Semaglutide Attenuates Anxious And Depressive-Like Behaviors and Reverses The Cognitive Impairment in a Type 2 Diabetes Mellitus Via The Microbiota-Gut-Brain Axis

doi:10.21203/rs.3.rs-3249869/v1

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Research Article

Semaglutide Attenuates Anxious And Depressive-Like Behaviors and Reverses The Cognitive Impairment in a Type 2 Diabetes Mellitus Via The Microbiota-Gut-Brain Axis

https://doi.org/10.21203/rs.3.rs-3249869/v1

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published 23 Jul, 2024

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Newly conducted research suggests that metabolic disorders, like diabetes and obesity, play a significant role as risk factors for psychiatric disorders. This connection presents a potential avenue for creating novel antidepressant medications by repurposing drugs originally developed to address antidiabetic conditions. Earlier investigations have shown that GLP-1 analogs exhibit neuroprotective qualities in various models of neurological diseases, encompassing conditions such as Alzheimer's disease, Parkinson's disease, and stroke. Moreover, GLP-1 analogs have demonstrated the capability to enhance neurogenesis, a process recognized for its significance in memory formation and the cognitive and emotional aspects of information processing. Nonetheless, whether semaglutide holds efficacy as both an antidepressant and anxiolytic agent remains uncertain. To address this, our study focused on a mouse model of depression linked to type 2 diabetes induced by a High Fat Diet (HFD). In this model, we administered semaglutide (0.05mg/Kg intraperitoneally) on a weekly basis to evaluate its potential as a therapeutic option for depression and anxiety. Diabetic mice had higher blood glucose, lipidic profile, and insulin resistance. Moreover, mice fed HFD showed higher serum IL-1β and LPS associated with impaired humor and cognition. The analysis of behavioral responses revealed that the administration of Semaglutide effectively mitigated depressive- and anxiety-like behaviors, concurrently demonstrating an enhancement in cognitive function. Additionally, Semaglutide treatment protected synaptic plasticity and reversed the hippocampal neuroinflammation induced by HFD fed, improving activation of the insulin pathway, demonstrating the protective effects of Semaglutide. We also found that Semaglutide treatment decreased astrogliosis and microgliosis in the dentate gyrus region of the hippocampus. In addition, Semaglutide prevented the DM2-induced impairments of POMC, and GPR43 and simultaneously increased the NeuN + and GLP-1R + neurons in the hippocampus. Our data also showed that Semaglutide increased the 5-HT and its receptor (5-HTT) and glutamatergic receptors in the hippocampus. At last, Semaglutide changed the gut microbiota profile (increasing Bacterioidetes, Bacteroides acidifaciens, and Blautia coccoides) and decreased leaky gut, improving the gut-brain axis. Taken together, Semaglutide has the potential to act as a therapeutic tool for depression and anxiety.

Diabetes mellitus 2

Depression

Cognition

Anxiety

Gut microbiota

Semaglutide

The increase in life expectancy in recent decades has promoted the growth of aging pathologies, such as neurodegenerative and metabolic diseases. Obesity is a multifactorial disease resulting from an imbalance between energy consumption and expenditure. Although genetics plays a role in its development, environmental factors are also responsible for the dramatic increase in its prevalence, being considered a worldwide epidemic and a public health problem due to difficulties in prevention and treatment(Perry and Shulman 2020). In 2016, approximately 1.9 billion adults aged 18 and over were overweight, and 650 million were obese (WHO 2020). Excess body fat predisposes individuals to develop chronic health conditions, including type 2 diabetes, hypertension, and cardiovascular diseases.

Type 2 diabetes (DM2) accounts for approximately 90–95% of diabetes cases and is a public health problem In DM2, hyperglycemia promotes some brain changes such as oxidative stress (Henson et al. 2023), mitochondrial dysfunction (Shokrzadeh et al. 2018), neuroinflammation (Wong et al. 2018), aggregation of amyloid β (Wang et al. 2014), tau phosphorylation (Platt et al. 2016), and mood changes (Simó et al. 2017)). Epidemiological studies demonstrated a strong correlation between DM2 and neurodegenerative diseases, with emphasis on Parkinson's disease (PD) (De Pablo-Fernandez et al. 2018; Biosa et al. 2018), on Alzheimer´s disease (AD) (Henson et al. 2023), and major depressive disorder (Detka et al. 2013).

In its turn, depression is emerging as a leading cause of mortality and morbidity worldwide, and the incidence of people reported with the disorder continues to rise (Ledford 2014). Approximately 50% of patients with depression have chronic diseases, and depressive symptoms can gradually worsen if patients are not treated appropriately. The occurrence of depressive symptoms and major depressive disorder (MDD) is higher among individuals with diabetes when compared to those without diabetes. Systematic review findings indicate that individuals with Type 2 diabetes have twice the likelihood of experiencing depression compared to control groups(Chireh et al. 2019).

Moreover, Diabetes mellitus significantly impacts the hippocampus, a crucial brain region involved in depressive disorders and cognitive processes (Bird and Burgess 2008). The dentate gyrus (DG) is a crucial brain region responsible for memory encoding. Its unique neuronal structure and function significantly differ from other cortical areas. One notable distinction is that the dentate gyrus generates new neurons, continually integrated into a fully functional neural circuit throughout an individual's lifespan (Piatti et al. 2013). Altered proliferation in the dentate gyrus has been associated with depression (Umschweif et al. 2021) and impairment of learning and memory processes (Oliveira et al. 2021). The involvement of pro-opiomelanocortin (POMC)-derived neuropeptides and the melanocortin 4 receptor (MC4R) in hippocampus-dependent synaptic plasticity suggests that the POMC/MC4R system plays a crucial role in the hippocampus and its dysfunction may contribute to cognitive disorders, such as AD (Shen et al. 2016). Additionally, behavioral and functional studies have shown that mice with mutations in POMC-Notch12/2 exhibit anxiety and depressive-like behavior, along with impaired synaptic transmission properties, specifically in the dentate gyrus (Feng et al. 2017).

Excessive activation of the immune system has been shown to play a key role in the pathogenesis of T2D and MMD. The association between inflammation and MDD is present in several central nervous system pathologies (CNS), such as bipolar disorder, Parkinson's, multiple sclerosis, and diabetes (Marrie et al. 2017). Although the available antidepressant drugs are effective in the treatment, more than 30% of depressed patients fail to achieve remission. The heterogeneity of depressive disorders can explain the limited effectiveness of current antidepressant therapies. In addition to monoaminergic neurotransmission, disturbances in neurogenesis, synaptogenesis, and inflammatory processes can largely contribute to depressive symptoms in patients (Malhi et al. 2018). Chronic neuroinflammation is characterized by excess glucocorticoids and glutamate, reactive microglial activation, increased production of pro-inflammatory cytokines, and reactive oxygen species (ROS) (Detka et al, 2013). This decline in mood is directly related to functional and structural changes in the hippocampus, whose neurons are sensitive to insulin.

The binding of insulin to their receptors (INSRs) leads to activation of IRS-1/2 sets off the phosphatidylinositol 3-kinase (PI3K)-dependent pathway, which phosphorylates proteins subsequently initiating several critical cross-talk pathways, including synaptic plasticity, cell survival and transcription of apoptosis regulators e growth factors. Moreover, the phosphorylation of IRS-1 leads to preferential activation of the mitogen-activated protein kinase (MAPK) pathway, which facilitates cellular growth, differentiation and regulates protein translation (Amoui et al. 2001). Accordingly, previous studies by our group showed that the action of the AMPK agonist (metformin) improved memory and learning in an animal model of type 1 diabetes through the modulation of insulin signaling, synaptic plasticity, and neurogenesis in the hippocampus and prefrontal cortex (Oliveira et al. 2021). Thus, induction of the insulin signaling cascades leads to complex cellular responses. Moreover, pre-clinical studies have shown an association between obesity and brain pro-inflammatory cytokine levels, possibly inhibiting insulin signaling and exacerbating depression and anxiety pathogenesis in Diabetes mellitus .

