Received: 20 December 2017 | First decision: 9 February 2018 | Accepted: 6 April 2018 DOI: 10.1111/apt.14689

Review article: short chain fatty acids as potential therapeutic agents in human gastrointestinal and inflammatory disorders

P. A. Gill1,2 | M. C. van Zelm2 | J. G. Muir1 | P. R. Gibson1

1Department of Gastroenterology, Central Clinical School, Monash University and Summary Alfred Hospital, Melbourne, Vic., Australia Background: Butyrate, propionate and acetate are short chain fatty acids (SCFA), 2Department of Immunology and Pathology, important for maintaining a healthy colon and are considered as protective in col- Central Clinical School, Monash University and Alfred Hospital, Melbourne, Vic, orectal carcinogenesis. However, they may also regulate immune responses and the Australia composition of the intestinal microbiota. Consequently, their importance in a variety Correspondence of chronic inflammatory diseases is emerging. Prof. PR Gibson, Department of Aims: To review the physiology and metabolism of SCFA in humans, cellular and Gastroenterology, Central Clinical School, Monash University and Alfred Hospital, molecular mechanisms by which SCFA may act in health and disease, and Melbourne, Vic., Australia. approaches for therapeutic delivery of SCFA. Email: [email protected] Methods: A PubMed literature search was conducted for clinical and pre-clinical Funding information studies using search terms: ‘dietary fibre’, short-chain fatty acids’, ‘acetate’, ‘propi- PAG is supported by a scholarship from the Central Clinical School, Monash University. onate’, ‘butyrate’, ‘inflammation’, ‘immune’, ‘gastrointestinal’, ‘metabolism’. MCvZ is supported by NHMRC Senior Results: A wide range of pre-clinical evidence supports roles for SCFA as modula- Research Fellowship GNT1117687. JGM is supported by NHMRC Senior Research tors of not only colonic function, but also multiple inflammatory and metabolic pro- Fellowship GNT1136988. cesses. SCFA are implicated in many autoimmune, allergic and metabolic diseases. However, translating effects of SCFA from animal studies to human disease is lim- ited by physiological and dietary differences and by the challenge of delivering suffi- cient amounts of SCFA to the target sites that include the colon and the systemic circulation. Development of novel targeted approaches for colonic delivery, com- bined with postbiotic supplementation, may represent desirable strategies to achieve adequate targeted SCFA delivery. Conclusions: There is a large array of potential disease-modulating effects of SCFA. Adequate targeted delivery to the sites of action is the main limitation of such appli- cation. The ongoing development and evaluation of novel delivery techniques offer potential for translating promise to therapeutic benefit.

The Handling Editor for this article was Professor Jonathan Rhodes, and this uncommis- sioned review was accepted for publication after full peer-review.

| Aliment Pharmacol Ther. 2018;48:15–34. wileyonlinelibrary.com/journal/apt © 2018 John Wiley & Sons Ltd 15 16 | GILL ET AL.

1 | INTRODUCTION quantitatively most important substrate for saccharolytic bacteria in the colon and the consumption of fermentable fibres increases colo- Short chain fatty acids (SCFA) comprise of 1-6 carbon based anions nic production of SCFA.8 produced during bacterial fermentation, of which, acetate (C2), pro- pionate (C3) and butyrate (C4) are most abundantly produced. SCFA 3.1 | SCFA production in vivo by fermentation of are produced naturally within the colon by fermentation of carbohy- dietary fibres drates, both dietary and endogenous, and protein that are accessible to the microbiota. Fermentation is a process that has also been There are many definitions of “dietary fibre”. Many countries, includ- exploited by humans for thousands of years in the production of ing Australia, Japan and those within the EU accept dietary fibre as foods and beverages, which consequently, contain SCFA. carbohydrates with a degree of polymerisation greater than 2 that Epidemiological evidence suggests there is an association fail to be hydrolysed or absorbed in the small intestine, as outlined between dietary fibre consumption and reduced risk of developing by Codex Alimentarius Alinorm.9 This definition is yet to be fully cardiovascular disease, diabetes and colon cancer.1-3 SCFA may play accepted in the USA, whereby dietary fibre is defined as non-digesti- a role in conferring such effects by directly affecting cell activity and ble carbohydrates and lignin that are intrinsic and intact in plants. function. A strong body of research, mostly in animal models demon- Irrespective of variations of definition, fibre is a crude term that rep- strates that SCFA affect outcomes of health and disease. However, resents a diverse group of non-digestible carbohydrates with varying this is yet to be conclusively translated in a clinical setting. The aims structural and functional properties that include ability to hold water of this review are: (1) to provide an overview of the putative mecha- (solubility), to be metabolised to SCFA by intestinal microbiota (fer- nisms by which SCFA can modulate human health and disease; (2) to mentation), to selectively stimulate the growth of health-promoting highlight the spectrum of diseases that have been linked to SCFA; bacteria (prebiotic effect) and to bind and modulate other events and (3) to discuss aspects of SCFA physiology and metabolism in the such as the inhibition of emulsifier-induced bacterial translocation context of utilising SCFA for therapeutic use. (post-biotic effect).10,11 Therefore, it is important to consider that not all subtypes of dietary fibre contribute to the SCFA pool and not all putative fibre-associated modulation of colonic health occurs via 2 | METHODS SCFA. For example, non-fermentable plant fibres such as cellulose cannot be utilised by the human gut microbiota, but increase faecal A literature review was conducted using PubMed, Medline, Scopus bulking, which stimulates peristalsis and hence reduces transit time and Google Scholar databases using combinations of search terms: through the colon.12 This effect presumably underlies its value for ‘short-chain fatty acids’, ‘acetate’, ‘propionate’, ‘butyrate’, ‘dietary treating constipation and contributes to reducing the tumour load in fibre’, ‘inflammation’, ‘immune’, ‘metabolism’, ‘fermentation’, ‘gastroin- rodent models of colorectal cancer.13,14 testinal’, ‘colon’, ‘Inflammatory bowel disease’, ‘colitis’, ‘autoimmune’, Fermentable fibres vary in the rapidity by which they are fer- ‘allergy’. Both clinical and pre-clinical studies were included for the mented12 and in the SCFA profiles resulting. Factors that modulate purposes of this explanatory review. Papers that were not in English or influence these characteristics are outlined in Table 1. The deliv- or did not have a full-text were excluded from the review. ery of SCFA to different regions of the colon also varies, and this has been of interest due to the rapid uptake of SCFA by epithelium and the subsequent local effects of such uptake. For example, the 3 | SOURCES OF SCFA preventive effect of SCFA on colorectal carcinogenesis via the ade- noma-carcinoma sequence in a rat model was clearly related to pro- Short chain fatty acids can be either ingested in the diet or produced motion of distal colonic fermentation rather than fermentation via metabolic processes within body tissues during fasting condi- per se.14 Most data available on regional SCFA production profiles tions, after consumption of alcohol, and within the bowel lumen.4,5 have been generated from animal studies and have been extrapo- By far the major source of SCFA in the colon is bacterial fermenta- lated to humans, where observations are largely restricted to studies tion of carbohydrates, which can be endogenous or dietary in origin. of faeces and blood. Interpretation of the findings in faeces, for The essential role of the microbiota in generating SCFA has been example, is limited as such measurements are unlikely to capture the highlighted in studies of germ-free mice, in which very low levels of dynamic processes of fermentation and absorption that occur more SCFA are produced compared to those in conventional mice, reflect- proximally along the colon. Information about concentrations of ing the relatively small amount of SCFA obtained directly from SCFA along the colon have relied on data from sudden death vic- dietary sources and endogenous, non-microbial production.6 Addi- tims, which indicated that SCFA concentrations are highest in the tionally, protein can also be utilised as a substrate for SCFA proximal colon, means values being 69, 25 and 25 mmol/L for acet- production by the gut microbiota during dissimilatory amino ate, propionate and butyrate, respectively.35 These high concentra- acid metabolism, producing branched-chain fatty acids such as iso- tions of SCFA are reflected by the low luminal pH in the ascending butyrate and iso-valerate.7 Dietary carbohydrates that reach the colon, which contrasts higher pH and lower SCFA concentrations in colonic lumen, usually referred to as dietary fibre, are the the faeces.36,37 The use of an ingestible pH-sensing device that GILL ET AL. | 17

