How to Take Butyrate Supplements - Tesseract

Evidence is increasing that disturbances in the gut microbiome may play a significant role in the etiology of obesity and type 2 diabetes. The short chain fatty acid butyrate, a major end product of the bacterial fermentation of indigestible carbohydrates, is reputed to have anti'inflammatory properties and positive effects on body weight control and insulin sensitivity. However, whether butyrate has therapeutic potential for the treatment and prevention of obesity and obesity'related complications remains to be elucidated. Overall, animal studies strongly indicate that butyrate administered via various routes (e.g., orally) positively affects adipose tissue metabolism and functioning, energy and substrate metabolism, systemic and tissue'specific inflammation, and insulin sensitivity and body weight control. A limited number of human studies demonstrated interindividual differences in clinical effectiveness suggesting that outcomes may depend on the metabolic, microbial, and lifestyle'related characteristics of the target population. Hence, despite abundant evidence from animal data, support of human data is urgently required for the implementation of evidence'based oral and gut'derived butyrate interventions. To increase the efficacy of butyrate'focused interventions, future research should investigate which factors impact treatment outcomes including baseline gut microbial activity and functionality, thereby optimizing targeted'interventions and identifying individuals that merit most from such interventions.

Whether the beneficial properties of butyrate can be translated to clinical practice and implemented to treat metabolic disturbances in humans still needs to be elucidated. Increasing colonic butyrate levels can be accomplished by various intervention strategies such as prebiotic and probiotic supplementation or transplantation of the intestinal microbiota. 13 , 27 Butyrate can also be administered as an end product itself either orally, intravenously, or rectally. 28 These interventions may mediate differential effects considering it may reach different metabolically active organs. To illustrate, orally administered free butyrate is taken up almost entirely by enterocytes in the proximal intestine and may not reach the colon. 29 Recently, an excellent review by Coppola et al. 30 already highlighted the potential protective role of butyrate in obesity and obesity'related disorders, predominantly by presenting animal data. Nevertheless, to exploit butyrate as a therapeutic intervention for obesity and disturbed glucose homeostasis in humans, it is crucial to characterize the conditions in which butyrate is (un)able to convey beneficial metabolic effects. Therefore, this review aims to assess the ability of butyrate to alleviate obesity'related chronic low'grade inflammation and impaired energy and substrate metabolism by integrating animal data with available human data to provide a comprehensive overview of the plethora of butyrate data that is out there. We summarize available literature on butyrate including its luminal production, absorption, and metabolism and discuss a mechanistic underpinning of its metabolic effects via interorgan cross talk. Thereafter, we discuss existing therapeutic strategies that aim to increase butyrate levels in the digestive system and/or the circulation and the current evidence regarding the putative effect of butyrate on body weight control and insulin sensitivity in humans. Lastly, this review intends to disentangle scientific inconsistencies and differences in the efficacy of human intervention trials to identify the hurdles that still need to be overcome in order to advance butyrate'focused intervention aimed at improving metabolic health.

However, the exact role of butyrate in the etiology of obesity remains controversial, since individuals with obesity appear to have higher fecal butyrate concentrations compared with their lean counterparts, even when a similar diet is consumed 14 , 15 and this difference is attenuated upon weight loss. 16 , 17 These observations have led some researchers to believe that butyrate may contribute to the obesogenic phenotype, for example, because microbial energy harvest from fibers is more efficient or because butyrate is used for de novo lipid synthesis. 18 , 19 Nevertheless, fecal concentrations may not accurately represent physiological concentrations because ˂10% of the total butyrate production is excreted in the feces. Mice studies suggest that the obese microbiota actually has a reduced capacity to ferment fibers 20 and produce butyrate. 21 Moreover, cross'sectional data indicate an inverse association between fasting plasma butyrate and body mass index (BMI), pointing towards reduced circulating butyrate levels in individuals with obesity. 22 The higher fecal butyrate levels observed in individuals with obesity may therefore merely reflect a difference in absorption or microbial utilization and not necessarily a higher production. Individuals with obesity or a disturbed glucose homeostasis actually seem to have a decreased abundance of butyrate'producing taxa and a decreased expression of genes involved in butyrate production in the gut microbiome, 23 , 24 , 25 , 26 supporting a significant role for butyrate in energy and glucose homeostasis.

The prevalence of obesity has been on the rise for the last 50 years and is currently still rising at an alarming rate. 1 , 2 Evidence is accumulating that hints towards a relationship between the gut microbiome and the development of obesity and obesity'associated complications such as type 2 diabetes (T2DM) and nonalcoholic fatty liver disease (NAFLD). 3 , 4 Consequently, therapeutic strategies to modulate the microbiome towards a more favorable profile have gained more interest in recent years. 5 The short chain fatty acids (SCFA) that are produced from the microbial fermentation of indigestible carbohydrates (e.g., dietary fibers), often referred to as saccharolytic fermentation, can mediate diverse local as well as peripheral effects. These metabolites are put forward as the gateway through which the gut microbiome is able to affect host physiology and metabolism. 6 The main three SCFA are butyrate, propionate, and acetate and are present in an estimated respective molar ratio of 20:20:60 in the colon and 4:5:91 in the systemic circulation. 7 , 8 All three SCFA have been recognized for their potential beneficial effects on metabolic health. 6 Acetate, for instance, may have beneficial metabolic effects in context of obesity and glucose homeostasis. 9 Although acetate is present at the highest concentration in intestine as well as systemic circulation, it is butyrate that has been under vigorous scientific scrutiny. Despite the extensive splanchnic extraction of butyrate, increased systemic butyrate concentrations in response to dietary fibers have been reported in healthy individuals 10 , 11 as well as individuals with metabolic syndrome (MetS). 12 Its presumed anti'inflammatory and weight'reducing properties coined the idea that butyrate may act as a helpful tool for obesity control. 13

Besides GPR41 and GPR43, butyrate is the only SCFA that can bind to GPR109A. This receptor is expressed in the small intestine, colon, adipose tissue, and several immune cells including macrophages. 134 , 135 , 136 , 137 In vitro work has shown that butyrate'mediated GPR109A signaling promotes interleukin'18 release from intestinal epithelial cells, 138 inhibits nuclear factor ĸB signaling pathways in macrophages, 139 , 140 and reinforces colonic macrophages and dendritic cells to promote the differentiation of naïve CD4+ T cells into regulatory T'cells and interleukin'10 producing T'cells, 110 , 138 which altogether reduce colonic inflammation. Interestingly, diabetic mice display increased GPR109A expression in the jejunum compared with nondiabetic controls. An explanation for this may be that GPR109A promotes glucose uptake, resulting in hyperglycemia. 141 Recently, studies have unveiled that butyrate also binds to the olfactory receptor: Olfr558. Next to its function as olfactory sensory neurons in the nose cavity, this receptor is enriched in renal and cardiac vasculature and suggested to be involved in blood pressure regulation and muscle regeneration. 142 , 143