The gut microbiota plays a significant role in the development of metabolic disorders, and it has become an area of interest for potential therapeutic interventions. Understanding how dysbiosis of the gut microbiota contributes to the pathophysiology of diabetes can provide new insights into novel therapeutic approaches for diabetes and help overcome treatment challenges(Homolak et al. 2021). Incretin hormone-based therapies are widely used to improve hyperglycemia in T2DM patients (Fonseca et al. 2010). GLP-1 is the principal hormonal incretin, an anorexigenic peptide secreted by L cells in response to nutrient intake. Interestingly, GLP-1R agonists (GLP-1RA) have been found to impact the intestinal environment, and changes in the gut microbiota have been associated with the use of GLP-1RA (Yusta et al. 2015). Moreover, several studies have demonstrated the beneficial effects of GLP-1, including its role in improving intestinal barrier functions by stimulating crypt cell fission, reducing proinflammatory cytokines produced by immune cells, and modulating the composition of the gut microbiota (May et al. 2019).

In CNS, GLP-1 receptor (GLP-1R) agonists also produced neuroprotective effects in diabetic patients (Gault and Hölscher 2018). The signaling of GLP-1R in the hippocampus plays a crucial role in memory function. Studies involving GLP-1R-deficient mice have shown that they exhibit impairments in spatial memory, which relies on the hippocampus. However, it has been found that this memory impairment can be reversed through the upregulation of hippocampal GLP-1R signaling, either through pharmacological interventions or genetic modifications (Hsu et al. 2015). In addition, treatment with the GLP-1 agonist liraglutide in patients with bipolar depression and other mood disorders improved cognitive function and relieved pathological depressive symptoms (Cuomo et al. 2018). As a result, there is currently a significant focus on investigating the potential of GLP-1R agonists in treating neurodegenerative diseases like AD and PD (Batista et al. 2019).

Few studies indicate that Semaglutide, a GLP-1R agonist, may potentially mitigate diabetes-induced behavioral abnormalities related to depression and cognitive impairments in diabetic mice. To investigate this further, we conducted a comprehensive study using diet-fed C57BL6 mice, a well-established model for type 2 diabetes mellitus (T2DM), intending to examine the impact of semaglutide on neurobehavioral deficits associated with diabetes, and exploring the underlying mechanisms this positive effect. We hypothesize that the GLP-1R agonist can restore the intestinal microbiota and improves the insulin brain pathway, thereby improving diabetes-induced depressive and anxious-like behavior as well as cognitive impairment in C57BL6 mice.

Animals

Sixty male C57BL/6 young adults (8–10 weeks) weighing 20g and bred in the vivarium of the Instituto Aggeu Magalhães (Recife, Pernambuco, BR). Before the protocol, the state of the health of mice (activity, interaction with cage mates, and general well-being) was verified. The mice were acclimatized for 7 days before the experiment.

Induced-diet type 2 diabettes

Initially, mice were distributed in the following groups: (A) control (n = 15) – mice that received a standard diet provided by PragSolucões® (www.pragsolucoes.com.br), according to the recommendations of the American Institute of Nutrition (AIN-93G)(Reeves et al. 1993) for 18 weeks; and (B) DM2 (n = 45) – type 2 diabetes mellitus based on obesity induced by high-fat diet (a diet rich in 60% fat) for 18 weeks produced by PragSolucões®, according to the recommendations of AIN-93M (The diet compositions are detailed in a previous study (Ludgero-Correia et al. 2012). 3–4 mice were kept in microisolators under standard conditions (12 h of light / dark cycle, 21º ± 2º C, humidity 60 ± 10%). Each animal was weighed weekly, and the daily food intakes were monitored into the last week of the experimental period (18 weeks). The diet was available at all times (ad libitum).

At the 13th week, the mice were later divided into the following experimental groups: (A) Control (n = 15); (B) DM2 (n = 15); (C) DM2 + Semaglutide (n = 15), (D) DM2 + Fluoxetine (n = 15). The DM2 + Semaglutide group received a high-fat diet and, at the 13th week, Semaglutide (Ozempic ® − 0.05mg/Kg – translational dose) was administered intraperitoneally every seven days for six weeks (Robinson et al., 2019). The DM2 + Semaglutide group received a high-fat diet and, at the 13th week, Fluoxetine (20mg/Kg – translational dose) was administered daily in drinking water for six weeks. Waist circumference was determined using an inelastic measuring tape around the abdomen of the mouse at the midpoint between the animal's mouth and pelvic limbs of the animals (He et al. 2016). The other groups received a vehicle solution i.p., once a week.

After 4 h of fasting, a tail puncture was performed to confirm diabetes induction using a One Touch Ultra Lifescan (Johnson & Johnson) glucometer. The glycemia was performed in the first, thirteenth, and eighteenth week. Then, mice received an intraperitoneal injection of Lidocaine (5%) and Thiopental (0.05g/ml) for euthanasia (Fig. 1). All experiments were conducted by the Ethical Principles in Animal Experimentation and were accepted by the Ethics Committee on the Use of Animals of the Aggeu Magalhães Institute (CEUA 135/2018 - IAM). Insulin resistance (HOMA-IR) was assessed using the homeostasis model assessment index, calculated using the subsequent formula: HOMA-IR = (fasting insulin in µU/ml) × (fasting glucose in mmol/l) / 22.5 (Obadia et al. 2022).

The Tail suspension test (TST)

The TST is a valuable mouse behavioral test in screening potential antidepressant drugs and assessing other manipulations that are expected to affect depression-related behaviors (Can et al. 2012a). Mice were placed upside down, about 40 cm above the floor, with the tape placed 1 cm from the tip of the tail in a position that could not escape or hold onto nearby surfaces. "Immobility" was considered when the animal remained immobile, and its body was vertically suspended. The immobility time was recorded in the last 5 minutes of the test (a total of 6 minutes).

The forced swim test (FST)

The FST is a rodent behavioral test used for the evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states (Can et al. 2012b). Briefly, mice are placed individually in an inescapable transparent container filled with water (22–25°C), and their escape-related mobility behavior is measured over six minutes. "Immobility" in the FST was defined as the absence of any movement other than those necessary to balance the body and keep the head above water. Furthermore, we analyzed the last five minutes because most mice are very active at the beginning of the FST, and potential treatment effects may not be evident during the first minute. We monitored the mice by video, and immobility was analyzed using on-screen stopwatch software (Xnote Stopwatch, dnSoft Research Group) for time measurements. In both tests, animals were acclimatized in the testing room for one hour before testing.

T Maze Spontaneous Alternation

The test was performed as described by previous study (RM and JN 2006). Briefly, The T-maze is a horizontally positioned apparatus, either elevated or enclosed, designed in the shape of a T. Mice begin from the base of the T and are given the option to select one of the goal arms connected to the opposite end of the stem. When two consecutive trials are conducted rapidly, the rodent tends to choose the arm that was not previously visited, demonstrating recollection of the initial choice. This phenomenon is referred to as 'spontaneous alternation'.

