Microbiota fasting-related changes ameliorate cognitive decline in obesity and boost ex vivo microglial function through the gut-brain axis

Abstract

Background: Obesity-related cognitive decline is linked to gut microbiota dysbiosis, with emerging evidence suggesting that dietary interventions may ameliorate cognitive impairment via gut-brain axis modulation. The role of microglial cells in this process remains underexplored.

Objective: To investigate how diet-induced changes in gut microbiota influence cognitive function in individuals with obesity and their microglial activity, and to determine the impact of specific dietary interventions.

Design: This study included 96 participants with obesity who were randomised into three dietary intervention groups: Mediterranean diet (Med), alternate-day fasting (ADF) and ketogenic diet (Keto). Cognitive performance and microbiota composition were assessed pre-intervention and post-intervention. The effects of microbiota-related changes on microglial function were further evaluated in mice models through faecal transplantation and in vitro model with microbiota exosome treatment.

Results: Both the Keto and ADF groups demonstrated significant weight loss, but cognitive performance improved most notably in the ADF group, in association with reduced inflammation. Diet-related microbiota composition was correlated with the cognitive outcomes in the human study. Mice models confirmed that the cognitive benefits of ADF were microbiota-dependent and linked to enhanced microglial phagocytic capacity and reduced inflammation, accompanied by changes in microglia morphology.

Conclusion: Fasting-induced modifications in gut microbiota contribute to cognitive improvement in individuals with obesity, with microglial cells playing a crucial mediatory role. Among the interventions, ADF most effectively enhanced microglial function and cognitive performance, suggesting its potential as a therapeutic strategy for obesity-related cognitive decline. Further studies are required to fully elucidate the underlying mechanisms.

Trial registration number: NCT04453150.

Keywords: diet; metabolomics; microbiome; neurobiology; obesity.

Figure 1. Participant flow diagram. Ninety-six healthy participants with obesity (32 per group, aged 18–65 years, body mass index (BMI) 30–45 kg/m²) were randomised into 3 months diet intervention: Mediterranean (Med), Ketogenic (Keto) and alternate-day fasting (ADF). Blood tests, faecal sample collection and cognitive assessments were performed. L&N, letter and number; TMT, trail making test.

Figure 2. Diet intervention based on ketone body increase reduced body weight in people with obesity and improved their cognitive performance associated with microbiota changes. (A) Body weight changes (ΔBW) in kilograms (kg) were higher in the ketogenic (Keto) and alternate-day fasting (ADF) groups compared with the Mediterranean diet (Med). (B) Body mass index changes (ΔBMI) were higher in Keto and ADF groups compared with Med. (C) No changes were found in fat mass changes (kg) between groups. (D) Basal metabolism rate (kcal) was increased in ADF compared with Keto group. (E) Better improvement in the Stroop cognitive test (Δinterference) was found in ADF compared with Med group. (F) Better improvement in the letter and number (L&N) test (Δ score) was found in Keto and ADF compared with Med group. (G) Worse improvement in the trail making test A (TMT A) score (Δ score) was observed in the Keto group compared with the ADF and Med groups. No changes were found in task B of this cognitive test. (H) Significant changes in enriched and depleted bacteria genus due to diet intervention in the individuals who underwent the cognitive test evaluations (n=11–17). (I) Heatmap represents positive (red) or negative (blue) correlation between microbiota diet-related changes and cognitive scores. One-way analysis of variance was used for the A–D dataset (n=23–34). Cognitive performance was analysed by one-way analysis of covariance using body weight loss as a covariable in E–G (n=10–17). ANCOM-bc analysis was used for H data representing log fold change±SE. Sperman’s correlation test was run for heatmap representation in I. *P<0.05, **p<0.01.

Figure 3. Alternate-day fasting (ADF) intervention improved systemic inflammation in people with obesity and promoted better functionality in human monocyte-derived microglia-like (MDMi) cells. Different inflammatory markers linked to obesity were analysed in serum samples from participants under the different diet interventions (Med: Mediterranean diet; Keto: ketogenic diet; ADF). In addition, human MDMi cells isolated from the participant’s blood before (obese group) and after (ADF group) diet intervention were cultivated to corroborate the inflammatory state of the individuals. (A) Ferritin increment (Δ) in ng/mL after diet intervention was significantly lower in ADF compared with Keto group. (B) No significant changes were found in Δ resistin (pg/ml), although ADF showed a trend towards a decrease after the intervention. (C) A significant decrease in Δ monocyte chemoattractant protein-1 (MCP-1; pg/mL) was found in the ADF group compared with Med group. (D) Representative micrographs of MDMi timelapse studies for latex beads engulfment as a measure of phagocytic capacity prior to and after ADF intervention. (E) The greatest number of beads/cells was engulfed by the ADF group. (F) Higher levels of oxidative stress were shown by ADF group. (G, H) Increased motility capacity (displacement and speed) was found in the ADF group. (I) Representative micrographs of Iba-1 (microglial marker) and latex beads staining from MDMi participant cells before and after ADF. (J) Functional improvement by ADF intervention in MDMi travelled distance during the wound healing assay. Data are expressed as the mean (±SEM). One-way analysis of variance was used for the A–C dataset (n=9–22). The Student’s t-test was performed in the cellular study (E–H: n=60–200 cells from 3/6 different participants for single cell analysis; J: n=5 participants). *P<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 4. Human monocyte-derived microglia-like (MDMi) cells increase their phagocytic capacity in response to acute microbiota-related changes associated with obesity, while they exhibit functional collapse under chronic stimulation. Human MDMi cells, isolated from the healthy donors’ blood were pretreated (1 hour) or treated (24 hours) with microbiota-derived exosomes (10 µg/mL) from participants before the dietary intervention to characterise microglial activation and function. (A) Representative micrographs of MDMi timelapse studies for latex beads engulfment as a measure of phagocytic capacity under short-term (1 hour) microbiota obesity-exosome pretreatment. (B) The greatest number of beads/cells was engulfed by the pretreated group. (C) Higher levels of oxidative stress were shown by the pretreated group. (D, E) No differences were found regarding motility capacity. (F) Representative micrographs of Iba-1 (microglial marker) and latex beads staining from the two experimental groups (control vs treated: 24 hours treatment). (G, H) Functional impairment (beads engulfment and travelled distance during the wound healing assay, respectively) in MDMi cells due to chronic exposure (24 hours) to the exosome treatment. (I–K) Increase in cytokines (tumour necrosis factor alpha (TNF-α); interleukin 6 (IL-6); interleukin 1 beta (IL-1β)) release from MDMi cells after the exosome 24 hours treatment. (L) Battery of different genes related to microglial activation and phenotype. Only a significant decrease was found in TREM2 expression due to the chronic exposure (24 hours) to microbiota exosomes. Data are expressed as the mean (±SEM). The Student’s t-test was performed for statistical analysis (n=60–200 cells from 3/6 different participants for single cell analysis, n=3–6 for the rest of analysis). *P<0.05, **p<0.01,****p<0.001.