TABLE 1 Factors that influence SCFA production via fermentation

Characteristic Factor Effects

Amount of fermentation Size and structure of the fibre • Speed and height of breath hydrogen rise following ingestion of short to long-chain 15 fibres is proportional to chain length. • Olligosaccharides are utilised in hours within in vitro fermentation systems, whilst 16,17 long-chain fibres are slowly fermented over days Structure and function of the • Rural African children with Bacteroidetes species Prevotella and Xylanibacter have 18 microbiota increased capacity to produce SCFA compared to rural European children. • Abundance of SCFA producing species: Bifidobacterium adolescentis, Eubacterium rectale, 19-22 Eubacterium hallii, Faecalibacterium prausnitzii and Ruminococcus bromii. • Hydrogen-metabolising bacteria prevent accumulation of hydrogen that inhibits fermen- 23 tation. • Inter-individual differences in faecal SCFA output in response to high RS and NSP diets 24 highlight inherent variation in the functional capacity of microbiota amongst humans Non-dietary, non-microbial • Indirect effects on microbiota: ageing, antibiotics, mucus production and colonic disease 25,26 states. 27 • pH—fermentation occurs ideally between 5.5 and 7.6 Profile SCFA produced Type of fibre • Pectin preferentially produces acetate, guar gum produces propionate and RS produces 28,29 butyrate Structure and function of the • Metabolic cross-feeding, for example, between B. adolecentis and E. hallii, B. adolecentis microbiota and Roseburia sp., causing conversion between acetate and butyrate, and lactate and 30,31 butyrate respectively. 32,33 • Butyrate-producing capacity of microbiota may decline with age and antibiotic use Colonic pH • Colonic pH of 5.5 stimulates butyrogenic fermentation, whilst acetate is promoted at pH 34 6.5

RS, resistant starch; NSP, non-starch polysaccharide.

sends information in real-time via telemetry has the potential to been reported to alter transit time in rodent models by acting on more accurately characterise this process, although there are other colonic smooth muscle tissue, releasing polypeptide YY (PYY).44 molecules that also influence pH such as lactate and succinate.38 This may occur via direct engagement of GPR43 (FFAR2) on colo- Production of a compact and robust bio-electrolytic sensor for real- nic L-enteroendocrine cells.45 A rise in post-prandial PYY has time measurement of colonic SCFA production is desirable, with pro- been observed in humans given SCFA, both via colonic enemas totype sensors in development.39 and through consumption of propionated-inulin.46,47 Colonic infu- There are two major factors that influence the regional produc- sion of PYY also led to a shortening in transit time, highlighting a tion of SCFA: dynamic relationship between SCFA production, PYY and gut transit time.48 1. The type of fibre: Slowly fermentable fibres may shift SCFA pro- duction from more rapidly fermented fibres from only the proxi- 3.2 | Endogenous metabolic processes that mal colon towards the distal colon. Consumption of wheat bran generate SCFA with a relatively rapidly fermented resistant starch increased SCFA concentrations in the distal colon of pigs and rats com- There are a few pathways for genesis of SCFA endogenously. First, pared to those fed only resistant starch.40,41 Healthy humans fat oxidation, predominantly during starvation-associated ketosis were able to increase faecal concentrations of SCFA, especially leads to the production of acetate, propionate and butyrate.49 Long- butyrate, by addition of wheat bran to resistant starch that failed chain fatty acids and pyruvate may be converted to acetyl-CoA, to alter faecal SCFA alone. This observation is considered strong which is then broken down to acetate through the actions of acetyl- evidence that fermentation had been shifted distally as in the CoA hydrolase. Animal studies have found that acetyl-CoA hydrolase animal studies.37 is expressed in all tissues, with highest activity found in liver and 2. Gut transit time: Changing the rate of transit along the colon will muscle.49 Secondly, acetate may also be produced in the liver on alter the time for fermentation and, hypothetically alter SCFA exposure to ethanol through the actions of alcohol dehydrogenase absorption in the proximal colon. For example, faecal butyrate and aldehyde dehydrogenase. Plasma acetate flux has been reported concentrations increase with more rapid colonic transit without a to increase 2.5 times in healthy males who consumed 24 g of radio- change in dietary fibre intake in humans.42 Drugs that slow or labelled ethanol, resulting in a rapid increase to serum concentra- hasten colonic transit, such as loperamide and cisapride, respec- tions.5 Serum acetate concentrations in healthy males and females tively, alter SCFA production.43 However, SCFA themselves have may rise to approximately 500 lmol/L, 75 min after a 0.5 g/kg dose 18 | GILL ET AL. of alcohol.50 Thirdly, propionate may be produced endogenously Hydrolysed lipids are a key substrate for SCFA production and thus, from branched-chain amino acids and odd-chain fatty acids via pro- the concentration at which they are present in the fermentation pionyl-CoA.51 mixture will determine the capacity of SCFA production. Addition The contribution of metabolically derived to plasma levels of of lipase to starter cultures is utilised in cheese manufacturing in SCFA varies according to the setting. In healthy humans, fatty acid order to improve SCFA production for enhanced flavour.63 Further- oxidation and ketosis may contribute to a 60 lmol/L increase in more, fibres such as inulin may also improve viability of bacterial plasma acetate levels in those fasting for three consecutive days, but cultures and has been reported to increase SCFA production in this was not seen in those who underwent a 12-h overnight fast.52 yoghurt.64 The contribution of metabolic processes to plasma SCFA levels is not thought to be significant in those living a normal lifestyle. For propionate and butyrate, utilisation in the tissues readily accounts 4 | PHARMACOKINETICS OF SCFA for any metabolic contribution.51,53 Thus, plasma concentrations are generally <10 lmol/L and butyrate <5 lmol/L.53 Variation in plasma Within the colon, approximately 60% of SCFA diffuse from the SCFA levels observed in healthy volunteers over the course of a day lumen through the epithelium to the lamina propria with the remain- is most likely due to changes in the rate of colonic fermentation ing portion taken up directly by epithelial cells.65 Epithelial uptake according to its exposure to dietary cabohydrates.53 However, there can be by diffusion of unionised SCFA, or receptor-mediated via by are two exceptions. First, diabetic individuals have elevated fasting both hydrogen- (MCT-1) and sodium-coupled (SMCT-1) transporters, plasma SCFA levels close to double that of healthy controls, a reflec- the latter of which has a higher affinity for SCFA.66-68 Receptor- tion of increased metabolic production from fatty acid oxidation and mediated uptake is a dynamic process that is sensitive to both physi- ketosis due to poor glycaemic control.54 Secondly, the plasma acet- ological and microbial factors in the colonic environment. While ate response to ethanol can be considerable as outlined above. expression of these transporters on the apical membrane of colonic epithelial cells is constitutive, it is also regulated by factors in the local environment. Butyrate has been shown to induce transcrip- 3.3 | SCFA intake via food tional modulation of MCT-1 on colonic epithelial cells within 30 min The fermentation process has been utilised for millennia by humans of exposure, resulting in increased abundance on the apical mem- in the production of various fermented foods and drinks. Unlike that brane.69 This effect is also seen in mice fed pectin. Bile acids may carried out by the gut microbiota, this process of fermentation must competitively inhibit both SMCT-1 and MCT-1, although less be tightly controlled to ensure the final product is free from patho- potently in the latter.70 Furthermore, microbiota may have both pos- genic bacteria and potentially toxic metabolites. Due to the odorous itive and negative effects on butyrate uptake. For example, entero- properties of SCFA, their concentrations are routinely quantified dur- pathogenic Escherichia coli induces endocytosis of MCT-1, whilst this ing quality control processes within the food and beverage industry. phenomenon is attenuated in the presence of Lactobacilli acidophilus, However, limited published information exists regarding SCFA quan- stimulating MCT-1 function.71 tities in foods.55 Short chain fatty acids delivered to the lamina propria enters the Within the dairy industry, predominately lactic acid producing blood vessels and are taken via the portal vein to the liver where bacteria are selected for starter cultures responsible for fermenta- the vast majority is metabolised on first pass; only acetate is detect- tion.56 Commonly selected cultures in commercial products include able at physiologically significant concentrations in the peripheral Bifidobacterium lactis, Streptococcus thermophilus, Lactobacillus aci- blood.72 It has been estimated that 36% of colonic-derived acetate dophilus and Lactobacillus bulgaris.57 These bacteria utilise the Emb- becomes systemically available, in contrast to only 9% of propionate den-Meyerhoff-Parnas pathway, fermenting hydrolysed sugars to and 2% of butyrate.73 In healthy subjects, fasting levels of acetate in pyruvate and lactate.56 These products may be further broken down peripheral blood have been reported to rise from a mean of 54 to to acetic acid during glycolysis in carbon restrictive environments. 169 lmol/L, 6-8 h after consumption of a 24-h high resistant starch Acetic acid producing bacteria from the Acetobacter and Gluconobac- diet, although this rise of >100 lmol/L has not been achieved ter genera are found in starter cultures used to create traditional fer- through the use of inulin, guar gum or XOS fibre supplements.74-77 mented products such as vinegar and kombucha.58,59 Consequently, Circulating concentrations of SCFA levels are likely to be tightly con- these products will or are likely to have higher levels of acetic acid trolled by energy levels within the body, as SCFA are known sub- than other fermented foods. The addition of the fungus, Penicillium strates for the production of glucose, fatty acids and ketone bodies, roqueforti to starter cultures of soft cheeses further enhances SCFA outlined in Figure 1.31 Intravenously delivered acetate is rapidly production from lipolysis, resulting in production of other SCFAs metabolised, creating a temporary increase in plasma levels that is such as propionate and butyrate.60 This occurs predominately during cleared within an hour.78 In studies using radiolabelled SCFA, the ripening process of blue cheeses. endogenous turnover of acetate is much lower in humans than in Bacterial starter cultures chosen for fermentation are highly sen- smaller animals such as dogs and rats, reflecting differences in the sitive to environmental factors. Oxygen levels, temperature and pH production and utilisation of acetate within cells and tissues of these must be tightly controlled to create optimal conditions.61,62 animals. Such endogenous variation highlights a major limitation to GILL ET AL. | 19