Elevated Plus Maze Test (EPM)

The T Maze is a behavioral assessment that measures animal exploratory behavior, specifically, rodent models utilized for studying CNS disorders. This test gauges the inclination of rodents to explore new environments, as they display a preference for visiting a new arm of the maze rather than a familiar one

The EPM is a rodent assay to identify anxiolytic pharmacological agents. The plus-maze was made of plywood and consisted of two enclosed arms (21.5 x 7.5 x 20 cm) and two open arms (21.5 x 7.5 cm), which extended from a central 7.5 x 7.5 cm platform. The plus-maze was elevated 38 cm above the floor. We placed the rodent in the center, and its behavior was observed and recorded in each arm. The test relies on the natural aversion of rodents to open spaces, thought to reflect their innate fear of heights and predation. A decrease in the time spent in the open arms and an increase in the time spent in the enclosed arms indicate an anxiety-like behavior (Komada et al. 2008).

Biochemical assessment

The weight of the animals was evaluated after the experiment, as well as the average food consumption (gram/week). All animals were euthanized (as previously described), and a chest incision was made to expose the heart of animals (n = 10–11) to collect the blood. Then, approximately 1 ml of the blood of each mouse was slowly collected by cardiac puncture. Soon after, the blood was centrifuged at 2,300 relative centrifugal force (RCF) for 15 minutes. The serum was applied in a 96-well ELISA plate, and the glucose level (Labtest Diagnótica S.A®, MG, Brazil - Ref. 133), Triglycerides (Labtest Diagnótica S.A®, MG, Brazil - Ref. 87), cholesterol (Labtest Diagnótica S.A®, MG, Brazil - Ref. 76), Insulin (Elabscience, China) and IL-1β (Abcam, catalog number ab100705) was determined by enzymatic systems, respectively. Results were expressed as mean ± standard deviation using GraphPad Prism V6.0.

Immunohistochemistry

The methodological processes for carrying out this technique were previously described(Paiva et al. 2023). The study involved the use of anesthetized animals for transcardiac perfusion. The perfusion process included physiological saline (20 ml) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS), pH 7.2. The brains were post-fixed overnight in the same fixative solution and later processed into paraffin to obtain coronal sections (5 µm) using a microtome. The sections were treated with 20 mM citrate buffer at pH 6.0 for antigen retrieval and heated to 100°C for 30 minutes. Subsequently, the sections were incubated with 1% BSA for 1 hour to block peroxidase and then 3% H2O2 for 30 minutes. Following this, the sections were exposed to primary antibodies, including GFAP (Novus Biological, catalog number NB300- 141) at a dilution of 1: 1000 and Iba1 (Wako, catalog number 016–20001) at a dilution of 1: 500, overnight at 4°C and then for 1 hour with biotin-conjugated secondary IgG antibody. Immunohistochemical reactions were amplified using the Kit Dako LSAB + System-HRP, revealed with 3'-3-diaminobenzidine (DAB), and counterstained with Harris Hematoxylin. The labeling intensity in the frontal cortex and hippocampus region was assessed by comparing the groups using the GIMP 2.6.2 program.

Immunofluorescence

The samples (3 animals/per group) were embedded in paraffin, and the sections (5 µm) were cut using a RM 2035 microtome (Reichert S, Leica) and mounted on glass slides, rehydrated, and treated with 20 mM citrate buffer (at pH 6.0) at 100 ° C for 30 min. According to the manufacturer's instructions, the hippocampus sections (5 µm) were submitted to immunohistochemical reactions using the Dako EnVision™ FLEX + kit (Dako, Code: K8002, USA). The primary antibodies were anti-POMC antibody (Abcam, catalog number ab32893) at a dilution 1:1000, anti-occludin (Santa Cruz, catalog number 133256) at a dilution 1:400, anti-GPR43 antibody (Biologic Science, catalog number bs13563) at a dilution of 1: 1000, GLP-1R antibody (Novus Biological, catalog number NLS1205) at a dilution of 1: 100, and NeuN antibody (Novus Biological, catalog number NBP1-77686) at a dilution of 1: 500. The primary antibodies were incubated overnight and then incubated with polyclonal fluor 488 and 546 conjugated secondary antibody against rabbit and mouse immunoglobulin for 1 h. The slices were washed, mounted in gelatin medium, observed under a fluorescence microscope Leica DMI8 system, and processed with the Leica Application Suite LAS software (Leica Microsystems, Wetzlar, Germany). Figures were exported as tiff files with Adobe Photoshop version 8. The number of hippocampal GPR43+/POMC + neurons were quantified from 8 images per group (2 images X 4 animals), and neurons were considered double-positive to POMC and GPR43 when they presented a yellowish-yellow stain (merge). NeuN and GLP-1R positive neurons were also quantified using the same protocol. The occludin expression was quantified in hippocampal areas from 8 images per group (2 images X 4 animals), and pixel density were quantified using the Gimp 2.10.18 program (GNU Image Manipulation Program, UNIX platforms).

Western blot

The hippocampi were rapidly dissected and homogenized in an extraction solution containing protease inhibitor cocktail (10 mM EDTA, Amresco, Solon, USA; 2 mM phenylmethane sulfonyl fluoride, 100 mM NaF, 10 mM sodium pyrophosphate, 10 mM NaVO4, 10 µg of aprotinin / mL and 100 mM Tris, pH 7.4 - Sigma - Aldrich). The samples (4 animals/per group) were mixed and homogenized to form a pool for each group. The homogenates were centrifuged and frozen at -80^oC. The proteins (20 µg/µL) were separated on sodium dodecyl sulfate (SDS) polyacrylamide by gel electrophoresis under reduced conditions and were then transferred electrophoretically onto the nitrocellulose membrane (OmniPage mini-vertical protein electrophoresis, Cleaver Scientific). After blocking with 3% BSA, the membranes were incubated overnight with antibodies against to: Phospho-IGF-I Receptor β (Tyr1135/1136 (dilution of 1:1000, Abcam, catalog number: ab6671), IL-1β (dilution of 1:500, Genway, catalog number: GWB-BBP232), p-JNK (dilution of 1:1000, Santa Cruz, catalog number: sc-6254), PI3K p85 alpha (phospho Y607) (dilution of 1:500, Abcam, catalog number: ab182651), AKT (phospho T308) (dilution of 1:500, Abcam, catalog number: ab38449), GLUT4 (dilution of 1:500, Abcam, catalog number: ab654), AMPKα-Phospho (Thr172) (dilution of 1:1000, Cell signaling, catalog number: #2535S), p-PKA ( dilution of 1 :1000, Abcam, catalog number: ab75991), CD220 (dilution of 1 :1000, bdbiosciences, catalog number: 611276), Nitrotirosin (dilution of 1 :1000, Abcam, catalog number: ab7048), ), p-GLP-1R ( dilution of 1 :1000, Novus Biological, catalog number: NLS1205), Serotonin transporter (5-HTT) (dilution of 1 :500, Alomone, catalog number: AMT-004). All primary antibodies were diluted in blocking solution (1,5% BSA, 0.02% Tris phosphate-buffered, and 0.01% Tween). After washing, the membranes were incubated with peroxidase-conjugated anti-rabbit horseradish (HRP) (dilution of 1:8000, Sigma-Aldrich, catalog number: A9169, USA), or anti-rat HRP (1:5000; Sigma-Aldrich, catalog number: A9037, USA), or anti-mouse HRP (1:5000; Sigma-Aldrich, catalog number: A0161, USA). An enhanced chemiluminescence reagent (Super Signal, Pierce, Ref. 34,080) was used to make the labeled protein bands visible in the iBright CL 1000 system (Thermo Fisher Scientific, catalog number: A44241). For quantification, densitometry values were obtained by measuring each band's pixel density using the Image J 1.38 software (NIH, MD, USA). After the visualization of the protein blots with enhanced chemiluminescence, the protein antibodies were stripped from the membranes, which were reprobed with the monoclonal anti-β-actin antibody (dilution 1: 5000, Sigma-Aldrich, catalog number A2228, USA) that was used as a loading control. Protein densitometry was performed using the ImageJ. The results were confirmed by three different repetitions for each investigated protein and statistical analyses were performed with these obtained values.

