglial M1 polarization during the acute phase after ischemic brain injury. Although the harmful effects of MetS on the CNS are less severe than those of stroke, they are subclinical and chronic, involving a long period of inflammation from a multifactorial source.

In individuals with MetS, high glucose levels can cause dysfunction in endothelial cells and oxidative stress in mitochondria [28]. Dysfunction in endothelial cells is characterized by a reduced ability to dilate blood vessels due to decreased availability of nitric oxide, dysregulated angiogenesis, increased inflammatory adhesion molecules, permeability, and low-density lipoprotein oxidation [28]. The activated endothelium in cerebral microcirculation plays an essential role in neuroinflammatory processes in individuals with MetS [8]. Activated leukocytes express adhesion molecules that help them interact with the vascular wall, making this a crucial step in developing vascular dysfunction, cell migration, and inflammatory perpetuation of cerebral damage [28]. In the intravital microscopy experiments, HFD-treated SAL-treated animals exhibited increased numbers of rolling and adhesive leukocytes on cerebral vessel walls. Treatment with semaglutide partially reversed the rolling of leukocytes and completely blocked their adhesion. In agreement with our results, a recent study demonstrated that semaglutide can decrease inflammation and oxidative stress in the hearts of obese mice [29] and suppress the expression of essential cytokine genes in neutrophils. This finding suggests that semaglutide can reduce inflammation and oxidative stress in cardiac tissue by inhibiting the expression of neutrophil inflammatory factors. In a study conducted by Luna-Marco et al. [30], it was observed that patients with type 2 diabetes had a decreased rolling velocity and increased adhesion of polymorphonuclear leukocytes (PMNs) to the human umbilical vein endothelial cell (HUVEC) monolayer. However, these interactions were prevented in PMNs from T2D patients treated with GLP-1 receptor agonists and were associated with decreased ICAM-1 and VCAM-1 levels in these patients. The endothelial adhesion molecules ICAM-1 and VCAM-1 play a central role in allowing white blood cells to stick to and move through the endothelium. ICAM-1 is a glycoprotein found on the surface of cells and acts as a receptor for adhesion. It is mainly produced by activated endothelial cells in response to inflammation. ICAM-1 is involved in various stages of white blood cell movement from sticking to the endothelium, rolling along its surface, passing through the endothelial layer, and moving within the tissue [31]. Our results showed that HFD SAL mice express more ICAM-1 than ND SAL mice, and semaglutide treatment prevented the HFD-induced ICAM-1 expression in the brain microvessels, indicating that HFD induces an inflammatory environment that affects the integrity of the blood–brain barrier [32, 33]. Semaglutide prevented leukocyte adhesion and rolling, as well as astrocyte detachment from the brain vessels, possibly through a mechanism involving inflammatory mediators within the brain [32] (Supplementary Fig. 1).

MetS has also been identified as the cause of microvascular rarefaction characterized by reduced microvascular density and endothelial dysfunction [9, 34]. Regular exposure to high lipid loads, even before structural changes occur in visceral adipose tissue, leads to inflammatory responses and microvascular dysfunction [34]. Microcirculation is responsible for supplying blood flow, oxygen, and nutrients to tissues through regulating vascular resistance at the precapillary level. On the other hand, postcapillary venules are preferential sites for the expression of adhesion molecules, where interactions between circulating leukocytes and the vascular endothelium occur during the inflammatory process [35].

The NVU comprises several types of cells, including brain endothelial cells, pericytes, arterioles, microglia, astrocytes, and neurons. These cells work together and communicate to influence each other's behavior, both under normal conditions and during inflammatory events.

The vascular protective effect of GLP-1 receptor agonists is partly attributed to improved metabolic impact and improved glycemic control. The patterns of cerebral neovascularization and remodeling in type 2 diabetes involve complex mechanisms. Research has demonstrated that diabetes affects neovascularization differently in various organ systems. Specifically, diabetes exacerbates brain damage in ischemic stroke [36, 37], as indicated in previous studies conducted by our group [38]. Moreover, research on type 2 diabetes models has indicated that managing blood sugar levels can help prevent abnormal growth of blood vessels in the brain. This suggests high blood sugar may contribute to this response [39]. These discoveries highlight the significance of comprehending and addressing the mechanisms behind problematic blood vessel growth in the brains of diabetic individuals, with a focus on the impact of high blood sugar on these abnormal responses. However, there is a possible explanation for the protective effect of semaglutide on capillary rarefaction in animals with metabolic syndrome, which involves pericytes and is independent of glycemic control. Pericytes are perivascular structures that surround vessels and divide the vascular membrane with capillaries, playing an important role in controlling angiogenesis and maintaining tissue perfusion. Diabetes can cause the nitration of GLP-1 receptors, leading to pericyte dysfunction and loss of cerebrovascular integrity. This can ultimately result in vascular cognitive impairments and dementia. However, administering GLP-1 receptor agonists can help restore pericyte function in diabetes by reducing inflammation and oxidative stress and increasing the survival rate of pericytes. Therefore, the restoration of pericyte function significantly improves cognitive impairment in diabetic mice [40].

