Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2 methods
Aim. Evidence-backed execution summary for Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2 methods from Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Colonic tissue and cell preparation.
reagent used in the protocol.
- Use
- Mice aged 6-26 weeks were killed by cervical dislocation, and colons were collected in ice-cold Leibovitz-15 medium. Cultures for secretion and calcium imaging experiments were prepared as previously described ( ). Colonic tissue used for RNA and protein extraction was washed in PBS and placed in RNA later or...
Colonic tissue and cell preparation.
reagent used in the protocol.
- Use
- GLUTag cells were cultured in Dulbecco's modified Eagle's medium (5.5 mmol/L glucose) supplemented with 10% FBS, 2 mmol/L L -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin.
RESEARCH DESIGN AND METHODS
reagent used in the protocol.
- Use
- SCFAs raise intracellular calcium in identified colonic L cells. A: Mixed colonic cultures were loaded with fura2-AM. Pseudocolor images of fura2 340:380 nm fluorescence ratio (reflecting [Ca 2+ ] i ) shown prior to (basal) and during the application of propionate (1 mmol/L), and after washing with saline. B: Iden...
Glucose-stimulated GLP-1 secretion in vivo.
reagent used in the protocol.
- Use
- Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in body weight were observed between the wild-type and knockout groups. Mice were fasted for 4 h and dosed per os with 20 mg/kg DPP4 inhibitor...
RNA extraction and quantitative PCR.
reagent used in the protocol.
- Use
- Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion). Total RNA from GLUTag cells and murine colonic tissue was prepared following the Tri-Reagent protocol (Sigma). All samples were reverse tr...
Colonic protein analysis.
reagent used in the protocol.
- Use
- Tissue was mechanically homogenized in lysis buffer. Active GLP-1 was assessed by ELISA (Millipore, Watford, U.K.) and expressed relative to total protein content, measured using a Bradford assay (Sigma).
Secretion from primary mixed colonic culture.
reagent used in the protocol.
- Use
- Secretion studies were performed 24-36 h after culture preparation. Where applicable, cultures were preincubated with 0.2 µg/mL pertussis toxin for 18 h. Cultures were washed with standard saline and incubated with test substances for 2 h at 37°C. Secreted and cellular GLP-1 was extracted as previous...
Ca 2+ imaging.
reagent used in the protocol.
- Use
- Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 mi...
RNA extraction and quantitative PCR.
Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion). Total RNA from GLUTag cells and murine colonic tissue was prepared following the Tri-Reagent protocol (Sigma). All samples were reverse tr...
- Use
- Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion). Total RNA from GLUTag cells and murine colonic tissue was prepared following the Tri-Reagent protocol (Sigma). All samples were reverse tr...
Ca 2+ imaging.
Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 mi...
- Use
- Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 mi...
ffar2 and ffar3 expression is enriched in primary L cells.
Expression of ffar2 and ffar3 was investigated by quantitative RT-PCR using mRNA from FACS-sorted intestinal L cells from transgenic GLU-Venus mice ( ). Nonfluorescent cells collected in parallel were used to represent the mixed non- L- cell population. As shown in, ffar2 and ffar3 were expressed in small int...
- Use
- Expression of ffar2 and ffar3 was investigated by quantitative RT-PCR using mRNA from FACS-sorted intestinal L cells from transgenic GLU-Venus mice ( ). Nonfluorescent cells collected in parallel were used to represent the mixed non- L- cell population. As shown in, ffar2 and ffar3 were expressed in small int...
ffar2 and ffar3 expression is enriched in primary L cells.
SCFA receptors FFAR2 and FFAR3 are expressed in L cells. Relative expression of ffar2 ( A ) and ffar3 ( B ) mRNAs relative to β-actin assessed by RT-PCR in FACS-sorted L cells and non-L cells from the small intestine (L + and L -, respectively) and colon (LC + and LC - ) and the GLUTag model...
- Use
- SCFA receptors FFAR2 and FFAR3 are expressed in L cells. Relative expression of ffar2 ( A ) and ffar3 ( B ) mRNAs relative to β-actin assessed by RT-PCR in FACS-sorted L cells and non-L cells from the small intestine (L + and L -, respectively) and colon (LC + and LC - ) and the GLUTag model...
Data analysis.
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Comparisons between conditions were made using Student t test (Microsoft Excel) or by one- or two-way ANOVA with post hoc Bonferroni correction or Dunnett test (Prism5; GraphPad), as indicated, with a threshold for significance of P < 0.05. All data are expressed as means ± SEM.
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Colonic tissue and cell preparation.
Mice aged 6-26 weeks were killed by cervical dislocation, and colons were collected in ice-cold Leibovitz-15 medium. Cultures for secretion and calcium imaging experiments were prepared as previously described ( ). Colonic tissue used for RNA and protein extraction was washed in PBS and placed in RNA later or a protein lysis buffer, respectively, and frozen until processed.