The expression of GPR41 is widespread, most abundantly in adipose tissue 118 and also in peripheral blood mononuclear cells, 117 enteroendocrine cells, enterocytes, 122 pancreas, spleen, bone marrow, and lymph nodes. 123 Experiments conducted in knockout GPR41 mice suggest the receptor to be involved in peptide YY (PYY) release, intestinal transit rate, and energy harvest from the diet. 124 GPR43 is predominantly expressed in immune tissues especially on polymorphonuclear cells such as neutrophils 118 , 119 , 125 and also in skeletal muscle tissue, liver, 126 white adipose tissue (WAT), 127 and on serotonin'containing mucosal mast cells and PYY'releasing L'enteroendocrine cells in the intestine. 128 Hence, butyrate may stimulate intestinal PYY and serotonin release through GRP43 signaling. These L'enteroendocrine cells simultaneously secrete proglucagon, which can act as a precursor for glucagon'like peptide 1 (GLP'1) production. 129 GLP'1 and PYY are gut'derived hormones that influence insulin secretion and glucose homeostasis, therefore sometimes referred to as incretins, and also regulate food intake and satiety as circulating hormones and through innervation of the gut'brain neural circuit. 130 GPR43 knockout mice display weight gain, increased adiposity, and reduced systemic insulin sensitivity even on a normal chow diet, whereas adipose tissue'specific GPR43 overexpression protects mice against the development of obesity even when a HFD is consumed. 131 Both GPRs have been implicated with beneficial effects on intestinal barrier integrity, inflammation, and immunity thereby maintaining gut health. 132 , 133

Secondly, butyrate can bind to receptors belonging to the G protein'coupled receptor (GPR) family (see Figure 2 ). All SCFA can bind to GPR41 and GPR43, but the receptor specificity varies per SCFA. Butyrate mainly activates GPR41 (ligand potency GPR41 = propionate = butyrate> acetate), whereas acetate and propionate prefer binding to GPR43 over butyrate (ligand potency GPR43 = propionate = acetate>butyrate) 117 , 118 , 119 , 120 albeit ligand specificity may be specie'specific and appears different for mice and humans. 121

Many of the effects of butyrate are mediated through the activation of two intracellular pathways 18 (see Figure 2 ). Firstly, butyrate is a histone deacetylase inhibitor (HDACi), specifically suppressing the activity of class I and II HDACs. 108 , 109 A HDACi inhibits the removal of acetyl groups from histones, making DNA more accessible for transcription and thereby increases the expression of downstream target genes. 109 Several in vitro studies have shown that butyrate'mediated HDAC inhibition may change T'cell polarization and effector function including a shift from CD4+ naïve cells towards regulatory T'cells 110 and a shift in gene expression of Tc17 cells towards a more CD8+ cytotoxic T'cell phenotype. 110 , 111 In this way, butyrate may regulate cytokine profiles, for example, by increasing the production of interleukin'10 and interleukin'17 and thereby decreases inflammation. 110 , 111 , 112 Many of the tumor suppressive effects of butyrate, extensively reviewed elsewhere, 113 , 114 , 115 , 116 have also been attributed to HDAC inhibition.

After absorption, butyrate can be transported to the mitochondria for subsequent β'oxidation. Here, butyrate is first converted back into butyryl'CoA, which eventually yields two acetyl'CoA molecules. 99 In the initial step of the tricarboxylic acid cycle, acetyl'CoA is converted to citrate which can by fully oxidized to generate adenosine triphosphate (ATP) or is shuttled out of the mitochondria and utilized for de novo lipogenesis. 71 Because butyrate is the main oxidative substrate for colonocytes, accounting for more than 70% of their total energy demand, 100 , 101 concentrations in the portal vein are reduced by approximately 'fold compared with colonic concentrations. 8 Sudden death autopsies of six victims performed in the late s revealed that butyrate concentrations in portal vein are approximately 29 μmol/L on average and decrease even further to 12 and 4 μmol/L in the hepatic and peripheral bloodstream, respectively. 8 A more recent study determined SCFA flux in patients undergoing abdominal surgery and found a butyrate concentration of 30.1, 12, and 7.5 μmol/L in the portal vein, hepatic vein, and radial artery, respectively. 102 Butyrate release appears highest in the distal intestine as butyrate concentrations were reported to be three times higher in the inferior mesenteric vein (approximately 62 μmol/L), which drains blood from the descending colon, sigmoid colon, and rectum, compared with the veins draining from proximal intestine (approximately 22 μmol/L). 103 Another study showed that systemic butyrate concentrations rapidly declined after intravenous infusion and returned to initial values 1 h after administration, highlighting its short half'life. 104 More than 95% of butyrate is absorbed by the intestinal tract 105 and for a large part is metabolized by enterocyte and colonocyte and thus excreted in expired breath in the form of CO 2. 106 The remaining part (~5%) is excreted in the feces, 107 and a negligible amount (<0.05%) can be traced back in urine. 104 , 106

Butyrate absorption can occur in the small and large intestine via different routes (see Figure 2 ). In lipid'soluble protonated form, butyrate is able to cross the apical membrane of the lumen through passive diffusion. However, since butyrate is a weak acid (pK ~4.8) and the colonic pH is between 5.5 and 6.7, >90% is present in ionized form and needs to be absorbed via an active transporter system. 85 , 86 Two main proteins involved in the transportation of anionic butyrate have been identified, both belonging to the monocarboxylate transporter family: the sodium'coupled monocarboxylate transporter 1 (SMCT'1) 87 , 88 , 89 and monocarboxylate transporter 1 (MCT'1). 90 , 91 SMCT'1 is put forward as the primary butyrate transporter. As the name implies, its transport depends on the sodium gradient, 87 , 88 , 89 whereas MCT'1 transport is coupled to the proton gradient. 90 , 91 Butyrate can also be absorbed via a carrier'mediated counter'transport system that exchanges butyrate for bicarbonate, but, so far, the exact proteins responsible for this exchange remain unidentified. 92 , 93 Butyrate absorption may vary along the gastrointestinal tract as the expression of both transporters appears to increase from the jejunum towards the distal colon in the human intestine. 94 , 95 Sodium'coupled butyrate transport probably plays a larger role in the distal colon as the SMCT'1 K m is considerably lower (~50 μM) than the MCT'1 K m (2.4'2.8 mM). The latter is therefore more active in the proximal colon where butyrate concentrations are high. 85 Interestingly, evidence suggests that inflammation may decrease butyrate'mediated uptake as well as the expression of both transporters. 95 , 96 , 97 Thus, one may speculate that the inflammatory state associated with obesity may downregulate transporter'mediated butyrate absorption. Limited literature is available on SCFA transport on the basolateral side of the membrane. Both SCFA'bicarbonate exchangers and SCFA'cation symport have been reported as plausible basolateral transport mechanisms. The kinetics of the SCFA'bicarbonate antiporter on the basolateral and apical side differ, implying that the transport is managed by two different proteins. 93 , 98

Depending on the location in gastrointestinal tract and individual differences in dietary intake, gut transit time, and gut microbiome composition, colonic butyrate concentrations may vary, but it is estimated to range between 10 and 20 mmol per kg intestinal content. 80 , 81 Yet, lower estimations (1'10 mmol/L of intestinal content) have also been reported. 82 Interestingly, recent work in animals and humans suggests that SCFA concentrations may fluctuate over the time span of a day. 83 , 84 Particularly later on the day, butyrate concentrations decreased as a result of a slight reduction in the abundance of several butyrate'producing strains. These butyrate oscillations may be explained by eating behavior and meal timing, but other factors, independent of food intake, such as the level of circadian hormones may also play a role. 83 , 84 Interestingly, high fat diet (HFD)'fed mice did not exhibit these diurnal butyrate fluctuation patterns, which indicates that microbial disturbances associated with the consumption of a westernized diet may disturb this circadian cycle of microbial butyrate production. 84