Lipids

β-oxidation

Acetate Butyrate Acetyl-CoA

Gluconeogenesis Ketone bodies Glucose Citric acid 2. cycle

3. Propionate 1.

FIGURE 1 Systemic metabolism of SCFA. (1) SCFA produced in the colon drains into the portal vein; (2) portal venous SCFA undergo first- pass metabolism by the liver; (3) within the liver SCFA may enter a number of metabolic pathways depending on the metabolic state. Acetate and butyrate may be converted to the central metabolite, acetyl-CoA. Consequently, these SCFA can be utilized to form lipids and ketone bodies. SCFA may also enter the citric acid cycle and become utilized for glucose production via gluconeogenesis. (3) Acetate may also pass through into the peripheral circulation and can be detected in peripheral blood extrapolation of peripheral effects of SCFA in animal models of the active site.87 Butyrate is the most potent HDAC inhibitor 4 human disease to the human situation. (HDACi), with half-maximal inhibitory concentration (IC50) of 10- 100 lmol/L in human cell lines.85 Propionate and, to a lesser extent acetate, also have inhibitory properties at a concentrations 4.1 | Molecular and metabolic interactions of SCFA of 100-1000 lmol/L.88 SCFA can modulate cellular activity both extra- and intracellularly. 3. Intracellular energy supply for colonic epithelium: Beta-oxidation of butyrate specifically is the preferred energy source for colonic 1. Extracellular activity via SCFA-specific G-protein coupled receptors epithelial cells (see below). (GPRs): GPR41 (also known as free fatty acid receptor 3 [FFAR3]), 4. Substrates for the Krebs cycle: The main site of SCFA metabolism GPR43 (FFAR2) and GPR109a (PUMA-G) are activated by ionised via the Kreb’s cycle is within hepatocytes of the liver, where pro- SCFA and signal downstream to the nucleus.79,80 SCFA-specific pionate is a major substrate for gluconeogenesis, and butyrate GPRs are expressed at a functional level on the surface of a wide for lipogenesis.31 Furthermore, cholesterol can be synthesized range of cells that include gut epithelial cells, adipocytes, enteroen- from acetate and butyrate. Alterations in cellular energy levels as docrine L-cells, innate immune cells, and neurons of the automatic a direct consequence of these metabolic changes will also influ- and somatic sensory ganglia.81,82 In transfected cell lines, human ence cell behaviour.89 GPR43 may be activated by acetate and propionate at an effective 5. Induction of apoptosis: At higher concentrations, butyrate and

half-maximal concentration (EC50) of approximately 50-200 lmol/ other SCFA induce apoptosis of cells. Extracellular concentrations L, depending on the expression system and cell line used.79,83 Propi- that have this action in vitro are in the range of 2-10 mmol/L; a 83 onate can activate GPR41 with an EC50 of 10 lmol/L. Given that concentration of 8 mmol/L induced apoptosis in normal colonic these concentrations may be found in the circulation, it is plausible crypt cells.90,91 Only surface colonic epithelial cells will be that GPR43 and 41 are biologically active systemically. GPR109a is exposed to such concentrations under physiological conditions, selective for butyrate but is relatively less potent, requiring a con- and such an action probably plays an important role in epithelial centration closer to 1 mmol/L, a concentration only found in the cell kinetics. This effect, however, may hypothetically occur in lumen of the terminal ileum and along the length of the colon.35,84 crypt cells when very high luminal concentrations are achieved; 2. Intracellular inhibition of histone deacetylases (HDACs): These this might at least partly underlie the increased colonic epithelial enzymes are involved in epigenetic regulation of gene expres- permeability, heightened translocation of Salmonella and sion.85 While HDACs are a family of enzymes constitutively increased mucus secretion induced by high doses of rapidly fer- expressed within all cells, SCFA appear to inhibit only type 1 and mentable substrates in rat and humans (to a lesser extent).92-94 type 2 HDAC.86 The mechanism of action for such inhibition has Furthermore, high doses of dietary uncooked potato starch that yet to be fully elucidated, but it has been proposed that a pair of achieved high SCFA luminal concentrations were associated with butyrate molecules could sit in the hydrophobic binding cleft of induced epithelial injury and promoted carcinogenesis.92,95,96 20 | GILL ET AL.