Colonic tissue and cell preparation.
GLUTag cells were cultured in Dulbecco's modified Eagle's medium (5.5 mmol/L glucose) supplemented with 10% FBS, 2 mmol/L L -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin.
Glucose-stimulated GLP-1 secretion in vivo.
Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in body weight were observed between the wild-type and knockout groups. Mice were fasted for 4 h and dosed per os with 20 mg/kg DPP4 inhibitor (cat. no. KR-62436; Sigma). Thirty minutes later, mice were dosed by gavage with 1.5 g/kg glucose solution (Fisher Scientific). For initial GLP-1 measurement, 150 µL blood was collected from awake mice via tail bleed into EDTA-coated capillary tubes (Bilbate) before glucose dosing. Thirty minutes after glucose dosing, mice were killed by CO 2 inhalation and blood was collected via cardiac puncture into tubes containing aprotinin (0.6 trypsin inhibiting units/mL). Blood samples were centrifuged immediately, and plasma was frozen on dry ice before assay in duplicate for...
Ca 2+ imaging.
Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 min at 37°C. Single-cell imaging was performed using an inverted fluorescence microscope (Olympus IX71; Southall, U.K.) with a 40 × oil-immersion objective. Fura2 was excited at 340/6 and 380/4 nm and Venus at 475/10 nm, using a 75-W xenon arc lamp and a monochromator (Cairn Research, Faversham, U.K.) controlled by MetaFluor software (Molecular Devices, Wokingham, U.K.). Emission was recorded with an Orca ER CCD camera (Hamamatsu, Welwyn Garden City, U.K.) using a dichroic mirror and a 510-nm long-pass filter. Test substances were added to the bath solution and perfu...
FFAR2 and FFAR3 affect GLP-1 release in vivo.
ffar2 and ffar3 knockout mice have impaired glucose tolerance. A: Glucose stimulated GLP-1 secretion in vivo. ffar2 -/- and ffar3 -/- mice and wild-type littermates ( n = 5 each) were dosed with DPP4 inhibitor at a dose of 20 mg/kg per os after a 4-h fast. Thirty minutes post-DPP4 inhibitor dosing, mice were dosed with 1.5 g/kg glucose per os Plasma active GLP-1 was assessed by a MesoScale assay at 0 and 30 min of the oral glucose tolerance test. Data represent means ± 1 SEM, and statistical significance was assessed by Student t test: * P < 0.05 and ** P < 0.01. B - E: Oral glucose tolerance test in ffar2 -/- mice ( n = 8) ( left panel ) and wild-type littermates ( n = 11) ( B and D ) or ffar3 -/- mice ( n = 7) ( right panel ) and wild-type littermates ( n = 6) ( C and E ). Following an overnight fast, mice were given...
Measurement outputs
What raw and processed outputs should exist?
For generation of the targeting vectors, homology arms ( ffar3, 1,250 base pairs [bp] 5′ and 3,914 bp 3′; ffar2, 1,761 bp 5′ and 4,199 bp 3′) were PCR...
- 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
Three- to four-month-old ffar2 -/-, ffar3 -/-, and wild-type littermate control 129/SvEv mice were fasted for 14 h and then dosed by gavage with 1.5 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
Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion)...
- 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
Tissue was mechanically homogenized in lysis buffer. Active GLP-1 was assessed by ELISA (Millipore, Watford, U.K.) and expressed relative to total protein content, measured usin...
- 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
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
SCFAs stimulate GLP-1 secretion.
from paperScoring or quantification
Quantify the primary readouts for this experiment: For generation of the targeting vectors, homology arms ( ffar3, 1,250 base pairs [bp] 5′ and 3,914 bp 3′; ffar2, 1,761 bp 5′ and 4,199 bp 3′) were PCR...; Three- to four-month-old ffar2 -/-, ffar3 -/-, and wild-type littermate control 129/SvEv mice were fasted for 14 h and then dosed by gavage with 1.5 g/...; Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion)...; Tissue was mechanically homogenized in lysis buffer. Active GLP-1 was assessed by ELISA (Millipore, Watford, U.K.) and expressed relative to total protein content, measured usin....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
SCFAs stimulate GLP-1 secretion. A: Acute stimulation of GLP-1 secretion. Mixed primary cultures from murine colon were incubated for 2 h in 10 mmol/L glucose (Con) or in the a...; SCFAs raise intracellular calcium in identified colonic L cells. A: Mixed colonic cultures were loaded with fura2-AM. Pseudocolor images of fura2 340:380 nm fluorescence ratio...; Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in...; Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion)...
