The mechanisms of Akkermansia muciniphila in regulating glucose homeostasis

Abstract

Obesity and overweight are associated with increased multiple pathologies including type 2 diabetes and cardiovascular diseases as well as cancer. One of the main features of obesity is systemic low-grade inflammation. Although the triggers of inflammation during obesity are debatable, gut microbiota has been considered to be associated with this onset of inflammation. Numerous of studies revealed that dysbiosis of gut microbiota is linked with obesity which may cause disruption of gut barrier leading to systemic inflammation. Advances of high throughput sequencing enabled to unveil profiles of gut microorganisms during obesity and current metagenomics and transcriptomics reveal the functionality of bacteria thus defining the mechanisms of single microbe effects in the obese. One of the recognized anti-obesity associated bacteria is Akkermansia muciniphila. This bacterium is found to be inversely correlated with glucose intolerance, plasma lipopolysaccharide levels and adipose tissue inflammation. Notably, recent report identified the anti-obesity feature-possessing membrane protein derived from A.muciniphila that have barrier strengthening functions as well as immune-regulatory features via toll like receptor 2 signaling pathway. However, these studies had been primarily focused on gut barrier functions.

Emerging evidences suggests that gut microbiota regulates brown adipose tissue (BAT) activities which increases glucose uptake and insulin sensitivity. Few studies, however, noted the relationships between A.muciniphila and this metabolically active organ. Additionally, previous study revealed that A.muciniphila has capacity to induce endocannabinoid like lipids which have potency to induce appetite regulating hormone, glucagon-like peptide 1 (GLP-1). Conversely, there has been no studies that has directly assessed the systemic and local induction of GLP-1 through A.muciniphila.

In this thesis, mechanisms of A.muciniphila in regulating glucose metabolism were analyzed in three aspects. First, BAT activation markers were analyzed to examine whether A.muciniphila has capacity to induce thermogenic effects. Moreover, systemic as well as local induction of GLP-1 were analyzed. Then, to validate if these effects were IL-6 dependent, we daily administrated with A.muciniphila in IL-6 knockout mice then compared with wild type mice. Secondly, gut microbiota of A.muciniphila fed high fat mice was compared with high fat mice then analyzed with IL-6 knockout (IL-6KO) mice to verify if the IL-6 dependent metabolic phenotypes according to this bacterium are potentially related with gut microbiome changes. Thirdly, in order to identify the protein derived from bacterium responsible for secretion of GLP-1, we narrowed down the candidate proteins via fractionations through fast-protein liquid chromatography and assessed with in vitro system followed by proteomic studies. Furthermore, effects of candidate proteins in regulating glucose homeostasis were analyzed in in vivo.

As previous studies, A.muciniphila fed obese mice improved glucose homeostasis compared to high fat fed mice. Notably, among the adipose tissues, we observed that mass of brown adipose tissue was significantly decreased and thermogenic molecular markers were significantly elevated. Moreover, ileal and systemic GLP-1 were significantly enhanced. Interestingly, previous studies demonstrated that thermogenesis and GLP-1 induction are associated with IL-6 induction. Indeed, A.muciniphila induced IL-6 in intestinal tissues and subsequently identified that A.muciniphila induced GLP-1 and brown fat activation were IL-6 dependent.

Dramatic changes in gut microbial communities were observed in A.muciniphila fed high fat mice versus high fat fed mice in both WT and IL-6KO mice. As expected high fat diet decreased the relative abundance of A.muciniphila but increased Desulfovibrionaceae, Mucispirillium schaedlerl, Oscillospira whereas A.muciniphila fed group increased A.muciniphila, Parabacteroides, Sutterella and Coprobacillus in WT mice. On the other hands, high fat fed IL-6KO mice with A.muciniphila supplemented mice were composed by Desulfovibrionaceae, Mucispirillum schaedlerl whereas in high fat fed IL-6KO mice were notably composed of A.muciniphila, S24-7, Rc4-4, Ruminoccaceae implying that distinct dysbiosis was occurred during IL-6 deficient and high fat diet condition and even with A.muciniphila supplementation. Correlation analysis revealed that relative abundances of A.muciniphila was positively correlated with BAT temperature as well as glucose tolerance. These results support the previous results that A.muciniphila induced GLP-1and brown fat activation were IL-6 dependent.

To identify the molecular marker derived from A.muciniphila that induces GLP-1, we used enteroendocrine L cells in vitro. We identified that A.muciniphila derived secreted proteins were responsible for inducing GLP-1. To narrow down the candidate proteins, we used fast protein liquid chromatography to fractionate supernatants of this bacterium. Eventually, we determined the effective fractionate that solely inducing GLP-1. Hence, we applied LC-MS/MS approach to identify target proteins. By screening candidate proteins in vitro, we confirmed a single protein which is responsible for inducing GLP-1 in vitro and validated its glucose regulatory function in in vivo. In conclusion, this thesis highlighted the mechanisms of A.muciniphila in regulating glucose metabolism by analyzing its thermogenic effect as well as GLP-1 secretory effect in high fat obese mice. Furthermore, these effects were dependent with host gene, IL-6 implying that gut microbiota crosstalk with host immune systems in order to exert beneficial effects on metabolically sensitive organ. Additionally, the novel molecule (Amuc_1631) derived from this bacterium responsible for inducing GLP-1 was identified and demonstrated to improve glucose tolerance suggesting the alternative therapeutic target to alleviate metabolic disorders.