Akkermansia muciniphila: is it the Holy Grail for ameliorating metabolic diseases?

ABSTRACT The increasing prevalence of metabolic diseases has become a severe public health problem. Gut microbiota play important roles in maintaining human health by modulating the host’s metabolism. Recent evidences demonstrate that Akkermansia muciniphila is effective in improving metabolic disorders and is thus considered as a promising “next-generation beneficial microbe”. In addition to the live A. muciniphila, similar or even stronger beneficial effects have been observed in pasteurized A. muciniphila and its components, including the outer membrane protein Amuc_1100, A. muciniphila-derived extracellular vesicles (AmEVs), and secreted protein P9. Hence, this paper presents a systemic review of recent progress in the effects and mechanisms of A. muciniphila and its components in the treatment of metabolic diseases, including obesity, type 2 diabetes mellitus, cardiovascular disease, and nonalcoholic fatty liver disease, as well as perspectives on its future study.


Introduction
The prevalence of metabolic diseases such as obesity, type 2 diabetes mellitus (T2DM), cardiovascular diseases (CVD), and nonalcoholic fatty liver disease (NAFLD) has becoming a severe public health problem: 1, 2 Gut microbiota can regulate host metabolism by influencing immune maturation and homeostasis, protecting against pathogen overgrowth, regulating intestinal endocrine functions and neurologic signaling, modulating energy metabolism, and producing functional metabolites. 3,4 The compositional and metabolic changes of intestinal microbiota (dysbiosis) is closely involved in the occurrence of metabolic diseases. 5,6 Thus, considerable attention has been paid to gut microbiota-targeted therapies on metabolic diseases with diverse approaches, including probiotics, prebiotics, fecal microbiota transplantation, and antibiotics. [7][8][9][10][11][12] Akkermansia muciniphila, a commensal bacterium, is an oval-shaped, non-motile bacterium with no endospore formation, and a microaerophilic microbe that was first isolated from human feces in 2004. [13][14][15] It colonizes in the intestinal tract early in life, and accounts for approximately 1-3% of the total intestinal microbiota in healthy adults. 16 A. muciniphila resides in the intestinal mucus layer, utilizing the mucin as the sole source of carbon, nitrogen, and energy. 13 In recent years, A. muciniphila has attracted much attention for its comprehensive roles in maintaining host wellbeing, 17 which is regarded as a promising "next-generation beneficial microbe" for metabolic disease prevention or therapy owing to its various properties, including producing shortchain fatty acids (SCFAs), 13,15,18 improving intestinal integrity, 19,20 and reducing endotoxemia through inhibiting the translocation of lipopolysaccharide (LPS) from the intestine to circulation. [19][20][21] However, the exact mechanisms underlying the benefits of A. muciniphila on metabolic diseases are complicated because similar effects have been observed on either live A. muciniphila or pasteurized A. muciniphila, their outer membrane proteins or secreted proteins, as well as extracellular vesicles.  In these papers, we systemically reviewed the updated progress of A. muciniphila with respect to its role in metabolic diseases, and discussion on its future research direction as well.

A. muciniphila and obesity
In recent years, with the changes in people's dietary habits and the increased availability of high calorie diet, overweightness and obesity have become one of the most serious health problems in the world. Manipulation of gut microbiota is a promising strategy for obesity prevention or therapy. [43][44][45][46] The relative abundance of A. muciniphila is significantly reduced in high-fat diet (HFD)-fed obese mice or rats compared with their lean littermates, and negatively correlated with body fat mass and glucose intolerance (Table 1). 19,[47][48][49][50][51][52] Nevertheless, inconsistent results have also been reported. For example, the copy number of A. muciniphila had an increased trend in high-fat and high-sucrose diet (HFHS)-fed mice without reaching a statistical significance when analyzed by qPCR. 53 In addition, by 16s rRNA sequencing, Arias et al. found significantly increased relative abundance of Akkermansia genus in HFD-fed female C3HeB/ FeJ mice. 54 Interestingly, the dietary impacts on Akkermansia levels were found to be genetically dependent in mice: Akkermansia was decreased in 129S1/SvImJ mice, but was increased in A/J, NOD/ LtJ, C57BL/6 J, and NZO/HILtJ mice after shortterm HFHS intake. 55 Thus, the divergent genetically susceptible to intestinal microenvironment might account for the different response of A. muciniphila abundance to diet in the host. In clinical studies, a decreased abundance of A. muciniphila occurred in adults and children with obesity of both sexes. [56][57][58][59][60][61][62][63][64][65][66][67][68] Furthermore, studies indicated that the reduced A. muciniphila in patients with obesity was independent of other metabolic diseases, including diabetes and NAFLD. 65,66,68,69 In contrast, an increase in A. muciniphila abundance was observed in children with obesity compared to children with normalweight. 70 Despite some inconsistent findings, the majority of studies in both animal and clinical studies support the negative correlation between A. muciniphila and obesity.
