Chapter Three - The Role of Short-Chain Fatty Acids in Health and Disease

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Abstract

There is now an abundance of evidence to show that short-chain fatty acids (SCFAs) play an important role in the maintenance of health and the development of disease. SCFAs are a subset of fatty acids that are produced by the gut microbiota during the fermentation of partially and nondigestible polysaccharides. The highest levels of SCFAs are found in the proximal colon, where they are used locally by enterocytes or transported across the gut epithelium into the bloodstream. Two major SCFA signaling mechanisms have been identified, inhibition of histone deacetylases (HDACs) and activation of G-protein-coupled receptors (GPCRs). Since HDACs regulate gene expression, inhibition of HDACs has a vast array of downstream consequences. Our understanding of SCFA-mediated inhibition of HDACs is still in its infancy. GPCRs, particularly GPR43, GPR41, and GPR109A, have been identified as receptors for SCFAs. Studies have implicated a major role for these GPCRs in the regulation of metabolism, inflammation, and disease. SCFAs have been shown to alter chemotaxis and phagocytosis; induce reactive oxygen species (ROS); change cell proliferation and function; have anti-inflammatory, antitumorigenic, and antimicrobial effects; and alter gut integrity. These findings highlight the role of SCFAs as a major player in maintenance of gut and immune homeostasis. Given the vast effects of SCFAs, and that their levels are regulated by diet, they provide a new basis to explain the increased prevalence of inflammatory disease in Westernized countries, as highlighted in this chapter.

Introduction

There is increasing evidence implicating the gut microbiota as critical contributors to host health and gut/immune homeostasis. This may be achieved, at least in part, through the release of short-chain fatty acids (SCFAs), which are the main bacterial metabolites produced following the fermentation of dietary fiber and resistant starches by specific colonic anaerobic bacteria. SCFAs are a subset of saturated fatty acids containing six or less carbon molecules that include acetate, propionate, butyrate, pentanoic (valeric) acid, and hexanoic (caproic) acid. Recent advances in the study of SCFAs, especially acetate, propionate, and butyrate, have highlighted their effects on various systems both at cellular and molecular levels. Indeed SCFAs or their deficiency may affect the pathogenesis of a diverse range of diseases, from allergies and asthma to cancers, autoimmune diseases, metabolic diseases, and neurological diseases.

SCFAs are carboxylic acids defined by the presence of an aliphatic tail of two to six carbons. Although SCFAs can be produced naturally through host metabolic pathways particularly in the liver, the major site of production is the colon which requires the presence of specific colonic bacteria explaining their absence in germ-free mice (Hoverstad & Midtvedt, 1986). Acetate (C2), propionate (C3), and butyrate (C4), being the major SCFA released through fermentation of fiber and resistant starches, are mostly released in the proximal colon in very high concentrations (70–140 mM) while their concentrations are lower in the distal colon (20–70 mM) and in the distal ileum (20–40 mM) (Wong, de Souza, Kendall, Emam, & Jenkins, 2006). The molar ratio of acetate, propionate, and butyrate production in the colon is 60:25:15, respectively (Tazoe et al., 2008), although proportions can vary depending on factors such as diet, microbiota composition, site of fermentation, and host genotype (Hamer et al., 2008). Butyrate is mostly utilized by colonocytes while acetate and propionate reach the liver through the portal vein. Propionate is subsequently metabolized by hepatocytes while acetate either remains in the liver or is released systemically to the peripheral venous system (Pomare, Branch, & Cummings, 1985). Thus, only acetate is usually detectable in peripheral blood. Extensive research has highlighted the beneficial effects of SCFAs on health, detailed below in this chapter. Health authorities have thus established a recommended daily intake of fiber, which according to the World Health Organization is 20 g per 1000 kcal consumed (in adults) and this quantity is reached through the daily consumption of grains as well as 400 g per day of fresh fruits and vegetables (www.who.int). Notably, the typical consumption of fiber in most Western countries is much less than this (King, Mainous, & Lambourne, 2012) and consumption of fiber is inversely related to premature death from all causes of disease (Park, Subar, Hollenbeck, & Schatzkin, 2011)