Figure 5. Microbiota changes associated with alternate-day fasting (ADF) ameliorate obesity-related cognitive decline by modulating hippocampal inflammation, neuronal activation and microglial morphology in healthy recipient mice. To directly assess the impact of these microbiota alterations on cognitive function and hippocampal physiology, faecal transplants were performed in healthy C57BL/6J mice using samples from human participants collected before (obese) and after the ADF intervention. (A, B) The novel object recognition (NOT) discrimination ratio (DR) was increased in ADF-recipient mice at short-term and long-term time points. (C, D) Higher hippocampal neuronal activation was shown by ADF-recipient mice. (E, F) Higher levels of interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) were shown by obese-recipient mice. (G) Immunoblots from pre-IL-1β, mature IL-1β and actin measurement. (H) Iba-1⁺ (microglial marker) cells and their densitometry showed an increase in the obese-recipient mice. (I) Representative micrographs of Iba-1 staining from the two experimental groups. (J) Mouse brain atlas (left) and representative micrographs showing DAPI staining. The analysed region in all immunohistochemistry assays—the dentate gyrus (DG)—is highlighted in orange. (K) Heatmap showing the different morphological parameters analysed in this study (Z-score). ADF-recipient mice differ completely from the obese-recipient group in terms of microglial morphology. (L) Representative three-dimensional microglial reconstruction from the two experimental groups. Data are expressed as the mean (±SEM). (M) Morphological parameters (soma size and number of branches per cell) in microglia from the two experimental groups. Obese-recipient mice showed larger soma size than ADF-recipient mice, while ADF-recipient mice showed a greater number of branches. The Student’s t-test was performed for statistical analysis (n=5–10). *P<0.05, **p<0.01, ****p<0.001.

Figure 6. Microglial overactivation due to microbiota obesity-related changes is associated with loss of function and microglial senescence. A faecal transplant from the human participant before (obese) and after alternate-day fasting intervention (ADF) was performed in healthy recipient mice (C57BL/6J) to better understand the direct effect of microbiota changes in microglial activation, function and state. (A) Representative micrographs of Iba-1 (microglial marker), Glut5 (glucose receptor 5), PFKFB3 (glycolytic enzyme) staining from the two experimental groups. (B) The greatest number of Glut5_PFKFB3_Iba-1⁺ cells were found in the obese-recipient mice, indicating a glycolytic state of microglial cells. (C) Representative micrographs of Iba-1, CD68 (lysosomal marker) and synaptophysin (Syn; presynaptic molecule) staining from the two experimental groups. (D) The greatest number of phagocytic microglia (Syn_CD68_Iba-1⁺ cells) were found in ADF-recipient mice. (E) Representative micrographs of Iba-1 and p16 (senescence marker) staining from the two experimental groups. (F) Higher number of senescent microglia (p16_Iba-1⁺ cells) were found in obese-recipient mice. (G) Representative micrographs of Iba-1 and ki67 (cell turnover marker) staining from the two experimental groups. (H) Higher number of ki67_Iba-1⁺ cells were found in ADF-recipient mice. Data are expressed as the mean (±SEM). The Student’s t-test was performed for statistical analysis (n=5). *P<0.05, ***p<0.005, ****p<0.001.

Figure 7. Metabolomic microbiota changes in response to the alternate-day fasting (ADF) intervention. Exosomes were isolated from the participants’ faecal samples before and after ADF intervention to analyse changes in different metabolites due to the diet. (A) Volcano plot showing the most significant differentiated metabolites due to the ADF intervention. (B) Upregulation of riboflavin and citrulline due to ADF intervention. (C) Upregulation of tryptophan, glutamine-alanine (Gln-Ala) and glycine-leucine (Gly-Leu) due to ADF intervention. (D) Threonine-threonine (Thr-Thr) and isoleucine-serine (Ile-Ser) were upregulated due to the ADF intervention. (E) N-epsilon-acetyllysine and thymidine were upregulated due to the ADF intervention. Data are expressed as the mean (±SEM). The Student’s t-test (paired) was performed for statistical analysis (n=9). *P<0.05, **p<0.01.