Incretin hormones, obesity and gut microbiota

obesity and its associated comorbidities.


Introduction
Obesity is a chronic relapsing disease associated with significant mortality and morbidity.Over the past 40 years, the prevalence of obesity has risen dramatically, reaching epidemic proportions [1].For several decades obesity has been viewed as the result of a long-term energy imbalance, but emerging evidence has revealed that its pathogenesis is much more complex [2].Several studies show that gastrointestinal hormones, namely incretins, may play a vital role in the pathogenesis of obesity and its comorbidities, as they are responsible for the regulation of body weight, maintenance of energy balance and glucose homeostasis [3,4].Moreover, a growing body of evidence supports the existence of a bidirectional signaling between the brain and gut bacterial community, in the pathophysiology of obesity.Indeed, the gut microbiota as been recently identified as a key regulator of incretin hormones [5][6][7][8].
Herein, we describe our current understanding of the interplay between incretins and gut microbiota in the context of obesity.We also provide a summary of the evidence that establishes the connection between gut microbiota and the modulation of incretins.

Obesity prevalence and pathophysiology
The global incidence of obesity has almost tripled over the last 50 years.An extensive analysis revealed that nearly one-third of the world's population is overweight, with approximately 10% affected by obesity.Furthermore, projections suggest that, by 2030, the number of people affected by obesity will reach 1.12 billion worldwide [1].The health risks associated with obesity have reached a concerning level, emerging as a major global issue [9].Indeed, obesity is not only characterized by the excessive accumulation of body fat but also by an imbalanced metabolism of lipids and glucose, chronic inflammation, and oxidative stress [10].
In addition, obesity significantly increases the risk of a variety of diseases associated with a higher mortality rate, including type 2 diabetes mellitus (T2DM), hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), metabolic syndrome and cardiovascular diseases (CVD) [9].
For years, obesity has been considered the result of a prolonged energy imbalance between calories consumed and calories expended [11,12].Nevertheless, weight loss interventions aimed at reducing calorie intake and increasing energy expenditure often fail to yield long-term results [13].Indeed, the pathophysiology of obesity has been proven to be more intricate, encompassing a combination of genetic predisposition and environmental factors.This interplay results in a positive energy balance, where fuel intake exceeds energy expenditure [14].
Traditional approaches for the treatment of obesity and its comorbidities include lifestyle interventions, medical therapy and metabolic surgery.However, lifestyle interventions and medical therapy are partially effective in promoting long-term weight loss [13].Metabolic surgery contributes to the improvement and remission of several obesity-related comorbidities, leading to sustained weight loss, improved quality of life, and prolonged survival [15].Several studies in animals and humans suggest that the improvement in glucose control after these procedures occurs rapidly, and may involve mechanisms independent of weight loss [16][17][18].Interestingly, a greater improvement of insulin resistance have been observed in metabolic procedures that bypass large portions of the small intestine [16,19].For these reasons, it has been suggested that some gut signals, including incretin hormones and gut-brain pathways, may be responsible for improved glycemic control, independent of weight loss.

The incretin system
The concept that intestinal hormones secreted from the gut play a role in the regulation of glucose metabolism was introduced at the beginning of the 20th century, and since then incretin hormones have garnered significant attention due to their crucial role in glucose homeostasis [20].
Incretin hormones are gut peptides that are secreted from enterondocrine cells (EEC) after nutrient intake and stimulate insulin secretion together with hyperglycaemia.GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) are responsible for the incretin effect: a greater insulin secretion when glucose is administered orally as opposed to being infused intravenously.GIP is synthesized and released from intestinal K-cells, which are mainly located in the duodenum and proximal jejunum [21].The K-cell is an open-type intestinal epithelial endocrine cell that reacts with luminal nutrients through its apical surface while its basolateral surface is connected with the neural and vascular tissue.Oral ingestion and subsequent absorption of nutrients such as glucose and long-chain fatty acids trigger the secretion of GIP.Subsequently it was demonstrated that the rate of nutrient absorption rather than the type of nutrients stimulates intestinal GIP release.
The major sources of GLP-1 are intestinal L-cells, which are relatively low in the proximal small bowel and increase in the distal gut, reaching their highest density in the ileum and colon [22].GLP-1 secretion from intestinal L-cells is triggered by a variety of nutrient and endocrine factors.The primary physiologic stimulus for GLP-1 secretion are meals rich in fats and carbohydrates [23].Moreover, oral, but not intravenous, glucose administration stimulates GLP-1 secretion in humans [24].Both GIP and GLP-1 exert their insulinotropic effects by binding to their receptors expressed on pancreatic β cells, stimulating insulin secretion in a glucose-dependent manner [25].

