The endocannabinoid system links gut microbiota to adipogenesis

Abstract

• We investigated several models of gut microbiota modulation: selective (prebiotics, probiotics, high-fat), drastic (antibiotics, germ-free mice) and mice bearing specific mutations of a key gene involved in the toll-like receptors (TLR) bacteria-host interaction (Myd88 ^−/−). Here we report that gut microbiota modulates the intestinal endocannabinoid (eCB) system-tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels.

• The activation of the intestinal endocannabinoid system increases gut permeability which in turn enhances plasma LPS levels and inflammation in physiological and pathological conditions such as obesity and type 2 diabetes.

• The investigation of adipocyte differentiation and lipogenesis (both markers of adipogenesis) indicate that gut microbiota controls adipose tissue physiology through LPS-eCB system regulatory loops and may play a critical role in the adipose tissue plasticity during obesity.

• In vivo, ex vivo and in vitro studies indicate that LPS acts as a master switch on adipose tissue metabolism, by blocking the cannabinoid-driven adipogenesis.

Abstract

Obesity and type II diabetes have reached epidemic proportions and are associated with a massive expansion of the adipose tissue. Recent data have shown that these metabolic disorders are characterised by low-grade inflammation of unknown molecular origin ( Hotamisligil and Erbay, 2008; Shoelson and Goldfine, 2009); therefore, it is of the utmost importance to identify the link between inflammation and adipose tissue metabolism and plasticity. Among the latest important discoveries published in the field, two new concepts have driven this study. First, emerging data have shown that gut microbiota is involved in the control of energy homeostasis ( Ley et al, 2005; Turnbaugh et al, 2006; Claus et al, 2008) Obesity is characterised by the massive expansion of adipose tissues and is associated with inflammation ( Weisberg et al, 2003). It is possible that both this expansion and the associated inflammation are controlled by microbiota and lipopolysaccharide (LPS) ( Cani et al, 2007a, 2008), a cell wall component of Gram-negative bacteria that is among the most potent inducers of inflammation ( Cani et al, 2007a, 2007b, 2008; Cani and Delzenne, 2009). Second, obesity is also characterised by greater endocannabinoid (eCB) system tone (increased eCB plasma levels, altered expression of the cannabinoid receptor 1 (CB ₁ mRNA) and increased eCB levels in the adipose tissue) ( Engeli et al, 2005; Bluher et al, 2006; Matias et al, 2006; Cote et al, 2007; D'Eon et al, 2008; Starowicz et al, 2008; Di Marzo et al, 2009; Izzo et al, 2009).

Several studies have suggested a close relationship between LPS, gut microbiota and the eCB system. Indeed, LPS controls the synthesis of eCB in macrophages, whereas macrophage infiltration in the adipose tissue occurring during obesity is an important factor in the development of the metabolic disorders ( Weisberg et al, 2003). We have shown that macrophage infiltration is not only dependent on the activation of the receptor CD14 by LPS, but is also dependent on the gut microbiota composition and the gut barrier function (gut permeability) ( Cani et al, 2007a, 2008). Moreover, LPS controls the synthesis of eCBs both in vivo ( Hoareau et al, 2009) and in vitro ( Di Marzo et al, 1999; Maccarrone et al, 2001) through mechanisms dependent of the LPS receptor signalling pathway ( Liu et al, 2003). Thus, obesity is nowadays associated with changes in gut microbiota and a higher endocannabinoid system tone, both having a function in the disease's pathophysiology.

Given that the convergent molecular mechanisms that may affect these different supersystem activities and adiposity remain to be elucidated, we tested the hypothesis that the gut microbiota and the eCB system control gut permeability and adipogenesis, by a LPS-dependent mechanism, under both physiological and obesity-related conditions.

First, we found that high-fat diet-induced obese and diabetic animals exhibit threefold higher colonic CB ₁ mRNA, whereas no modification was observed in the small intestinal segment (jejunum). Moreover, selective modulation of gut microbiota using prebiotics (i.e. non-digestible compounds fermented by specific bacteria in the gut) (Gibson and Roberfroid, 1995) reduces by about one half this effect. Similarly, in genetically obese mice ( ob/ob), prebiotic treatment decreases colonic CB ₁ mRNA and colonic eCB concentrations (AEA) ( Figure 2A). In addition, we have observed a modulation of FAAH and MGL mRNA ( Figure 2A). Furthermore, we have found that antibiotic treatment decreasing the number of gut bacteria content was associated with a strong reduction of the CB ₁ receptor levels in the colon of healthy mice.