Indigestible saccharides are the major substrates leading to SCFA production. Polysaccharides are subdivided into three categories: starch, starch-like, and nonstarch polysaccharides (NSPs). Starch, such as amylose, and starch-like polysaccharides, such as glycogen, consist of polymers of glucose linked by alpha 1–4 and alpha 1–6 glycosidic bonds. These bonds are broken down by salivary, pancreatic, and intestinal brush barrier enzymes and are thus digestible by mammals. Under healthy conditions, starch and starch-like polysaccharides are fully digested in the small intestine yielding glucose. Polysaccharides that are undigested or partially digested in the small intestine are able to undergo a process of fermentation by specific colonic anaerobic bacteria leading to the release of SCFAs in addition to gases and heat. These polysaccharides are called fermentable polysaccharides and are subclassified as NSPs, or dietary fibers, and resistant starch (RS). Depending on their degree of solubility, fibers are subclassified into insoluble or soluble fibers and in both cases are found in plant cell walls. Cellulose and lignin are examples of insoluble fibers while pectin substances or gums forming a gel in water are classified as soluble fibers. Insoluble fibers are highly fermentable and hence generate greater quantities of SCFA in the colon while soluble fibers have a rather low fermentability but increase fecal bulking and decrease colonic transit time. RS can be subdivided into four types: physically trapped starch (in coarse grains), RS granules naturally rich in amylose (i.e., raw potato flour), retrograded starch (i.e., cooked and cooled potato), and chemically modified starch (i.e., processed foods) (Englyst, Kingman, & Cummings, 1992). RS is considered as the most powerful butyrogenic substrate where fermentation of RS in vitro as well as in vivo generally results in a significant higher level of butyrate production compared to NSP (Englyst et al., 1992). Oligosaccharides, defined by a short chain of monosaccharide units, such as galactooligosaccharides, fructooligosaccharides, mannanoligosaccharides, and chitooligosaccharides are also substrates for SCFAs (Pan, Chen, Wu, Tang, & Zhao, 2009). Finally, to a lesser extent, some SCFAs such as isobutyrate and isovalerate are produced during the catabolism of branched chain amino acids valine, leucine, and isoleucine and intermediate of fermentation in the microbiota such as lactate or ethanol can also be metabolized into SCFA (Macfarlane & Macfarlane, 2003).

The process involved in the production of SCFAs from fiber involves complex enzymatic pathways that are active in an extensive number of bacterial species. The most general pathway of SCFA production in bacteria is via the glycolytic pathway, although certain groups of bacteria such as the Bifidobacteria can utilize the pentose phosphate pathway to produce the same metabolites (Cronin et al., 2011, Macfarlane and Macfarlane, 2003). Other pathways utilizing a variety of substrates are also able to produce SCFAs. Radioisotope analysis by Miller and Wolin (1996) demonstrated that a major pathway of acetate production by bacteria was via the oxygen-sensitive Wood–Ljungdahl pathway and is regarded as the most efficient pathway of acetate production (Fast & Papoutsakis, 2012). Using similar methods they show that propionate was generally generated by a carbon dioxide fixation pathway while butyrate was most commonly formed by conventional acetyl-S coenzyme A condensation (Miller & Wolin, 1996). Other pathways, such as the Bifidobacterium pathway (fructose-6-phosphate phosphoketolase pathway) found in the Bifidobacterium genus are able to utilize monosaccharides in a unique manner to ultimately generate SCFAs (Pokusaeva, Fitzgerald, & van Sinderen, 2011). These results suggest that different species possessing specific enzymes are involved in the production of the various SCFAs. Indeed, the Wood–Ljungdahl pathway is typically found in acetate-producing bacteria (known as acetogens) where the majority are of the Firmicutes phylum (Ragsdale & Pierce, 2008). On the other hand, the major groups involved in the production of butyrate are of the Cytophaga and Flavobacterium group belonging to the Bacteroidetes phylum (Guilloteau et al., 2010). Specific species of bacteria characterized by their high levels of butyrate production include Clostridium leptum, Roseburia species, Faecalibacterium prausnitzii, and Coprococcus species belonging to both the Firmicutes and Bacteroidetes phyla (Guilloteau et al., 2010).