from paperReporting output
Report representative outputs alongside summary comparisons for For generation of the targeting vectors, homology arms ( ffar3, 1,250 base pairs [bp] 5′ and 3,914 bp 3′; ffar2, 1,761 bp 5′ and 4,199 bp 3′) were PCR..., Three- to four-month-old ffar2 -/-, ffar3 -/-, and wild-type littermate control 129/SvEv mice were fasted for 14 h and then dosed by gavage with 1.5 g/..., Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion)..., Tissue was mechanically homogenized in lysis buffer. Active GLP-1 was assessed by ELISA (Millipore, Watford, U.K.) and expressed relative to total protein content, measured usin....
inferred from protocolStructured statistical methods
SCFAs stimulate GLP-1 secretion. A: Acute stimulation of GLP-1 secretion. Mixed primary cultures from murine colon were incubated for 2 h in 10 mmol/L glucose (Con) or in the a...; SCFAs raise intracellular calcium in identified colonic L cells. A: Mixed colonic cultures were loaded with fura2-AM. Pseudocolor images of fura2 340:380 nm fluorescence ratio...; Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in...; Total RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice ( ) was isolated using a microscale RNA isolation kit (Ambion)...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Mice aged 6-26 weeks were killed by cervical dislocation, and colons were collected in ice-cold Leibovitz-15 medium. Cultures for secretion and calcium imaging experiments were prepared as previously described ( ). Colonic tissue used for RNA and protein extraction was washed in PBS and placed in RNA later or a protein lysis buffer, respectively, and frozen until processed.
GLUTag cells were cultured in Dulbecco's modified Eagle's medium (5.5 mmol/L glucose) supplemented with 10% FBS, 2 mmol/L L -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin.
Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in body weight were observed between the wild-type and knockout groups. Mice were fasted for 4 h and dosed per os with 20 mg/kg DPP4 inhibitor (cat. no. KR-62436; Sigma). Thirty minutes later, mice were dosed by gavage with 1.5 g/kg glucose solution (Fisher Scientific). For initial GLP-1 measurement, 150 µL blood was collected from awake mice via tail bleed into EDTA-coated capillary tubes (Bilbate) before glucose dosing. Thirty minutes after glucose dosing, mice were killed by CO 2 inhalation and blood was collected via cardiac puncture into tubes containing aprotinin (0.6 trypsin inhibiting units/mL). Blood samples were centrifuged immediately, and plasma was frozen on dry ice before assay in duplicate for active GLP-1 (MesoScale, Gaithersburg, Maryland).
Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 min at 37°C. Single-cell imaging was performed using an inverted fluorescence microscope (Olympus IX71; Southall, U.K.) with a 40 × oil-immersion objective. Fura2 was excited at 340/6 and 380/4 nm and Venus at 475/10 nm, using a 75-W xenon arc lamp and a monochromator (Cairn Research, Faversham, U.K.) controlled by MetaFluor software (Molecular Devices, Wokingham, U.K.). Emission was recorded with an Orca ER CCD camera (Hamamatsu, Welwyn Garden City, U.K.) using a dichroic mirror and a 510-nm long-pass filter. Test substances were added to the bath solution and perfused at ∼1 mL/min. Fura2 fluorescence measurements were taken every 2 s, background corrected, and expressed as the 340:380 nm ratio. Average fluorescence ratios from individual cells were determined over 20-s periods before addition and during perfusion of a test agent. Peak responses to a tes...
ffar2 and ffar3 knockout mice have impaired glucose tolerance. A: Glucose stimulated GLP-1 secretion in vivo. ffar2 -/- and ffar3 -/- mice and wild-type littermates ( n = 5 each) were dosed with DPP4 inhibitor at a dose of 20 mg/kg per os after a 4-h fast. Thirty minutes post-DPP4 inhibitor dosing, mice were dosed with 1.5 g/kg glucose per os Plasma active GLP-1 was assessed by a MesoScale assay at 0 and 30 min of the oral glucose tolerance test. Data represent means ± 1 SEM, and statistical significance was assessed by Student t test: * P < 0.05 and ** P < 0.01. B - E: Oral glucose tolerance test in ffar2 -/- mice ( n = 8) ( left panel ) and wild-type littermates ( n = 11) ( B and D ) or ffar3 -/- mice ( n = 7) ( right panel ) and wild-type littermates ( n = 6) ( C and E ). Following an overnight fast, mice were given 1.5 g/kg glucose per os, and blood glucose ( B and C ) and insulin ( D and E ) were measured at the time points indicated. F and G: Insulin tolerance test in ffar2 -/- mice ( n = 11) ( left panel ) and wild-type littermates ( n = 6) ( F ) or in ffar3 -/- mice ( n = 7) ( rig...
Machine-readable layer
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