Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice methods
Aim. Evidence-backed execution summary for Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice methods from Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice.
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What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- Eighty-four male C57BL/6JOlaHsd mice (Harlan Laboratories, Horst, The Netherlands) were individually housed under controlled conditions (temperature 21 °C, 12 h/12 h light-dark cycle, 55 ± 15 % humidity), with ad libitum access to food and water. At arrival, the mic...
HPLC analysis of flavonoid levels in serum and diet
reagent used in the protocol.
- Use
- Flavonoid levels in serum were measured using HPLC with coulometric array detection as described (Hoek-van den Hil et al. ). Anthocyanins could not be detected by our method. Before analysis, samples were hydrolysed by β-glucuronidase/sulphatase to obtain deconjugated flavonoids, resulting in total flavonoid le...
Lipid determination in serum and liver
reagent used in the protocol.
- Use
- Because flavonoids were previously shown to interfere with commonly used commercially available enzymatic lipid assays (Hoek-van den Hil et al. ), alternative methods were used to measure the amount of lipids in serum and liver, as described (Hoek-van den Hil et al. ). Serum lipids were extracted and analysed with 1...
RT-qPCR
reagent used in the protocol.
- Use
- RNA from liver was isolated using RNeasy columns (Qiagen, Venlo, The Netherlands), RNA from epiWAT was extracted with Trizol (Invitrogen, Breda, The Netherlands), and quality was verified [as published (Hoek-van den Hil et al.; Hoek-van den Hil et al. )]. RT-qPCR was performed and analysed as described [6]. Data we...
Serum leptin levels
reagent used in the protocol.
- Use
- Serum leptin levels were determined using a leptin ELISA kit (Crystal Chem Inc., Chicago, IL, USA) according to the manufacturer's instructions.
Statistical analysis
reagent used in the protocol.
- Use
- All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurement...
Serum lipids
reagent used in the protocol.
- Use
- HF significantly induced higher levels of 'MUFA and PUFA', 'PUFA', '18:2 fatty acids', '18:1 and 16:1 fatty acids' and 'omega 3 fatty acids' in serum compared with NF. No significant differences between HF and HF + flavonoid were observed in the s...
White adipose tissue and leptin
reagent used in the protocol.
- Use
- HF feeding significantly induced relative mesWAT and epiWAT weights compared with NF feeding. Supplementation with the flavonoids prevented this induction of relative mesWAT weight, which was significantly lower for HF + quercetin, HF + hesperetin and HF + anthocyanins compared with HF...
Indirect calorimetric and activity measurements
Indirect calorimetry and activity were measured in weeks 1, 5 and 11. Indirect calorimetry was performed by an open-circuit LabMaster Metabolism Research Platform (TSE systems GmbH, Bad Homburg, Germany) and analysed as described previously (Hoevenaars et al. ) with minor adaptations. A reference cage was measured,...
- Use
- Indirect calorimetry and activity were measured in weeks 1, 5 and 11. Indirect calorimetry was performed by an open-circuit LabMaster Metabolism Research Platform (TSE systems GmbH, Bad Homburg, Germany) and analysed as described previously (Hoevenaars et al. ) with minor adaptations. A reference cage was measured,...
Motor performance
Balance and motor coordination was assessed by Rotarod (IITC Life Science, Woodland Hills, USA) in week 9. Latency to fall was recorded on an accelerating rod (3-38 rpm in 300 s); mice were placed on the rod four times with an inter trial rest period of 30 min; and the average of two longest run...
- Use
- Balance and motor coordination was assessed by Rotarod (IITC Life Science, Woodland Hills, USA) in week 9. Latency to fall was recorded on an accelerating rod (3-38 rpm in 300 s); mice were placed on the rod four times with an inter trial rest period of 30 min; and the average of two longest run...
Motor performance
Several parameters of gait were assessed in week 10 by CatWalk analysis (Noldus Information Technology, Wageningen, The Netherlands) using the reflection of light projected on a glass walking area. Each mouse made at least six compliant runs, being defined as a maximum speed variation of 40 %, minimum run durat...
- Use
- Several parameters of gait were assessed in week 10 by CatWalk analysis (Noldus Information Technology, Wageningen, The Netherlands) using the reflection of light projected on a glass walking area. Each mouse made at least six compliant runs, being defined as a maximum speed variation of 40 %, minimum run durat...
Histology of epididymal white adipose tissue
Paraffin-embedded epididymal white adipose tissue (epiWAT) was cut into 5-µm sections and stained using Periodic Acid Schiff Haematoxylin (PASH). Per animal, circumference of at least 400 adipocytes was measured using AxioVision software v4.8 (Carl Zeiss Microscopy GmbH, Jena, Germany). To estimate macrophage i...
