Short-chain fatty acids and G-protein coupled receptors

The host has a very limited repertoire of glycoside hydrolases needed to digest complex dietary plant polysaccharides. One important activity of the gut’s microbiota is its potential to digest such dietary fibres.39 Dietary fibres constitute the indigestible portion of plant foods containing two fractions: insoluble fibre such as cellulose or lignin and soluble fibre such as galacto-oligosaccharides or fructo-oligosaccharides. These soluble fibres are digested by enzymes derived from the gut microbiota into short chain fatty acids (SCFAs) such as acetate, butyrate and propionate. Interestingly, SCFAs constitute approximately 5–10% of the energy source in healthy people. Fibre-enriched diets have been demonstrated to improve insulin sensitivity in lean and obese diabetic subjects.40–43 SCFAs diffuse passively, are recovered via monocarboxylic acid transporters, or act as signalling molecules by binding to G-protein-coupled receptors such as G-protein coupled receptor 41 (Gpr41) (free fatty acid receptor 3, FFAR3) and G-protein coupled receptor 43 (Gpr43) (FFAR2).44 ,45 These G-protein-coupled receptors are expressed by many cell types including gut epithelial cells, adipocytes and immune cells. Kimura et al recently demonstrated that Gpr43-deficient mice are obese even when consuming a normal diet, whereas mice overexpressing this receptor specifically in the adipose tissue remain lean independent of calorie consumption.46 Importantly, and this proved a key role for the microbiota, when mice were raised germ-free or were treated with certain antibiotics both types of mice exhibited a normal phenotype. The authors could furthermore demonstrate that SCFA-mediated activation of Gpr43 resulted in suppression of insulin signalling in the adipose tissue subsequently preventing fat accumulation. High-fat diet-induced insulin resistance, as assessed by insulin tolerance and glucose tolerance testing, was significantly increased in Gpr43-deficient mice, compared with wild type mice, an effect which was also abolished after antibiotic therapy. Gpr43 activation also enhances insulin sensitivity by promoting glucagon-like peptide 1 secretion in the gut.47 Gpr43 is neither expressed in liver nor in muscle and therefore it seems that adipose tissue-derived Gpr43 is able to modulate all the metabolic effects after engagement with microbiota-derived products such as SCFAs. Therefore SCFAs are an important energy source for the host, and act as signalling molecules especially in the adipose tissue thereby maintaining energy balance. Data also suggest that the microbiota is the major source for Gpr43 agonists and biological functions of Gpr43 are completely dependent on the gut microbiota (figure 1).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Metabolic pathways affected by the intestinal microbiota. (1) Bacterial glycoside hydrolases cleave complex carbohydrates derived from dietary fibre to produce short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate. SCFAs affect the host's metabolism in several ways. (2) SCFAs-dependent activation of G-protein coupled receptor 41 (Gpr41) induces the expression of peptide YY, an intestinal hormone that inhibits gut motility, increases intestinal transit rate, and reduces the harvest of energy from the diet.48 (3) Engagement of G-protein coupled receptor 43 (Gpr43; and Gpr41) by SCFAs has been shown to trigger the incretin hormone glucagon-like peptide 1 (GLP-1) to increase insulin sensitivity.47 (4) SCFAs-mediated activation of Gpr43 on adipocytes suppresses insulin signalling and inhibits fat accumulation in adipose tissue.46 (5) The SCFAs butyrate and propionate activate intestinal gluconeogenesis (IGN); butyrate through a cAMP-dependent mechanism, propionate via a gut-brain neuronal circuit involving Gpr41.49 (6) The intestinal microbiota suppresses the expression of fasting-induced adipose factor (Fiaf), an intestinal epithelial cell (IEC)-derived circulating inhibitor of the lipoprotein lipase (LPL).56