The SCFA concentration along the gastrointestinal tract has two gradients: one from the proximal towards the distal colon and another from the base towards the top of the colonic crypt. 71 Several studies have estimated that, on a daily basis, theoretically, 100'400 mmol SCFA can be produced from the consumption of 10 g of fiber. 72 , 73 Butyrate accounts for approximately 20% of the total SCFA production. 74 The proximal colon, in particular the cecum, has the highest SCFA concentrations since the availability of substrates for saccharolytic fermentation is the highest here. As the availability of carbohydrate based'substrates decreases towards the distal colon, SCFA concentrations decline and the amount of SCFA obtained from proteolytic fermentation increases. Proteolytic fermentation yields other by'products besides SCFA including ammonia and branched SCFA such as isobutyrate and isovalerate. 75 Hence, even though isobutyrate is an isoform of butyrate, it is formed from other substrates, mainly valine, by different microbial pathways and therefore may have distinct metabolic effects from butyrate. Isobutyrate is less readily absorbed and metabolized compared with butyrate but may act as an alternative energy source when butyrate levels are low or when butyrate oxidation is abberant. 76 Little is known about the effect of branched SCFA on host health, but increased proteolytic fermentation has mostly been associated with detrimental health effects. 77 , 78 , 79

The production of butyrate from indigestible carbohydrates and its mechanism of action in the colon. Hexose sugars derived from complex indigestible carbohydrates are broken by specific bacterial strains and either produce butyrate directly (e.g., Faecalibacterium prausnitzii, Eubacterium Rectale , and Roseburia intestinalis) or via cross'feeding pathways in which lactate or acetate is converted to butyrate. Butyrate is then absorbed into intestinal cells, predominantly by active transport systems including SMCT'1 and MCT'1. Thereafter, butyrate is either oxidized in the TCA cycle to generate ATP or cytosolic acetyl'CoA is generated, which can be utilized for lipid synthesis or can activate HATs thereby influencing gene expression whereas intracellular butyrate can directly inhibit HDACs. 5'HT, serotonin; acetyl'CoA, acetyl coenzyme A; ATP, adenosine triphosphate; GLP'1, glucagon'like peptide 1; GPR109A, G protein'coupled receptor 109A; GPR41, G protein'coupled receptor 41; GPR43, G protein'coupled receptor 43; HATs, histone acetylases; HDACs, histone deacetylases; MCT'1, monocarboxylate transporter 1; PYY, peptide YY; SMCT'1, sodium'coupled monocarboxylate transporter 1; TCA, tricarboxylic acid cycle. Created with BioRender.com

The microbial synthesis of butyrate in the colonic lumen. First, dietary fibers (carbohydrates) are broken down to monosaccharides and, subsequently, phosphoenolpyruvate through the Embden'Meyerhof'Parnas pathway. Thereafter, acetyl'CoA is produced via pyruvate, which eventually gets converted to butyryl'CoA. Butyryl'CoA can be converted to butyrate through two pathways. The most common one uses acetate as a cosubstrate to generate butyrate and an acetyl'CoA molecule and the other, less common, pathway, produces butyrate via butyrate'phosphate. Both pathways are regulated by different enzymes (indicated in red in the figure). Created with BioRender.com

Butyrate can be produced in the gut from hexose sugars by the condensation of by two acetyl coenzyme A (acetyl'CoA) molecules. In postprandial conditions, the Embden'Meyerhof'Parnas pathway breaks down the hexose sugars derived from complex indigestible polysaccharides to produce phosphoenolpyruvate. 6 Phosphoenolpyruvate acts as a precursor for acetyl'CoA, which, by a succession of four rapid reactions, gets converted to butyryl'CoA. The final step, transforming butyryl'CoA into butyrate, can be performed by two different metabolic pathways, using different terminal enzymes: either phosphotransbutyrylase and butyrate'kinase via butyryl'phosphate or butyryl'CoA:acetate CoA'transferase (see Figure 1 ). The latter uses acetate as a cosubstrate and appears to be the most common pathway. 65 , 66 Metagenomic data indicate that these two acetyl'CoA pathways together account for approximately 80% of total butyrate production, followed by the lysine pathway (11%). Glutarate and 4'aminobutyrate, although only to a minor extent, can also serve as substrates for butyrate synthesis. 31 Some strains including Eubacterium hallii and Anaerostipes spp have the ability to convert lactate or acetate into butyrate. Thus, some dietary fibers induce butyrogenic effects indirectly by increasing lactate or acetate production which in turn can be utilized by other bacteria to synthesize butyrate, a phenomenon referred to as cross'feeding (see Figure 2 ). 32 , 67 , 68 , 69 Furthermore, a study comparing two in vitro gut models, one with both luminal and mucosal microbial niches and one without the mucosal niche, showed that the presence of a mucosal environment induced a shift from acetate towards butyrate production. 70 This shift may be explained by certain butyrate'producing strains that only adhere to the mucosal layer or because mucins, via cross'feeding pathways, can act as a substrate for mucin'converting microbes thereby generating acetate and lactate, which thereafter can be converted to butyrate.

Two key bacterial strains are inferred with a capacity for butyrate production: Faecalibacterium prausnitzii (Clostridial cluster IV) and Eubacterium rectale/Roseburia spp (Clostridial cluster XIVa), both gram'positive anaerobic bacteria belonging to the Firmicute family. 61 Nevertheless, butyrate'producing bacteria constitute a functional group rather than a specific phylogenetic family, as many other butyrate'producing strains have been identified among various clostridial clusters. 18 , 61 The mildly acidic intestinal milieu in the proximal colon appears to promote butyrate'producing bacteria, which thrive at a lower luminal pH, and thereby outcompete gram'negative carbohydrate'utilizing bacteria from the Bacteroides species. 62 , 63 Recently, an in vitro study using human fecal samples emphasized how colonic acidity can affect butyrate production. A pH ˃7.5 reduced the abundance of butyrate'producing taxa, subsequently decreasing butyrate production, even when pectin was provided as a substrate. 64

Butyric acid is also present in several food products that contain bovine milk fat, such as butter and cheese, in which the SCFA is esterified at the α (sn'3) position. 51 , 52 This binding positioning in milk triacylglycerols strongly influences its catabolic rate since pancreatic lipase is able to cleave triacylglycerols at this position resulting in rapid free fatty acids (FFA) release in the small intestine. 53 , 54 Butyric acid can also be found in several triglyceride mixtures belonging to the short' and long'chain acyl triglyceride molecule family. These food additives, also referred to as salatrims, are commonly used as a fat calorie replacer. In these mixtures, butyric acid is interesterified with a long chain fatty acid moiety such as stearic acid. 55 In human clinical studies as well as rodent models for obesity and diabetes, butyrate is mainly supplied orally, in the form of sodium butyrate. Sodium butyrate is well'known for its unpalatable flavor and odor and, since it does not require cleavage by lipase, is rapidly taken up in the upper gastrointestinal tract. 56 At present time, novel strategies exist that improve the edibility and palatability of butyrate and/or increase the absorption and/or release of butyrate in the digestive tract. To illustrate, the use of a special coating made from hydroxy propyl methyl cellulose and Shellac on sodium butyrate tablets can delay its release in the intestinal tract by approximately 2 to 3 h, thereby delivering the product more distally. 57 Furthermore, esterifying butyrate to a dietary fiber such as butyrylated starch prevents digestion in the upper part of the gastrointestinal tract and has shown to increase colonic butyrate concentrations in individuals with low 58 and normal 59 fecal butyrate concentrations. Tributyrin, in which butyrate is esterified to triglycerides, and other butyric acid derivatives such as 4'phenylbutyric acid have an increased palatability and bioavailability compared with butyrate but may induce substantial side'effects and therefore warrant caution if used in context of improving metabolic health. 60

