Gut microbiota mediates intermittent-fasting alleviation of diabetes-induced cognitive impairment methods
Aim. Evidence-backed execution summary for Gut microbiota mediates intermittent-fasting alleviation of diabetes-induced cognitive impairment methods from Gut microbiota mediates intermittent-fasting alleviation of diabetes-induced cognitive impairment.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- The second set of mice (same grouping with first set, n = 10-11) were treated with the same IF regimen schedule and then were killed to collect the serum, hippocampus, gut, and fecal samples for multi-OMICS study. Fecal, serum, and hippocampus samples were collected to analyze gut microbiome, meta...
Methods
reagent used in the protocol.
- Use
- For the first set animals, the db/m mice and the db/db mice were divided in two subgroups ( n = 10), including AL feeding (db/db) and IF (db/db-IF), wherein the IF mice were deprived of food for 24 h, every other day and were fed ad libitum on the intervening day for 28 days (Fig. ). Bodywei...
Methods
reagent used in the protocol.
- Use
- After IF regimen, the antibiotics treatment, and metabolites treatment, the cognitive behavioral assessment was determined with a water-maze test. After behavioral tests, mice were killed, then the serum and tissues samples were collected by either snap-frozen by liquid nitrogen and store at -80 °C...
Electron microscopy for the structural analysis of the hippocampus
reagent used in the protocol.
- Use
- A transmission electron microscope (TEM) analysis was done after the collection of CA1 region of hippocampus. The hippocampus was split and treated in a cold fixative solution made of 2.5% glutaraldehyde (pH 7.2) at 4 °C for 4 h. After washing with PBS (0.1 M, pH 7.2) thrice. Then the specimen...
qRT-PCR
reagent used in the protocol.
- Use
- The total RNA extracted from frozen tissues using TRIzol reagent (Jingcai Bio., Xi'an, Shaanxi, China) was determined and reverse transcribed for real-time PCR. Relative mRNA expression was quantified using SYBR Green dye (TB Green Premix Ex Taq II) and specific primers. Real-time PCR was carried out in a CFX9...
16S rRNA Microbiome sequencing
reagent used in the protocol.
- Use
- Fecal samples were collected from respective groups either at the beginning or before the behavioral tests. The total cellular DNA was extracted with the E.Z.N.A. Stool DNA Kit (Omega) according to the company instructions. The bacterial hypervariable V3-V4 region of 16S rRNA was chosen for MiSeq (Illumina, CA...
Results
Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control mice, the food/energy intake was higher in db/db mice (Fig. ) (ANOVA with Tukey's test, p < 0.01). IF decreased the...
- Use
- Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control mice, the food/energy intake was higher in db/db mice (Fig. ) (ANOVA with Tukey's test, p < 0.01). IF decreased the...
Gut microbiota are required for the beneficial effects of IF
An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the drinking water starting 14 days before the 4-week IF regimen and throughout the experiment, and the behavioral changes were assessed after...
- Use
- An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the drinking water starting 14 days before the 4-week IF regimen and throughout the experiment, and the behavioral changes were assessed after...
Methods
The third set of animals were divided into eight groups, including db/m ( n = 10), db/m-IF ( n = 10), db/m-antibiotics ( n = 10), db/m-antibiotics-IF ( n = 10), db/db ( n = 7), db/db-IF ( n = 13), db/db-antibiotics ( n = 7), d...
- Use
- The third set of animals were divided into eight groups, including db/m ( n = 10), db/m-IF ( n = 10), db/m-antibiotics ( n = 10), db/m-antibiotics-IF ( n = 10), db/db ( n = 7), db/db-IF ( n = 13), db/db-antibiotics ( n = 7), d...
Methods
After IF regimen, the antibiotics treatment, and metabolites treatment, the cognitive behavioral assessment was determined with a water-maze test. After behavioral tests, mice were killed, then the serum and tissues samples were collected by either snap-frozen by liquid nitrogen and store at -80 °C...
- Use
- After IF regimen, the antibiotics treatment, and metabolites treatment, the cognitive behavioral assessment was determined with a water-maze test. After behavioral tests, mice were killed, then the serum and tissues samples were collected by either snap-frozen by liquid nitrogen and store at -80 °C...
Electron microscopy for the structural analysis of the hippocampus
A transmission electron microscope (TEM) analysis was done after the collection of CA1 region of hippocampus. The hippocampus was split and treated in a cold fixative solution made of 2.5% glutaraldehyde (pH 7.2) at 4 °C for 4 h. After washing with PBS (0.1 M, pH 7.2) thrice. Then the specimen...