16S rRNA gene sequence-based microbiota analysis

After euthanasia, feces content (6 mice/group) were collected and processed. The Total DNA extraction was performed. The DNA extracted using magnetic beads(Christoff et al. 2017). Sequencing library preparation for bacterial identification was prepared using the V3/V4 16S rRNA gene 341F (CCTACGGGRSGCAGCAG)(Wang and Qian 2009) and 806R (GGACTACHVGGGTWTCTAAT)(Caporaso et al. 2012) primers and the samples were sequenced in a MiSeq system (Illumina, USA), using the standard Illumina primers provided by the manufacturer kit. Raw data from MiSeq was processed using a pipeline owned by Neoprospecta (Christoff et al., 2017). Illumina FASTQ files had the primers trimmed and their accumulated error evaluated (Phred < 20). Besides, clusters with abundances lower than 2 were removed. Taxonomic ranks were allocated using a 16S rRNA accurate sequence database set at a 99% identity level (Christoff et al., 2017) using blastn v.2.6.0+ (Altschul et al. 1990). The resulting oligotype tables, analogous to traditional OTU tables, were used for calculating alpha-diversity metrics using R (version 4.1.0) and the Phyloseq package. Relative abundance plots were made in R (version 4.1.0).

Statistical analysis

For Microbiome Statistical Analysis the Alpha diversity was computed using the Hill diversity series (Hill, 1973), where q = 0 is the richness, q = 1 is the Shannon’s entropy, and q = 2 is the Reciprocal Simpson index. To evaluate differences in taxonomic profiles between experimental groups, beta diversity was analyzed by using principal coordinate analysis (PCoA). The PCoA was performed on log-ratio transformed values, using Bray-curtis. The analyses were performed in R environment (R Core Team, 2023), using the package vegan (Oksanen, et al. 2020). The parametric data were analysed using analysis of variance (ANOVA-one way) followed by Tukey’s post-test or Holm-Sidak multiple comparisons tests. The data were represented by mean ± SD or mean ± confidence interval, and probability values lower than 0.05 were considered significant.

Semaglutide treatment reduces body weight gain in diabetic animals fed a high-fat diet

The DM2 mice had a significantly higher mean weight (p < 0.05) than the control group (12th week). In the 18th week of treatment, the DM2 + Semaglutide group had a significantly lower weight (30.66g ± 3.54) when compared to the DM2 group (41.19g ± 5.33). The group with DM2 treated with fluoxetine showed no significant difference compared to the DM2 group (Fig. 2A).

We also monitored the food intake of these animals in the 18th week. We observed no significant difference in food intake between the control group, DM2 group, and DM2 + Fluoxetine group. However, we noticed that the diabetic animals treated with semaglutide exhibited a reduced food intake compared to the other groups (p < 0.001) (Fig. 2B).

Semaglutide treatment improves the biochemical profiles in diabetic animals

Before euthanasia, the measurement of the length of the abdominal waist was performed, and the animals in the DM2 group had a significantly greater length (10.16cm ± 0.65) compared to the group treated with Semaglutide (8.21cm ± 0.96 cm)(p < 0.0001). There were no significant differences between the diabetic group and the fluoxetine treated group (Fig. 3A).

After euthanasia, blood was collected by transcardiac puncture and posteriorly centrifuged to obtain serum. The DM2 group showed a significant increase (p < 0.0001) in blood glucose (212.6mg/dL ± 32.14) when compared to the control group (148mg/dL ± 32.44). Glycemia was significantly reduced (p < 0.0001) in the Semaglutide group (113.1mg/dL ± 19.32) (Fig. 3B). Similarly, animals with diabetes showed a significant increase (p < 0.0001) in the lipid profile compared with the control group. In contrast, the administration of Semaglutide improved significantly (p < 0.001) the lipid profile in diabetic animals (Fig. 3C-F). There were no significant differences between the DM2 and the DM2 + Fluoxetine group.

We observed that the DM2 group had significantly higher levels of serum insulin (294.2 pg/ml ± 80.48) when compared to the other groups (p < 0.01), suggesting possible insulin resistance in peripheral tissues. In turn, treatment with semaglutide significantly decreased serum insulin levels when compared to the T2DM group (137.4 pg/ml ± 24.02) (p < 0.001) (Fig. 3G). Besides, we observed marked increased in HOMA-IR in the DM2 group (3 ± 0.8) compared to the control group (0.9 ± 0.4). However, the Semaglutide group presented significant difference in this parameter compared to the DM2 group (0.7 ± 0.1) (Fig. 3H).

5.3 Treatment with Semaglutide and Fluoxetine decreases serum levels of IL-1β, LPS and improves brain 5-HT levels in animals with diabetes.

The animals in the DM2 group had a significantly higher serum concentration of IL-1β (23,75pg/mL ± 0,72) (p < 0.05), reflecting the peripheral inflammatory process. On the other hand, the expression of this cytokine in the serum of the groups treated with Semaglutide and Fluoxetine (8,875pg/mL ± 1,450; 7,58 pg/mL ± 2,71, respectively) was significantly lower when compared to the T2DM group (p < 0.001) (Fig. 4A).

Intestinal permeability is increased in obese patients and patients with diabetes mellitus, favoring the translocation of microbiota-derived LPS into the bloodstream (Rosadini and Kagan 2017). As expected, we observed a significantly higher increase in LPS concentration in the DM2 group (0,67µg/mL ± 0,035) compared to the control, thus indicating an increase in intestinal permeability. However, treatments with Semaglutide and Fluoxetine significantly decreased the concentration (0,22 µg/mL ± 0,19 ; 0,102 µg/mL ± 0,17) of LPS in the HFD group (p < 0.05 and p < 0.01, respectively) (Fig. 4B).

Some studies have shown the importance of determining serotonin levels to diagnose somatic and psychiatric disorders. Here, the T2DM group had brain 5-HT levels (7.38ng/mL ± 0.29) significantly reduced compared to the control (p < 0.01). However, treatment with semaglutide (8.800 ng/mL ± 0.92) and fluoxetine (8.66 ng/mL ± 0.582) significantly reversed the low levels of serotonin in the brain of animals with DM2 (p < 0.05) (Fig. 4C).

5.4 Diet-induced type 2 diabetes promotes anxious and depression-like behaviors, and cognitive deficits, which are reversed by treatment with Semaglutide and Fluoxetine

The performance of FST (Fig. 5A) and TST (Fig. 5B), showed that obese mice presented a behavior similar to depression since they had longer immobility times (131.6s ± 31.21 and 164.5s ± 17) in the behavioral tests when compared to the control group (44.63s ± 28.43 and 105.8s ± 32,35) (p < 0.0001). However, the administration of Semaglutide and Fluoxetine significantly reduced the immobility times in Forced Swimming (64.62s ± 32.83 and 36.75s ± 29.03) (Fig. 5A) and Tail Suspension (130.8s ± 26,37 and 99.9s ± 30.21) (Fig. 5B), respectively. Furthermore, in the EPM we observed that animals with type 2 diabetes showed a longer time in the closed arm (187.6s ± 19,59) (p < 0.0001) compared with the control group. Moreover, the DM2 group had a decreased time in the open arm (76.62s ± 14), indicating a behavior similar to anxiety (Fig. 5C-D). Treatment with Semaglutide induced a significant reduced time (129.7s ± 29.38) in the closed arm (p < 0.01), and a significantly increased time in the open arm (114.6s ± 35,84) (p < 0.0001), revealing an anxiolytic effect (Fig. 5C-D). Treatment with fluoxetine had similar results (p < 0.0001).