Given this negative correlation, the role of A. muciniphila in obesity has been widely investigated in both mice and human subjects. Everard et al. reported that administration of A. muciniphila reversed a series of disorders in HFD-fed mice, including reducing body weight, relieving insulin resistance, and fasting hyperglycemia, as well as increasing the mRNA expression of genes involved in the regulation of adipocyte differentiation and lipid oxidation. 19 From that on, many research teams further found live or even pasteurized A. muciniphila as well as its components, including the outer membrane protein Amuc_1100, A. muciniphila-derived extracellular vesicles (AmEVs), and secreted protein P9, were effective in improving diet-induced obesity. 19,21,22,24,26,27,29,[32][33][34]36,39,42 These findings were summarized in Table 2. The benefits of A. muciniphila in obesity intervention has also been investigated in one clinical trial. Depommier et al. compared the safety and efficacy of live and pasteurized A. muciniphila in adults with overweight or obesity. Their results showed that daily oral supplementation of 10 10 CFU of live or pasteurized A. muciniphila for three months was safe and well tolerated. However, improved insulin sensitivity, reduced insulinemia, and plasma total cholesterol was only present in patients given pasteurized A. muciniphila supplementation. 37 This prospective study showed the feasibility to administer A. muciniphila to obese humans, however, further study is needed to demonstrate the relationship between supplement of A. muciniphila and improvement of metabolic parameters on a larger scale of subjects.

A. muciniphila and T2DM
T2DM is a common metabolic disease, which is genetic susceptible and obesity-oriented. [73][74][75] In addition to well-recognized genetic and environmental risk factors, gut dysbiosis has emerged as a new risk factor for T2DM development, 2,76 in which the decreased abundance of A. muciniphila was frequently observed in either diabetic mice, 19 or patients with pre-diabetes or T2DM. 77 Our previous study demonstrated that administration of A. muciniphila reduced the fasting blood glucose level in western diet-fed mice, suggesting that A. muciniphila contributes to T2DM recovery. 26 Moreover, a series of studies have shown that A. muciniphila supplementation may regulate host lipoprotein metabolism, improve insulin sensitivity, and alleviate hepatic metabolic inflammation in The 16S rRNA sequencing result showed the relative abundance of Akkermansia had an increased trend in high-fat and high-sucrose group without reaching a statistical significance. However, qPCR analysis showed the copy number of A. muciniphila was comparable between two groups.
Wang et al (2015) 52 HFD induced obesity Feces qPCR 1. Normal (n = 8)2. HFD (n = 8) The level of A. muciniphila in the HFD-fed rats was lower than that in the normal group rats. There was a significantly higher abundance of A. muciniphila in patients with short and medium durations than those with long duration of diabetes.

Human
(Continued) The relative abundance of A. muciniphila in children with obesity was higher than that in children with normal-weight.
Li  (Continued) 1. HFD + PBS (n = 4) 2. HFD + AmEVs (n = 3) AmEVs significantly decreased the body weight gain, when compared with HFD-fed group. The total cholesterol level in the AmEVs-fed group was lower than that in the HFD-fed group. The epididymal fat pad weight in the AmEVs-fed group was lower than that in the NC group, albeit not significantly so.