Glial cells can rapidly alter their structure and function in response to changes in metabolic activity. This can result in damage to nerve cells and changes in synaptic plasticity. Astrocytes contribute to the stability of the neuronal microenvironment [41] and react to tissue injury by increasing the expression of GFAP, an intermediate filament protein expressed by astrocytes [42, 43]. This property makes GFAP a valuable marker of astrogliosis according to immunohistochemistry, a phenomenon that occurs in brains subjected to neuronal loss and vascular and microvascular changes [42]. Astrocytic endings are closely linked to the vascular wall, and this interaction is significant in the physiology of the neurovascular unit. Furthermore, once altered, this connection can lead to dysregulation of cerebral blood flow, glucose transport and metabolism, and impairment of the BBB. We have already shown that high-fat diet-induced metabolic syndrome impacts the coverage of astrocytes in vessels, which is related to neuroinflammation and microvascular rarefaction [10]. Research provides strong evidence that the interaction between astrocytes and blood vessels is vital for maintaining the integrity of the blood–brain barrier (BBB). Astrocytes have been shown to regulate BBB integrity through various mechanisms, such as mitochondrial transfer [44] and adhesion to laminins via the alpha7 integrin subunit [45]. Additionally, studies have demonstrated that astrocyte reactivity can lead to BBB breakdown, as seen in prion diseases, where reactive astrocytes were found to be detrimental to BBB integrity [46]. The findings emphasize the importance of the complex relationship between astrocytes and blood vessels in maintaining the function of the blood–brain barrier (BBB). Our findings suggest semaglutide might have neuroprotective effects beyond managing blood sugar levels. Strengthening this interaction could be a promising approach to support the integrity of the blood–brain barrier and overall brain health. In this study, semaglutide notably increased the presence of astrocytes in cerebral blood vessels and reversed the decreased number of capillaries, as well as the leakage of IgG into the brain tissue in the cortex in the HFD SAL group (Supplementary Fig. 2).

Microglia are a specific type of immune cell in the central nervous system. Their primary function is to protect the CNS against infections, injuries, and degenerative diseases. These cells constantly monitor their surrounding environment and display phagocytic characteristics in response to brain injury. They play a significant role in neuroinflammation. When activated, these cells undergo morphological changes that enable them to increase and perform phagocytosis. However, microglial activation can have significant pathophysiological consequences in infectious and neurodegenerative diseases. TLR4 is highly expressed in microglia, and its signaling pathway is linked to activated microglial phenotypes, which are responsible for the adverse effects of obesity on the hippocampus and other brain regions [47]. Under normal physiological conditions, microglia are essential for promoting neuronal survival rates by releasing neurotrophins. They also help maintain synaptic terminals and regulate neuronal circuits through synaptic pruning [48]. Other results revealed hypothalamic microglial activation is induced by a HFD independent of body weight [49]. These studies suggest that this microglial activation can be suppressed by GLP-1 receptor agonism while improving other metabolic abnormalities, such as high-fat diet consumption. In contrast, semaglutide was ineffective at completely reversing microglial activation in our MetS model. Although the 4-week treatment with semaglutide in animals that already had MetS effectively reduced the weight and metabolic profile of these animals, we believe that changing the microglial profile to the resting state may take longer than controlling the metabolic effects. Furthermore, we understand that it is not imperative that the protective effect of a drug is reflected immediately and only in microglial alterations. Other cellular mechanisms, which are not yet known, may contribute to the neuroprotective effect of this drug, or a longer treatment time to reverse this phenomenon may be necessary. One limitation of our study is that we only used male mice. This oversight might have overlooked potential gender-specific differences that could impact the outcomes. In the future, research should strive to examine both male and female mice to strengthen the reliability and applicability of the findings. Another limitation is including a pair-fed group to distinguish the direct effects of semaglutide from those resulting from weight loss. Future experiments will include pair-fed groups to address this limitation, allowing us to differentiate the pharmacological effects of semaglutide from those mediated by weight loss.