Incretin hormones in obesity
Obesity and other metabolic disorders are associated with dysregulation of incretin secretion and action, in particular a hypersecretion of GIP during both fasting and after oral glucose challenge was observed in subjects affected by obesity [26][27][28].Genetically obese mice (ob/ob) display an increased density of K cells in the small intestine compared to those on normal diet [29][30][31], while disrupting GIP signaling prevents diet-induced obesity and insulin resistance [32][33][34][35].However, in humans the causal relationship between increased GIP signaling and obesity is still unclear.A decrease in meal-related GLP-1 response [36][37][38] and a negative correlation with body mass index (BMI) [37] has been observed in individuals with obesity, suggesting that low GLP-1 levels may contribute to obesity by reducing anorexigenic signaling [36,39].It has been also suggested that GLP-1 decrease in subjects with obesity may be related to the insulin resistance and/or reduced L-cell responsiveness to carbohydrates [40].Of note, an increase in GLP-1 secretion and a decrease in hunger was described after a moderate or intense exercise [41] or in individuals who have lost weight following life-style interventions [42].This increase, along with the impact on satiation, likely contributes to the observed weight reduction resulting from these interventions.
Overall, the reduction of the incretin effect in obesity could be explained by a reduced responsiveness to GIP and/or to a lower contribution of GLP-1 to the insulin secretory response and the anorexigenic signaling.However, in both human and rodent studies it is challenging to determine whether observed changes in the enteroendocrine axis are caused by diet or by other factors related to obesity.

Gut microbiota and obesity
The intestinal microbiota consists of more than 1500 species and more than 50 different phyla.The most dominant phyla of bacteria in the gut microbiota are Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Verrucomicrobia.Firmicutes and Bacteroidetes are the most frequent phyla accounting for at least 90% of the total microbial population in human gut [43].Maintaining the heterogeneity and stability within the gut microbiota community is essential for promoting host health.Alterations in diversity and microbiota community structure can impact host metabolism, potentially leading to obesity.A growing body of evidence suggests that people affected by obesity exhibit significant differences in their gut microbiota compared to healthy people [44][45][46][47][48][49][50].In some studies, an increased Firmicutes-to-Bacteroides ratio has been associated with obesity, while in others, this relationship was not observed [51][52][53][54].However, most studies consistently report a reduced phylogenetic diversity and a low microbial gene richness (MGR) associated with obesity [55].Low levels of MGR are associated with increased BMI, low-grade inflammation, adiposity, dyslipidemia, T2DM, and insulin resistance [56,57] and can be found in up to 40% of people with overweight.Other studies explored the association between obesity and specific gut bacteria (Table 1).At the genus level, subjects with obesity show higher levels of Lactobacillus reuteri, Fusobacteria Alistipes and Anaerococcus compared to lean subjects, while Akkermansia, Lactobacillus plantarum and Bifidobacterium are less abundant [44][45][46][48][49][50].These findings suggest a specific association between certain bacterial species and obesity.Furthermore, bacteria from the same genus exhibited contrasting functions in relation to obesity which could be partially explained by the complexity of the disease.
Akkermansia muciniphila is a key bacterium for weight loss and its supplementation improves metabolic parameters in subjects with obesity [58].Lactobacillus and Bifidobacterium are traditional probiotics that play an important role in the balance of the human intestinal microecology.Reduced abundance of Lactobacillus paracasei and Bifidobacterium in the gut is associated with obesity [59], while the abundance of Lactobacillus reuteri and Lactobacillus gasseri is positively correlated with obesity.The precise mechanisms through which the microbiota influence the onset of obesity are still being unraveled and studies on germ-free (GF) mice provide partial evidence supporting its potential causative role [52,60].Indeed, several studies report conflicting results, showing that GF mice are partially protected from diet-induced obesity [61][62][63][64][65].These mixed results are likely influenced by dietary sucrose levels, with low-sugar lard-based high-fat diets amplifying the effects of microbiota-induced obesity [66].Some studies showed that conventionalized mice develop obesity on a high-fat, high-sucrose diet, whereas GF mice are protected [61,65], while G. Angelini et al. others showed a dramatic weight increase when GF were fed a low-sucrose lard-based HFD [67][68][69].While these studies have provided some evidence supporting a potential role for microbiota in obesity development, these conflicting results indicate that further research is needed to fully understand the mechanisms involved.