Bodogai et al (2018)   A. muciniphila sub reduced body weight and food consumption, improved blood glucose control, and prevented memory decay but not depression induced by high fat diet.
A. muciniphila sub can also decrease systemic inflammation and improve tryptophan metabolism in mice fed HFD, produce high concentrations of acetic acid, propionic acid and isovaleric acid, and restore gut microbiota altered by HFD.
Alive (n = 9) The supplemention of A. muciniphila was safe and well-tolerated, it can reduce the levels of relevant blood markers of liver dysfunction and inflammation while the overall gut microbiome structure was unaffected.
(Continued) e1984104-10    mice. 23,29,42 A recent clinical trial of a new probiotic formulation WBF-011, which contains A. muciniphila and another four bacterial strains as well as inulin, found that WBF-011 improved postprandial blood glucose in T2DM patients. 38 This is the first randomized controlled trial to show the effect of A. muciniphila on improving T2DM in human subjects. In addition, the baseline abundance of A. muciniphila also affected the metabolic outcomes of calorie restriction: individuals with higher baseline A. muciniphila showed better responses toward calorie restriction than those who had low baseline A. muciniphila. 78 Interestingly, the alteration of A. muciniphila was also found to be involved in the anti-T2DM effect of metformin, a widely used first-line medicine for T2DM. 79 Shin et al. reported that metformin significantly increased the abundance of A. muciniphila in HFD-fed mice, while oral supplementation of A. muciniphila to HFD-fed mice without metformin also improved glucose tolerance and reduced inflammation in adipose tissue. 22 Cuesta-Zuluaga et al. found higher abundance of A. muciniphila in T2DM patients with metformin therapy than healthy subjects (Table 3). 80 These results suggest that the elevated A. muciniphila contributes to the anti-T2DM effect of metformin, providing new understanding on the role of A. muciniphila. Overall, existing evidence highlights the significance of A. muciniphila in T2DM development, as well as its involvement in the anti-T2DM activity of clinical medicines.

A. muciniphila and CVD
CVD remains the leading cause of death worldwide, especially in western countries. 81,82 The relationship between gut microbiota dysbiosis and CVD has been well determined. 83,84 Dietary phosphatidylcholine or L-carnitine can be metabolized into trimethylamine (TMA) by the gut microbiota, [85][86][87] and then transported to the liver, where TMA is converted into trimethylamine N-oxide (TMAO) by hepatic flavin monooxygenase 3 (FMO3). [88][89][90] TMAO has been shown to be a potent trigger and biomarker for CVD. 91,92 Recently, Plovier et al. reported that supplementation with A. muciniphila significantly increased the excretion of TMAO and TMA in urine, resulting in decreased plasma TMAO and TMA levels. 21 In addition, they found that HFD induced two-fold higher FMO3 expression compared with that in control-diet fed mice, whereas treatment with pasteurized A. muciniphila could offset this change, suggesting pasteurized A. muciniphila intervention may also reduce TMAO production. 21 Li et al. discovered that oral administration of live A. muciniphila reduced exacerbation of atherosclerotic lesion formation, as well as aortic and systemic inflammation induced by a western diet, and improved intestinal integrity in antherosclerotic Apoe −/− mice. 20 These evidences indicate that A. muciniphila, live or pasteurized, has a protective effect against CVD development.
In addition to the co-metabolized TMA/TMAO pathway by host and gut microbiota, short-chain fatty acids (SCFAs), which can be generated by A. muciniphila, are also essential metabolites for bridging the crosstalk between A. muciniphila and host. 93,94 The beneficial effects of SCFAs on host metabolism have been extensively investigated and reviewed in CVD. 95,96 In summary, A. muciniphila may play a protective role in CVD development directly or through producing metabolites, and via crosstalk with host and commensal bacteria as well.