Combining various fibers may provide a more optimal substrate or microbial environment for butyrate production than each fiber separately. To illustrate, a mixture of guar gum (propiogenic) and pectin (acetogenic) enhanced butyrate production in the caecum of mice after 6 weeks of supplementation. 42 In general, the extent and rate of SCFA production from fibers depends on its fermentability, which can be influenced by numerous factors including: degree of polymerization, variations in esterification and saccharide linkage, the preparation method e.g., cooking and cooling, 37 whether it is provided as a concentrate or in a whole'grain matrix, 33 and manufacturing methods e.g., entrapping the starch in microspheres. 43 , 44 Next to stimulating butyrate production, many of these fibers also influence other intestinal processes including alterations in intraluminal pH, gastric emptying, fecal bulking, and the production of bile acids along with systemic effects such as the feeling of fullness and direct effects on the immune system and glycaemic control. 45 Although butyrogenic fibers may predominantly increase butyrate levels in the intestine, it usually also promote the production of other SCFA. Isotope tracing studies have revealed that inulin consumption, for example, significantly increased carbon enrichment of all three SCFA in the circulation in healthy individuals 46 as well as individuals that were overweight or obese, 47 although enrichment was highest for circulating butyrate. Hence, it is important to bear in mind that the beneficial metabolic effects of these fibers cannot be attributed to butyrate alone. 48 Moreover, dietary intake and production of other SCFA can even potentiate the production and effect of butyrate itself. Functional metagenomic analysis showed an increase in butyrate production after resistant starch type 2 intervention in humans was predominantly dictated by the presence of Ruminococcus bromii, which produces the acetate necessary for butyrate production (as described in the following section). 49 Furthermore, esterifying exogenous acetate to resistant starch, thereby delivering acetate to the colon, increased fecal and systemic butyrate concentrations and augmented weight loss and insulin sensitivity in obese mice compared with resistant starch alone. 50

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Butyrate, a four carbon SCFA, is mainly formed from microbial saccharolytic fermentation in the colon and, to a minor extent, can also be produced from the fermentation of residual peptides or proteins (also referred to as proteolytic fermentation). 31 Dietary fiber intake can lead to butyrate production in multiple ways: butyrogenic fibers increase butyrate production by acting as a substrate for bacterial fermentation, whereas bifidogenic fibers increase the abundance of bifidobacteria, which cannot produce butyrate themselves but increase butyrate production indirectly. 32 Examples of dietary fibers that stimulate butyrate production include resistant starch and nonstarch polysaccharides such as arabinoxylans, β'glucans, oligofructose, and inulin. 33 , 34 , 35 , 36 Resistant starch is naturally present in among others legumes, unripe bananas, and cooled'down cooked potatoes but can also be added or fortified into bread and cereals. 34 , 37 Arabinoxylans are mainly found in wheat'based products such a breakfast cereals and bread. 38 Some of these breakfast cereals such as oats and barley may also contain β'glucans, which is also naturally present in edible mushrooms and seaweed. 39 Inulin can be found in a diverse set of plants and vegetables including Jerusalem artichoke, onion, and chicory root and is used as a fat replacer in many food products and, similar to oligofructose, can serve as replacement for sugar. 35 Studies have shown that specifically resistant starch is potent in stimulating butyrate production and yields more butyrate compared with nonstarch polysaccharides. 40 , 41

3. MECHANISTIC UNDERPINNING: BUTYRATE AND INTERORGAN CROSSTALK

3.1. Local intestinal and whole'body effects

One of the primary functions of butyrate is to provide fuel to the cells lining the intestinal epithelium. Numerous studies have demonstrated that butyrate plays a crucial role in the energy homeostasis and mitochondrial functioning of colonocytes. 144 , 145 , 146 , 147 Colonocytes of germfree mice are energy'deprived, but butyrate administration can restore this impaired mitochondrial respiration. 144 Cytosolic acetyl'CoA derived from butyrate via tricarboxylic acid cycle'derived citrate can be utilized to form lipids or can transfer its acetyl groups to histone acetylases (HATs) (see Figure 2), which increase the expression of genes involved in cell proliferation and differentiation. 71 Butyrate also appears to increase the expression of genes involved in fat and energy metabolism in human colonic mucosa. 148 Additionally, butyrate plays an important role in maintaining gut health and gut functioning. It facilitates colonic transit and stimulates neuronal excitability of the colonic circular muscles, 149 , 150 presumably by promoting serotonin release, a well'known stimulator of peristalsis. 151 Butyrate can also promote intestinal gluconeogenesis in enterocytes through gene expression modulation. 152 This butyrate'induced gluconeogenic effect plays a significant role in its observed beneficial metabolic effects since butyrate administration was unable to enhance glucose tolerance or prevent weight gain in intestinal gluconeogenesis knockout mice. 152

Besides its role in energy homeostasis, evidence suggests that oral butyrate supplementation modulates the composition and functionality of the gut microbiome 153 , 154 , 155 , 156 , 157 , 158 , 159 and restores intestinal barrier integrity in diabetic as well as obese mice. 153 , 158 , 160 , 161 , 162 Endotoxemia may play a crucial role in the chronic low'grade inflammation observed in individuals with T2DM and/or obesity. Mice studies have shown an association between increased fat intake and endotoxemia, and this endotoxemia is associated with deteriorated glucometabolic parameters. 163 , 164 Human data seem to corroborate a relationship between intestinal leakage and metabolic health. People with T1DM and T2DM have significantly higher endotoxin levels than nondiabetic controls, which can be reduced by antidiabetic medication. 165 Furthermore, a study showed that dietary fat intake acutely increased endotoxin levels in healthy individuals as well as individuals with obesity, yet a more pronounced elevation was observed in individuals with T2DM and obesity. 166 A recent study reported a significant negative association between BMI and colonic permeability, and several 'leaky' gut markers including zonulin were positively associated with metabolic health parameters in plasma. 167 Butyrate may act as an intestinal barrier'strengthening agent by regulating the expression, localization, and assembly of tight junction proteins 162 , 168 , 169 , 170 , 171 , 172 and promoting the production of antimicrobials 172 and mucin glycoproteins 173 , 174 , 175 and thereby could potentially counteract intestinal leakage. Nevertheless, these effects are mainly derived from animal and in vitro experiments and are not substantiated by human data yet.

