Conformations, vibrational frequencies and Raman intensities of short-chain fatty acid methyl esters using DFT with 6-31G(d) and Sadlej pVTZ basis sets

Density functional calculations, using B3LPY/6-31G(d) methods, have been used to investigate the conformations and vibrational (Raman) spectra of three short-chain fatty acid methyl esters (FAMEs) with the formula CnH2nO2 (n = 3-5). In all three FAMEs, the lowest energy conformer has a simple 'all-trans' structure but there are other conformers, with different torsions about the backbone, which lie reasonably close in energy to the global minimum. One result of this is that the solid samples we studied do not appear to consist entirely of the lowest energy conformer. Indeed, to account for the 'extra' bands that were observed in the Raman data but were not predicted for the all-trans conformer, it was necessary to add-in contributions from other conformers before a complete set of vibrational assignments could be made. Provided this was done, the agreement between experimental Raman frequencies and 6-31G(d) values (after scaling) was excellent, RSD = 12.6 cm(-1). However, the agreement between predicted and observed intensities was much less satisfactory. To confirm the validity of the approach followed by the 6-3 1 G(d) basis set, we used a larger basis set, Sadlej pVTZ, and found that these calculations gave accurate Raman intensities and simulated spectra (summed from two different conformers) that were in quantitative agreement with experiment. In addition, the unscaled Sadlej pVTZ, and the scaled 6-3 1 G(d) calculations gave the same vibrational mode assignments for all bands in the experimental data. This work provides the foundation for calculations on longer-chain FAMEs (which are closer to those found as triglycerides in edible fats and oils) because it shows that scaled 6-3 1 G(d) calculations give equally accurate frequency predictions, and the same vibrational mode assignments, as the much more CPU-expensive Sadlej pVTZ basis set calculations.

The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism

Increased intake of dietary carbohydrate that is fermented in the colon by the microbiota has been reported to decrease body weight, although the mechanism remains unclear. Here we use in vivo11C-acetate and PET-CT scanning to show that colonic acetate crosses the blood–brain barrier and is taken up by the brain. Intraperitoneal acetate results in appetite suppression and hypothalamic neuronal activation patterning. We also show that acetate administration is associated with activation of acetyl-CoA carboxylase and changes in the expression profiles of regulatory neuropeptides that favour appetite suppression. Furthermore, we demonstrate through 13C high-resolution magic-angle-spinning that 13C acetate from fermentation of 13C-labelled carbohydrate in the colon increases hypothalamic 13C acetate above baseline levels. Hypothalamic 13C acetate regionally increases the 13C labelling of the glutamate–glutamine and GABA neuroglial cycles, with hypothalamic 13C lactate reaching higher levels than the ‘remaining brain’. These observations suggest that acetate has a direct role in central appetite regulation.

Effect of Short-Chain Fatty Acid Acetate on Colon Cancer

Acetate is a short chain fatty acid produced as a result of fermentation of ingested fibers by the gut microbiota. While it has been shown to reduce cell proliferation in some cancer cell lines1,2, more recent studies on liver3 and brain4 tumours suggest that acetate may actually promote tumour growth. Acetate in the cell is normally converted into acetyl-coA by two enzymes and metabolized; mitochondrial (ACSS1) and cytosolic (ACSS2) acetyl-coA synthetase. In the mitochondria acetyl-coA is utilized in the TCA cycle. In the cytosol it is utilized in lipid synthesis. In this study, the effect of acetate treatment on the growth of HT29 colon cancer cell line and its mechanism of action was assessed. HT29 human colorectal adenocarcinoma cells were treated with 10mM NaAc and cell viability, cellular bioenergetics and gene expression were investigated. Cell viability was assessed 24 hours after treatment using an MTT assay (Sigma, UK, n=8). Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured by XFe Analyzer (Seahorse Bioscience, USA). After a baseline reading cells were treated and OCR and ECAR measurements were observed for 18 hours (n=4). Total mRNA was isolated 24 hours after treatment using RNeasy kit (Qiagen, USA). Quantitative PCR reactions were performed using Taqman gene expression assays and Taqman Universal PCR Master Mix (ThermoFisher Scientific, UK) on Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, USA) and analysed using ΔΔCt method (n=3). Acetate treatment led to a significant reduction in cell viability (15.9%, Figure 1). OCR, an indicator of oxidative phosphorylation, was significantly increased (p<0.0001) while ECAR, an indicator of glycolysis, was significantly reduced (p<0.0001, Figure 2). Gene expression of ACSS1 was increased by 1.7 fold of control (p=0.07) and ACSS2 expression was reduced to 0.6 fold of control (p=0.06, Figure 3). In conclusion, in colon cancer cells acetate supplementation induces cell death and increases oxidative capacity. These changes together with the trending decrease in ACSS2 expression suggest suppression of lipid synthesis pathways. We hypothesize that the reduced tumor growth by acetate is a consequence of the suppression of ACSS2 and lipid synthesis, both effects reported previously to reduce tumor growth3–5. These effects clearly warrant further investigation.