A. muciniphila and NAFLD
NAFLD is a chronic liver disease and hepatic manifestation of metabolic syndrome. The homeostasis of commensal bacteria and bacteria-derived molecules have been increasingly recognized as a key determinant of NAFLD. 6 The association of A. muciniphila with NAFLD development was recently investigated in obese mice with NAFLD, in which a decreased abundance of A. muciniphila was observed. 97 The administration of anti-obesity drug, such as liraglutide, decreased the levels of total cholesterol and triacylglycerol in the liver while increasing the abundance of A. muciniphila. 98 A. muciniphila supplementation also decreased the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and alleviated liver histopathological damage in a mouse model . 99 Kim et al. recently reported that oral administration of A. muciniphila prevented fatty liver disease by regulating the expression of genes that regulate fat synthesis and e1984104-16 inflammation in the liver. 34 Moreover, different genotypes of A. muciniphila, isolated from human stool samples, played different roles in HFDinduced hyperlipidemia and liver steatosis. Specifically, A. muciniphila I (Amuc_GP01, strain GP01 of A. muciniphila I) was more effective for alleviating hyperlipidemia, liver steatosis, and glucose tolerance than A. muciniphila II (Amuc_GP25, strain GP25 of A. muciniphila II) in dietary obese mice. Both two genotypes could improve the intestinal barrier, but the effect of A. muciniphila II on improving endotoxemia was not apparent, possibly because they have different characteristics of genes and functions, leading to the identification of specific target pathways and disparate roles. 42 Overall, these results indicate that A. muciniphila may alleviate NAFLD by regulating lipid metabolism and reducing inflammation.

Production of SCFAs and cross-feeding with butyrate-producing bacteria
SCFAs, mainly acetate, propionate, and butyrate, are the principal products of carbohydrate and protein fermentation by gut microbiota. 100 There are a large number of investigations on the diverse roles of SCFAs in host metabolism. 101,102 A. muciniphila is also a potent generator of acetate, propionate, and oligosaccharides by fermenting mucin, 13,18 resulting in the activation of fatty acid receptors FFAR2/GPR41 and FFAR3/GPR43. 103 Interestingly, GPR41 is involved in the microbiotaassociated adiposity process, as regularly raised Gpr41 −/− mice are leaner than wild-type mice, whereas this difference is not observed under germfree (GF) conditions. 104 Activation of GPR41 and GPR43 induces intestinal L cells to produce peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 (GLP-2). 105-107 PYY acts on the gastrointestinal tract by modulating a series of physiological actions. It is a satiety signal released following meals, and decreasing food intake. 108 GLP-1, one of the principal incretin hormones, promotes glucose-dependent insulinotropic activity, inhibits appetite and food intake, delays gastric emptying, and restores the impaired "incretin effect" in T2DM patients. [108][109][110] Acetate could also promote anti-lipolytic activity through GPR43 in white adipose tissue (WAT). 111 GPR43 stimulation by acetate in the WAT, rather than muscle or liver, also improves glucose and lipid metabolism. 112 Propionate can be converted into glucose by intestinal gluconeogenesis (IGN), resulting in satiety and reduced hepatic glucose production. 113 In addition, Lukovac et al. found that many transcription factors regulating lipid metabolism and proliferation, such as Hnf4α and p53 family members (Tp53 and Tp73), were affected by both A. muciniphila and propionate. 114 The biological function of A. muciniphila is also associated with cross-feeding activity with other butyrate-producing bacteria such as Faecalibacterium prausnitzii and Anaerostipes caccae, resulting in the increased production of butyrate. 115,116 Moreover, acetate can also stimulate the growth of butyrate-producing bacteria within the same mucosal niche. 117 Butyrate is not only a preferred energy source for colon cells, 118 it also has various beneficial functions for the host, especially in metabolic diseases, [119][120][121] and is a more potent agonist for GPR41 than acetate or propionate. 103 Overall, considering the facts that pasteurization of A. muciniphila enhanced its capacity to improve body weight, reduce fat mass development and dyslipidemia, 21 and administration of Amuc_1100, AmEVs, and secreted P9 protein replicated part of the biological functions of live bacteria (Table 2), the beneficial effects of A. muciniphila might depend in part on its capacity of SCFAs production, as well as the cross-feeding relationship with other butyrate-producing bacteria in the gut.