We further determined whether Semaglutide could prevent cognitive decline induced by DM2. In the spatial memory test, chronic HF diet resulted in a decrease in alternation (44.06% ± 10.47) compared with the control mice (66.36% ± 5.81) (p < 0.01), suggesting that the recognition memory of DM2 mice was impaired. However, semaglutide and fluoxetine administration significantly increased the alternation index ( 69.34 ± 14.0 ; 66.54% ± 11.0) (p < 0.01) (Fig. 5E).

5.5 Diabetic animals show increased expression of pro-inflammatory cytokines.

TLR4 activation stimulates the release of pro-inflammatory cytokines such as nuclear factor kappa B (NF-κB), TNF-α and IL-1β via myeloid differentiation factor 88 (MyD88) (Zhong et al. 2019). Diabetic animals showed significantly higher protein expression of TLR4 (87.33% ± 7.5), MyD88 (76% ± 8,544), p-NF-Κb (75.67% ± 6), TNF-α (100% ± 4.4), IL-6 (82.33 ± 7), IL-1β (90.67% ± 10.97), Nytrotirosin (161,7% ± 8,5) than control animals. However, intervention with semaglutide and fluoxetine reduces the protein expression of these markers significantly, contributing to a decrease in hippocampal neuroinflammation (Fig. 6).

5.6 Diabetic animals show dysregulation of neurotransmitter receptors and brain GLP-1R

Pharmacological evidence suggests that abnormal glutamatergic and serotonergic neurotransmission may be associated with the pathophysiology of depression (He et al. 2023). Here, we observed that the diabetic animals had significantly lower levels of glutamate receptors like NMDAR1 (50.61% ± 9.19), NMDAR2 (88.3% ± 6.2), Glu2R (31.33% ± 7.3) and reduced levels the 5-HTT (37% ± 9.9) than the control group. Interestingly, treatment with semaglutide and fluoxetine significantly restores the expression of these receptors (Fig. 7).

Chronic antidepressant treatment leads to CREB upregulation, and raising CREB levels in rodent models produces behaviors similar to those seen with antidepressants. The diabetic animals significantly lower levels of pCREB (35.12% ± 13) than the control group (69% ± 12.72). However, Semaglutide (93.5% ± 1.73) and Fluoxetine (89% ± 4) increased significantly the levels of pCREB in diabetic animals compared to untreated animals.

GLP-1R plays many crucial roles in the CNS. In particular, GLP-1 improves memory and learning in the hippocampus, stimulates the generation of new neurons, lowers inflammation and cell death, influences reward-related behavior, and diminishes food consumption. Diabetic animals showed a significantly reduced expression of GLP-1R (38.56% ± 5.6) in the hippocampus compared to control animals (61% ± 5.5). In contrast, treatments with Semaglutide (65% ± 3.6) and Fluoxetine (81% ± 2.8) significantly increase GLP-1R receptor expression in the hippocampus of diabetic mice, confident for improvement in mood and cognition symptoms (Fig. 7).

5.7 Effect Semaglutide treatment reduced gliosis in the hippocampus in diabetic mice

The DG of control animals showed typical latent microglia with branched and thin filaments and basal Iba1 + cells (5 cells ± 1.08) (Fig. 9). In contrast, the DM2 group showed a significant expression of Iba1 + cells (p < 0.001) (11 cells ± 3.11), exhibiting amoeboid phenotype. On the other hand, treatment with semaglutide or Fluoxetine treatment reduced Iba1 + cells (5.88 cells ± 1.2, and 6 cells ± 2, respectively) (p < 0.01), showing phenotype similar the control group (Fig. 8).

Astrocytes express the glial fibrillary acid protein (GFAP), and its increase is related to the activation of these cells, often called reactive gliosis (RG). In addition, astrocytic activation showed morphological changes, such as hypertrophy in cell body size and thickening of cellular processes (Robel et al. 2011). In the dentate gyrus, the control group mice showed astrocytes with pattern morphology and basal GFAP + cells (12 cells ± 3.1) (Fig. 10). Diabetic mice showed a significantly (p < 0.001) higher number of GFAP + Cells (20.7 cells ± 3.45) compared to the control group. Besides, the astrocytes of the DM2 group had hypertrophied cell bodies and thicker cellular processes. In contrast, Semaglutide and Fluoxetine treatment significantly reduced reactive astrogliosis in the DM2 group (11.45 cells ± 1.9, p < 0.01 and 14.75 cells ± 3.49, p < 0.05, respectively) (Fig. 9).

5.8 Semaglutide treatment stimulates PI3K/AKT signaling in the hippocampus of DM2 mice

The PI3K/AKT pathway plays a significant role in preventing and treating mood disorders. Here, we investigated whether diabetic mice could exhibit symptoms similar to depression and anxiety and whether the cognitive decline was related to a decreased insulin signaling pathway. We observed that animals in the DM2 group had significantly (p < 0,05) lower expression of CD220 (161.7% ± 8.5), IGF-1R (131.8% ± 15.11), p-PI3K (134.1% ± 26.19), p-AKT (50.61% ± 8.53), GLUT4 (57.72% ± 6.6), p-PKA (138.1% ± 4.87), and p-AMPK (76.5% ± 10.97) when compared with the control group. Furthermore, we evidenced that diabetic mice had significantly (p < 0.001) higher p-JNK (179.5% ± 38.69) compared with the control group (90.5% ± 19.05), leading to inhibition of the PI3K/AKT pathway. Semaglutide treatment, otherwise, reversed the DM2-impairment of the insulin pathway. Animals treated with Semaglutide showed significantly higher expression of CD220 (106.4% ± 12.7), IGF-1R (138.1% ± 15.11), p-PI3K (185.3.1% ± 22.77), p-AKT (155% ± 19.62), GLUT4 (108.2% ± 6.0), p-PKA (174% ± 13.33), and p-AMPK (101.5% ± 17.9) when compared with the DM2 group. Fluoxetine treatment induced only significant effects on the AMPK (49% ± 4.61) and AKT (99.66% ± 14.25 ) levels (Fig. 10).

5.9 Semaglutide treatment reverses changes in GPR43, POMC, GLP1-R and NeuN hipocamppal neurons induced by DM2

SCFAs function as signaling molecules, inhibiting histone deacetylases (HDACs) and activating orphan G protein-coupled receptors (GPRs). The GPR activation by SCFAs exerts several effects to improve metabolic homeostasis and specific cellular signaling pathways, which potentially regulate T2DM pathogenesis (Tang and Li 2021). Otherwise, neuropeptides derived from pro-opiomelanocortin (POMC) and their interaction with the melanocortin 4 receptor (MC4R) influence hippocampal synaptic plasticity, and disruptions in this system may contribute to cognitive deficits in neurodegenerative diseases such as Alzheimer´s disease (Bo et al. 2020). Our results showed that DM2 animals presented a reduction of POMC+/GPR43 + neurons (5.66 cells ± 1.52) compared to the control group (77 cells ± 4.58) (p < 0.05). However, treatment with Semaglutide significantly enhanced POMC+/GPR43 + neuron numbers (40.25 cells ± 8.42) (p < 0.05) compared to the diabetic group. Interestingly, Fluoxetin treatment also improved POMC+/GPR43 + neuron numbers (26.25 cells ± 3.8) (p < 0.05) (Fig. 11).