In contrast, an earlier study observed that conventionally raised Gpr41^–/– mice or germ-free Gpr41^–/– mice colonised with Bacteroidetes theatiotaomicron and Methanobrevibacter smithii were significantly leaner than wild type littermates, while there were no genotype-related differences in germ-free mice.48 A fascinating study from France indicated that SCFAs might exert some of their functions by directly regulating intestinal gluconeogenesis.49 Here, they reported that butyrate upregulated intestinal gluconeogenesis via a cAMP-dependent mechanism and independently from Gpr43 (FFAR2), and propionate affected intestinal gluconeogenesis via a gut-brain neural circuit involving FFAR3 (Gpr41). In mice deficient for intestinal gluconeogenesis (intestinal specific deletion of G6Pase catalytic subunit), the beneficial metabolic effects of SCFAs and soluble fibre were absent clearly highlighting the need for this mechanism. Overall, many studies have now demonstrated that the microbiota in lean mice produces greater amounts of SCFAs, especially propionate and butyrate, thereby promoting leanness by inhibiting fat accumulation in adipose tissue and raising energy expenditure (figure 1).

SCFAs might constitute an attractive link between the microbiota and systemic inflammatory diseases as demonstrated by various recent studies showing that SCFAs, especially butyrate direct the development of extrathymic anti-inflammatory regulatory T cells.50 SCFAs also control the generation of colonic regulatory T cells and protect against colitis in a Gpr43-dependent manner51 (figure 2). Marsland et al recently demonstrated that mice fed a high-fibre diet have an altered microbiota and are protected from allergic airway inflammation. The SCFA propionate regulated allergic inflammation, bone marrow haematopoiesis and dendritic cell function. The effects of propionate were dependent on Gpr41 but not Gpr43. Microbiota-derived propionate has also been recently shown to limit cancer cell proliferation in the liver suggesting antineoplastic functions.52 Taken together, these findings suggest that metabolites produced by the gut microbiota can influence haematopoiesis and immune responses in the lung.53 These data are supportive for the current notion that microbiota-derived products are important players in the generation of local and systemic immunity/inflammation. As studies in T2D especially and consistently revealed that production of SCFAs, especially butyrate is impaired it is reasonable to assume that such mechanisms might contribute to low-grade inflammation observed in those disorders.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Crosstalk between the intestinal microbiota and the host modulates mucosal immunity and systemic inflammation. (1) Short-chain fatty acids (SCFAs), particularly butyrate, promote the formation of peripheral regulatory T cells (Treg).50 ,51 ,93 Mechanistically, butyrate constitutes a natural occurring inhibitor of the histone deacetylases 6 and 9. In such a way butyrate could result in acetylation of Histone H3 to promote the expression of the Treg-specific forkhead transcription factor FoxP3. (2) Toll-like receptor (TLR) 5 is a pattern recognition receptor for flagellin, the protein monomer of bacterial flagella. Signals derived from TLR5 are important for the maintenance of intestinal homoeostasis.58 (3) Accordingly, intracellular signals derived from the NLR family pyrin domain containing 3/NLR family pyrin domain containing 6 (Nlrp3/6) inflammasome act in a similar way.60 Loss of these signals results in detrimental changes in the composition of the intestinal microbiota that promotes insulin resistance and the metabolic syndrome. The features are transmissible into wild type animals with the dysbiotic microbiota. (4) Loss of intestinal homoeostasis and dysbiosis foster insufficiencies in the gut barrier function that result in translocation of bacterial components, for example, lipopolysaccharides (LPS) or even whole organisms into the portal and systemic circulation. There, these factors could promote local inflammation in organs such as the adipose tissue and the liver. AMP, antimicrobial peptides; ASC, apoptosis associated speck-like protein containing a CARD; HDAC, Histone deacetylase.

Studies investigating SCFAs, their metabolism and potential functions have played a major role recently in a better understanding of the interaction of beneficial diets and the host-microbiotal interplay. Several other mechanisms have been discussed in the past years and can only be mentioned briefly. The gut microbiota affects the composition and abundance of certain bile acid species through a variety of mechanisms resulting commonly in low levels of various bile acids in case of obesity.54 Obese mice demonstrate increased expression of farnesoid X receptor and fibroblast growth factor 15, which expression is regulated by bile acids, and directly regulate various metabolic effects. Conventionally raised mice contain much more total body fat compared with those raised under germ-free conditions.21 Conventionalisation of mice suppresses intestinal expression of fasting-induced adipose factor (Fiaf) specifically in differentiated villous epithelial cells in the ileum. Fiaf is a circulating lipoprotein lipase inhibitor produced by the intestine and by liver and adipose tissue.55 Indeed, germ-free Fiaf^−/− mice have the same amount of total body fat weight as conventionalised (Fiaf-suppressed) mice, establishing Fiaf as a key factor of microbial regulation of peripheral fat storage.21 Another metabolic pathway apart from Fiaf involves antimicrobial peptides (AMP)-activated protein kinase (AMPK) that protects mice from obesity produced by consumption of a high-fat high-sugar Western diet.56 Germ-free mice remain lean despite high calorie intake and this state is accompanied by increased activity of phosphorylated AMPK levels in liver and skeletal muscle and enhanced insulin sensitivity, in the liver.56