Maintaining the integrity of gut barrier
A number of studies have revealed the correlation between obesity-related metabolic diseases and increased gut permeability, which induces metabolic endotoxemia and inflammation. [122][123][124][125] A. muciniphila can considerably improve gut barrier integrity in obese mice by restoring the thickness of the intestinal mucus layer, 19 and oral supplementation of A. muciniphila can increase the number of goblet cells, normalize the mucus thickness of the inner layer, and increase the expression of tight-junction proteins in the gut of both HFD-induced obese mice and mice with alcoholic fatty liver. 19,41,126 Moreover, Li et al. discovered that A. muciniphila reduced intestinal permeability by increasing the expression of occludin and ZO-1 in Apoe −/− mice. 20 Similarly, Zhao et al. reported that administration of A. muciniphila Figure 1. Effects of Akkermansia muciniphila and its derived parts on ameliorating metabolic disorders. The level of A. muciniphila decreased in several metabolic diseases, including obesity, type 2 diabetes mellitus (T2DM), cardiovascular diseases (CVD), and nonalcoholic fatty liver disease (NAFLD). Many interventions based on the diet and surgery have been reported for improving the human health in context of metabolic disorder, which accompanied by the increase of A. muciniphila. A. muciniphila and its different parts, including live or pasteurized A. muciniphila, Amuc_1100, P9, as well as AmEVs, have shown to reduce body weight and fat mass gain, and regulate glucose homeostasis and intestinal barrier. Mechanistically, A. muciniphila and its different parts have shown to improve the intestinal barrier through up-regulating the expression of tight-junction proteins and reducing the leakage of LPS, thus reducing inflammation. In addition, live A. muciniphila produces acetate, propionate, and 1,2-propandiol through the fermentation of mucin. It has a nutritional interaction with butyrate-producing bacteria to stimulate the production of butyrate. These SCFAs can active GPR41 and GPR43 to affect glucose and lipid metabolism. The activation of GPR41 and GPR43 induces the intestinal L cells producing peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 (GLP-2) to decrease food intake. Butyrate promotes the epithelial barrier function by increasing the expression of hypoxia-inducible factor-1α (HIF-1α). Moreover, A. muciniphila, live or pasteurized, can normalize the mucus thickness and increase the number of goblet cells. Pasteurized A. muciniphila specifically decreases the expression of hepatic flavin monooxygenase 3 (FMO3), increases the excretion of TMAO and TMA in urine, and decreases the level of plasma TMAO. It may also increase fecal energy excretion to reduce obesity. Amuc_1100 can act on TLR2 to regulate intestinal homeostasis. Furthermore, the newly identified secreted protein P9 can bind to ICAM-2 to trigger the secretion of GLP-1 by L cells. Both P9 and AmEVs can simulate IL-6, leading to further secretion of GLP-1. The above mechanism was summarized based on existing articles; however, there may be other mechanisms. could reduce chronic low-grade inflammation by decreasing the permeability of the gut and lipopolysaccharide (LPS)-binding protein (LBP) downstream signaling in the liver and muscle. 24 Reunanen et al. found that A. muciniphila could adhere to the intestinal epithelium and enhance enterocyte monolayer integrity in vitro, suggesting the ability of A. muciniphila to repair the damaged gut barrier. 14 Furthermore, a large number of studies have shown that bacteria-derived SCFAs maintain the integrity of the intestinal tract and prevent the translocation of LPS across the intestinal wall to alleviate the systemic inflammatory response. 63,127,128 Not only live A. muciniphila, but also pasteurized A. muciniphila has been found to enhance the gut barrier function, leading to attenuation of metabolic endotoxemia. 21 In addition, Amuc_1100 elevated the development of transepithelial electrical resistance in Caco2-cells and increased the expression of tight junction genes in HFD-fed mice to improve intestinal barrier function. 21,129 Moreover, administration of AmEVs also protected mice from HFD-induced leaky gut (Table 2). 29,41 In addition, gut barrier function is disrupted in inflammatory bowel disease (IBD). As a mucindegrader, A. muciniphila decreased significantly in dextran sulfate sodium (DSS)-induced colitis in mice and in IBD patients, 130,131 while administration of A. muciniphila or AmEVs have been reported to protect the progression of DSSinduced colitis. [132][133][134][135] On the contrary, the abundance of A. muciniphila was found to be increased in the spontaneous colitis in the Il10 −/− mice model of IBD, while supplementation of A. muciniphila further promoted colitis in this model. 136 However, it should be noticed that these findings in immune compromised or genetic editing mouse models cannot be translated into the human situation directly. Although the role of A. muciniphila in colitis is somewhat contradictory based on these reports, most studies supported the potential benefits of A. muciniphila or its derived metabolites in respect to their functions of reducing metabolic endotoxemia and systemic inflammation of host, or improving the integrity of gut barrier. Further studies are also needed to determine the exact role of A. muciniphila, live or pasteurized, or its metabolites, in colitis.