Given the range of molecular and metabolic interactions charac- 5.2 | Anti-tumour effects terised for SCFA, it remains unclear how or why SCFA show pref- erence for HDAC inhibition or GPR activation within different cell Butyrate has been extensively studied for its anti-tumour effects in 14,111 types, before being metabolised. The kinetics between GPR and both human colonic carcinoma cell lines and in animal models. SCFA transporters on the surface of cells, and what potential fac- Its topical delivery to the colonic epithelium reduced early tumouri- tors regulate interaction with cognate SCFA also remains unknown. genic effects (aberrant crypt formation) in the rat azoxymethane 112 Variation in SCFA concentration at different sites of the body, in model. To date, however, the development of butyrate as a SCFA transporter expression (for example, expression of SMCT-1 is chemotherapeutic agent has not progressed beyond phase I stud- 113 almost exclusively limited to brush border of colonic epithelial cells, ies. A major limitation has been delivering butyrate to the correct while MCT-1 is expressed by almost all tissues97,98) and in the con- location at sufficient concentrations. It needs to be delivered topi- centration required to elicit an effect via GPR/HDAC, results in a cally. If the rectum is used as the portal of entry, it needs to be scenario where SCFA-mediated effects are extremely tissue spe- given frequently, since twice daily enemas failed to specifically 112 cific. Regardless, the aforementioned mechanisms have been heavily reduce aberrant crypt formation in rats, but five times daily did. implicated in health and disease, both within the colon and at The use of oral preparations of butyrate itself failed to deliver it to peripheral sites. the distal colon. Fibre combinations can enhance distal colonic deliv- ery, but such combinations have not shown consistent benefits in human intervention studies. Modified forms of resistant starch com- 5 | SCFA IN HEALTH AND DISEASE posed of butyrate molecules esterfied to the sugar backbone can increase butyrate delivery to the distal colon and have anti-tumouri- 5.1 | Maintenance of the colonic epithelium genic effects in rats, but the effects of this strategy in humans have 114 It is well-established that SCFA play a critical role in colonic health, not yet been clearly reported. ’ particularly in maintenance of the epithelial lining. Their plentiful The complexity of butyrate s effects is compounded by confusion “ ” presence is critical for the turnover and differentiation of colonic over the butyrate paradox by which this SCFA has seemingly both 115 epithelial cells. Very low luminal SCFA exposure to the colonic pro-tumour and anti-tumour properties. The effects of butyrate epithelium is associated with mucosal hypoplasia, reduced epithelial have been shown to be altered by the degree of differentiation of 116 proliferation and differentiation of the epithelium, and mucosal the cell. Migration of intestinal epithelial crypt cells to the surface atrophy in experimental animals and humans when luminal sub- epithelium is increased in the presence of SCFA, therefore promot- 117 strates are reduced via parenteral nutrition, elemental diet or diver- ing epithelial cell proliferation. It has been suggested that butyrate sion of the faecal stream.99-101 In animal models, consumption of a may confer its anti-tumour effect via inhibition of HDACs that con- diet high in resistant starch induces changes in metabolic gene trol gene expression within the cell. Indeed, the butyrate paradox is expression towards glucose and fatty acid metabolism within the likely to be a result of the complex role of SCFA in regulating cellular 118 colonic epithelial cells, likely through increased uptake of SCFA.102 metabolism and epigenetics, seen in the Warburg effect. Cells in There is also evidence that high concentrations might have toxic a cancerous state primarily undergo anaerobic glycolysis. This causes effects on the epithelium in rats (as discussed above), just as SCFA butyrate to accumulate within the cell to a concentration whereby it do on cell lines in vitro.103 Thus, colonic epithelium starved or may inhibit HDAC, resulting in increased expression of apoptotic swamped with SCFA have impaired structure and function. How- genes. Conversely, healthy cells metabolise butyrate whilst undergo- ever, whether differences in SCFA delivery to the epithelium within ing oxidative phosphorylation. Within this metabolic pathway it is the physiological range have specific modulating effects on epithe- converted to acetyl-CoA, a co-factor that promotes expression of 119 lial homeostasis, such as affecting epithelial turnover and differenti- cell proliferation genes. This may drive polyp formation in vivo. In min/+ ation or barrier function, has not been convincingly demonstrated the adenomatous polyposis coli (APC) MutS homolog 2 À/À in humans and animals. This is despite multiple actions of butyrate (MHS2) mouse model of colorectal cancer, butyrate acted as an particularly in many cellular processes. Some of these included oncometabolite driving polyp formation, when administered as a l 120 modulation of specific enzyme systems such as urokinase, reduction colonic enema at concentrations between 50 M and 0.5 mM. in paracellular permeability or promotion of epithelial cell restitution However, engagement of butyrate-specific GPR109a on colonic after injury in the Caco-2 model of colonic epithelium,104-106 stimu- epithelial cells may also confer protection from cancer by upregulat- 121 lation of mucus production in another cell line, which in turn ing tumour suppressor genes Slc5a8, Msh2 and Msh3. prevented epithelial adherence of pathogenic bacteria,107 HDAC1- mediated promotion of expression of structural proteins responsible 5.3 | Anti-inflammatory effects on the colonic for maintaining tight junctions,108,109 and increased epithelial epithelium biosynthesis of retinoic acid, an important mediator of immune homeostasis in the gut.110 All of these phenomena are consistent Short chain fatty acids, particularly butyrate, have effects on colonic with a positive effect of butyrate in epithelial turnover and barrier epithelial cells that are regarded as anti-inflammatory. Studies function. in vitro have highlighted that SCFA can control expression of GILL ET AL. | 21 inflammatory cytokines such as IL-8, IL-17, IL-1b, IL-6, IL-12 and effects on the host by inhibiting pro-inflammatory activity. Addition TNF-a by colonic epithelial cells.115,122-125 This may be through acti- of SCFA to the site of subcutaneous infection in mice was found to vation of the MEK-ERK and p38 MAPK intracellular signalling path- impair phagocytosis by neutrophils recruited to the site of infection ways by SCFA engaging GPR41 and GPR43 on the surface of and was also associated with increases in bacterial load.137 During colonic epithelial cells.123 Murine models have implicated both a colonic Citrobacter rodentium infection in mice, however, provision of pro- and anti-inflammatory role for SCFA dependent effects on the acetate enhances IL-6-dependent recruitment of neutrophils to sites colonic epithelium, ultimately reflecting the complex nature of home- of infection.123 Taken together, these observations highlight that ostasis within the gut.123,126 This may also involve SCFA-dependent SCFA may modulate neutrophilic activity. The extent to which this is activation of the NLRP3 inflammasome via GPR43 and GPR109a, pro- or anti-inflammatory is likely to be dependent on the immuno- promoting production of IL-18.127 Indirect effects of butyrate on logical milieu within the site of action. inflammation via the maintenance of a healthy colonic epithelial bar- rier as outlined above are also of likely importance. 5.6 | Effects regulatory T cells