Besides beneficial effects on the gastrointestinal barrier, butyrate stimulates the production of gut'derived neuropeptides involved in energy homeostasis and food intake behavior such as glucose'dependent insulinotropic polypeptide (GIP), GLP'1, PYY, and serotonin in obese mice models. 176 , 177 In obese mice, acute intragastric butyrate administration but not intravenous butyrate administration significantly decreased 24'h food intake, 155 implying that the anti'obesity effect of butyrate is achieved by regulatory processes that occur before reaching the periphery. Human cross'sectional data from a cohort covering individuals with a wide range of BMI and glucometabolic status demonstrated that fasting plasma butyrate concentration was significantly associated with circulating GLP'1 but not PYY. 22 Nevertheless, human experimental data remain limited. In patients with T2DM, 45'days of oral butyrate supplementation (600'mg/day) significantly increased serum GLP'1 levels compared with placebo. 178 Yet, acute rectal administration of SCFA mixtures, containing physiological amounts of butyrate, in men with overweight/obesity did not alter GLP'1 but significantly increased fasting and postprandial plasma PYY concentrations. 179 The effect of butyrate on the release of gut hormones warrants more investigation and may depend on intervention duration, mode of administration, and metabolic phenotype or pathological state of the sample population. To illustrate, a mice study comparing the effect of 12'weeks of supplementation with oral sodium butyrate, resistant starch, or a combination of the two reported that resistant starch (coincided by an increase cecal butyrate production) supplementation increased systemic PYY and GLP'1 levels, whereas oral butyrate and the combination intervention did not alter or significantly decreased the levels of both incretins, respectively. These observations suggest that exogenous butyrate uptake in the upper GI'tract may activate a negative feedback loop, thereby inhibiting incretin release from endogenous colonic butyrate. 180

Despite low systemic butyrate concentrations, the effects of butyrate extend beyond the intestine. Animal work has shown that orally administered butyrate may increase energy expenditure, 181 , 182 , 183 change systemic inflammatory marker profiles, 153 , 160 , 183 , 184 , 185 , 186 and alter energy substrate metabolism, promoting a shift from carbohydrate to fat utilization. 155 , 182 The weight'reducing properties of butyrate are supported by abundant evidence from animal studies in which butyrate supplementation prevents diet'induced weight gain. 153 , 154 , 155 , 156 , 158 , 161 , 177 , 182 , 183 , 187 , 188 , 189 , 190 , 191 In addition, animal studies using butyrate'producing probiotic strains 192 , 193 or butyrogenic fibers 42 , 50 showed comparable beneficial results on weight control. Butyrate is known to act on the opioidergic system by epigenetically stimulating the expression of μ'opioid receptor, which may be involved in reward'related pathways that reduce food intake. 194 Similarly, in obese rodent models, oral and intragastric butyrate administration improves insulin sensitivity and glucose tolerance. 155 , 156 , 181 , 182 , 183 , 191 , 195 Besides the weight loss'associated beneficial effects on glucose homeostasis, butyrate supplementation also attenuated oxidative stress and inflammation in nonobese diabetic mice and therefore may have additional antidiabetic properties besides weight loss. 185 Nonetheless, the therapeutic effect of butyrate appears cohort'dependent. To illustrate, the effects on inflammatory processes and intestinal homeostasis of monobutyrin (a glycerol ester of butyrate) treatment varied between two rat cohorts from identical strains kept under exactly the same experimental circumstances as a result of differential microbial composition and, subsequently, microbial metabolite production. 196 Moreover, preliminary data indicate that obesity prone rats need a higher oral butyrate dose than obesity resistant rats (rats were categorized based on their weight gain after 8'weeks of HFD) to elicit the same response on body weight and glucometabolic parameters. 187 Together, this emphasizes that microbial composition and metabolic phenotype can profoundly impact experimental outcomes.

Human evidence supporting the beneficial metabolic effects of butyrate remains limited. The proposed anti'inflammatory potential of butyrate observed in animal studies is, for instance, not as evident in humans. A study evaluating the peripheral blood mononuclear cells of individuals with MetS after 4 weeks of daily oral 4'g sodium butyrate supplementation did not reveal overt effects on inflammatory cytokines production when stimulated by a diverse set of pathogenic stimuli. 197 In contrast, butyrate intervention did significantly improve anti'inflammatory response in the context of trained innate immunity, in which monocytes are capable of enhanced cytokine production upon secondary stimulation with an unrelated stimulus. 197 Peripheral blood mononuclear cells of patients with T2DM that were supplemented 600'mg of sodium butyrate for 45'days displayed reduced markers for diabetes'associated pyroptosis, a form of programmed cell death that promotes inflammation, compared with individuals that received placebo. Butyrate intervention upregulated the expression of several microRNAs that are known to inhibit inflammatory gene expression, potentially explaining this effect. 198 Another study demonstrated that the incubation of monocytes, derived from patients with T2DM, with a supraphysiological butyrate concentration decreased monocyte migration and resulted in a more favorable tumor necrosis factor'α/interleukin'10 production ratio. 199

In summary, butyrate is a pleiotropic metabolite that can induce a wide array of physiological functions and interorgan crosstalk may form the basis for its beneficial effects (see Figure 3). To compose a mechanistical framework, evidence regarding the effect of butyrate on insulin sensitivity and weight control on the liver, adipose tissue, skeletal muscle, pancreas, and brain will be discussed below.

FIGURE 3.

The putative metabolic effects of butyrate on different organs based on evidence from mainly animal and in vitro data. When butyrate is produced from the fermentation of dietary fibers in the intestine it mediates direct local effects on the intestinal barrier. Here, butyrate binds to G protein'coupled receptors, thereby stimulating the synthesis of several neuropeptides that can signal to the brain. Butyrate that is not utilized by intestinal cells is transported to the liver via the portal vein. Thereafter, minor quantities of butyrate reach the circulation, consequently affecting other organs such as the adipose tissue, skeletal muscle tissue and pancreas. 5'HT, serotonin; GIP, glucose'dependent insulinotropic polypeptide; GLP'1, glucagon'like peptide 1; GPR109A, G protein'coupled receptor 109A; GPR41, G protein'coupled receptor 41; GPR43, G protein'coupled receptor 43; PYY, peptide YY. Created with BioRender.com

3.2. Butyrate and liver metabolism

The first organ butyrate encounters after release from the intestine is the liver (except when butyrate is absorbed in the rectum region). Since hepatocytes extensively extract and metabolize butyrate, it may exert considerable effects here. Researchers have identified a liver'specific butyrate transporter: organic anion transporter 7, which takes up butyrate in exchange for the sulfate'conjugated steroid: oestrone sulfate. Consequently, hepatic butyrate transport may play a role in liver steroid hormone metabolism and detoxification processes. 200 Additionally, an isotope tracing study revealed that butyrate infused in the caecum of mice can be traced back in the liver, where its carbon is incorporated into cholesterol and palmitate. 201 Similarly, incubating isolated rat hepatocytes with butyrate'induced cholesterolgenesis and lipogenesis. 202 In human adolescents with obesity, plasma butyrate concentrations were associated with an increase in hepatic de novo lipogenesis after consumption of a high carbohydrate load. 203 Together, these observations imply that butyrate may contribute to the accumulation of fat in the liver, also described as hepatic steatosis. Hepatic steatosis and hepatic inflammation are linked to the development of insulin resistance, a critical hallmark in the pathogenesis of T2DM and NAFLD. Intriguingly, in contrast to above indications that acute butyrate supplementation promotes hepatic steatosis, many studies report (chronic) that butyrate interventions mediate hepatoprotective effects in obese mice models 153 , 155 , 158 , 181 , 182 , 183 , 188 , 195 , 204 , 205 (see Figure 3). These butyrate'fed mice exhibited significant reductions in the development of diet'induced hepatic inflammation, intrahepatic fat accumulation, and liver injury. 153 , 158 , 182 , 188 , 204 , 205 However, a human study that evaluated intrahepatic triglyceride content by 1H'liver magnetic resonance spectroscopy in individuals with MetS after 4'weeks of oral butyrate intervention did not observe any alterations in liver fat. 206