Pasteurized A. muciniphila in metabolic disease
Although the beneficial effects of A. muciniphila on metabolic diseases have been extensively investigated, 19,20,78 their clinical application is still challenging, owing to its microaerophilic requirements and the loss of activity after heat-killing. 19 Meanwhile, its growth media probably contain animal-derived compounds, which may have viruses, allergens or bacterial contaminants, thus limiting the usage for clinical study. Plovier et al. showed that A. muciniphila retained its efficacy in improving metabolic disorders when grown on a synthetic medium, a replacement for animal derived mucins. 21 Ottman et al. identified 79 putative outer membrane and membrane-associated extracellular proteins and 23 of those had different abundance between cells of A. muciniphila grown on mucin-containing media and those grown on the non-mucus glucose-containing media. 137 Moreover, Shin et al. found that A. muciniphila grown under mucin-containing media upregulated genes encoding mucin-degrading enzymes. In contrast, A. muciniphila grown under mucin-depleted conditions upregulated the genes involved in glycolysis, energy metabolic pathways, and 79 genes encoding extracellular protein candidates including Amuc_1100, which, in turn, reduced obesity and improved intestinal barrier more efficiently than administration of A. muciniphila grown under mucin-containing conditions. 39 These findings by different teams suggest mucin in the medium might affect the expression of outer membrane protein and subsequently influence the function of A. muciniphila. Interestingly, the recent study discovered that pasteurized A. muciniphila was more potent than live A. muciniphila for reducing body weight and improving glucose tolerance in HFDinduced obese mice. 21 This is of great significance for clinical applications, and therefore, increased attention has been paid to the effect of pasteurized A. muciniphila on metabolic diseases in recent years. Zhang et al. found that oral administration of live or pasteurized A. muciniphila significantly increased the levels of plasma high-density lipoprotein (HDL) and decrease hepatic glycogen, as well as reduced inflammatory markers of LPS and TNFα to alleviate systemic inflammation. However, oral administration of live or pasteurized A. muciniphila did not improve glucose levels in diabetic rats. 138 Depommier et al. reported that daily oral supplementation of pasteurized A. muciniphila in a small number of subjects improved insulin sensitivity and decreased insulinemia and plasma total cholesterol compared to the placebo group, and the effects of pasteurized A. muciniphila were better than those of live A. muciniphila. 37 Although this study only included a small number of participants, the results highlight the potential of pasteurized A. muciniphila in clinical applications. It is hypothesized that the effects of pasteurized A. muciniphila are attributed to increased energy excretion in feces, reduced carbohydrate absorption, and enhanced intestinal epithelial turnover, but without impacts on intestinal lipid absorption or chylomicron synthesis. 32 In conclusion, these studies demonstrate that pasteurized A. muciniphila is superior to live ones for improving metabolic disorders in mice, rats, and humans; however, the underlying mechanism warrants further investigation.