Second, we show that the endocannabinoid system controls gut barrier function ( in vivo and in vitro) and endotoxaemia. More precisely, we designed two in vivo experiments in obese and lean mice ( Figure 2). In a first experiment, we blocked the CB ₁ receptor in obese mice with a specific and selective antagonist (SR141716A) and found that the blockade of the CB ₁ receptor reduces plasma LPS levels by a mechanism linked to the improvement of the gut barrier function ( Figure 2C) as shown by the lower alteration of tight junctions proteins (zonula occludens-1 (ZO-1) and occludin) distribution and localisation, and independently of food intake behaviour ( Figures 2D and 3). In a second set of experiments performed in lean wild-type mice, we mimicked the increased eCB system tone observed during obesity by chronic (4-week) infusion of a cannabinoid receptor agonist (HU-210) through mini-pumps implanted subcutaneously. We found that cannabinoid agonist administration significantly increased plasma LPS levels. Furthermore, increased plasma fluorescein isothiocyanate-dextran levels were observed after oral gavage ( Figure 2F and G). These sets of in vivo experiments strongly suggest that an overactive eCB system increases gut permeability. Finally, in a cellular model of intestinal epithelial barrier (Caco-2 cells monolayer), we found that CB ₁ receptor antagonist normalised LPS and the cannabinoid receptors agonist HU-210-induced epithelial barrier alterations.

Third, we provide evidence that adipogenesis is under the control of the gut microbiota, through the modulation of the gut and adipose tissue endocannabinoid systems in both physiological and pathological conditions. We found that the higher eCB system tone (found in obesity or mimicked by eCB agonist) participates to the regulation of adipogenesis by directly acting on the adipose tissue, but also indirectly by increasing plasma LPS levels, which consequently impair adipogenesis and promote inflammatory states. Here, we found that both the specific modulation of the gut microbiota and the blockade of the CB ₁ receptor decrease plasma LPS levels and is associated with higher adipocyte differentiation and lipogenesis rate. One possible explanation for these surprising data could be as follows: plasma LPS levels might be under the control of CB ₁ in the intestine (gut barrier function); therefore, under particular pathophysiological conditions in vivo (e.g. obesity/type II diabetes), this could lead to higher circulating LPS levels. Furthermore, CB ₁ receptor blockade might paradoxically increase adipogenesis because of the ability of CB ₁ antagonist to reduce gut permeability and counteract the LPS-induced inhibitory effect on adipocyte differentiation and lipogenesis (i.e. a disinhibition mechanism). In summary, given that these treatments reduce gut permeability and, hence, plasma LPS levels and inflammatory tone, we hypothesised that LPS could act as a regulator in this process. This hypothesis was further supported in vitro and in vivo by the observation that cannabinoid-induced adipocyte differentiation and lipogenesis were directly altered (i.e. reduced) in the presence of physiological levels of LPS. In summary, because these treatments reduce gut permeability, hence, plasma LPS and inflammatory tone, we hypothesised that LPS acts as a regulator in this process. Altogether, our data provide the evidence that the consequences of obesity and gut microbiota dysregulation on gut permeability and metabolic endotoxaemia are clearly mediated by the eCB system, those observed on adiposity are likely the result of two systems interactions: LPS-dependent pathways activities and eCB system tone dysregulation ( Figure 9).

Our results indicate that the endocannabinoid system tone and the plasma LPS levels have a critical function in the regulation of the adipose tissue plasticity. As obesity is commonly characterised by increased eCB system tone, higher plasma LPS levels, altered gut microbiota and impaired adipose tissue metabolism, it is likely that the increased eCB system tone found in obesity is caused by a failure or a vicious cycle within the pathways controlling the eCB system.

These findings show that two novel therapeutic targets in the treatment of obesity, the gut microbiota and the endocannabinoid system, are closely interconnected. They also provide evidence for the presence of a new integrative physiological axis between gut and adipose tissue regulated by LPS and endocannabinoids. Finally, we propose that the increased endotoxaemia and endocannabinoid system tone found in obesity might explain the altered adipose tissue metabolism.

Abstract

Obesity is characterised by altered gut microbiota, low-grade inflammation and increased endocannabinoid (eCB) system tone; however, a clear connection between gut microbiota and eCB signalling has yet to be confirmed. Here, we report that gut microbiota modulate the intestinal eCB system tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels. The impact of the increased plasma LPS levels and eCB system tone found in obesity on adipose tissue metabolism (e.g. differentiation and lipogenesis) remains unknown. By interfering with the eCB system using CB ₁ agonist and antagonist in lean and obese mouse models, we found that the eCB system controls gut permeability and adipogenesis. We also show that LPS acts as a master switch to control adipose tissue metabolism both in vivo and ex vivo by blocking cannabinoid-driven adipogenesis. These data indicate that gut microbiota determine adipose tissue physiology through LPS-eCB system regulatory loops and may have critical functions in adipose tissue plasticity during obesity.