A wide range of in vitro and in vivo studies have provided mechanisms by which butyrate may positively affect liver function and metabolism. First, a NAFLD mice model showed intragastric butyrate supplementation impeded HFD'induced hepatic GLP'1 receptor downregulation, which was independently associated with improved hepatic steatosis. 207 Since hepatic GLP'1 resistance may develop in patients with T2DM and NAFLD, 207 , 208 a GLP'1 synthesizer like butyrate may work better than exogenous GLP'1 or GLP'1 agonists. Secondly, butyrate appears to avert diet'induced hepatic proinflammatory cytokine and enzyme production in obese mice. 153 , 205 These anti'inflammatory responses may partly be mediated by inhibiting an important pro'inflammatory transcriptional regulator'nuclear factor'κB. 205 Thirdly, butyrate may increase the expression of nuclear factor erythroid 2'related factor 2 and its downstream antioxidant enzymes including glutathione and thereby prevent diet'induced hepatic oxidative stress. 183 , 195 Lastly, evidence suggests that butyrate may induce a switch from hepatic lipogenesis to β'oxidation in obese mice, thereby improving hepatic insulin sensitivity. 182 , 209 This may be attributed to an effect on peroxisome proliferator activated receptor γ and fibroblast growth factor 21 (FGF21) expression, as butyrate was unable to convey beneficial hepatic effects peroxisome proliferator activated receptor γ 182 and FGF21 209 knockout mice. Butyrate activates FGF21 in vitro, 209 and FGF21 overexpression in transgenic mice has shown to prevent diet'induced obesity. 210 FGF21 is a hepatokine (albeit also produced in minor amounts by other tissues such as skeletal muscle tissue) involved in the lipolysis and β'oxidation of long'chain fatty acids 209 and may upregulate GLUT1 expression and glucose uptake in extrahepatic tissues such as the adipose tissue. 210 Nevertheless, increased levels of serum FGF21 are reported in individuals with obesity and/or T2DM, 211 , 212 suggesting FGF21 resistance may have developed over time. Indeed, clinical trials using FGF21 analogs in individuals with obesity did not show any weight'reducing effects although lipid profiles, glucose homeostasis, and whole'body insulin sensitivity were improved. 213 , 214

Ex vivo experiments suggest that butyrate promotes hepatic gluconeogenesis 215 , 216 and has adverse effects on hepatic mitochondrial energy homeostasis. 217 , 218 However, these observations do not translate to the in vivo situation. In diabetic mice, butyrate supplementation reduced gluconeogenesis, glycated hemoglobin (HbA1c), and insulin resistance 219 and these restorative effects on hepatic glycolipid metabolism and liver histology are supported by numerous other studies using diabetic mice. 157 , 160 , 161 , 220 , 221 Animal studies showed that administration of sodium butyrate improved hepatic mitochondrial dynamics and efficiency, 183 increased phosphorylation of the AMP'activated protein kinase/acetyl'CoA carboxylase pathway, 183 , 207 and increased expression of glucose transporter 2 183 and the insulin receptor, 207 which may explain improved hepatic insulin sensitivity.

Altogether, an intriguing amount of animal data suggests that butyrate ameliorates diet'induced hepatic insulin resistance, fat deposition, and inflammation, whereas human data are lacking. Future studies should use ultrasound'based technology and magnetic resonance imaging techniques to assess liver histology and the amount and distribution of liver fat after butyrate'focused interventions in humans. 222

3.3. Butyrate and adipose tissue metabolism

One key function of the adipose tissue is to store dietary fatty acids in the postprandial state, to be released in times of increased energy requirement. In individuals with obesity and insulin resistance, adipose tissue functioning appears impaired. This dysfunction is characterized by immune infiltration, a reduced storage capacity, and lipid spillover, contributing to systemic low'grade inflammation and ectopic fat accumulation, respectively, which eventually disturbs insulin signaling. 223 Adipocytes may interact with macrophages and other immune cells, and this interaction may contribute to the observed chronic low'grade inflammation. 224 , 225 Butyrate supplementation has shown to attenuate diet'induced adiposity in obese 153 , 155 , 181 , 226 , 227 , 228 as well as (pre)diabetic rodent models 161 , 219 and may reduce adipocyte hypertrophy associated with a HFD. 155 , 182 , 189 , 228 , 229 Moreover, protein analysis of the adipose tissue of butyrate'fed obese mice demonstrated increased expression of the insulin receptor 189 as well as downstream targets such as glucose transporter 4 156 , 189 compared with HFD'controls, suggesting improved adipose'tissue insulin sensitivity (see Figure 3). Butyrate is proposed to influence adipose tissue function in several ways, by affecting intracellular adipogenesis, lipolysis, and adipose tissue inflammation.

In vitro and in vivo work propose that butyrate triggers adipocyte hyperplasia by stimulating adipogenesis. 189 , 230 This effect is supported by increased expression of adipose tissue'specific proliferating cell nuclear antigen, an essential protein for DNA replication, in butyrate'treated versus control'fed obese mice. 189 In contrast, in lean mice and piglets, adipogenesis appears reduced after long'term butyrate treatment, 231 , 232 suggesting that butyrate'induced alterations in adipogenesis may differ in lean and obese animal models. The effect of butyrate on lipolysis still remains under debate as some studies suggest it stimulates lipolysis, whereas others report antilipolytic effects. Several in vitro studies suggest that supraphysiological 230 , 233 , 234 as well as physiological butyrate concentrations increase basal and β'adrenergically mediated glycerol release, which is a measure for adipose tissue lipolysis. 234 Butyrate may mediate lipolytic effects through gene modification. 233 Specifically increased acetylation and activation of the β3'adrenergic receptor, a key regulator in lipolysis, have been reported after butyrate intervention in the WAT of obese mice. 228 Evidence suggests that β'adrenergic'mediated lipolysis is blunted in context of obesity which may, among other things, be explained by a reduced level and sensitivity of the β3'adrenergic receptor. 235 , 236 Hence, if butyrate'mediated activation of the β3'adrenergic receptor also occurs in humans, this may potentially (partially) restore sensitivity. Yet, in contrast to the increased lipolysis after incubation with butyrate alone, a SCFA mixture high in butyrate concentration (35%) did not affect basal nor β'adrenergically mediated glycerol release in a human adipocyte model. 234 Moreover, opposed to lipolytic effects in monoculture, butyrate appears to diminish lipolysis concurrent with reduced inflammatory responses in a differentiated adipocyte'macrophage co'culture. 140 , 237 Thus, it is crucial to evaluate adipocytes in context of macrophages. These adipocyte interactions highlight that in vivo studies need to be conducted in order to investigate the effects of butyrate on adipose tissue in context of other tissues (as they may affect one another). In obese rodent models, chronic butyrate treatment attenuated diet'induced elevations in systemic lipid profiles including triglycerides and cholesterol. 155 , 181 , 187 , 188 , 190 , 191 , 238 These improved lipid markers hint towards enhanced adipose tissue storage capacity but may also be the result of improved liver functioning (or both). Human cross'sectional data showed that fasting plasma butyrate levels were negatively associated with plasma FFA levels. Yet, no significant associations were observed with plasma triacylglycerols and glycerol. 22 Rectal administration of a SCFA mixture containing high butyrate concentrations significantly increased lipid oxidation and reduced fasting plasma free glycerol compared with placebo in men that were obese or overweight. 179 However, this increase in lipid oxidation was significantly correlated to plasma acetate but not to butyrate concentrations. A 4week intervention study in individuals with MetS from both sexes showed 4'g/day of oral butyrate supplementation significantly reduced total cholesterol and triglycerides levels compared with baseline. 206 In contrast, another comparable study with men with MetS observed a significant increase in plasma total cholesterol and low'density lipoprotein cholesterol and no alterations in FFA and triglycerides compared with initial levels. 159 In patients with T2DM receiving 600'mg/day for 6'weeks, a similar increase in plasma total and low'density cholesterol was observed albeit only compared with baseline levels and not compared with placebo. 239