Direct effects of SCFA on Treg cells has been of interest as deficien- 5.4 | Effects of SCFA on immune cells cies in these cells have been implicated in both mucosal and sys- As illustrated in Figure 2, SCFA may directly affect immune cells, not temic immune disease.138,139 Indeed, transfer into germ-free mice of only via long-recognised direct inhibitory effects on cell division, but a group of SCFA-producing strains of Clostridia isolated from the also via modulation of cell signalling, epigenetic regulation and meta- human intestinal microbiota promoted the generation of protective bolism, the molecular mechanisms of which have now also been colonic Treg cells.140 Mice given 150 mmol/L SCFA in the drinking described.128,129 These could occur during the innate response by water have increased colonic T cell expression of Foxp3, the lineage affecting macrophages and granulocytes, as well as antigen presenta- specification factor of Treg cells.141 The mechanism through which tion by dendritic cells, and also during adaptive immune responses this occurs remains unclear. Initial murine studies reported that this by affecting T and B cell function. Foxp3 induction was dependent on engagement of GPR43 by SCFA, both individually and in combination.141 However, as T and B cells do not express functional levels of this receptor, SCFA-mediated 5.5 | Effects on innate immune cells HDAC inhibition has been implicated.88 Within Treg cells, the Butyrate regulates many subsets of innate cells of the mucosal HDAC9 isoform has been proposed to directly contribute to 142 immune system via inhibition of NF-KB nuclear translocation and increased transcription of Foxp3. As a consequence, increased HDAC.88,130 First, CD11b+CD11cÀ intestinal macrophages downreg- acetylation of signalling protein S6 Kinase (S6K) within the mTOR ulate production of inflammatory IL-6 and IL-12 in response to buty- pathway may also increase cellular metabolism, contributing to rate.131 Secondly, polarisation of macrophages to the alternative M2 enhanced suppressive function.88,143 phenotype in the presence of IL-4 may be enhanced, attenuating Although SCFA increase expression of the colonic homing recep- DSS-induced inflammation in a murine colitis model.132 Thirdly, tor, GPR15 on Treg cells,144 they also appear to boost peripheral butyrate down-regulates genes involved in activation of lamina pro- Treg numbers in murine models.133 This effect is yet to be studied pria-residing CD103+ dendritic cells, thus giving them a tolerance- in humans, but the ratio of Treg:Th17 cells increases in human promoting phenotype.133 In addition, engagement of GPR109a by peripheral blood mononuclear cells incubated in vitro with SCFA.145 butyrate on this dendritic cell subset appears to control the activity Numerous studies have highlighted similar anti-inflammatory changes of the tolerogenic retinoic acid-producing enzyme, RALDH.134 to peripheral blood mononuclear cells.146-149 As reported concentra- Indeed, co-culture of CD4+ helper (Th) cells with SCFA-treated den- tions of SCFA used in these in vitro studies are upwards of 50-fold dritic cells drives the conversion of T cells towards a Foxp3+ regula- greater than physiological concentration in the peripheral blood,35 it tory T (Treg) cell phenotype. Conversely, SCFA-treated GPR109À/À is unlikely that such effects could be validly translated to the same dendritic cells fail to induce Treg formation and, instead promote the situation in vivo. SCFA may also confer anti-inflammatory effects on inflammatory Th17 phenotype.121,133 immune cells in the dermis, a site where SCFA-producing commensal Neutrophils in vitro chemotactically respond to acetate at con- bacteria are present. Topical application of butyrate induced the for- centrations typically found in human peripheral blood. However, it is mation of Foxp3+ Treg cells in the skin of mice and was also associ- not known if this could modulate neutrophil function in the periph- ated with reduced swelling in a model of contact hypersensitivity.150 ery, as the presence of such a concentration gradient has not been characterised in vivo.135 Indeed, neutrophils exposed to SCFA appear 5.7 | Effects on B cells and antibody production to downregulate expression of the inflammatory receptors C5aR and CXCR2 via a GPR43-dependent mechanism.140 Furthermore, buty- Increased appreciation for effects of SCFAs on B cells is also emerg- rate decreases neutrophilic expression of the inflammatory mediators ing. Increased delivery of acetate to the colon via acetylated- TNF-a and nitric oxide.136 It is important to consider that, in the starches resulted in a tolerogenic B cell phenotype in non-obese context of infection, SCFA could potentially exert unfavourable diabetic (NOD) mice; with splenic B cells from these mice found to 22 | GILL ET AL.

(A)

(B)

FIGURE 2 Immune-modulating effects of SCFA. A, Mechanisms of SCFA action within cells. SCFA can engage G-protein coupled receptors on the surface of cells altering intracellular signalling pathways. SCFA may also pass into the nucleus, altering gene expression and mTOR signalling by inhibiting activity of histone deacetylases. Within the cell, SCFA may also be used as an energy source, altering cellular metabolism. B, The spectrum of immune cell targets with reported changes to phenotype GILL ET AL. | 23 have reduced expression of MHCII and co-stimulatory molecules satiety hormone leptin by adipocytes.165 In contrast, enhanced CD80 and CD86.151 SCFA may both indirectly and directly enhance SCFA delivery might promote obesity. Comparative studies of IgA and IgG antibody responses. GPR43 engagement by SCFA on faecal microbiota have highlighted that obese and overweight dendritic cells upregulates retinoic acid production, which in turn has individuals have different bacterial profiles and faecal concentra- been found to induce IgA production by B cells.152 SCFA have also tions of SCFA, particularly propionate, are increased in the been reported to directly activate the mTOR pathway in B cells, obese.166 However, as no assessment of the diets of the subjects leading to, increased glucose uptake, glycolytic activity and terminal studied was made, the results cannot be interpreted in terms of differentiation into antibody-secreting plasma cells.153 Such observa- enhanced capacity to harvest energy in the form of increased tions highlight an important role for SCFA in regulating B cell func- SCFA production, as was deduced from microbiota studies in lean tion via metabolic changes within the cell. and obese mice.167 2. Control of glucose homeostasis: A role for SCFA in maintaining glucose homeostasis has also been documented; consumption 5.8 | SCFA in immune disorders of an arabinoxylan-rich diet increases plasma SCFA and improve Short chain fatty acids have now been implicated in a wide spectrum glucose tolerance in type II diabetics.168,169 Mechanistically, of inflammatory diseases shown in Table 2. Although the majority of SCFA can directly stimulate GPR43 on colonic enteroendocrine studies show the association of SCFA with protection from develop- cells to produce glucagon-like peptide (GLP)-1, improving glu- ment of disease, the opposite may occur. Thus, provision of SCFA in cose homeostasis during feeding.170 Propionate has also been the drinking water caused levels of SCFA to accumulate in the renal reported to elicit a similar effect on ileal cells by activating a tissue, resulting in severe T-cell mediated inflammation.154 Further- GLP-1 dependent neuronal network via GPR43, highlighting more, the potency of each SCFA varies across disease models and that direct consumption of SCFA may also improve glucose tol- has resulted in a varying array of proposed mechanisms put forward. erance.171 Studies in Gpr43À/À mice have also implicated a role For example, the protective effect of SCFA in asthma models has for direct stimulation of GPR43 in promoting pancreatic b-cell been reported to occur via propionate-mediated effects on dendritic proliferation.172 However, SCFA were not observed to stimulate cell development and subsequent antigen-presenting function in the this pathway. Propionate may also signal via a GPR41-mediated house-dust mite model, but also via acetate-mediated promotion of gut-brain pathway to increase intestinal gluconeogenesis, shown protective Treg cells in utero in the allergic airways disease to be associated with improved glucose homeostasis and model.142,155 Collectively, these studies lack supportive data in decreased weight gain in mice.173 To confuse the issue, Perry humans, in whom many studies have been unable to provide an ade- et al have proposed that increased colonic acetate production quate dose of fermentable fibre or have failed to examine the puta- could promote increased glucose stimulated b-cell insulin tive mechanisms put forward by animal studies.156,157 There is a release via the parasympathetic nervous system, leading to insu- need for increased collaboration between immunologists, nutrition lin resistance.174 scientists and dietitians to ensure studies are correctly designed to 3. Appetite regulation: GPR41 activation by propionate stimulates translate into clinical findings. production of the satiety hormone, leptin, by adipocytes.165 Inu- lin challenge, purported to specifically increase colonic propi- onate production, was reported to be associated with reduced 5.9 | Metabolic effects desire for high energy foods and led to decreased energy intake Short chain fatty acids can be used for de novo synthesis of glucose in healthy men.175 However, inulin has multiple effects, includ- and lipids, providing a rationale for studying a potential role for ing intestinal distension with its potential for inducing abdomi- SCFA in modulating energy homeostasis and metabolism.31 Multiple nal symptoms and elevating production of other SCFA. Given metabolic effects of SCFA have been reported and these include the that acetate can cross the blood-brain barrier,176 SCFA may act following: as signalling molecules in a bi-directional gut-brain pathway responsible for control of energy homeostasis. Indeed, acetate 1. Control of obesity: Epidemiological evidence suggests a protective has also been reported to have a role in modulating central role for fibre in the development of obesity.161 If causal, SCFA appetite regulation in humans.78 Murine studies have elucidated may be involved. SCFA activation of GPR43 in white adipose tis- that this may be via inhibition of neuronal circuits within the sue decreases fatty acid uptake, supressing fat accumulation.162 brain that control release of the PYY in the gut.177 Moreover, Furthermore, GPR43 activation on brown adipocytes promotes GPR41 has been found to be expressed on sympathetic ganglia mitochondrial biogenesis, increasing the capacity of the cell to of humans.178 Taken together, these observations suggest that utilize energy for thermogenesis.163 Although these effects are SCFA may regulate neuronal pathways that control feeding difficult to observe in vivo, a distal colonic infusion of acetate behaviours. was shown to increase fasting fat oxidation in overweight 4. Cardiovascular effects: SCFA may also play a role in regulating men.164 Control of energy intake may also be mediated by SCFA. blood pressure. Provision of acetate in the drinking water GPR41 activation by propionate stimulates production of the reduces cardiac hypertrophy, systolic and diastolic blood pressure 24 | GILL ET AL.