Innate immune system

Metabolic syndrome is thought to develop through the interaction of various genetic and environmental factors, and includes a complex and yet poorly understood interaction between the intestinal microbiota and the innate system.57 An important role in the development of a metabolic syndrome has recently been demonstrated for the pattern recognition receptor such as the toll-like receptor (TLR)5.58 TLR5^–/– mice exhibit hyperphagia, developed hyperlipidaemia, hypertension, insulin resistance and obesity, and an altered microbiota. Transfer of intestinal microbiota of TLR5^−/− mice into germ-free mice led to metabolic syndrome. These data suggest that innate immune signalling is critical in the development of the metabolic syndrome and alterations in the intestinal microbiota are able to induce the metabolic syndrome (figure 2).

Inflammasomes consist of an upstream sensor nod-like receptors protein, the adaptor protein Asc (apoptosis associated speck-like protein containing a CARD) and the effector protein caspase-1. Various groups have recently shown that the inflammasome may play an important role in metabolic inflammation and some inflammasomes might affect the intestinal microbiota, metabolic syndrome and fatty liver disease.59 ,60 Henao-Mejia J et al observed that inflammasome-deficient mice on a methionine-choline deficient diet develop hepatic steatosis and liver inflammation as well as T2D. Disease could again be transferred between mice upon cohabitation, supporting that the microbiota from inflammasome-deficient mice has the potential to induce inflammatory liver disease. Whether such phenomena have any relevance in human disease, however, remains unclear.

Endotoxin—a major gut-derived player in metabolic inflammation

Several recent studies have suggested that gut-derived endotoxin (lipopolysaccharide, LPS) might be crucially involved in chronic inflammation observed in T2D. A first paper was published by Cani et al showing that a high fat diet (HFD) increased LPS content of the gut’s microbiota and resulted in metabolic endotoxaemia.61 Here, they also observed that subcutaneous infusions of LPS into mice resulted in insulin resistance and obesity similar as after feeding a HFD. In support of such a concept, antibiotic and prebiotic therapy improved metabolic inflammatory parameters in high-fat fed and ob/ob mice.62 ,63 In an animal model of metabolic adaptation where C57Bl/6 mice receiving a HFD became diabetic or resisted diabetes, the diabetes phenotype was associated with an increased gut permeability, endotoxaemia and a specific gut microbial profile.64 These preclinical findings were recently corroborated by clinical studies demonstrating that patients with metabolic syndrome and T2D exhibited endotoxaemia.15 ,16 Endotoxin could therefore indeed reflect one relevant player derived from the gut’s microbiota affecting inflammation-related metabolic functions.

The concept of ‘metabolic infection’

The adipose tissue is the major source of proinflammatory cytokines in case of obesity and associated hepatic steatosis, exhibiting a much higher expression of cytokines, compared with their liver expression.65 ,66 Burcellin et al recently suggested that a so called ‘metabolic infection’ might exist, suggesting that the gut microbiota might be a relevant factor in low-grade systemic inflammation and development of insulin resistance.17 In their studies, the authors demonstrated that prior to the onset of diabetes and early after the mice are switched to a HFD, intestinal bacteria translocate from the gut to the adipose tissue and blood where they might induce low-grade inflammation. Human studies using 16S rDNA pyrosequencing demonstrated that blood levels of certain bacterial DNA (>85% derived from Proteobacteria) were enhanced in prediabetes.67 Furthermore, a recent investigation showed that patients from the DESIR study exhibited a blood microbiota dysbiosis with an increased detection of Proteobacteria in subjects with cardiovascular complications.68 The discovery of bacterial DNA within various tissues such as blood, liver and adipose tissue allows a new perspective on the evolution of metabolic inflammation including adipose tissue inflammation69 (figure 2).

