The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism methods
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Acetate reduces acute food intake
We went on to test whether acetate was itself an anorectic signal using acute peripheral administration in C57BL/6 mice. Intraperitoneal (i.p.) injection of acetate (500 mg kg -1 ) produced an increase in serum acetate ( ), similar to those that have been associated with suppression of free fatty acids, which was associated with a significant reduction in acute food intake at both 1 and 2 h post injection ( ). An acetate tolerance test demonstrated that there was no impact of i.p. acetate at 500 mg kg -1 body weight on blood glucose concentrations ( ). Acute behavioural observations post injection showed that this was also not associated with aversive behaviour ( ). Furthermore, at 60 min post injection, there were no significant differences in circulating concentrations of plasma PYY and GLP-1 ( ). We encapsulated acetate into liposomes. Us...
Methods
Animal experiments were performed at Imperial College London except for high resolution magic angle spinning (HMRS) experiments which were performed at Instituto de Investigaciones Biomédicas 'Alberto Sols' (IIB). Experiments conducted at Imperial College London were approved by Imperial College London, and all animal procedures were performed in accordance with the UK Animals Scientific Procedures Act (1986). IIB experiments were approved by the ethical committee of the Instituto de Investigaciones Biomedicas 'Alberto Sols' (and met the guidelines of the national (R.D. 53/2013) and the European Community (2010/62/UE) for care and management of experimental animals. Unless otherwise stated, all experiments were performed in C57BL/6 male mice (6-8 weeks old, Charles River, Margate, UK) that were single-housed under controlled temperature (21-23&...
Serum SCFA measurement
A 100-500-µl aliquot of serum was filtered through a 30-kDa micropartition system (Vivaspin RC VS02H22 filters, Sartorius Inc., Mississauga, ON, Canada) by centrifugation at 14,000 g at 4 °C for 90 min. An internal standard solution (25 µl) consisting of 100 mM ethylbutyrate and 100 mM formic acid was added to 225 µl of the protein-free filtrate supernatant in a 2-ml Hichrom vial (Agilent Technologies, South Queensferry, UK). To measure SCFA, 1 µl of each sample was injected into a 5,890 Series II GC system (HP, Crawley, UK) fitted with a NukolTM Capilllary Column (30 m × 0.53 mm × 1.0 µm, SUPELCOTM Analytical, UK) and flame ionization detector. The carrier gas, helium, was delivered at a flow rate of 14 ml min -1. The head pressure was set at 10 p.s.i. w...
Hypothalamic quantitative PCR
This was carried out using the methods described by Bewick et al. Quantitative reverse transcriptase PCR (RT-qPCR) was used to study the expression of the different target genes. Total RNA was extracted from the whole hypothalami using TRIZOL (Invitrogen) according to the manufacturer's protocol. All samples were treated by DNaseI (Invitrogen) before the reverse transcription. First-strand cDNAs were prepared using 1 µg RNA and SuperScriptII Reverse Transcriptase (Invitrogen) in the presence of random hexamer and oligo(dT) primers (Promega, Charbonnières-les-Bains, France). The qPCRs were performed using the Light Cycler Fast Start DNA Master SyBR Green I kit (Roche, Meylan, France) in the presence of specific primer pairs that were selected to amplify small fragments (100-200 bp). PCR products were checked for specificity by measuring their melting tempera...
Effect of i.c.v. acetate on energy intake
Male Wistar rats 190-240 g (specific pathogen free; Charles River) were placed in a stereotaxic frame (David Kopf Instruments) under 0.5-2.5% isoflurane anaesthesia. A hole was drilled using a stereotactically mounted electric drill using coordinates calculated from the Rat Brain Atlas as described previously, according to the coordinates of Paxinos and Watson (0.8 mm posterior to the Bregma in the midline and 6.5 mm below the surface of the skull). A permanent 22-gauge stainless steel cannula (Plastics One Inc., Roanoke, Virginia, USA) projecting to the third cerebral ventricle was implanted. Dental cement was used to hold the cannula in position anchored by three stainless steel screws inserted into the skull. The skin was approximated using nylon sutures. After a 7 day recovery period, animals were handled daily and acclimatized to the injection procedure....