- Use
- A transmission electron microscope (TEM) analysis was done after the collection of CA1 region of hippocampus. The hippocampus was split and treated in a cold fixative solution made of 2.5% glutaraldehyde (pH 7.2) at 4 °C for 4 h. After washing with PBS (0.1 M, pH 7.2) thrice. Then the specimen...
qRT-PCR
The total RNA extracted from frozen tissues using TRIzol reagent (Jingcai Bio., Xi'an, Shaanxi, China) was determined and reverse transcribed for real-time PCR. Relative mRNA expression was quantified using SYBR Green dye (TB Green Premix Ex Taq II) and specific primers. Real-time PCR was carried out in a CFX9...
- Use
- The total RNA extracted from frozen tissues using TRIzol reagent (Jingcai Bio., Xi'an, Shaanxi, China) was determined and reverse transcribed for real-time PCR. Relative mRNA expression was quantified using SYBR Green dye (TB Green Premix Ex Taq II) and specific primers. Real-time PCR was carried out in a CFX9...
Integrated multi-OMICs analysis on IF regimen
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Having identified different omics signatures that were upregulated or downregulated by IF in diabetic mice, the next step was to assess the interplay across gene expressions in the hippocampus (Fig. ), gut microbiota compositions (Fig. ), and microbial metabolites (Fig. ), in relation to the observ...
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Results
Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control mice, the food/energy intake was higher in db/db mice (Fig. ) (ANOVA with Tukey's test, p < 0.01). IF decreased the bodyweight of db/db mice (Fig. ) (ANOVA with Tukey's test, p < 0.01) without changing their total energy intake (Fig. ). In addition, IF treatment substantially reduced water intake per day in db/db mice (Supplementary Fig. ) (ANOVA with Tukey's test, p < 0.01) with less urine at the end of the treatment (Supplementary Fig. ). Fig. 1 Intermittent fasting alleviated insulin resistance and cognitive impairment in db/db mice. a Timeline depicting the diet of IF or ad libitum in each group; b bodyweight ( n =&...
Methods
The second set of mice (same grouping with first set, n = 10-11) were treated with the same IF regimen schedule and then were killed to collect the serum, hippocampus, gut, and fecal samples for multi-OMICS study. Fecal, serum, and hippocampus samples were collected to analyze gut microbiome, metabolome, and RNA sequencing, respectively.
Gut microbiota are required for the beneficial effects of IF
An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the drinking water starting 14 days before the 4-week IF regimen and throughout the experiment, and the behavioral changes were assessed after 28 days (Supplementary Fig. ). The antibiotics treatment weakened the weight loss effects of the IF treatment (Fig. ) (ANOVA with Tukey's test, p < 0.05). The antibiotics treatment had no impact on the eWAT weight, but enhanced the liver and cecum weight (Supplementary Fig. ). Both IF and antibiotics treatment reduced food and water intake in db/db mice (Supplementary Fig. ). However, the administration of antibiotics had no impact on the beneficial effects of IF on insulin resistance (Supplementary Fig. ). Of note, the cogni...
Methods
Male BKS.Cg- Dock7 m + / + Lepr db /J (stock no: 000642) Homozygous Lepr db/db mice were diabetic, and heterozygous Lepr db/m mice were used as controls (denoted as db/db and db/m in the text) in this study. All mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the Northwest A&F University animal facility under standard conditions with a strict 12 h:12 h light:dark cycle, humidity at 50 ± 15%, temperature 22 ± 2 °C. The animals were fed ad libitum before the IF was initiated at 4 months of age. Mice were fed a regular chow (AIN-93M, purchased from TROPHIC Animal Feed High-tech Co. Ltd, Nantong, China) and pure water.
Methods
For the first set animals, the db/m mice and the db/db mice were divided in two subgroups ( n = 10), including AL feeding (db/db) and IF (db/db-IF), wherein the IF mice were deprived of food for 24 h, every other day and were fed ad libitum on the intervening day for 28 days (Fig. ). Bodyweight, food intake, and water consumption were recorded on fasting day. These animals performed behavioral tests, and then were killed to collect serum and tissues.
Methods
The third set of animals were divided into eight groups, including db/m ( n = 10), db/m-IF ( n = 10), db/m-antibiotics ( n = 10), db/m-antibiotics-IF ( n = 10), db/db ( n = 7), db/db-IF ( n = 13), db/db-antibiotics ( n = 7), db/db-antibiotics-IF ( n = 13). The different sample size was due to requirement to distinguish the impact of IF on feeding and fasting day. The IF regimen schedule was the same as the previously mentioned sets. The antibiotics cocktail (Penicillin G sodium 0.4 g L -1, metronidazole 0.4 g L -1, neomycin sulfate 0.4 g L -1, streptomycin sulfate 0.4 g L -1, vancomycin hydrochloride 0.25 g L -1 for db/db mice, half concentration of same antibiotics for db/m mice) were give...