The GLP-1R labeling was decreased in the DM2 group (34.8 cells ± 8.1) compared to the control group (59.8 cells ± 11.8). Treatment with Semaglutide (83.4 cells ± 10.2) and Fluoxetine (63.2 cells ± 3.8) significantly increased the GLP-1 expression in hippocampal neurons (Fig. 11). These data confirmed the results obtained by western blot analyses seen in Fig. 7.

NeuN is a neuronal nuclear marker used to index neuronal viability (Fig. 11). The extent of NeuN immunostaining in HC was significantly decreased in the DM2 group (18 cells ± 6.3) compared to the control group (89 cells ± 3.8). However, Semaglutide (70 cells ± 3.8) and Fluoxetine (47 cells ± 7) treatment significantly reverted this effect in diabetic mice (Fig. 11).

5.10 Semaglutide treatment preserves colonic integrity in DM2 mice and restores the GLP-1R levels

Gut microbiota dysbiosis is positively associated with intestinal barrier damage and endotoxemia. Therefore, epithelium tight junction proteins were examined in response to Semaglutide treatment. We found by immunofluorescence analyses that colonic occludin levels decreased in DM2 mice (15,622 ± 4,084) compared to the control group (80,867 ± 6,822), while semaglutide treatment increased colonic mucosal occludin (78,143 ± 4,955) (p < 0.05), indicative of enhanced tight junctions of colonic epithelial tissues (Fig. 12A).

Here, we also observed that the diabetic animals had significantly lower levels of gut GLP-1 expression 28.14% ± 2.14) than the control group. Interestingly, treatment with Semaglutide (96% ± 16) and Fluoxetine (76% ± 7.3) significantly restores the GLP-1 levels (Fig. 12B).

5.11 Semaglutide treatment attenuates the gut microbiota dysbiosis induced by HF diet

Our previous study has shown that a chronic HF diet led to microbial dysbiosis regarding composition (de Paiva et al. 2023). It is unknown if Semaglutide could prevent HF diet-induced microbiota shift. We performed the gut microbiota profiles after a long-term HF diet with or without Semaglutide for 6 weeks by 16S rRNA sequencing. The four groups had a significant difference in bacterial diversity (Exponential Shannon, Richness, and reciprocal Simpson indices – Fig. 13A-C). Besides, beta diversity analyses (principal coordinate analysis – PcoA – Fig. 13D) demonstrated that gut microbiota of the DM2 + Semaglutide group exhibited significant differences in species diversity, composition, and abundance. In terms of phylum analyses, an increase in the mean proportion of Bacteroidetes and a decrease in Firmicutes was observed in DM2 mice compared to control mice. In contrast, Semaglutide treatment reverted these alterations, enhancing Bacteriodetes and decreasing Firmicutes (Fig. 14A-C).

The semaglutide intervention increased the relative abundance of the genera Bacteriodes and Blautia compared to DM2 and control mice (Fig. 15). At the species level, DM2 mice showed an reduced of the relative abundance of Bacteroides acidifaciens, and the Semaglutide treatment enhanced Bacteroides acidifaciens and Blautia coccoides compared to all groups (Fig. 16).

Animal models aid in understanding the various stages of type 2 diabetes mellitus (DM2) development and elucidating the genetic factors associated with insulin-sensitive tissues, revealing the underlying molecular mechanisms of disease progression (Kottaisamy et al. 2021). To induce pre-DM, several animal models have been established, such as high-fat/high-calorie rodent diets and streptozotocin (STZ)-injected models fed a high-fat diet (HFD) (Islam; Venkatesan., 2016). According to Lee et al. ((Lee et al. 2022), experimentally induced progressive stages of type 2 diabetes mellitus by HFD are classified as 6 weeks (pre-DM), 12 weeks (DM2), and 20 weeks (advanced-stage DM2). The present study utilized an 18-week induction model of HFD, considered an advanced stage of DM2. In its turn, weight loss is one of the most important therapeutic goals and is associated with improvements in all cardiovascular risk factors, including cholesterol, triglycerides, and blood pressure, as well as enhanced cardiac function and overall prognosis (ESC 2021) (Siqueira et al. 2007). GLP1-RAs were the first hypoglycemic medications to induce weight loss due to their pleiotropic actions, which include central nervous system interactions with reward circuits and food intake (Harder et al. 2004). The effect of semaglutide has been shown to be superior to that of dulaglutide (SUSTAIN 7) and liraglutide (SUSTAIN 10) in therapeutic doses for type 2 diabetes mellitus (Capehorn et al. 2020). As shown in our study, serum lipid levels, such as TG and cholesterol, increased in the DM2 group fed a high-calorie diet. Previous studies have highlighted an association between DM2 and elevated TG levels (Zhao et al. 2019). High TG levels indicate DM2 as an indirect sign of insulin resistance, which is also responsible for the development of non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) (Caturano et al. 2021). Furthermore, serum glucose levels were increased in DM mice. In contrast, treatment with Semaglutide improved the lipid and glycemic profile in DM2 mice. Previous studies have demonstrated that decreased serum glucose and insulin levels reduce lipogenic gene expression and serum lipid levels, decreasing the risk of developing DM2 (Di Folco et al. 2022).

Currently, the pathogenesis of depression includes not only alterations in monoaminergic transmission but also endocrine and immune disturbances, particularly excessive activation of pro-inflammatory cytokines and insulin resistance in the central nervous system (CNS). Accordingly, DM2 is not only a risk factor for the development of cardiovascular diseases but also neurological and psychiatric disorders such as depression. Multiple investigations have revealed shared pathogenic mechanisms between diabetes and neurodegenerative and mood disorders (Gault and Hölscher 2018). The prevalence of type 2 diabetes mellitus is on the rise, resulting in an increased focus on diabetes-related cognitive dysfunction and depressive-like behavior. The impairment of hippocampal structure and function lies at the core of neurological symptoms associated with type 2 diabetes. Factors such as hyperglycemia-induced insulin resistance, reduced synaptic plasticity, glutamatergic signaling system oxidative stress, reactive gliosis, and inflammatory response contribute to changes in hippocampal structure and function (Lyra e Silva et al. 2019)

Multiple studies have highlighted the neuroprotective effects of GLP-1-mediated signaling. It has been demonstrated that GLP-1 can protect synapses, enhance synaptic function, promote neurogenesis, mitigate oxidative stress in neurons, and reduce neuronal apoptosis. These findings have led to extensive research into the potential use of GLP-1R agonists to treat neurodegenerative disease (Long-Smith et al. 2013) Additionally, there are indications from a few studies that these compounds may also hold promise for treating neuropsychiatric disorders, including depression (Kim et al. 2020). To evaluate the effect of a 6-week treatment with Semaglutide on depression and anxiety-like behavior, mice were subjected to the FST, TST, and EPM. We observed a significant decrease in immobility time in both tests following treatment compared to untreated animals. A similar study showed that administration of liraglutide alleviates depressive and anxiety-like behaviors in a mouse model of depression induced by chronic administration of corticosterone, preventing corticosterone-induced impairments and simultaneously increasing the level of phosphorylated GSK3β in the hippocampus (Weina et al. 2018).