The production of SCFAs is a highly complex and dynamic process. For example, butyrate and propionate may be degraded into the smaller two carbon chain acetate by sulfate- or nitrate-reducing acetogenic bacteria such as Acetobacterium, Acetogenium, Eubacterium, and Clostridium species (Westermann, Ahring, & Mah, 1989). However, increased proportion of butyrate-producing or -consuming species such as F. prausnitzii and Roseburia species can reverse this process (Duncan et al., 2004). Such interactions can involve the mutualistic production of SCFAs as demonstrated by the cocolonization of Bacteroides thetaiotaomicron and Eubacterium rectale where acetate produced by B. thetaiotaomicron acted as a substrate for butyrate generation by E. rectale (Mahowald et al., 2009).

In addition to enzymatic requirements, expression of protein transporters is also imperative for SCFA production. For example, the presence of ATP-binding cassette (ABC) transporters in Bifidobacterium longum is crucial for the uptake and transport of substrates, such as fructose, required for acetate production (Davidson and Chen, 2004, Fukuda et al., 2011). Another transporter, the PEP translocation group or the phosphotransferase system (PTS) is able to transport carbohydrates which can be subsequently metabolized to produce SCFAs (Postma et al., 1993, Zoetendal et al., 2012). Genomic analysis revealed that Bacteroidetes possesses more polysaccharide-degrading enzymes but less ABC transporters and fewer PTS than the Firmicutes (Mahowald et al., 2009) suggesting that despite having the machinery to produce SCFAs they might not efficiently uptake the substrate necessary for their production. However, Firmicutes may be excellent scavenger of acetate through their ABC transporters and can uptake acetate to produce butyrate and propionate as fermentative by-products. It has therefore been hypothesized that the two predominant phyla could exist in a balance whereby acetate from Bacteroidetes is used to produce butyrate and propionate by Firmicutes (Mahowald et al., 2009). Therefore, the complex and delicate interaction within the microbiota may also control the proportion and levels of SCFAs in the gut lumen. Accordingly, prebiotics (agents favoring the growth of beneficial bacteria) or probiotic (introduction of beneficial bacteria) agents altering such balance may modulate the production of SCFAs.

Dietary changes can alter the composition of the gut microbiota in as little as a day (Wanders, Graff, & Judd, 2012) and even minute alteration of dietary factors such as fiber content could shape microbial communities (Donohoe et al., 2011). The biggest issue presented by a Western diet typically high in fat and digestible saccharides is that nutrients are mostly absorbed in the duodenum leaving very few substrates for the colonic bacteria. Consequently, this results in dysbiosis, the impairment of microbiota composition and increased susceptibility to inflammatory diseases such as inflammatory bowel diseases (IBDs) or colon cancer. On the other hand, in rural areas where the diet is closer to the Paleolithic diet comprising of fruit and vegetables enriched in fibers and RS, the prevalence of these inflammatory diseases is low while SCFA and presence of SCFA-producing bacteria are significantly more elevated (De Filippo et al., 2010). These data aligns with a “diet hypothesis” which suggests that adequate intake of fiber promotes a healthy microbiota that significantly reduces the prevalence of inflammatory diseases, notably through the release of SCFA (Macia et al., 2012, Maslowski and Mackay, 2011). Despite intense public health efforts to promote the beneficial effects of a healthy diet in Western countries, the incidence of obesity and inflammatory diseases are still increasing suggesting that other approaches must be explored. One alternative could be to provide food supplements such as the prebiotic inulin-type fructans, which have been shown to promote Bifidobacteria at the expense of Roseburia species and of Clostridium cluster XIVa in mice (Dewulf et al., 2011). The other alternative would be to directly introduce a cocktail of beneficial bacteria including the SCFA producer Bifidobacteria into solution, such as yogurt, similar to how some currently available probiotics products are consumed. One study has shown that gavage of mice with B. longum increased the production of acetate (Xiong et al., 2004) and reduced their susceptibility to infection. Another study showed that mice inoculated with VSL#3 (commercial formula containing eight naturally occurring probiotic strains of bacteria) showed protection against acute DSS-induced colitis (Mennigen et al., 2009). This suggests that even if all the mechanisms behind the use of probiotics are not fully understood, such as their rate of survival or site of action, they remain to be a very promising therapeutic strategy.