The type and quantity of dietary fat and carbohydrate alter faecal microbiome and short-chain fatty acid excretion in a metabolic syndrome ‘at-risk’ population syndrome.

An obese-type human microbiota with an increased Firmicutes:Bacteroidetes ratio has been described that may link the gut microbiome with obesity and metabolic syndrome (MetS) development. Dietary fat and carbohydrate are modifiable risk factors that may impact on MetS by altering the human microbiome composition. We determined the effect of the amount and type of dietary fat and carbohydrate on faecal bacteria and short chain fatty acid (SCFA) concentrations in people ‘at risk’ of MetS.

Biological significance of short-chain fatty acid metabolism by the intestinal microbiome

Purpose of review Evidence suggests that short-chain fatty acids (SCFAs) derived from microbial metabolism in the gut play a central role in host homeostasis. The present review describes the current understanding and physiological implications of SCFAs derived from microbial metabolism of nondigestible carbohydrates. Recent findings Recent studies indicate a role for SCFAs, in particular propionate and butyrate, in the metabolic and inflammatory disorders such as obesity, diabetes and inflammatory bowel diseases, through the activation of specific G-protein-coupled receptors and modification of transcription factors. Established prebiotics, such as fructooligosaccharides and galactooligosaccharides, which support the growth of Bifidobacteria, mainly mediate acetate production. Thus, recent identification of prebiotics which are able to stimulate the production of propionate and butyrate by benign saccharolytic populations in the colon is of interest. Summary Manipulation of saccharolytic fermentation by prebiotic substrates is beginning to provide information on structure–function relationships relating to the production of SCFAs, which have multiple roles in host homeostasis.

The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism

Increased intake of dietary carbohydrate that is fermented in the colon by the microbiota has been reported to decrease body weight, although the mechanism remains unclear. Here we use in vivo11C-acetate and PET-CT scanning to show that colonic acetate crosses the blood–brain barrier and is taken up by the brain. Intraperitoneal acetate results in appetite suppression and hypothalamic neuronal activation patterning. We also show that acetate administration is associated with activation of acetyl-CoA carboxylase and changes in the expression profiles of regulatory neuropeptides that favour appetite suppression. Furthermore, we demonstrate through 13C high-resolution magic-angle-spinning that 13C acetate from fermentation of 13C-labelled carbohydrate in the colon increases hypothalamic 13C acetate above baseline levels. Hypothalamic 13C acetate regionally increases the 13C labelling of the glutamate–glutamine and GABA neuroglial cycles, with hypothalamic 13C lactate reaching higher levels than the ‘remaining brain’. These observations suggest that acetate has a direct role in central appetite regulation.