Our research revealed that treatment with semaglutide had protective effects similar to those of eplerenone in MetS animals, as previously reported in SHR animals [50]. In the groups treated with semaglutide, we observed a significant reversal of the harmful changes caused by long-term HFD consumption. Specifically, we noted that the hippocampal pyramidal cell layer appeared denser, with cells arranged in a more ordered, hierarchical, and sequenced manner. This restoration was further confirmed by the clear difference between the nucleus and cytoplasm in hippocampal cells, indicating a notable recovery of cellular structure and function. These findings suggest semaglutide may have a neuroprotective effect on hippocampal disorganization and potential apoptotic pathways triggered by a high-fat diet (HFD). The protective effects were supported by the behavioral improvements observed in the HFD SEMA group. The animals in this group demonstrated better performance in the Light/Dark test, showing fewer signs of anxiety. Additionally, when tested using the Fear Conditioning Memory Test, the HFD SEMA group spent more time in the freezing position compared to the HFD SAL group, indicating cognitive protection (Supplementary Fig. 3). Therefore, semaglutide could be a promising therapeutic agent for mitigating diet-induced neurodegeneration.

Conclusions

This study provides compelling evidence that semaglutide can mitigate the adverse effects of metabolic syndrome induced by a high-fat diet on neurovascular and metabolic function in mice. A notable finding was the substantial improvement in the interaction between astrocytes and blood vessels, resulting in enhanced protection of the BBB. This effect suggests that semaglutide may reverse microvascular rarefaction, common in metabolic syndrome, by restoring the structural and functional integrity of the neurovascular unit. However, despite these beneficial effects, the persistence of the high-fat diet in the experimental model appears to have limited the ability of semaglutide to completely reverse the pathological activation of microglia. This phenomenon suggests that although semaglutide offers significant protection against neurovascular and metabolic changes, the maintenance of adverse dietary stimuli may compromise the effectiveness of semaglutide in restoring microglial homeostasis. Thus, therapeutic interventions targeting neurovascular health in the context of metabolic syndrome may require a combined approach, emphasizing the pharmacological activation of GLP-1 receptors and dietary modification to achieve optimal results in treating and preventing neurodegenerative and metabolic dysfunctions. To visually summarize these insights, we propose an illustration that succinctly represents the key outcomes of our study (Fig. 8).

Fig. 8
figure 8

This illustration summarizes the systemic metabolic effects of semaglutide treatment and its specific impact on the cerebral microenvironment in an experimental animal model of metabolic syndrome. Semaglutide, a GLP-1 receptor agonist (GLP-1 RA), has profound metabolic benefits. It induces weight loss, reduces adipose tissue, lowers fasting glycemia, and decreases levels of insulin, leptin, resistin, and triglycerides. These systemic actions are accompanied by significant cerebral effects, particularly an enhancement in the coverage of astrocytes on brain vessels. This improvement suggests a healthy blood–brain barrier, which reduces inflammation and promotes neurogenesis. The increased cortical capillary density further implies improved cerebral blood flow and nutrient delivery. Together, these findings emphasize the dual role of semaglutide in modulating metabolism and protecting the brain, highlighting its therapeutic potential for conditions characterized by metabolic dysregulation and blood–brain barrier dysfunction

Full size image

Availability of data and materials

No datasets were generated or analysed during the current study.

Change history

Abbreviations

BBB:

Blood–brain barrier

CNS:

Central Nervous System

GFAP:

Glial fibrillary acidic protein

GLP-1R:

Glucagon-like peptide-1 receptor

HFD:

High‑fat diet

IB4:

Biotinylated isolectin B4

Iba-1:

Ionized calcium‑binding adaptor molecule-1

ND:

Normolipid diet

NVU:

Neurovascular unit

SAL:

Saline solution

SEMA:

Semaglutide

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Acknowledgements

The authors thank Edson Fernandes de Assis for performing the cytokine assays with the multiplex platform and the Fiocruz network of technological platforms and the confocal platform from the Oswaldo Cruz Foundation, Fiocruz, Rio de Janeiro—Manguinhos Maré campus.

Funding

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Universidade Estácio de Sá (Scholarship for Students: Pibic-UNESA and Pibic-IDOMED).

