{'fraction_non_alphanumeric': 0.02549817870152132, 'fraction_numerical': 0.019891436325976716, 'mean_word_length': 5.644323170008304, 'pattern_counts': {'":': 0, '<': 4, '<?xml version=': 0, '>': 4, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Butyrate to combat obesity and obesity‐associated metabolic disorders: Current status and future implications for therapeutic use

Summary 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.

(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 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, crosssectional 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.
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. 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 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 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

| Butyrate biosynthesis
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 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 butyrylphosphate 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 van DEUREN ET AL. 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). 32,67-69 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.

| Butyrate concentration in the human gut
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] 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

| Butyrate absorption, metabolism, distribution, and excretion
Butyrate absorption can occur in the small and large intestine via different routes (see Figure 2). In lipid-soluble protonated form, butyrate F I G U R E 1 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 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 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 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 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 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.
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) 117-120 albeit ligand specificity may be specie-specific and appears different for mice and humans. 121 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 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 Thus, butyrate, as a HDACi, can directly influence gene expression and, through GPR activation, modulates appetite neurocircuitry and anti-inflammatory immune responses. The production, absorption, metabolism, and mechanism of action of butyrate in the gut are summarized in Figure 2.

| 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  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  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 glucosedependent 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 antiobesity 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 interven-  [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 van DEUREN ET AL.
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 antiinflammatory response in the context of trained innate immunity, in which monocytes are capable of enhanced cytokine production upon secondary stimulation with an unrelated stimulus. 197 198 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 factorα/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.

| 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- 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. 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   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 (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 crosssectional 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 lowdensity 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 lowdensity 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 wellknown 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. 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

| Butyrate and pancreatic insulin functioning
The pancreas is a crucial organ for energy and substrate metabolism,

| 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  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. increased peripheral insulin sensitivity. 291 Table 1). No effect on fasting glucose, homeostatic model assessment for insulin resistance (HOMA-IR), or body weight was observed for either of the probiotic mixtures. Strain-specific qPCR showed fecal that A. halli was detected more often after 4 and 12 weeks of WBF-011 supplementation. Unfortunately, fecal C. Butyricum and C. beijerinckii were below detection limit at all time points and it is therefore uncertain whether these bacterial strains were engrafted in the gut microbiome.