Besides adipogenesis and lipolysis, butyrate also alters the expression of proteins involved in adipose tissue inflammation, also referred to as adipokines. 240 Butyrate administration has shown to attenuate the production of several diet'induced pro'inflammatory markers including tumor necrosis factor'α in the adipose tissue of obese mice 153 , 158 , 162 and diabetic mice. 241 Additionally, evidence from obese mice models suggests that chronic butyrate supplementation decreases systemic and adipose tissue'specific leptin 177 , 183 , 189 , 227 , 228 , 229 and increases adiponectin 183 , 189 concentrations, two other well'known adipokines, towards a similar range as those of lean mice. Leptin is associated with inflammatory processes, increasing in proportion to body fat, whereas adiponectin has an inverse relationship with adipocyte size and may contribute to anti'inflammatory processes, adipose tissue vascularization, and insulin sensitivity. 242 , 243 Recent work in an obesity mice model also showed that sodium butyrate supplementation may reinforce a more anti'inflammatory immune cell profile in the adipose tissue, shifting towards increased levels of M2 macrophages and regulatory T'cells relative to the population of M1 macrophages and naïve CD4+ T'cells. 162

Next to effects on the WAT, butyrate treatment may stimulate mitochondrial activity, lipid oxidation, and thermogenic capacity, evidenced by elevated uncoupling protein'1 protein levels, in the brown adipose tissue (BAT) of obese 155 , 181 and microbiota depleted mice. 244 Additionally, BAT and subcutaneous WAT may metabolize butyrate because the adipocytes of butyrate'treated mice exhibit increased mRNA acyl'CoA medium'chain synthetase 3 expression, the enzyme for the initial step of butyrate oxidation, and carnitine palmitoyltransferase 1α expression, suggesting elevated fatty acid oxidation. 245 These effects may underpin butyrate's presumed beneficial effect on energy expenditure in animal models. Nevertheless, 4'weeks of 4'g/day butyrate intervention did not alter metabolic BAT activity or resting energy expenditure in lean men nor men with MetS. 159

Overall, animal studies suggest that butyrate may restore adipose tissue inflammation and activate BAT. Yet, its effect on lipogenesis and lipolysis remains inconsistent, and human data, so far, do not solidify the observations of animal studies.

3.4. Butyrate and skeletal muscle metabolism

Skeletal muscle may account for approximately 80% of the insulin'stimulated glucose clearance under hyperinsulinemic'euglycemic clamp conditions. 246 , 247 In postprandial conditions, this is considerably lower, accounting for 23% of total glucose disposal 248 , 249 but still plays an important part in regulating energy flux. The obese insulin'resistant phenotype is characterized by impaired mitochondrial functioning and metabolic inflexibility, in which the skeletal muscles can no longer match lipid oxidation to the increased lipid supply. 223 , 235 Moreover, both T2DM and obesity have been associated with relative loss of muscle mass and strength. 250 Few studies have investigated the specific effect of butyrate on muscle metabolism, but sodium butyrate treatment has shown to reduce lipid accumulation 191 , 227 and improve mitochondrial functioning in the skeletal muscle of obese rodents 181 , 187 , 227 (see Figure 3). These effects might be mediated by increased expression of antioxidant enzymes and peroxisome proliferator'activated receptor γ isoform α and mitochondrial transcription factor A, two transcriptional regulators involved in mitochondrial biogenesis. 187 , 232 Additionally, chronic butyrate interventions have shown to increase the percentage of slow'twitch type I muscle fibers in obese mice 181 , 226 and lean piglets. 251 These fibers are oxidative and contain more mitochondria than fast'switch type II muscle fibers. Short'term butyrate supplementation may enhance mitochondrial lipid oxidation in the gastrocnemius muscle of obese mice, indicated by increased expression of genes and proteins involved in lipid oxidation and oxidative phosphorylation compared with control. 181 , 227 A mice model investigating the effect of chronic butyrate administration in aging mice supports above reported effects as butyrate'reduced intramuscular fat accumulation and increased markers of mitochondrial biogenesis, antioxidant activity, and oxidative metabolism in the skeletal muscle. 252 A human cross'sectional study using mendelian randomization analysis has identified a causal relationship between the production of microbial butyrate and appendicular lean mass in Chinese menopausal women, suggesting that butyrate may play a role in maintaining muscle mass in humans as well. 253

Butyrate may also increase muscle'specific insulin sensitivity, 181 , 187 evidenced by enhanced phosphorylation of the insulin receptor substrate 1 181 and increased mRNA expression of insulin receptor substrate 1 and glucose transporter 4 187 in the gastrocnemius muscle of butyrate'treated obese rodents compared with controls. Nevertheless, the insulin'sensitizing effect of butyrate is probably also mediated indirectly, via the production of gut'derived incretins. GLP'1 is known to alter muscle microvasculature increasing both blood volume and blood flow in insulin sensitive healthy humans 254 and rats, 255 and these responses remain preserved in insulin'resistant rats. 256 In this way, butyrate may enhance insulin action and glucose oxidation in the muscle because insulin delivery is increased as a result of enlarged endothelial myocyte surface. Furthermore, incubating primary myocytes derived from individuals with obesity with GLP'1 increased glucose uptake and restored the activity of enzymes involved in muscle metabolism. 257 Similar effects have been reported for PYY. 258

Altogether, butyrate may counteract obesity'associated mitochondrial dysfunction and muscle atrophy and can indirectly increase insulin'mediated glucose disposal in the muscle tissue. Future butyrate'focused intervention studies in humans should evaluate transcriptomics from muscle biopsies and changes in muscle mass, for example, by a dual X'ray absorptiometry scan. 259