- Use
- Paraffin-embedded epididymal white adipose tissue (epiWAT) was cut into 5-µm sections and stained using Periodic Acid Schiff Haematoxylin (PASH). Per animal, circumference of at least 400 adipocytes was measured using AxioVision software v4.8 (Carl Zeiss Microscopy GmbH, Jena, Germany). To estimate macrophage i...
Statistical analysis
All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurement...
- Use
- All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurement...
Motor performance
HF mice performed significantly poorer on the Rotarod than NF mice, likely due to their higher body weight. Performance on Rotarod was, however, not significantly different between any HF + flavonoid group and HF (Supplementary Figure S2). There were also no significant differences between the HF +...
- Use
- HF mice performed significantly poorer on the Rotarod than NF mice, likely due to their higher body weight. Performance on Rotarod was, however, not significantly different between any HF + flavonoid group and HF (Supplementary Figure S2). There were also no significant differences between the HF +...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurement...
Correlations
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Pearson correlations between all parameters measured showed significant correlations between body weight gain and all parameters for HF and all HF + flavonoid groups (Fig. a), except for the indirect calorimetry measurements (RER and EE). Strongest correlation was found between body weight gain and l...
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Methods
Eighty-four male C57BL/6JOlaHsd mice (Harlan Laboratories, Horst, The Netherlands) were individually housed under controlled conditions (temperature 21 °C, 12 h/12 h light-dark cycle, 55 ± 15 % humidity), with ad libitum access to food and water. At arrival, the mice were 9 weeks of age. During the first 5 days of a 3-week adaptation period, mice were fed a standard Harlan chow diet, followed by a standardised semi-synthetic normal-fat diet [NF, 10 energy% (en%) fat] with the same dietary constituents as the intervention high-fat diet (HF, 40 en%) in which carbohydrates were substituted with fats (Hoevenaars et al. ) (Research Diets Services B.V., Wijk bij Duurstede, The Netherlands). At the start of the 12-week intervention period, mice were stratified based on body weight over 7 groups ( n = 12), to obtain ident...
Motor performance
Several parameters of gait were assessed in week 10 by CatWalk analysis (Noldus Information Technology, Wageningen, The Netherlands) using the reflection of light projected on a glass walking area. Each mouse made at least six compliant runs, being defined as a maximum speed variation of 40 %, minimum run duration of 0.5 s and maximum run duration of 10 s. Quantitative gait parameters were analysed using the CatWalk XT 10.0 software (Noldus Information Technology).
Lipid determination in serum and liver
Because flavonoids were previously shown to interfere with commonly used commercially available enzymatic lipid assays (Hoek-van den Hil et al. ), alternative methods were used to measure the amount of lipids in serum and liver, as described (Hoek-van den Hil et al. ). Serum lipids were extracted and analysed with 1 H nuclear magnetic resonance ( 1 H-NMR), and neutral lipids were stained in frozen liver sections with Oil red O (Sigma) and quantified.
Statistical analysis
All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurements ( n = 6), subsets of mice as indicated between the brackets were randomly selected, because of limited equipment or practical reasons. GraphPad Prism version 5.03 (GraphPad software, San Diego, CA, USA) was used for statistical analysis. Data were checked for normality and if needed log transformed ( Acot3, Cyp4a14, Por, Fasn and Cyp2b9 in liver and Cpt1a in EpiWAT). One-way ANOVA was used to compare the groups, followed by Dunnett's post hoc test to compare the different HF + flavonoid groups to HF, and HF to NF. For the analysis of data of...
Measurement outputs
What raw and processed outputs should exist?
## Indicates significant difference of HF to NF ( p < 0.01), ### p < 0.001, ** indicates a significant difference of HF + flavonoid to HF (...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Cyp2b9, a target gene of the transcription factor Constitutive Androgen Receptor (CAR also known as NR1I3), was strongly upregulated by HF versus NF. Interestingly, this gene w...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Flavonoid levels in serum were measured using HPLC with coulometric array detection as described (Hoek-van den Hil et al. ). Anthocyanins could not be detected by our method. Be...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Indirect calorimetry and activity were measured in weeks 1, 5 and 11. Indirect calorimetry was performed by an open-circuit LabMaster Metabolism Research Platform (TSE systems G...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
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
## Indicates significant difference of HF to NF ( p < 0.01), ### p < 0.001, ** indicates a significant difference of HF + flavonoid to HF ( p < 0.01), *** p < 0.001 Body weight was significantly increased due to HF compared with NF feeding during the whole intervention period, with a cumulative body weight gain of HF mice being four times higher than the weight gain of NF mice (Fig. ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: ## Indicates significant difference of HF to NF ( p < 0.01), ### p < 0.001, ** indicates a significant difference of HF + flavonoid to HF (...; Cyp2b9, a target gene of the transcription factor Constitutive Androgen Receptor (CAR also known as NR1I3), was strongly upregulated by HF versus NF. Interestingly, this gene w...; Flavonoid levels in serum were measured using HPLC with coulometric array detection as described (Hoek-van den Hil et al. ). Anthocyanins could not be detected by our method. Be...; Indirect calorimetry and activity were measured in weeks 1, 5 and 11. Indirect calorimetry was performed by an open-circuit LabMaster Metabolism Research Platform (TSE systems G....