Akkermansia muciniphila

Knowledge in this area is still sparse and somewhat controversial.33 A muciniphila has been recently characterised as a mucin-degrading bacterium residing in the mucus layer.70 A muciniphila, a Gram-negative bacterium, is highly prevalent and constitutes 3–5% of the gut’s microbiota and its concentrations are inversely correlated with the presence of overweight and diabetes in murine and human studies.63 ,71 ,72 Dietary supplementation of fibres, that is, oligofructose to genetically obese mice dramatically increases abundance of A muciniphila.63 Several reports have recently demonstrated that A muciniphila might play a key role in the integrity of the mucous layer and has the potential to reduce inflammation and offer protection against the development of obesity and T2D. The most convincing report suggesting protective functions for Akkermansia was recently presented by Everard et al73 In this study, the authors showed that in genetic and dietary models of murine obesity concentrations of A muciniphila were highly decreased. A prebiotic therapy with oligofructose restored basal levels of A muciniphila and improved metabolic functions including metabolic endotoxaemia. When treating these animals via administration of A muciniphila, body weight was reduced and hyperglycaemia reversed. These studies suggest that A muciniphila controls fat storage, adipose tissue inflammation and glucose metabolism. Data were further corroborated by the fact that heat-killed A muciniphila lacked the above beneficial effects. Interestingly, in this study A muciniphila restored the decreased mucus layer probably improving gut permeability. This study is also supported by recent findings demonstrating that metformin, a key antidiabetic drug results in an increase in Akkermansia concentrations and again as observed in the Everard study administration of A muciniphila resulted in an improvement of various metabolic functions including glucose tolerance and adipose tissue inflammation.74

Early life therapy in the non-obese diabetic mouse model of diabetes using vancomycin results in an increase in the abundance of Akkermansia and improves diabetes further supporting a protective function for this bacterium.75 A muciniphila might exert anti-inflammatory functions also in other disease models as it has been shown that administration of this bacterium improves dextrane sulfate colitis-induced colitis.76

Data on Akkermansia are still somewhat controversial as, for example, in the Chinese MGWAS study a potential protective role for this bacterium could not be demonstrated.33 Qin et al reported as stated before that A muciniphila-related genes were more abundant in patients with T2D; this association, however, needs to be reassessed as Qin et al studied only 337 of the 2176 A muciniphila genes. HFD in various rodent models reduces butyrate and in contrast enhances inflammation and liver fat, and these effects are counteracted by the ingestion of dietary fibre.77 In this particular study when investigating the intestinal microbiota, concentrations of Akkermansia were highest with the fibre-free diet suggesting that Akkermansia expands in the colon of rats fed a HFD. Akkermansia supports mucin degradation and could facilitate intestinal inflammation as shown in Salmonella Typhimurium infected mice.78 Higher abundance of A muciniphila has been observed in healthy controls in some studies discussed previously.36 The still conflicting results on A muciniphila need further studies and clarification.

Faecalibacterium prausnitzii

F prausnitzii is considered the protoypical anti-inflammatory microbiota component. Several studies demonstrated that F prausnitzii concentrations are lower in patients with metabolic syndrome and diabetes.33 ,34 ,36 ,79 The diversity of the microbiota as well as the abundance of F prausnitzii is significantly lower in obese patients and patients with T2D compared with lean individuals.80 Functional studies using F prausnitzii in various disease models of metabolic inflammation and tests of its beneficial functions have yet to be performed.