| HUMAN BUTYRATE-FOCUSED THERAPEUTIC INTERVENTIONS TO TREAT OBESITY AND RELATED METABOLIC DISORDERS
Intervention-induced changes in fecal SCFA concentrations were highly variable between participants and not significantly different between groups. Cross-feeding pathways may partially explain why WBF-011 mediates stronger metabolic effects. A. muciniphila may provide acetate which C. beijerinckii, C. butyricum, and A. hallii can utilize to form butyrate. Besides this cross-feeding pathway, A. halli is also able to convert the lactate produced by B. infantis to butyrate.
Remarkably, participants taking a sulfonylurea agent along with metformin appeared to respond less to WBF-011 intervention compared with metformin use alone. 286 These individuals are usually characterized by a longer duration or severity of T2DM. Additionally, metformin is known to modulate the gut microbiome resulting in increased butyrate production and researchers suggest a synergistic relationship between the two. 292 Hence, one could speculate that WBF-011 may be more effective in the initial stage of T2DM or that the dose of metformin in these patients was lower, resulting in less synergism.
Recently published work revealed that fasting plasma butyrate levels were significantly increased after WBF-011 intervention compared with placebo, and this was associated with a decrease in HbA 1c in individuals that were not using a sulfonylurea agent. 287  can also be provided orally as an end product itself. Interestingly, 4 weeks of daily oral sodium butyrate supplementation did not affect peripheral nor hepatic insulin sensitivity, measured using the golden standard measurement, in males with MetS (n = 10). In contrast, both parameters were significantly increased in healthy males (n = 9) 159 (see Table 2). The provided dose may have been insufficient for individuals with MetS, potentially explaining this discrepancy. After (4 week of supplementation), respectively, also do not report overt changes in glucose metabolism upon oral sodium butyrate supplementation (see Table 2). 178,283 Remarkably, butyrate supplementation in patients with T1DM decreased the abundance of butyrate-producing bacteria and fecal SCFA concentrations. 283 In patients with T2DM, within-group analysis revealed the combination of inulin and oral sodium butyrate administration was able to significantly reduce fasting blood sugar and hip-to-waist ratio compared with baseline albeit not compared with placebo intervention. Since the use of first-line medication for T2DM, which could include metformin, prior and during the study was allowed, a synergistic effect with butyrate may have occurred. 178 Another RCT including patients with T2DM showed that 6 weeks of sodium butyrate supplementation increased fasting plasma insulin compared with baseline albeit not compared with placebo.
Remarkably, HOMA-IR increased significantly compared with initial levels as well as placebo, yet this was no longer significant after adjusting for potential confounding factors including a significant difference in T2DM duration and concentration of antidiabetic medication between the two intervention groups. 239 Unfortunately, the latter two studies performed with individuals with T2DM did not evaluate a biomarker for butyrate production. None of the above described clinical studies indicate changes in body weight after butyrate intervention, but it remains uncertain whether this may be attributed to the short intervention period (<8 weeks).
In conclusion, the efficacy of butyrate-focused human interventions appears modest and is only apparent in within-group analyses.
Nonetheless, the efficacy may depend on the target population and baseline characteristics such as microbiome composition and further investigations are warranted. Besides assessing butyrate levels directly in the circulation, future studies should focus on bacterial activity to study changes in butyrate production and pathways involved in more detail, for example, by using multi-omics approaches such as metagenomics and metabolomics. 296 Noninvasive ingestible capsules that enable direct sampling of luminal content may be used to acquire important bioinformation on microbial butyrate production in different regions of the gastrointestinal tract. 297,298 Numerous studies have reported heterogeneity in the production and kinetics of butyrate after probiotic and prebiotic supplementation, [299][300][301][302][303][304] which may depend on microbial phenotype and absorption capacity of the host, and this may partially explain interindividual variation in metabolic response towards these interventions. To illustrate, after probiotic intervention, fecal butyrate concentrations were substantially increased in individuals with a low butyrate production at baseline. 301 [307][308][309][310] Next to interindividual differences, several other components may influence clinical efficacy including intervention duration, concentration and type/form of butyrate or fiber supplied, mode and frequency of administration, and whether butyrate is provided fasted or in the postprandial state (see Table 2). For prebiotic interventions, This fiber combination may potentially reach the colon more distally, explaining the observed increased systemic butyrate levels.

| CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Overall, solid statements about the potential metabolic benefits of butyrate-focused interventions in humans remain premature and are likely highly context specific. In order to tilt microbial disturbances and impaired metabolic processes, interventions may require a van DEUREN ET AL.
personalized approach and a longer intervention period. Future studies should specify whether the optimal dose of butyrate differs for specific target populations, for example, individuals with obesity and individuals using metformin and elucidate the optimal mode, frequency, and (dietary) context of butyrate intake. Lastly, the controversy on the role of butyrate in individuals with metabolic disturbances needs to be disentangled. Future research should elucidate whether butyrate is an important etiological factor in the prevention and management of obesity and obesity-related complication and determine which processes in carbohydrate fermentation and SCFA handling are altered. Whether obesity and T2DM dysregulates butyrate production, absorption, clearance, and/or alters the sensitivity towards butyrate provides crucial information that can be fundamental for improving the efficacy of butyrate-focused clinical trials.

ACKNOWLEDGMENTS
We would like to thank you for your interest in this review. We aim to incite discussion on how to ingrate current available literature and bridge existing knowledge gaps, which may eventually contribute to the development of better and more targeted intervention strategies to combat obesity and obesity-related metabolic diseases. This review was made possible because of the work conducted by many great scientists, and we acknowledge their effort, dedication, and compiled work, which has greatly contributed to our scientific understanding so far. Especially the last decade, research in this field accelerated and emphasized its complexity, suggesting future health interventions can no longer comply a "one approach fits all" strategy.

CONFLICT OF INTEREST
The authors declare no conflict of interest.