As discussed, while the majority of SCFAs are generated and utilized within the vicinity of the gut, a small proportion of propionate and acetate reaches the liver where they can be used as substrates for the energy-producing tricarboxylic acid cycle and efficiently metabolized to produce glucose. A small percent of SCFAs in the gut exists as unionized forms and can directly cross the epithelial barrier. However, most exists in an ionized state and requires specialized transporters for their uptake. Therefore, the passage of the majority of SCFAs across the mucosa involves active transport mediated by two main receptors: the monocarboxylate transporter 1 (MCT-1) and the sodium-coupled monocarboxylate transporter 1 (SMCT-1) receptors. Both MCT-1 and SMCT-1 are highly expressed on colonocytes and also along the entire gastrointestinal tract including the small intestine and the cecum (Iwanaga, Takebe, Kato, Karaki, & Kuwahara, 2006). Additionally, MCT-1 is also highly expressed on lymphocytes suggesting the importance of intracellular SCFA uptake by these cells (Halestrap & Wilson, 2012). Additionally, SMCT-1 is expressed on the kidney and thyroid gland. SMCT-1 binds SCFAs in order of affinity butyrate > propionate > acetate (Ganapathy, Gopal, Miyauchi, & Prasad, 2005). Unabsorbed SCFAs are excreted.

Section snippets

SCFA Sensing and Signal Transduction

The ability of SCFAs to modulate biological responses of the host depends on two major mechanisms. The first involves the direct inhibition of histone deacetylases HDACs to directly regulate gene expression. Intrinsic HDAC inhibitor (HDACi) activity is particularly characteristic of the SCFAs butyrate and propionate. The second mechanism for SCFA effects is signaling through G-protein-coupled receptors (GPCRs). The major GPCRs activated by SCFAs are GPR41, GPR43, and GPR109A.

Varied Functions of SCFAs

SCFAs, particularly butyrate, are key promoters of colonic heath and integrity. Butyrate is the major and preferred metabolic substrate for colonocytes providing at least 60–70% of their energy requirements necessary for their proliferation and differentiation (Suzuki et al., 2008). As such, colonocytes of germ-free mice, deficient in SCFAs, are highly energy deprived, as indicated by decreased expression of key enzymes involved in fatty acid metabolism in mitochondria (Tazoe et al., 2008).

Integrative View of the Gut Microbiota, SCFAs, and Disease

The incidence of both inflammatory and autoimmune diseases has increased dramatically in Westernized countries over the past several decades. While both genetic and environmental factors influence the induction of such diseases, the contribution of diet and the relevance of SCFAs have only been appreciated recently. The effect of SCFAs on various inflammatory and autoimmune diseases will be discussed below.

IBDs such as Crohn’s disease (CD) and ulcerative colitis (UC) are characterized by

Perspective

The incidence of autoimmunity, IBD, and allergy has increased dramatically in Western and Westernized countries. This increase parallels a decrease in the consumption of fiber and indigestible starches. Carefully designed studies are now required to evaluate the effect of diet, independent of other possible contributing factors (i.e., hygiene, infection, sunlight, etc.). These studies will be critical for determining the role of diet, particularly fiber and SCFAs, in the development of Western

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