A. muciniphila outer membrane protein enhances the gut barrier
In addition to the A. muciniphila itself, the identification of active components of A. muciniphila for the treatment of metabolic diseases is also valued recently. Cell derived fragments of A. muciniphila have been shown to activate Toll-like receptor 2 (TLR2), in which a highly abundant outer membrane pili-like protein of A. muciniphila, named Amuc_1100, with specific activating capacity for TLR2 has been identified by proteomics. TLRs regulate bacterial recognition, intestinal homeostasis, and shape host metabolism. 129 Ottman et al. found that Amuc_1100 activated TLR 2 and TLR4 and significantly increased transepithelial electrical resistance in vitro. 129 In line with the enhanced effects of pasteurized A. muciniphila, Amuc_1100 was found to be active after pasteurization. 21 It has also been found that the expression of Cnr1, which codes cannabinoid receptor 1 (CB1) in the jejunum, was lower in Amuc_1100 treated mice. 21 The downregulation of CB1 was associated with improved gut integrity and lipid accumulation induced by LPS in both liver and adipose tissue. 139 Therefore, Amuc_1100 might contribute in part to the beneficial effect of live or pasteurized A. muciniphila on gut barrier function.
In addition to Amuc_1100, several other proteins of A. muciniphila have also been identified including Amuc_1434, Amuc_1686, Amuc_0771, and Amuc_1666. Meng et al. reported that Amuc_1434, a member of the aspartic protease family, 140 degraded mucin2 protein secreted by LS174T and suppresses LS174T cell viability, 141 suggesting its potential involvement in controlling colon cancer. Nevertheless, the roles of some βgalactosidases with mucin degradation capacity, such as Amuc_1686, Amuc_0771, and Amuc_1666, in regulating metabolic disorders remain unclear so far. 142,143

A. muciniphila-derived extracellular vesicles (AmEVs) improve metabolic disorders
Emerging evidence shows that bacteria-derived extracellular vesicles, especially AmEVs, play important roles in mediating host-bacteria interactions. 41,144,145 Chelakkot et al. analyzed the fecal extracellular vesicles of healthy people and individuals with obesity, and discovered that the feces of healthy individuals contained higher levels of AmEVs than individuals with obesity. They also revealed that oral gavage of AmEVs decreased HFD-induced body weight gain and fat mass, and improved metabolic functions and gut integrity. 41 Ashrafian et al. also found that AmEVs ameliorated intestinal barrier impairment in obese mice. 29 Moreover, AmEVs regulated inflammation and energy homeostasis in the colon of obese mice. Compared with A. muciniphila, oral gavage of AmEVs (10 μg/mouse) alleviated more body and fat weight gain as well as blood glucose and cholesterol levels in HFD-induced obese mice. In addition, AmEVs administration significantly reduced the expression of TLR-4 and induced lower TLR-2 expression in the colon tissue of obese mice. 29 It is, however, improtant to note that AmEVs administration reducd daily food intake in this study. Additionally, the dose of AmEVs derived from how many A. muciniphila is unclear, and the relevant of the oral dose of AmEVs to physiological e1984104-20 levels of AmEVs secreted by A. muciniphila in the gut is also unknown. Thus, the rationale for the dose used for AmEVs administration need to be further explored. Moreover, whether AmEVs contain Amuc_1100 or other effectors need further investigation. It has been reported that oral administration of AmEVs alleviated DSS-induced inflammatory bowel disease, characterized by reduced infiltration of inflammatory cells through the colon wall. 132 Overall, these results suggest that AmEVs may protect the host by decreasing intestinal permeability and reducing inflammation in the gut. Therefore, the beneficial effects of the live bacteria may be, at least partly, due to AmEVs.