Diet

Dietary modification has been demonstrated in previous years to rapidly affect the gut microbiota.81 Wu et al observed that short-term diets had no or minor influence on their enterotypes but long-term diets indeed were able to influence the enterotype of individuals.31 Diets enriched in animal protein and saturated fat favoured a ‘Bacteroides’ enterotype, a carbohydrate enriched diet the ‘Prevotella’ enterotype. The Bacteroides enterotype was highly associated with animal protein, a variety of amino acids and saturated fats suggesting that meat consumption as in a Western diet characterised this enterotype. Vegetarians demonstrated enrichment in the Prevotella enterotype. Phyla positively associated with fibre were Bacteroidetes and Actinobacteria, whereas Firmicutes and Proteobacteria showed the opposite association. A study comparing European and African children observed that a Western diet led to a dominance of Bacteroides whereas a more vegetarian diet in Africa was dominated by the Prevotella enterotype.32 The discussion on the issue of ‘enterotypes’ is still open as other studies failed so far to see such associations.

Another important diet study was recently published by David et al.82 Here, the authors could show that short-term dietary interventions were also able to modify the gut’s microbiota substantially. Interestingly, the similarity of each study subjects’s gut microbiota compared with their baseline communities (ß diversity, Jensen-Shannon distance) decreased on the animal-based diet even within 1 day. Analysis revealed that an animal-based diet had a greater impact on the gut microbiota than the plant-based diet. The most abundant taxon changes were observed after an animal-based diet with the clusters Bilophila wadsworthia, Alistipes putredinis and Bacteroides sp, all characterised by the feature of bile resistance. In contrast, an animal-based diet resulted in a decrease in Firmicutes that are able to metabolise dietary plant polysaccharides such as Roseburia, E rectale or Ruminococcus bromii. Animal-based diets resulted in lower levels of SCFAs compared with a plant-based diet. The increase in the abundance of B wadsworthia is of special interest. HFDs in rodents result in an increase in enteric deoxycholic acid (DCA) concentrations, which is produced under the control of commensals such as B wadsworthia and has recently been demonstrated to promote development of liver cancer.83 In the study discussed here, faecal bile acid levels including DCA levels increased during an animal-based diet. DCA is able to affect the growth of various members of the microbiota such as Bacteroidetes and Firmicutes.84 B wadsworthia also has sulfite-reducing capacities and the end product of this pathway, that is, H2S might exert proinflammatory effects at the mucosa level.85 Growth of B wadsworthia is stimulated by selected bile acids in rodents after the consumption of saturated fatty acids. Overall the authors could demonstrate that the human gut microbiome can rapidly switch between herbivorous and carnivorous functional profiles.

Nevertheless all these studies suggest that diet has a major impact on the microbiota and raise hope that selective diets might also be able to affect dysbiosis observed in T2D and thereby modify and improve metabolic control. A thorough discussion of the effects of various diets on the microbiota is beyond the scope of this article.

Drugs

There is increasing evidence that certain drugs might unexpectedly affect the gut’s microbiota. The biguanide drug metformin is widely used in the therapy of T2D. Metformin is able to slow aging in Caenorhabditis elegans by metabolic alteration of the E coli with which it is cultured.86 This effect involves disruption of the bacterial folate cycle, resulting in decreased levels of s-adenosylmethionine synthase and decelerated aging in the worm. Importantly, the effect on C elegans life span was completely dependent on the presence of bacteria. These findings might have human implications because of the widespread use of this drug. Common side effects include GI complaints such as diarrhoea and bloating, reduced folate levels and increased homocysteine levels.87 Indeed administration of metformin alters composition of the microbiota in rats.88 These data are in accordance with a recent study demonstrating that metformin is able to affect the mouse microbiota and increases concentrations of A muciniphila.74 Importantly, metformin increased the number of mucin-producing goblet cells and subsequent studies convincingly showed that administration of A muciniphila had beneficial effects on metabolic functions. This study therefore highlights that medications such as metformin might exert their functions at least partially via modulation of the gut’s microbiota.

Faecal transplant

One report recently showed that faecal transplant via a gastroduodenal tube from lean donors into obese subjects with metabolic syndrome demonstrated a small improvement of insulin sensitivity in recipients.89 Faecal transplant resulted in an increase of the microbial diversity after 6 weeks and a 2.5-fold increase, for example, in the butyrate producer R intestinalis whereas SCFA levels overall decreased. Despite this first evidence that such an approach could be attractive, much more information is needed to advance with larger, well-designed studies to prove whether such a crude approach is overall beneficial for patients with metabolic syndrome/T2D.