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short-chain fatty-acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short-chain fatty-acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post-operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p = 0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). CONCLUSION: Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Short-chain fatty acid acetate stimulates adipogenesis and mitochondrial biogenesis via GPR43 in brown adipocytes

Short-chain fatty acids play crucial roles in a range of physiological functions. However, the effects of short-chain fatty acids on brown adipose tissue have not been fully investigated. We examined the role of acetate, a short-chain fatty acid formed by fermentation in the gut, in the regulation of brown adipocyte metabolism. Our results show that acetate up-regulates adipocyte protein 2, peroxisomal proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1 expression and affects the morphological changes of brown adipocytes during adipogenesis. Moreover, an increase in mitochondrial biogenesis was observed after acetate treatment. Acetate also elicited the activation of ERK and cAMP response element-binding protein, and these responses were sensitive to G(i/o)-type G protein inactivator, Gβγ-subunit inhibitor, phospholipase C inhibitor, and MAPK kinase inhibitor, indicating a role for the G(i/o)βγ/phospholipase C/protein kinase C/MAPK kinase signaling pathway in these responses. These effects of acetate were mimicked by treatment with 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide, a synthetic G protein-coupled receptor 43 (GPR43) agonist and were impaired in GPR43 knockdown cells. Taken together, our results indicate that acetate may have important physiological roles in brown adipocytes through the activation of GPR43.

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short-chain fatty-acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short-chain fatty-acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post-operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p = 0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). CONCLUSION: Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short-chain fatty-acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short-chain fatty-acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post-operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p = 0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). CONCLUSION: Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Short-chain fatty acids stimulate the migration of neutrophils to inflammatory sites

SCFAs (short-chain fatty acids) are produced by anaerobic bacterial fermentation. Increased concentrations of these fatty acids are observed in inflammatory conditions, such as periodontal disease, and at sites of anaerobic infection. In the present study, the effect of the SCFAs acetate, propionate and butyrate on neutrophil chemotaxis and migration was investigated. Experiments were carried out in rats and in vitro. The following parameters were measured: rolling, adherence, expression of adhesion molecules in neutrophils (L-selectin and beta 2 integrin), transmigration, air pouch influx of neutrophils and production of cytokines [CINC-2 alpha beta (cytokine-induced neutrophil chemoattractant-2 alpha beta), IL-1 beta (interleukin-1 beta), MIP-1 alpha (macrophage inflammatory protein-1 alpha) and TNF-alpha (tumour necrosis factor-alpha)]. SCFAs induced in vivo neutrophil migration and increased the release of CINC-2 alpha beta into the air pouch. These fatty acids increased the number of rolling and adhered cells as evaluated by intravital microscopy. SCFA treatment increased L-selectin expression on the neutrophil surface and L-selectin mRNA levels, but had no effect on the expression of beta 2 integrin. Propionate and butyrate also increased in vitro transmigration of neutrophils. These results indicate that SCFAs produced by anaerobic bacteria raise neutrophil migration through increased L-selectin expression on neutrophils and CINC-2 alpha beta release.

Short-chain fatty acids cause an IL-8 response in cystic fibrosis airways via increased GPR41

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune/inflammatory processes.

OBJECTIVES: To investigate the capacity of anaerobes to contribute to CF airway pathogenesis via SCFAs.

METHODS: Samples from 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFAs levels in anaerobe supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of SCFAs receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings, and 16HBE14o- and CFBE41o- cells were evaluated using RT-PCR, western blot, laser scanning cytometry and confocal microscopy. SCFAs-induced IL-8 secretion was monitored by ELISA.

MEASUREMENTS AND MAIN RESULTS: Fifty seven of 109 (52.3%) PWCF were anaerobe-positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF under (n=24) and over 6 years (n=85). All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic and butyric acid. SCFAs levels were higher in BAL samples from adults than children. GPR41 levels were elevated in; CFBE41o- versus 16HBE14o- cells; CF versus non-CF bronchial brushings; 16HBE14o- cells after treatment with CFTR inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells with a higher production of IL-8 in CFBE41o- than 16HBE14o- cells.

CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via upregulated GPR41.