Methods
The fourth set of animals were divided into six groups ( n = 8), including db/m, db/db, db/db-IPA, db/db-5-HT, db/db-TUDCA, db/db-SCFAs. For db/db-IPA, db/db-5-HT, db/db-TUDCA groups, mice were intraperitoneally injected with IPA (10 mg kg -1 d -1 ), 5-HT (1 mg kg -1 d -1 ), and TUDCA (250 mg kg -1 d -1 ) dissolved in saline, respectively. The mice in other groups were injected with same volume saline. For the SCFAs group, the SCFAs (acetate 67.5 mM, propionate 40 mM, butyric acid 25 mM) were dissolved in the drinking water of the mice. The mice were treated for 14 days. The treatment was also performed during the behavioral tests.
qRT-PCR
The total RNA extracted from frozen tissues using TRIzol reagent (Jingcai Bio., Xi'an, Shaanxi, China) was determined and reverse transcribed for real-time PCR. Relative mRNA expression was quantified using SYBR Green dye (TB Green Premix Ex Taq II) and specific primers. Real-time PCR was carried out in a CFX96TM real-time system (Bio-Rad). The following conditions were used: 95 °C for 10 min, then 95 °C for 15 s, and 60 °C for 1 min in 40 cycles. The 2 -ΔΔCT method was used to analyze the relative changes in gene expression. For the mitochondrial biogenesis, the total DNA extracted from brain tissue using a DNA extraction kit (Bioteke Co., Beijing, China) were also determined by real-time PCR. The mitochondria number were indicated by the lower ratio of mitochondrial DNA (mtDNA, COX2) to nuclear DNA (nDNA, globi...
Measurement outputs
What raw and processed outputs should exist?
Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control m...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Having identified different omics signatures that were upregulated or downregulated by IF in diabetic mice, the next step was to assess the interplay across gene expressions in...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The third set of animals were divided into eight groups, including db/m ( n = 10), db/m-IF ( n = 10), db/m-antibiotics ( n = 10), db/m-ant...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control m...; Having identified different omics signatures that were upregulated or downregulated by IF in diabetic mice, the next step was to assess the interplay across gene expressions in...; An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the...; The third set of animals were divided into eight groups, including db/m ( n = 10), db/m-IF ( n = 10), db/m-antibiotics ( n = 10), db/m-ant....
from paperStatistical comparison
Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control m...; Having identified different omics signatures that were upregulated or downregulated by IF in diabetic mice, the next step was to assess the interplay across gene expressions in...; An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the...
from paperReporting output
Report representative outputs alongside summary comparisons for Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control m..., Having identified different omics signatures that were upregulated or downregulated by IF in diabetic mice, the next step was to assess the interplay across gene expressions in..., An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the..., The third set of animals were divided into eight groups, including db/m ( n = 10), db/m-IF ( n = 10), db/m-antibiotics ( n = 10), db/m-ant....
inferred from protocolStructured statistical methods
Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control m...; Having identified different omics signatures that were upregulated or downregulated by IF in diabetic mice, the next step was to assess the interplay across gene expressions in...; An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Three-month-old db/db mice were fed either an ad libitum diet or fasted at 24 h intervals for 28 days (Fig. ). Compared with the db/m group, the littermate control mice, the food/energy intake was higher in db/db mice (Fig. ) (ANOVA with Tukey's test, p < 0.01). IF decreased the bodyweight of db/db mice (Fig. ) (ANOVA with Tukey's test, p < 0.01) without changing their total energy intake (Fig. ). In addition, IF treatment substantially reduced water intake per day in db/db mice (Supplementary Fig. ) (ANOVA with Tukey's test, p < 0.01) with less urine at the end of the treatment (Supplementary Fig. ). Fig. 1 Intermittent fasting alleviated insulin resistance and cognitive impairment in db/db mice. a Timeline depicting the diet of IF or ad libitum in each group; b bodyweight ( n = 10 mice per group); c energy intake; d food intake; e insulin-tolerance test; f fasting glucose; g fasting insulin level ( n = 7 mice per group); h HOMA-IR ( n = 7 mice per group). The animals' cognitive functions were assessed by the Morris water-maze test as d...
The second set of mice (same grouping with first set, n = 10-11) were treated with the same IF regimen schedule and then were killed to collect the serum, hippocampus, gut, and fecal samples for multi-OMICS study. Fecal, serum, and hippocampus samples were collected to analyze gut microbiome, metabolome, and RNA sequencing, respectively.