3.5. Butyrate and pancreatic insulin functioning

The pancreas is a crucial organ for energy and substrate metabolism, responsible for among others the secretion of insulin, a key hormone in the regulation of postprandial substrate metabolism. Butyrate might be able to prevent pancreatic dysfunction associated with the insulin'resistant obese phenotype. Animal data indicate that butyrate may increase insulin secretion 161 , 177 , 187 and reduce pancreatic fat deposition and β'cell damage, thereby preserving islet functioning 161 , 187 , 190 , 219 , 238 (see Figure 3). Several studies have shown that chronic butyrate treatment decreased fasting insulin levels in T2DM rats 260 and obese rodents 156 , 177 , 183 , 191 , 238 compared with their respective controls. One of these studies showed that acute butyrate administration rapidly increased insulin release compared with a saline control, whereas the same dose of other fatty acids including acetic acid did not significantly alter insulin secretion. 177 In vitro studies performed a couple of decades ago suggest that butyrate induces an acute stimulatory effect on insulin release. 261 , 262 Nevertheless, these studies used supraphysiological concentrations (2'10 mM), and recent work with rat islets only demonstrated a significant effect on pancreatic β'cell functioning after 24'h incubation with 5'mM of sodium butyrate, whereas an acute insulinotropic effect was not observed. 263 Since pancreatic β'cells express GPR41 and GGPR43, 264 butyrate may directly regulate insulin secretion through G protein mediated signaling, yet whether this occurs remains controversial. 265 The observed insulin release pattern after acute butyrate administration in HFD mice overlapped with GLP'1, PYY, and GIP release, while other SCFA were unable to induce gut'derived hormones (with exception of propionate'induced GIP stimulation), pointing towards indirect regulation of insulin production. GLP'1 can influence pancreatic β'cells by accelerating the glucose'dependent closure of ATP'regulated potassium channels, which provokes postprandial insulin secretion 266 and simultaneously inhibits glucagon release. 267 Moreover, butyrate may also stimulate the antioxidant defense system in the pancreas 187 and inhibit pancreatic β'cell apoptosis through gene expression modulation 187 , 190 , 260 thereby indirectly contributing to enhanced pancreatic functioning.

Altogether, animal data suggest that butyrate may potentially improve pancreatic insulin response but the acute effect of butyrate on glycaemic control and insulin release (in dietary context) remains to be investigated in humans. Future studies could study the effect of different doses of butyrate on postprandial substrate metabolism by using a cross'over design.

3.6. Butyrate and the brain

The brain plays an important regulatory role in energy homeostasis as a master regulator of food intake behavior and serving as a thermostat for energy expenditure. Evidence suggests that obesity is characterized by vagal afferent signaling dysregulation, indicated by a diminished ability to switch off orexigenic responses in the fed state as well as a reduced sensitivity to endocrine satiety proteins. 268 Moreover, besides the commonly known obesity'associated metabolic complications, obesity is associated with neuropathic pain, alterations in brain structure, impaired cognitive functioning, and increased risk of developing neurogenerative diseases including Alzheimer's. 269 , 270 Mice studies have demonstrated that butyrate may have the ability to counteract these obesity'associated neurological changes, 188 , 271 , 272 and the pain'reducing properties of butyrate have been corroborated by a cross'over randomized controlled trial (RCT) using rectal butyrate enemas in healthy adults. 273

As systemic butyrate levels are relatively low, it is unlikely that note'worthy amounts of butyrate reach the brain. Positron emission tomography in nonhuman primates confirmed that butyrate can cross the blood barrier, but uptake is extremely low (˂0.006%). 274 In contrast, substantial increases in butyrate concentration in the brain were reported after administration of butyrate'producing bacterial strains in mice, 275 , 276 and an isotope tracing study in mice suggests that butyrate contributes to tricarboxylic acid metabolites in the brain. 244 Despite these observations, the effect butyrate may have on the brain is probably predominantly indirect. Butyrate may act as a sensor to provide intestinal information to the brain by signaling brain regions involved in food intake including the nucleus tractus solitaries. A mice study demonstrated supraphysiological intraperitoneal butyrate injection (1'6'mmol/kg) dose'dependently and time'dependently decreased food intake and induced the strongest anorexigenic effect out of the three major SCFA. This anorexigenic effect was completely abolished by capsaicin pretreatment, an inhibitor of afferent vagal nerve innervation. Selective inhibition of the hepatic branch of the vagus nerve resulted in a similar inhibitory effect, so a hepatic'portal'vagal route may be at play. The authors postulated butyrate may regulate satiety via GPR41 or GPR109A signaling on nodose ganglion neurons in the brain, 277 but this remains to be investigated.

As stated previously, butyrate stimulates the production of gut'derived neuropeptides including serotonin, GIP, GLP'1, and PYY as well as adipose tissue'derived leptin. Similarly, monobutyrin supplementation preserved the sensitivity to cholecystokinin, another well'known vagal anorexigenic stimulator, and strengthened the response to cholecystokinin'induced energy intake reduction in HFD'fed mice. 196 These endocrine proteins and neurotransmitters can signal various hypothalamic nuclei in the brain resulting in an increased feeling of satiety and a reduced drive to eat 130 , 278 , 279 (see Figure 3). Intriguingly, a mice study showed intragastric butyrate administration but not intravenous administration led to a significant decrease in 24'h food intake compared with control, and this was abolished by subdiaphragmatic vagotomy. Intragastric butyrate supplementation reduced FOS'positive neurons, a marker for neuronal activity, in the nucleus tractus solitaries and dorsal vagal complex in the brainstem and reduced c'FOS expression of neuropeptide Y positive orexigenic neurons in the hypothalamus. 155 Taken together, this study suggests that the gut'brain axis is necessary for butyrate to elicit a significant effect on food intake behavior.

Both WAT and BAT depots interact with the brain through distinct sympathetic neuronal axon projections (and the WAT also via leptin production). Whereas the WAT is predominantly involved with energy storage, the BAT may modulate energy expenditure. 280 Butyrate'fed mice exhibit elevated tyrosine hydroxylase expression in the BAT, a marker for peripheral sympathetic nerve activity, compared with control obese mice. The butyrate'induced thermogenic effect and increased lipid oxidation observed in the BAT were diminished after vagotomy, which also points towards (partial) regulation by the vagus nerve. 155 However, the quantification of metabolically active BAT and its contribution to energy expenditure in humans remains uncertain and sympathetically mediated thermogenesis is probably predominantly generated by skeletal muscle tissue. 281 , 282

How butyrate may affect the activity of reward'related pathways in humans remains to be investigated. Clinical studies have demonstrated that 4'weeks of 4'g/day sodium butyrate did not alter total energy intake compared with the start of the intervention in lean individuals, 159 patients with T1DM, 283 nor individuals with MetS. 159 , 206 One of these studies also evaluated if butyrate had any satietogenic effects, by using the Visual Analog Scale for appetite and hunger, but no changes were observed post intervention. 206 Despite these findings, the same study revealed that butyrate supplementation modulated neural pathways in the brain. Butyrate intervention had a tendency to reduce cerebral dopamine transporters binding in the striatum of individuals with MetS. 206 This transporter has been linked to reward processing and glucose homeostasis and appears downregulated in people with a higher BMI. Hence, a reduced dopamine transporter binding may appear counterintuitive since butyrate is considered an anorexigenic stimulator. Heart rate variability, a marker of autonomic nervous system activity, was also significantly increased post butyrate intervention. 206 Both observations advocate butyrate can affect the human brain dopaminergic system and vagal nerve innervation, respectively. However, additional research is required to elucidate how these pathways are affected and if they translate to actual dietary changes in humans.

Overall, animal data suggest that butyrate may provide a therapeutic strategy to innervate the central nervous system and combat obesity'associated impaired sympathetic signaling. Yet, reductions in food intake or satiety in response to chronic butyrate intervention in humans have not been reported so far. A summary of the effects of butyrate derived from animal studies on organ level and the mediated crosstalk between organs is displayed in Figure 3.

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