TABLE 2 Spectrum of autoimmune and allergic diseases implicated to be modulated by SCFAs based on animal models

SCFA Mechanism Effect on Disease Animal model C2 C3 C4 GPR HDAC inhibition Other Cell(s) of interest inflammation Ref. Autoimmunity Inflammatory DSS colitis x X Neutrophil Anti 135 bowel disease CD4+ T cell adoptive x x Foxp3+ Treg Anti 158 transfer Multiple sclerosis MOG-induced EAE x x Treg & Th17 cells Anti 159 Type 1 diabetes Non-obese diabetic x x x x Splenic B cells, Anti 151 mice Marginal zone B cells, Follicular B cells, Foxp3+ Treg Graft-versus-host MHC-mismatched x x Intestinal epithelial Anti 109 disease bone marrow cells, T cells transplant Arthritis K/BxN inflammatory x x Neutrophil Anti 135 arthritis MSU crystal-induced x x Macrophage Pro 160 gout Renal disease SCFA-induced x x x x Th1 & Th17 cells Pro 154 Allergy Asthma House dust mite x x Precursor DCs, Anti 155 Lung DCs, Th2 cells Allergic airways x x Neutrophil Anti 135 disease x Foxp3+ Treg Anti 142 Food allergy Peanut extract-cholera x x x CD103+ DCs, Anti 134 toxin-induced allergy Foxp3+ Treg

DSS, dextran sulphate sodium; MOG, myelin oligodendrocyte glycoprotein; EAE, experimental autoimmune encephalomyelitis; MSU, monosodium urate.

in deoxycorticosterone acetate-treated mice.179 Although the 6.1 | Delivery by a pro-drug approach mechanism behind such observations are not fully elucidated, GPR41 is expressed on the surface of murine endothelial cells, Tributyrin, a butyrate prodrug, may increase serum butyrate to a potentially implicating a role for propionate in regulating blood median level of 52 lmol/L with an oral dose of 200 mg/kg given pressure.180 three times daily.181 However, clinical use of tributyrin has been lim- ited by large intra-individual variation in serum butyrate and the impractical number of capsules required. Incorporation of tributyrin 6 | UTILISING SCFA AS A THERAPEUTIC in microcapsules made from the 8-membered sugar ring molecule, ɣ- FOR INFLAMMATORY DISEASE cyclodextrin appears to protect the compound from release in the upper gastrointestinal tract.182 This reduces the unpleasant sensory Human randomised control trials are needed to confirm the wide characteristics of tributyrin and promotes delivery to the small intes- range of therapeutic and protective effects of SCFA reported in tine and colon. There are as yet no reports of its application in a animal studies. However, these animal studies do not directly clinical trial. translate into human intervention studies owing to the huge quan- tities of dietary fibre used. Daily fibre intake by a mouse on a 6.2 | Fermentable fibre murine high fibre diet has been estimated to be equivalent to 274 g/day for a human, approximately 9.7 times that of the rec- Highly fermentable fibres, such as inulin and guar gum can produce ommended intake of 30 g/day.127 Such doses in humans would a significant rise in plasma SCFA levels within 6 h when consumed neither be possible or tolerated. Therefore, alternative approaches as a 20-30 g supplement.76,77 Furthermore, longer term consumption are needed to obtain adequate levels of SCFA in the colon and in studies ranging from days to weeks, of a similar dose of resistant peripheral tissues in humans. Several delivery methods can be starch increases faecal levels of total SCFA and butyrate.24,183,184 applied as shown in Figure 3. These fibre doses are reported as tolerable within study cohorts but GILL ET AL. | 25