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We went on to test whether acetate was itself an anorectic signal using acute peripheral administration in C57BL/6 mice. Intraperitoneal (i.p.) injection of acetate (500 mg kg -1 ) produced an increase in serum acetate ( ), similar to those that have been associated with suppression of free fatty acids, which was associated with a significant reduction in acute food intake at both 1 and 2 h post injection ( ). An acetate tolerance test demonstrated that there was no impact of i.p. acetate at 500 mg kg -1 body weight on blood glucose concentrations ( ). Acute behavioural observations post injection showed that this was also not associated with aversive behaviour ( ). Furthermore, at 60 min post injection, there were no significant differences in circulating concentrations of plasma PYY and GLP-1 ( ). We encapsulated acetate into liposomes. Using this method of peripheral administration of liposome-encapsulated acetate over a 4-h period, we did not detect liposomes in the hypothalamus or changes in food intake ( ). We therefore hypothesized that the anorectic effects of acetate may result from a direct effect on the central nervous syste...
Animal experiments were performed at Imperial College London except for high resolution magic angle spinning (HMRS) experiments which were performed at Instituto de Investigaciones Biomédicas 'Alberto Sols' (IIB). Experiments conducted at Imperial College London were approved by Imperial College London, and all animal procedures were performed in accordance with the UK Animals Scientific Procedures Act (1986). IIB experiments were approved by the ethical committee of the Instituto de Investigaciones Biomedicas 'Alberto Sols' (and met the guidelines of the national (R.D. 53/2013) and the European Community (2010/62/UE) for care and management of experimental animals. Unless otherwise stated, all experiments were performed in C57BL/6 male mice (6-8 weeks old, Charles River, Margate, UK) that were single-housed under controlled temperature (21-23 o C) and light conditions (12 h light-dark cycle; lights on at 07:00 hours).
A 100-500-µl aliquot of serum was filtered through a 30-kDa micropartition system (Vivaspin RC VS02H22 filters, Sartorius Inc., Mississauga, ON, Canada) by centrifugation at 14,000 g at 4 °C for 90 min. An internal standard solution (25 µl) consisting of 100 mM ethylbutyrate and 100 mM formic acid was added to 225 µl of the protein-free filtrate supernatant in a 2-ml Hichrom vial (Agilent Technologies, South Queensferry, UK). To measure SCFA, 1 µl of each sample was injected into a 5,890 Series II GC system (HP, Crawley, UK) fitted with a NukolTM Capilllary Column (30 m × 0.53 mm × 1.0 µm, SUPELCOTM Analytical, UK) and flame ionization detector. The carrier gas, helium, was delivered at a flow rate of 14 ml min -1. The head pressure was set at 10 p.s.i. with split injection. Run conditions were: initial temperature 60 °C, 1 min; +20 °C min -1 to 145 °C; +4 °C min -1 to 200 °C, hold 25 min. Peaks were integrated using Agilent ChemStation software (Agilent Techn...
This was carried out using the methods described by Bewick et al. Quantitative reverse transcriptase PCR (RT-qPCR) was used to study the expression of the different target genes. Total RNA was extracted from the whole hypothalami using TRIZOL (Invitrogen) according to the manufacturer's protocol. All samples were treated by DNaseI (Invitrogen) before the reverse transcription. First-strand cDNAs were prepared using 1 µg RNA and SuperScriptII Reverse Transcriptase (Invitrogen) in the presence of random hexamer and oligo(dT) primers (Promega, Charbonnières-les-Bains, France). The qPCRs were performed using the Light Cycler Fast Start DNA Master SyBR Green I kit (Roche, Meylan, France) in the presence of specific primer pairs that were selected to amplify small fragments (100-200 bp). PCR products were checked for specificity by measuring their melting temperature. Samples (in duplicate) were quantified by comparison with a standard curve obtained by dilutions of purified-specific cDNAs.
Male Wistar rats 190-240 g (specific pathogen free; Charles River) were placed in a stereotaxic frame (David Kopf Instruments) under 0.5-2.5% isoflurane anaesthesia. A hole was drilled using a stereotactically mounted electric drill using coordinates calculated from the Rat Brain Atlas as described previously, according to the coordinates of Paxinos and Watson (0.8 mm posterior to the Bregma in the midline and 6.5 mm below the surface of the skull). A permanent 22-gauge stainless steel cannula (Plastics One Inc., Roanoke, Virginia, USA) projecting to the third cerebral ventricle was implanted. Dental cement was used to hold the cannula in position anchored by three stainless steel screws inserted into the skull. The skin was approximated using nylon sutures. After a 7 day recovery period, animals were handled daily and acclimatized to the injection procedure. To ensure that cannulae were correctly positioned, rats received i.c.v. 150 ng angiotensin II in a 5-µl volume via a 28-gauge stainless steel injector placed into and projecting 1 mm below the tip of the cannula. Rats were observed for a prompt drinking response. A total volume of 5...
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