A. muciniphila-secreted protein ameliorates metabolic disease
Since administration of either the cell-free supernatantlive or live A. muciniphila, but not bacterial pellet, increased systemic GLP-1 secretion, Yoon et al. identified an 84 kDa protein in the culture supernatant, named P9, which accounts for the induction of GLP-1 secretion in HFD-fed mice and L cells. 36 Administration of P9 to HFD-fed mice prevented obesity and improved glucose tolerance by regulating GLP-1 secretion and inducing brown adipose tissue thermogenesis. In terms of mechanism, ICAM-2 can bind to P9 and modulate P9-induced secretion of GLP-1. In addition, P9 strongly induced IL-6 expression and IL-6 dosedependently increased GLP-1 secretion, wheares IL-6 deficiency downregulated the expression of ICAM-2 and blocked the response toward P9induced GLP-1 secretion in mice, demostrating that P9 may improve metabolic diseases through an IL-6-GLP-1 signaling axis. 36 Since pasteurized A. muciniphila and Amuc_1100 also have beneficial effects on regulating blood glucose, 21,32 P9 is not the only way for this bacterium to regulate glucose homeostasis. Cani and Knauf commented on this research and raised several important questions, such as how P9 acts on L cells to stimulate GLP-1 secretion and whether P9 is specific to A. muciniphila, etc. 146 Further, in addtion to stimulating GLP-1 secretion, whether P9-mediated induction of IL-6 promotes inflammtaion in the gut and how it affects gut barrier function need to be illustrated.

Conclusions and perspectives
The current focus on improving health using gut microbiota-targeted strategies is overwhelming in the context of accumulating experimental and clinical evidence. A. muciniphila has emerged as a uniquely promising "next-generation beneficial microbe", especially for metabolic disease management. A large number of studies have confirmed the alteration of A. muciniphila in both animal models and human patients with metabolic diseases (summarized in Table 1), its therapeutic benefits (summarized in Table 2), as well as the efficiency of interventions to boost its abundance (summarized in Table 3). However, most current animal studies with A. muciniphila supplementation were performed with A. muciniphila grown under mucincontaining conditions. The animal-derived mucin may introduce contaminants and cause compromised beneficial effect of A. muciniphila on alleviating metabolic diseases. Therefore, mucin-depleted media should be explored and given more attention for both animal and human investigations. Notably, most of the mechanistic studies on the effects of A. muciniphila were performed in animal models. Given the differences between animal models and humans in genetic and environmental elements, it is critical to investigate the real effects and mechanisms of A. muciniphila in clinical study. Recently, two randomized controlled trials confirmed that administration of A. muciniphila or A. muciniphila containing formulation WBF-011 to human subjects with obesity or T2DM were safe and well tolerated in a 12-week period with significant improvement in several metabolic paramaters. 37,38 This paves the way for more clinical applications of A. muciniphila in the near future. The mechanisms underlying the effects of A. muciniphila on metabolic diseases have been extensively investigated and are summarized in Figure 1.
Given the similar, or even superior efficacy of pasteurized A. muciniphila and its outer membrane proteins such as Amuc_1100 or extracellular vesicles and secreted proteins, the exact mechanisms of A. muciniphila activity in the real world of complicated commensal systems is only beginning to be discovered. In this sense, we envisage several critical aspects for future studies on A. muciniphila. First, a complete understanding is required with regards to the common or differential mechanisms between live A. muciniphila and its derived products, including pasteurized A. muciniphila, or the active components such as proteins, vesicles, or metabolites released from the bacteria. Since it is unclear the equivalence of the doses used for AmEVs, P9, and other effectors with the physiological levels of A. muciniphila found in the gut, the physiological relevance of exact mechanisms of A. muciniphila activity need further exploration. Second, more efforts should be focused on elucidating the complex crosstalk between A. muciniphila and commensal bacteria, which may help to explain the discrepant results that have been observed in preclinical and clinical studies. Third, a deeper exploration of the relationship between the specificity in various conditions and the strains of A. muciniphila, rather than at the species level. Finally, scientists should always hold a reasonable dose of expectation and skepticism in terms of the overwhelming "good effects" of any potential beneficial microbe, including A. muciniphila, if the scientific basis has not been well established. Altogether, given the accumulating evidence of A. muciniphila on ameliorating metabolic disorders in both animals and humans, A. muciniphila is widely supposed to be one of the most promising microbes with multiple benefits for host metabolism. Although the mechanisms underlying the effects of A. muciniphila are largely unclear, identification and isolation of specific effectors and biomolecules that derived from A. muciniphila will pave the way for understanding the mechanisms of their action, which is essential and full of challenges for translation of the positive findings in animals to clinic application.