from paperStatistical comparison
## Indicates significant difference of HF to NF ( p < 0.01), ### p < 0.001, ** indicates a significant difference of HF + flavonoid to HF (...; Body weight was significantly increased due to HF compared with NF feeding during the whole intervention period, with a cumulative body weight gain of HF mice being four times h...; All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measure...; Respiratory exchange ratio (RER) and EE were not significantly different between any HF + flavonoid group and HF in week 11, the 24-h patterns are shown in Fig....
from paperReporting output
Report representative outputs alongside summary comparisons for ## Indicates significant difference of HF to NF ( p < 0.01), ### p < 0.001, ** indicates a significant difference of HF + flavonoid to HF (..., Cyp2b9, a target gene of the transcription factor Constitutive Androgen Receptor (CAR also known as NR1I3), was strongly upregulated by HF versus NF. Interestingly, this gene w..., Flavonoid levels in serum were measured using HPLC with coulometric array detection as described (Hoek-van den Hil et al. ). Anthocyanins could not be detected by our method. Be..., Indirect calorimetry and activity were measured in weeks 1, 5 and 11. Indirect calorimetry was performed by an open-circuit LabMaster Metabolism Research Platform (TSE systems G....
inferred from protocolStructured statistical methods
## Indicates significant difference of HF to NF ( p < 0.01), ### p < 0.001, ** indicates a significant difference of HF + flavonoid to HF (...; Body weight was significantly increased due to HF compared with NF feeding during the whole intervention period, with a cumulative body weight gain of HF mice being four times h...; All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measure...; Respiratory exchange ratio (RER) and EE were not significantly different between any HF + flavonoid group and HF in week 11, the 24-h patterns are shown in Fig....
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
Eighty-four male C57BL/6JOlaHsd mice (Harlan Laboratories, Horst, The Netherlands) were individually housed under controlled conditions (temperature 21 °C, 12 h/12 h light-dark cycle, 55 ± 15 % humidity), with ad libitum access to food and water. At arrival, the mice were 9 weeks of age. During the first 5 days of a 3-week adaptation period, mice were fed a standard Harlan chow diet, followed by a standardised semi-synthetic normal-fat diet [NF, 10 energy% (en%) fat] with the same dietary constituents as the intervention high-fat diet (HF, 40 en%) in which carbohydrates were substituted with fats (Hoevenaars et al. ) (Research Diets Services B.V., Wijk bij Duurstede, The Netherlands). At the start of the 12-week intervention period, mice were stratified based on body weight over 7 groups ( n = 12), to obtain identical groups for this important parameter. Male mice were used in order to enable comparison of outcomes with previous findings (Hoek-van den Hil et al. ). One group of mice continued on NF, while the other six groups of mice received HF with or without supplementation of different flavonoids (HF...
Several parameters of gait were assessed in week 10 by CatWalk analysis (Noldus Information Technology, Wageningen, The Netherlands) using the reflection of light projected on a glass walking area. Each mouse made at least six compliant runs, being defined as a maximum speed variation of 40 %, minimum run duration of 0.5 s and maximum run duration of 10 s. Quantitative gait parameters were analysed using the CatWalk XT 10.0 software (Noldus Information Technology).
Because flavonoids were previously shown to interfere with commonly used commercially available enzymatic lipid assays (Hoek-van den Hil et al. ), alternative methods were used to measure the amount of lipids in serum and liver, as described (Hoek-van den Hil et al. ). Serum lipids were extracted and analysed with 1 H nuclear magnetic resonance ( 1 H-NMR), and neutral lipids were stained in frozen liver sections with Oil red O (Sigma) and quantified.
All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurements ( n = 6), subsets of mice as indicated between the brackets were randomly selected, because of limited equipment or practical reasons. GraphPad Prism version 5.03 (GraphPad software, San Diego, CA, USA) was used for statistical analysis. Data were checked for normality and if needed log transformed ( Acot3, Cyp4a14, Por, Fasn and Cyp2b9 in liver and Cpt1a in EpiWAT). One-way ANOVA was used to compare the groups, followed by Dunnett's post hoc test to compare the different HF + flavonoid groups to HF, and HF to NF. For the analysis of data of liver weight, hepatic gene expression of Acacb and Cpt1a, and WAT gene expression of Pparg, a Kruskal-Wallis test was used, because also after log transformation these data were not normally distributed. Curve fitting was used to analyse body weight gain during the 12-week intervention period...
Machine-readable layer
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