It has been demonstrated that peripheral pro-inflammatory cytokines are capable of crossing the blood-brain barrier (BBB) through distinct saturable transport systems and, therefore, can act in the central nervous system (CNS). Moreover, studies involving a high-fat diet in mice have shown increased brain cytokine levels associated with obesity (Felger and Lotrich 2013). Certain cytokines, such as IL-1β and TNF-α, are involved in the pathogenesis of depression (Deverman and Patterson 2009; Pace and Miller 2009). Our previous study also confirmed that reducing IL-1β, IL-6, and TNF-α levels in the hippocampus alleviated learning and memory deficits in obese mice (Paiva et al., 2023). Additionally, the innate immune response mediated by TLR4 is associated with neuroinflammation and represents an important pathway for the pathogenesis of depression (Wang et al. 2013; Obadia et al. 2022). Upon ligand binding to TLR4, various signaling transduction pathways are activated, among which the TLR4/NF-κB axis is the most important. This signaling can be activated through the MyD88-dependent signaling pathway, and its activation can lead to downstream production of inflammatory cytokines such as TNF-α and IL-6, exacerbating neuroinflammation. High-fat and high-sugar diets are associated with increased levels of LPS, which activates the TLR4 signaling pathway contributing to oxidative stress and inflammation (Beutler 2000). GLP-1, as well as its therapeutic analogs, have been shown to decrease levels of inflammatory markers in peripheral tissues and brain cells (Shan et al. 2019). Specifically, Semaglutide was able to significantly reduce glial activation, as well as the expression of NF-κB and pro-inflammatory cytokines (IL-1β, IL-6, IL-18) (Simó et al. 2017). Similarly, here we showed that diet-induced diabetic animals presented elevated levels of TLR4, MyD88, p-NFKB, TNF-α, IL-6, IL-1β, and nitrotyrosine. However, GLP-1R activation by semaglutide can reduce neuroinflammation by decreasing the activation of the TLR4/NF-κB pathway.

It is possible that insulin resistance, combined with the pro-inflammatory environment, inhibits insulin signaling in the CNS, thereby exacerbating the pathogenesis of anxiety and depression and the cognitive deficits observed in diabetic patients. The regulation of insulin in anxiety and depression-like behavior has been widely reported. An increasing body of research has revealed that the stimuli associated with the PI3K/AKT signaling pathway protect hippocampal and cortical neurons while inhibiting microglia activation. As a result, these stimuli play a significant role in preventing and treating mood disorders (Zou et al. 2020). Besides, the phosphorylation of IRS-1 induced by INSR promotes the activation of the PI3K pathway, whereas the phosphorylation of IRS-1 induced by IGF-1R leads to a preference for activating the MAPK pathway. It is important to note that mTOR is the primary downstream target of the PI3K/AKT signal transduction and represents a crucial regulatory factor in cellular metabolism (Long et al. 2021). Here, we observed that diabetic animals showed a decrease in insulin receptors and, consequently, in activating the PI3K/AKT pathway. However, treatment with Semaglutide improved the INSR/PI3K/AKT signaling, improving mood and cognition deficits caused by DM2. Similarly, previous studies have shown that obese or diabetic mice present a decrease in INSR activation, leading to impaired learning, memory, and cognition (Xie et al. 2019; Oliveira et al. 2021; de Paiva et al. 2023). Other studies demonstrated that exenatide and liraglutide reduce the levels of IRS-1pS616 and IRS-1pS36 (putative biomarkers of neuronal insulin resistance in AD) in the diabetes model, resulting not only in the facilitation of insulin signaling but also in the restoration of normal tissue responses to insulin, leading to improvements in AD pathology and functional improvements in cognition (Moloney et al. 2010).

Indeed, in addition to the processes described above, it has been well-established that neurotransmitters play a crucial role in regulating behavior (Liu et al. 2018). There is a clear relationship between the imbalance of neurotransmitters and specific symptoms of depressive and anxiety disorders. Biological evidence suggests that MDD results from changes in 5-HT-mediated activity in various brain regions, including the ventral hippocampus (HPC) and frontal cortex (Hamon and Blier 2013). Diabetes and brain insulin signaling have been associated with modifications of the 5-HT system. For example, the destruction of insulin-secreting beta cells in the pancreas of rats led to a significant reduction in brain 5-HT levels (Zemdegs et al. 2016). Interestingly, the present results showed an increase in the 5-HT and its receptor (5-HTR) after Semaglutide treatment. Another study showed that rats receiving intracerebroventricular administration of exendin-4 had a reduction in 5-HT and its metabolite, 5-HIAA, while administration of GLP-1R antagonists increased brain levels of serotonin and its metabolites (Owji et al. 2002); however, these animals were normoglycemic and not diabetic. The regulation of serotonin levels is a complex process influenced by various factors, including GLP-1 signaling (Mietlicki-Baase et al. 2014). Further research is needed to fully understand the intricate interactions and effects of GLP-1 on neurotransmitter systems.

On the other hand, a growing body of evidence implicates a role for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in major depression. Alterations in AMPA and other ionotropic glutamate receptors have been reported in depression and after antidepressant treatment (Bleakman et al. 2008). Compounds that enhance signaling through AMPA receptors (AMPA receptor potentiators) exhibit antidepressant-like behavioral effects in animal models and produce neuronal results similar to those produced by currently available antidepressants, including induction of neurotrophins and increased proliferation of hippocampal progenitor cells (Bleakman et al. 2008). Besides AMPA receptors, the metabotropic glutamate receptors or the co-agonist glycine site of NMDA receptors may be promising candidates for anxiolytic therapy (Freudenberg et al. 2015). Knocking out the insulin receptor leads to a reduction in the NMDA receptor subunit phosphorylation, as well as downstream targets such as PI3K and AKT (Maqsood and Stone 2016).

Physiopathological changes in the intestine have been reported in clinical trials and studies in various animal models of type 2 diabetes (Ding et al. 2020). Even though the current understanding of the GLP-1-mediated brain-gut axis regulation in the gastrointestinal (GI) system is limited, GLP-1 has considerable importance as an essential component of intestinal function. GLP-1R agonists have been found to improve intestinal epithelial integrity, reduce increased intestinal permeability, and activate Brunner's glands, which release mucins into the intestinal lumen to create a barrier against pathogens and stomach acids (Hunt et al. 2021). Furthermore, Grunddal et al., described that in vivo administration of GLP-1 agonists stimulates mucin production (Holst et al. 2022). Loss of GLP-1R signaling exacerbates the effects of dextran sulfate sodium-induced intestinal damage. A recent study on human jejunal L cells revealed dysregulation of enteroendocrine differentiation markers associated with lower L cell density in individuals with obesity and type 2 diabetes, resulting in a decreased density of GLP-1-positive cells and potentially reducing the capacity to produce GLP-1 in response to food (Osinski et al. 2021). Additionally, the lack of GLP-1R signaling in GLP-1R knockout mice (GLP-1R KO) worsens the intestinal damage effect (Yusta et al. 2015). Here, we demonstrate that semaglutide administration for six weeks could significantly reduce intestinal permeability in diabetic mice, indicating that GLP-1R activation contributed to intestinal homeostasis.

In addition to the secretion of incretin hormones such as GLP-1 and GIP (Wang et al. 2020), the intestine harbors the largest microbial population in the human body, containing over 100 trillion microbial cells with 3.3 million intestinal microbial genes, collectively known as the microbiome. The gut microbiota plays essential roles and significantly contributes to human health and disease. It has been demonstrated that dysbiosis alters intestinal barrier permeability and increases the secretion of metabolic endotoxins, contributing to various diseases, including obesity and type 2 diabetes, as well as playing a role in the development of neuropsychiatric and neurodegenerative diseases (Slyepchenko et al. 2017). GLP-1R agonists have been shown in clinical and preclinical trials to induce changes in the gut microbiota (Liu et al. 2018; Madsen et al. 2019). GLP-1R agonists affect the composition of the microbiota by altering gastric emptying rate and timing, as well as the intestinal lumen's internal environment, including local pH levels and nutrient availability (Montandon and Jornayvaz 2017). Through alpha and beta diversity analysis, we demonstrated that semaglutide treatment led to a clear separation of the diabetic mice's microbiota compared to the other groups, indicating that semaglutide treatment altered the fecal microbiota composition. A similar study showed that in obese and type 2 diabetic/obese rat models, liraglutide administration significantly increased the Bacteroidetes/Firmicutes ratio and reduced the abundance of obesity-related Families, such as Romboutsia, Ruminiclostridium, and Erysipelotrichaceae, while increasing lean meat-related genus, such as Prevotella (Zhao et al. 2018).