efficient and preferential delivery of each SCFA to the colon when 189 1. Prodrug compared to unmodified resistant starch. Thus, consumption of these starches produces a significant increase in SCFA levels in stoma digesta from Ileostomy patients and faeces from healthy con- trols.190,191 Moreover, consumption of 40 g of butyrylated starch Esterfication per day protected healthy controls from the development of colonic 4. Endogenous tumourigenic adducts induced by concurrent consumption of a 300 g/day high red meat diet.192 These starches confer significant 2. Dietary protection against autoimmune T1 diabetes in NOD mice.151 Esterifi- cation of propionate to inulin has also been reported, with the syn- thetic process proposed as a more efficient alternative to using starch, with 10 g of propionated inulin estimated to deliver the 3. Postbiotic equivalent amount of propionate to the colon as 90 g of non-starch polysaccharide.46,193 This dose has also been observed to increase post-prandial plasma PYY and GLP-1, with long-term ingestion reducing weight gain in obese individuals.46 These studies highlight FIGURE 3 Potential routes of SCFA delivery to increase therapeutic effects achieved through targeted and efficient delivery concentrations in the colonic lumen and/or systemic circulation by; of SCFA to the colon, representing a potentially desirable approach (1) using a butyrate prodrug; (2) through the diet with fermentable for use in future randomised control trials. fibres that can be chemically modified via esterification; (3) via postbiotic SCFA containing solutions; and (4) by increasing There are potential short-comings of these approaches. Practi- endogenous production cal aspects such as taste and smell may be an issue, despite being reported as tolerable when consumed as a drink in healthy sub- gastrointestinal symptoms, are generally increased, making it unlikely jects.156 Whether they can be incorporated into food without that regular consumption of such doses would be tolerated amongst imparting unpleasant taste needs also to be addressed. Their all members of the general public. safety has received limited attention. As discussed above, SCFA Combining these fibres with slowly fermentable wheat bran may may be toxic at very high luminal concentrations in the colonic promote the generation of SCFA along the entire length of the lumen. The presence of SCFA in defined gut microbiota growth colon.37 Of particular interest has been high amylose maize starch medium inhibits the growth of key Bacteroidetes species of the (HAMS), a source of type 2 resistant starch that is commercially human gut microbiota.194 Furthermore, the lowering of the luminal available and can provide high levels of resistant starch after cooking pH is associated with inhibited metabolic activity of luminal bacte- and freezing.185 Consumption of breads and baked goods containing ria, which potentially may have deleterious effects on other func- HAMS increased faecal SCFA output in human subjects with hyper- tions of the microbiota.27 However, little is known of what triglyceridaemia.186 Arabinoxylan oligosaccharides (AXOS), another luminal concentrations of SCFA and of what pH profiles are type of highly fermentable fibre produced by enzymatic hydrolysis of achieved by these compounds. On the other hand, inhibition of arabinoxylan, can be produced in bread by the addition of xylanase fermentation will lead to more starch being available for fermenta- into the dough prior to baking.187 Consumption of a 140 g serving tion in the distal colon, which could result in reduced protein fer- of white bread containing 18 g of added AXOS increased peripheral mentation.7 Clearly, more studies are needed to determine the serum levels of acetate, butyrate and total SCFA in young adults and physiological and microbiological effects of such modified carbohy- was also observed to improve overnight glucose tolerance.169 A drate use. major limitation of utilising a dietary approach to delivering SCFA in the colon is the variable nature of SCFA production seen in response 6.4 | Direct “postbiotic” delivery of SCFA to consumption of fermentable fibre, as discussed earlier.24 Further- more, encouraging the general public to consume an adequate dose Direct delivery of SCFA in the gut without the need for fermenta- of fermentable fibre that will achieve sufficient boosting of SCFA tion has been explored as a “postbiotic” treatment for disease.195 production has been challenging.188 This has highlighted a need for There has been particular focus in IBDs and colitis, whereby more targeted approaches that are compatible with a wider variety impaired SCFA-epithelial interaction may contribute to disease of gut microbiota profiles. pathology.196 A number of open-label and RCTs trialling postbiotic butyrate and SCFAs within IBD and colitis patients have been con- ducted, the results of which are summarised in Table 3. Treatment 6.3 | Chemically modified resistant starch with SCFA enemas appeared to improve histological and endoscopy Esterfication of SCFAs to resistant starch protects them from release disease scores in an initial open label study in diversion colitis and absorption in the small intestine. Such chemical modification patients.197 This effect was less clear in randomised placebo-con- combines both postbiotic and dietary approaches, generating trolled clinical trials in larger cohorts of patients with active distal 26 | GILL ET AL.

TABLE 3 Human clinical trials of SCFA in inflammatory bowel disease and colitis

Treatment arms Results

Secondary Disease No. Design Active ComparatorDose duration Primary end-point end-points Ref.

Enema Diversion colitis 4 Observation SCFA Saline (sequential) 60 mL b.d. Improved Saline had 197 (46:23:31) 2-60 weeks endoscopic no benefits 130 mM appearance + (pH 7) histological scores 13 RCT SCFA Isotonic 60 mL b.d. No differences 201 (46:23:31), saline 2 weeks in histological 130 mM endoscopic (pH 7) scores 17 RCT Butyrate Saline + 30 mL b.d. No differences Butyrate- 203 600 mM 3mM 30 days in end-points mediated butyrate vs placebo up-regulation Endoscopic of gene score expression improved associated compared with with baseline mucosal (P < 0.01) repair Distal ulcerative 12 Observation SCFA 100 mL b.d. Improved 207 colitis—active (80:30:40) disease activity (1 remission, 9 improved) and histology scores 47 RCT #1 SCFA (46:23:31) Isotonic 60 mL b.d. No differences in No differences 199 3 arms 130 mM saline 8 weeks disease activity across groups (pH 5.5) score for partial #2 Butyrate Response: SCFA clinical response, 100 mM 60%, butyrate or endoscopic (pH 5.5) 44%, placebo and histological 56% scores 40 RCT SCFA Saline 100 mL b.d. No difference in SCFA better 208 (54:20:27), 6 weeks overall clinical than placebo for 150 mM score or stool frequency (pH not proportion (P = 0.05), urgency stated) of responders (P = 0.02) and patient self- evaluation (P = 0.05) No differences in endoscopic or histological scores) 38 RCT Butyrate Saline + 60 mL nocte No differences No differences 200 80 mM 0.8 mM 6 weeks in disease in complete (pH 7) butyrate response- response, or in butyrate disease activity, 7/19 vs endoscopic or placebo 9/19) histological (P = 0.51) scores 91 RCT SCFA Saline 100 mL b.d. No differences No differences 198 (53:20:27), 6 weeks in change in in change in 150 mM disease stool frequency, (pH 7) activity scores rectal bleeding, (P = 0.23) endoscopic appearance, histological indices (except mucin depletion less with SCFA; P = 0.03)

(Continues) GILL ET AL. | 27

TABLE 3 (Continued)

Treatment arms Results

Secondary Disease No. Design Active ComparatorDose duration Primary end-point end-points Ref.