Author information

Author notes

  1. Vanessa Estato and Nathalie Obadia contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Immunopharmacology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation–Fiocruz, Campus Maré. Centro de Pesquisa, Inovação e Vigilância em Covid-19 e Emergências Sanitárias. Endereço: Av. Brasil, 4036-Bloco 2. Manguinhos, Rio de Janeiro, RJ, CEP 21040-361, Brazil

    Vanessa Estato, Nathalie Obadia, Marcelo Gomes Granja & Hugo Caire de Castro Faria-Neto

  2. Medical School, Estácio–IDOMED, Rio de Janeiro, Brazil

    Vanessa Estato, Paulo Henrique Chateaubriand, Vivian Figueiredo, Marcela Curty, Mariana Costa Silva, Renata Gabriela Lustosa Ferreira, Marcela Campos Baroni, Alessandra Aragão, João Oliveira Góes Neno, Clara Avelar Mendes Vasconcellos & Marcelo Gomes Granja

  3. Pharmacy School, Universidade Estácio de Sá, Rio de Janeiro, Brazil

    Nathalie Obadia & Juliane Santa-Ritta

  4. Laboratory of Pre-clinical Research, Iguaçu University, Rio de Janeiro, Brazil

    Joana Costa D’Avila

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Contributions

VE wrote the main manuscript; VE, NO and HN conceived the study and design; VE and NO conducted the experiments; VE, NO and MG supervised in vivo experiments; JR, MC, MCS, AA, JN and CV executed in vivo experiments; VE, NO, PC, VF, MC, MCS and JD executed immunohistochemistry experiments; VE analyzed immunofluorescence; VE and HN assisted with funding, experimental design, analyzed the data, and edited the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Vanessa Estato.

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Ethics approval and consent to participate

The Animal Welfare Committee of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ) approved the experimental protocol under license number L-012/2021.

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All authors have agreed to publish this article.

Competing interests

The authors declare no competing interests.

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The original online version of this article was revised: Fig 5 has been updated.

Supplementary Information

13098_2024_1528_MOESM1_ESM.tif

Supplementary Material 1. Supplementary Fig. 1: Semaglutide prevented HFD-induced ICAM-1 expression in the endothelium of brain capillaries. Confocal images of representative images of ICAM-1 expression on blood vessels in the parietal cortex and hippocampus CA1 region. Arrows indicate blood vessels. Scale bar 50 μm. The brains of 3 animals of each group were perfused and fixed with PFA 4%. Frozen slices 12 mm thick were blocked with PBS + 5% NGS, labeled with a Rat Monoclonal Antibody for CD54eBioKAT-1from eBioscience™, Invitrogen™ diluted 1:200 in blocking buffer, then with an Alexa 488 Goat anti-Rat IgG secondary antibody diluted 1:500 in blocking buffer and counterstained with DAPI, and mounted in Vectashield mounting media for fluorescence. Images of equivalent brain regions of each animal were acquired in a confocal Zeiss microscope.

13098_2024_1528_MOESM2_ESM.tif

Supplementary Material 2. Supplementary Fig. 2: Semaglutide treatment improves the integrity of the blood–brain barrier in a high-fat diet-induced model. Confocal images of the BBB were used to assess blood-to-brain extravasation of IgG using semi-quantitative fluorescence microscopy in the cortex. A) Representative micrographs (green = IgG, blue = DAPI, scale bar = 100 μm. B) Quantification of fluorescence intensity expressed as percentual area). ***p < 0.001 versus the HFD SAL group, n = 6.

13098_2024_1528_MOESM3_ESM.tif

Supplementary Material 3. Supplementary Fig. 3: Behavioral assessment of anxiety and memory in HFD SEMA and control groups. (A) Fear Conditioning Memory Test results, with the HFD SEMA group demonstrating significantly better learning and memory retention, indicated by a longer hesitation time before stepping onto the electrified grid, compared to the untreated HFD group. (B, C and D) Light/Dark Box Test results show the time for transition between the light and dark sides of the box, the number of entries and exits in the lit compartment, and the time spent on a lightbox. The HFD SEMA group exhibited more frequent entries and exits, suggesting a trend towards reduced anxiety, although this was not statistically significant. Data are presented as mean ± SEM, with significance determined by Student’s t-test (*p < 0.05).

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Estato, V., Obadia, N., Chateaubriand, P.H. et al. Semaglutide restores astrocyte–vascular interactions and blood–brain barrier integrity in a model of diet-induced metabolic syndrome. Diabetol Metab Syndr 17, 2 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13098-024-01528-0

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Keywords

Diabetology & Metabolic Syndrome

ISSN: 1758-5996

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