An antibiotics cocktail treatment was applied to establish the role of the microbiota in the neuroprotection afforded by IF. The mice were administrated with antibiotics in the drinking water starting 14 days before the 4-week IF regimen and throughout the experiment, and the behavioral changes were assessed after 28 days (Supplementary Fig. ). The antibiotics treatment weakened the weight loss effects of the IF treatment (Fig. ) (ANOVA with Tukey's test, p < 0.05). The antibiotics treatment had no impact on the eWAT weight, but enhanced the liver and cecum weight (Supplementary Fig. ). Both IF and antibiotics treatment reduced food and water intake in db/db mice (Supplementary Fig. ). However, the administration of antibiotics had no impact on the beneficial effects of IF on insulin resistance (Supplementary Fig. ). Of note, the cognition improvement assessed using the Morris water-maze test associated with IF was partly abolished by antibiotics treatment, with longer escape times on the 5th day of the test and lower times in the target quadrant on the probe test day (Supplementary Fig.; Fig. ). Moreover, mitochondr...
Male BKS.Cg- Dock7 m + / + Lepr db /J (stock no: 000642) Homozygous Lepr db/db mice were diabetic, and heterozygous Lepr db/m mice were used as controls (denoted as db/db and db/m in the text) in this study. All mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the Northwest A&F University animal facility under standard conditions with a strict 12 h:12 h light:dark cycle, humidity at 50 ± 15%, temperature 22 ± 2 °C. The animals were fed ad libitum before the IF was initiated at 4 months of age. Mice were fed a regular chow (AIN-93M, purchased from TROPHIC Animal Feed High-tech Co. Ltd, Nantong, China) and pure water.
For the first set animals, the db/m mice and the db/db mice were divided in two subgroups ( n = 10), including AL feeding (db/db) and IF (db/db-IF), wherein the IF mice were deprived of food for 24 h, every other day and were fed ad libitum on the intervening day for 28 days (Fig. ). Bodyweight, food intake, and water consumption were recorded on fasting day. These animals performed behavioral tests, and then were killed to collect serum and tissues.
The third set of animals were divided into eight groups, including db/m ( n = 10), db/m-IF ( n = 10), db/m-antibiotics ( n = 10), db/m-antibiotics-IF ( n = 10), db/db ( n = 7), db/db-IF ( n = 13), db/db-antibiotics ( n = 7), db/db-antibiotics-IF ( n = 13). The different sample size was due to requirement to distinguish the impact of IF on feeding and fasting day. The IF regimen schedule was the same as the previously mentioned sets. The antibiotics cocktail (Penicillin G sodium 0.4 g L -1, metronidazole 0.4 g L -1, neomycin sulfate 0.4 g L -1, streptomycin sulfate 0.4 g L -1, vancomycin hydrochloride 0.25 g L -1 for db/db mice, half concentration of same antibiotics for db/m mice) were given in the drinking water starting 14 days before the IF regimen and throughout the experiment (Fig. ). The 16S rRNA copies in the feces of the animals after antibiotics treatment were detected with qPCR (Fig. ), as previous described. All the behavior studies and biochemical samples were...
The fourth set of animals were divided into six groups ( n = 8), including db/m, db/db, db/db-IPA, db/db-5-HT, db/db-TUDCA, db/db-SCFAs. For db/db-IPA, db/db-5-HT, db/db-TUDCA groups, mice were intraperitoneally injected with IPA (10 mg kg -1 d -1 ), 5-HT (1 mg kg -1 d -1 ), and TUDCA (250 mg kg -1 d -1 ) dissolved in saline, respectively. The mice in other groups were injected with same volume saline. For the SCFAs group, the SCFAs (acetate 67.5 mM, propionate 40 mM, butyric acid 25 mM) were dissolved in the drinking water of the mice. The mice were treated for 14 days. The treatment was also performed during the behavioral tests.
The total RNA extracted from frozen tissues using TRIzol reagent (Jingcai Bio., Xi'an, Shaanxi, China) was determined and reverse transcribed for real-time PCR. Relative mRNA expression was quantified using SYBR Green dye (TB Green Premix Ex Taq II) and specific primers. Real-time PCR was carried out in a CFX96TM real-time system (Bio-Rad). The following conditions were used: 95 °C for 10 min, then 95 °C for 15 s, and 60 °C for 1 min in 40 cycles. The 2 -ΔΔCT method was used to analyze the relative changes in gene expression. For the mitochondrial biogenesis, the total DNA extracted from brain tissue using a DNA extraction kit (Bioteke Co., Beijing, China) were also determined by real-time PCR. The mitochondria number were indicated by the lower ratio of mitochondrial DNA (mtDNA, COX2) to nuclear DNA (nDNA, globin). Reagent information and primer sequences are shown in Supplementary Table.
Machine-readable layer
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"headline": "Gut microbiota mediates intermittent-fasting alleviation of diabetes-induced cognitive impairment",
"datePublished": "2020",
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