Distal ulcerative 51 RCT 4 g 5-ASA + 4 g 5-ASA + 80 mL b.d. No difference in Improvement with 209 colitis— butyrate, saline 6 weeks proportion in butyrate in stool refractory 80 mM clinical remission/ frequency (P < 0.01), to oral 5- (pH not improved: Butyrate: urgency/tenesmus ASA + stated) 6/12 vs control (P < 0.05) and patient steroids 1/13 self-evaluation (P < 0.01) Oral butyrate Diverticulosis 73 RCT 2 9 150 mg Placebo 2 tablets o.d. Greater number of Better subjective 205 with [52a] butyrate 12 months episodes of diverticulitis symptom improvement previous (microencapsulated) with butyrate with butyrate than diverticulitis vs placebo: clinically placebo (P = 0.014) diagnosed 2/30 vs 7/22 (P = 0.04); ultrasound diagnosed 6vs1 (P = 0.02) Mild ileo-colonic 13 Observation 2 g butyrate (enteric 2/day Clinical remission Improved endoscopic 206 Crohn’s disease coated) 8 weeks in 7, response in 2 and histological scores Mild active UC 30 [52a] RCT 2.4 g 5ASA 2.4 g 5ASA Daily No differences in No differences in 210 + 4g 6 weeks clinical remission/or improvement in clinical butyrate (Eudragit- response: Butyrate activity, histological and S- (n = 12) 7/5 vs endoscopic scores coated) control (n = 13) 5/5 aNumber analysed per-protocol. ulcerative colitis and diversion colitis, with SCFA enemas not improv- Food containing SCFA is another potential way of delivering ing clinical indices of disease when compared to placebo.198-201 SCFA to the systemic circulation. SCFA ingested orally in solution Although enemas improved clinical indices of disease when com- are rapidly absorbed into the circulation and have been reported to pared to pre-treatment, a higher than expected response to placebo cause a transient increase in blood acetate levels.211 Furthermore, occurred in many trials. Saline enemas, the usual placebo control, do they are likely to be adsorbed by epithelial surfaces in the oesopha- alter rectal epithelial proliferative kinetics in healthy subjects202 and gus, stomach and intestine. Consumption of vinegars, which are nat- when infused twice daily into the distal colon of rats treated with urally high in acetic acid, is anecdotally believed to promote health azoxymethane, reduced the development of aberrant crypts compared benefits.212 In small human studies, vinegar consumption reduces with no-infusion controls.112 Furthermore, variation in concentration post-prandial insulin and glucose fluctuations in healthy and insulin and frequency of the enemas provided in these studies have likely resistant subjects.213,214 Long-term consumption of fermented foods contributed to mixed results seen. For example, butyrate infusion into such as yoghurt has been associated with a reduction in symptoms the distal colon of rats had no specific effect over saline infusions on of allergy.215,216 Indeed, murine splenocytes incubated with Lacto- aberrant crypt formation when given twice a day, but have a signifi- bacilli from traditional Korean kimchi decreased production of IL-4, a cant effect when delivered five times per day.112 Hence, positive bio- mediator of Th2 hypersensitivity reactions, highlighting a potential logical effects of saline (control) enemas reduce the power of immunomodulatory mechanism.217 Given that these foods are likely detecting specific effects and twice-daily use may well be insufficient to contain SCFA, further studies are warranted to elucidate a possi- as a therapy. In other words, clinical efficacy of enemas are likely to be ble role for “postbiotic” SCFA in this mechanism. impractical to achieve as an extended period of contact between SCFA and mucosa may be required for therapeutic effects. This has damp- 6.5 | Promotion of endogenous SCFA ened enthusiasm for the enema approach, despite recent studies high- lighting that they may reduce inflammation by inducing changes to Utilising endogenous metabolic processes that produce SCFA to attain expression of mucosal genes involved in cytokine expression and health benefits is an intriguing possibility. Low-level alcohol consump- metabolism of reactive oxidative species.203,204 tion, that creates a transient increase in plasma acetate levels, is asso- Slow-release preparation of oral butyrate, utilising pH-dependent ciated with a lower risk of heart disease and diabetes.218 Pownall et al release mechanisms, is an alternative approach to colonic enema that proposed that protective effects of alcohol may be mediated by acet- had benefits in patients with diverticulosis and Crohn’s disease in ate acting via GPR43 on adipocytes, inhibiting lipolysis and decreasing pilot studies.205,206 However, this approach has not been further plasma triglyceride levels.219 Long-term fasting that promotes ketoge- developed and requires testing in larger placebo-controlled RCTs. nesis and increases circulating SCFA is associated with reduced 28 | GILL ET AL. circulating inflammatory cytokines and improved glucose toler- ORCID ance.220,221 The ketone body b-hydroxybutyrate, is structurally similar P. A. Gill http://orcid.org/0000-0001-8579-8493 to that of butyrate and inhibits NLP3-inflammasome mediated inflam- M. C. van Zelm https://orcid.org/0000-0003-4161-1919 mation in mouse models.222 Further studies are required to define J. G. Muir https://orcid.org/0000-0002-6669-4004 whether increasing endogenous SCFA is causally associated with P. R. Gibson https://orcid.org/0000-0001-9108-1712 reducing disease before utilising such a strategy.

REFERENCES 7 | CONCLUSIONS 1. Threapleton DE, Greenwood DC, Evans CE, et al. Dietary fibre A wide range of pre-clinical evidence supports a role for SCFA as intake and risk of cardiovascular disease: systematic review and meta-analysis. BMJ. 2013;347:f6879. a modulator of colonic and overall health. However, fundamental 2. Bingham SA, Day NE, Luben R, et al. Dietary fibre in food and pro- differences between animal and human physiology dictate caution tection against colorectal cancer in the European Prospective Inves- in extrapolating animal effects to human diseases and create a tigation into Cancer and Nutrition (EPIC): an observational study. need to conduct clinical trials to validate these effects in humans. Lancet. 2003;361:1496-1501. 3. Montonen J, Knekt P, Jarvinen R, Aromaa A, Reunanen A. Whole- A major issue relates to differences in the dose of SCFA that can grain and fiber intake and the incidence of type 2 diabetes. Am J be delivered to the target, whether it be, for example, the distal Clin Nutr. 2003;77:622-629. colon or systemic circulation. Additional factors might include 4. Pouteau E, Nguyen P, Ballevre O, Krempf M. Production rates and inter-individual variability in microbiota and metabolism. Major limi- metabolism of short-chain fatty acids in the colon and whole body tations in progress have included inability to measure SCFA pro- using stable isotopes. Proc Nutr Soc. 2003;62:87-93. 5. Siler SQ, Neese RA, Hellerstein MK. De novo lipogenesis, lipid duction in vivo other than in peripheral sites of faeces or blood. kinetics, and whole-body lipid balances in humans after acute alco- However, emerging technologies may provide insight into this hol consumption. Am J Clin Nutr. 1999;70:928-936. dynamic process. Furthermore, development of novel targeted 6. Hoverstad T, Midtvedt T. Short-chain fatty acids in germfree mice approaches for colonic SCFA delivery represents a desirable strat- and rats. J Nutr. 1986;116:1772-1776. 7. Yao CK, Muir JG, Gibson PR. Review article: insights into colonic egy that might achieve more consistent and reliable dosing. These protein fermentation, its modulation and potential health implica- may also be combined with dietary supplementation of SCFA to tions. Aliment Pharmacol Ther. 2016;43:181-196. maximise delivery. With these emerging technologies at hand, this 8. Cummings JH, Macfarlane GT. The control and consequences of gives enormous potential for SCFA to finally be translated thera- bacterial fermentation in the human colon. J Appl Bacteriol. 1991;70:443-459. peutically in a wide range of intestinal, metabolic and inflammatory 9. Stephen AM, Champ MM, Cloran SJ, et al. Dietary fibre in Europe: diseases. current state of knowledge on definitions, sources, recommendations, intakes and relationships to health. 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McIntyre A, Gibson PR, Young GP. Butyrate production from diet- cation and booklets on the low FODMAP diet. They have published ary fibre and protection against large bowel cancer in a rat model. an educational/recipe book on diet. Funds raised contribute to Gut. 1993;34:386-391. 15. Christl SU, Murgatroyd PR, Gibson GR, Cummings JH. Production, research of the Department of Gastroenterology and to the Univer- metabolism, and excretion of hydrogen in the large intestine. Gas- sity. The authors receive no personal remuneration. MCvZ has no troenterology. 1992;102:1269-1277. conflicts of interest to declare. 16. Stewart ML, Timm DA, Slavin JL. Fructooligosaccharides exhibit more rapid fermentation than long-chain inulin in an in vitro fer- mentation system. Nutr Res. 2008;28:329-334. AUTHORSHIP 17. Kaur A, Rose DJ, Rumpagaporn P, Patterson JA, Hamaker BR. In vitro batch fecal fermentation comparison of gas and short-chain Guarantor of the article: P.R. Gibson. fatty acid production using “slowly fermentable” dietary fibers. J Author contributions: P.A Gill performed the literature search. P.A Food Sci. 2011;76:H137-H142. 18. De Filippo C, Cavalieri D, Di Paola M, et al. Impact of diet in shaping Gill and P.R Gibson wrote the manuscript. J.G Muir, M.C van Zelm gut microbiota revealed by a comparative study in children from and P.R Gibson critical reviewed the manuscript. All authors Europe and rural Africa. Proc Natl Acad Sci USA. 2010;107:14691- approved of the final version of the manuscript. 14696. GILL ET AL. | 29

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