The relative abundance analysis also revealed different compositions and levels of phyla, genera, and species among the experimental groups. Treatment with Semaglutide significantly increased the abundance of Bacteroidetes and decreased that of Firmicutes. The phyla Firmicutes and Bacteroidetes together constitute over 80% of the total gut microbiota and comprise more than 100 bacterial species (Kedees et al. 2013). Although the importance of the Firmicutes to Bacteroidetes ratio (F/B ratio) remains inconclusive, the proportion of Firmicutes is significantly increased in patients with type 2 diabetes compared to healthy individuals (Craciun et al. 2022). Bacteroides acidifaciens is a gram-negative anaerobic commensal bacterium known for its growth in bile acids. Its induction is of interest because this strain has the potential for treating metabolic diseases such as diabetes and obesity. Mice fed with this bacterium have shown elevated serum insulin levels, accompanied by an increase in glucagon-like peptide-1 (GLP-1) and a decrease in intestinal dipeptidyl peptidase-4, an enzyme that degrades GLP-1 (Wang et al. 2022). In this study, we found that B. acidifaciens levels were increased in Semaglutide-treated mice with type 2 diabetes, suggesting that Semaglutide significantly increases the population of intestinal microorganisms associated with leanness. We also observed that diabetic animals treated with semaglutide showed higher levels of Blautia coccoides than the other groups. Blautia is a genus of anaerobic bacteria with probiotic characteristics widely present in the mammalian intestine (Liu et al. 2015a, 2021). Moreover, there is a disparity in the association of this genus with human diseases, with fewer Blautia observed in individuals with diabetes/obesity (Ozato et al. 2019). Similarly,(Aoki et al. (2021) observed that Bacteroides acidifaciens and Blautia producta colonization in germ-free mice synergized acetate production from inulin. Furthermore, the absence of GPR43 abolished the protective effect of inulin in non-alcoholic fatty liver disease in HFD-fed mice.

The gut microbiota plays a role in health and disease by producing short-chain fatty acids (SCFAs). SCFAs, such as acetate, propionate, and butyrate, are primary byproducts synthesized by the gut microbiota in the large intestine through anaerobic fermentation of indigestible polysaccharides found in dietary fibers (Silva et al. 2020). These SCFAs have multiple functions in intestinal homeostasis by activating G protein-coupled receptors (GPCRs) 41 and 43 (also known as free fatty acid receptors - FFAR-2 and 4). GPR43 is abundantly expressed in numerous gastrointestinal tract cells where they modulate the intestinal permeability by increasing tight junctions proteins, such as zonnula occludens and occludin (Ang and Ding 2016).

SCFAs can enter the systemic circulation as signaling molecules that affect the host's metabolism (Tang and Li 2021). Within the CNS, SCFAs play a role in influencing neuroinflammation by affecting the structure and function of glial cells, promoting the generation of new neurons, regulating neurotrophic factor levels, contributing to serotonin production, and enhancing overall neuronal stability and function (Razazan et al. 2021). These effects can be achieved through the activation GPR41 and 43 (Fock and Parnova 2023), however, their function in neuronal cells is not well-known. In the brain, GPR43 mediates the anti-inflammatory effects of SCFAs, as demonstrated in neuropathological mouse models such as sepsis-associated encephalopathy, perioperative neurocognitive disorder, and Alzheimer's disease (Ang and Ding 2016; Razazan et al. 2021). Interestingly, B. coccoides and B. acidifaciens produce acetate, reducing obesity by regulating hepatic GPR41/43 levels (Liu et al. 2015b; Yang et al. 2016). In this study, we observed that diabetic animals treated with semaglutide were able to increase the expression of this receptor in hippocampal POMC + neurons of the dentate gyrus, suggesting that it may play a significant role in neuron proliferation and survival. Overall, the interaction between SCFAs and the gut-brain pathways can directly or indirectly affect emotions, cognition, and the development of brain-related disorders.

The antidiabetic drug Semaglutide can potentially function as a therapeutic approach to treating depression and anxiety. This potential stems from its ability to decrease hippocampal neuroinflammation and increase neurogenesis through the insulin/GLP-1 pathway and modulation of the gut microbiota. Semaglutide is widely used and considered safe for individuals without diabetes. However, a clinical trial is needed to investigate the impact of semaglutide in depressed and anxious diabetic patients. Additional research is required to compare GLP-1 mimetics with existing depression treatments and to clarify the underlying biochemical mechanisms in greater depth.

Data Availability

The data that support the findings of this study are available from the corresponding author (Peixoto C.A.) upon request.

Funding

This work was supported by the Research Excellence Program-Instituto Aggeu Magalhães (IAM-PROEP#400208/2019-9), the Knowledge Generation Program of the Fundação Oswaldo Cruz (FIOCRUZ; #VPPCB-007-FIO-18-2-17), the Institute of Science and Technology of Neuroimmunomodulation (INCT- NIM; #465489/2014-1) and the National Council for Scientific and Technological Development (CNPq; #301777/2012-8). This study was partially funded by the Coordination for the Improvement of Higher Education Personnel-Brazil (CAPES).

Contributions

I.H.R.P. and C.P. conceived the study and design the experiment. I.H.R.P. performed the main experimental work, analyzed the data, created the figures and drafted the manuscript. All other authors performed experiments, and J.R.B.S performed the statistical analysis. C.P. extensively and critically reviewed the manuscript. All authors accepted the final version of the paper.

ETHICS DECLARATIONS

Ethics Approval and Consent to Participate

All experiments were conducted by the Ethical Principles in Animal Experimentation and were accepted by the Ethics Committee on the Use of Animals of the Aggeu Magalhães Institute (CEUA 135/2018-IAM).

Consent for Publication

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing Interests

The authors declare that they have no competing interests.

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Semaglutide Attenuates Anxious And Depressive-Like Behaviors and Reverses The Cognitive Impairment in a Type 2 Diabetes Mellitus Via The Microbiota-Gut-Brain Axis

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Abstract

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1. INTRODUCTION

2. MATERIALS AND METHODS

16S rRNA gene sequence-based microbiota analysis

RESULTS

5.5 Diabetic animals show increased expression of pro-inflammatory cytokines.

5.6 Diabetic animals show dysregulation of neurotransmitter receptors and brain GLP-1R

5.7 Effect Semaglutide treatment reduced gliosis in the hippocampus in diabetic mice

5.8 Semaglutide treatment stimulates PI3K/AKT signaling in the hippocampus of DM2 mice

5.9 Semaglutide treatment reverses changes in GPR43, POMC, GLP1-R and NeuN hipocamppal neurons induced by DM2

5.10 Semaglutide treatment preserves colonic integrity in DM2 mice and restores the GLP-1R levels

5.11 Semaglutide treatment attenuates the gut microbiota dysbiosis induced by HF diet

DISCUSSION

CONCLUSION

Declarations

References

Additional Declarations

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