Gut metabolites and incretins
Several of the metabolites produced by the colonic microbiota have been shown to modulate the activity of the EECs population (Fig. 1).Short-chain fatty acids (SCFAs), are key candidates involved in the crosstalk between microbes and host cells.In addition to serving directly as energy substrates, SCFAs have been shown to directly stimulate the release of incretins from colonic EECs [70][71][72].Indeed, SCFAs can initiate cell-specific signaling cascades through the activation of the G-protein-coupled free fatty acid receptor FFAR2 (GPR43) [73,74].According to several studies, binding of SCFA to GPR43 increases GLP-1 secretion from intestinal L-cells while intestinal cells isolated from total body GPR43 knockout mice showed reduced GLP-1 secretion [75][76][77][78].
Non-digestible, fermentable polysaccharides supplementation enhance gut microbiota production of SCFAs and reduces host food intake by improving satiety [79][80][81][82][83][84].The reduction in food intake is partly explained by the increase secretion of GLP-1, which decreases appetite and energy intake [85,86].Furthermore, it has been suggested that the fermentation of non-digestible carbohydrates in the gut may promote the differentiation of L-cells in the small intestine and proximal colon, thereby increasing their numbers [87][88][89].
Although the link between gut microbiota fermentation of specific non-digestible carbohydrates and the modulation of incretins is becoming widely accepted in rodent studies [90][91][92], the evidence that similar links exist in humans remains a matter of debate.
A study conducted in healthy volunteers found that the ingestion of lactulose (20 g/day) or intracolonic administration of SCFAs were associated with an increased production of incretins [93], while another study reported that dietary oligofructose (20 g/day) was associated with a significant increase in plasma GLP-1 following a meal [94].Moreover, 2 weeks administration of oligofructose (16 g/day) in healthy volunteers promoted satiety and reduced food consumption [95].Two other studies reported that gut microbiota fermentation of non-digestible carbohydrates was associated with increased satiety and reduced food intake, which impacted energy intake, however gut peptides were not measured in these studies [79,96].A follow-up study examined the impact of gut microbiota fermentation of oligofructose on appetite sensations, and related these findings to higher plasma GLP-1 levels [84], while another study demonstrated that a single dose of inulin-type fructans increases postprandial plasma GLP-1 [97].Despite some studies found a link between gut microbiota fermentation, SCFAs, gut peptides, and appetite regulation, additional studies are required to determine whether specific alterations of the gut microbiota and/or the production of endogenous gut peptides are the sole responsible of these effects.
In addition to SCFAs, several gut bacteria metabolites including secondary BAs, and indole derivatives, can modulate EECs secretory activity.
Bile acids stimulate GLP-1 release by crossing the epithelium to access G protein-coupled bile acid receptor 1 (GPBAR1) located basolaterally on L-cells [98,99].The microbiota plays a crucial role in bile  acid-dependent L-cell activation by deconjugating bile acids to enhance their permeability and by dehydroxylating them to increase their action on GPBAR1 [100].Indole, produced by bacterial breakdown of dietary tryptophan, has been found to have opposite effects on GLP-1 release, a stimulatory action through blockage of voltage-gated K+ channels and an inhibitory effect due to suppression of mitochondrial metabolism [101].While it is established that microbial metabolites modulate EECs function in the distal gut, it is unclear whether these effects are the cause, or the consequence of the metabolic dysregulation associated with obesity.

Bariatric surgery and gut microbiota modulation
Strategies to manage obesity, such as increased physical activity and changes in dietary habits, result in a moderate amount of weight reduction, making it challenging to sustain over the long term.Bariatric surgery has been proven as the most effective approach to treat severe obesity, leading to significant and long-term weight loss as well as the improvement of obesity-related comorbidities [15].Currently, common surgical techniques include sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) [102].SG is a restrictive procedure, which removes a large portion of the stomach, whereas RYGB is both restrictive and malabsorptive and results in the creation of a gastric pouch that is connected to the distal jejunum by a Roux limb.
Bariatric surgery leads to a decrease in post-meal GIP secretion [103], an effect that seems to be more pronounced in individuals with diabetes [104,105] and to an increase of GLP-1 postprandial levels [106][107][108].The reasons for the increase of GLP-1 after surgery are not fully understood but have been linked to faster delivery of intact nutrients to the ileum or an increased intestinal transit [109,110].In addition to the physiological changes induced by the surgery itself, there are other factors that may contribute to weight loss which have not been fully elucidated.One of these factors is the gut microbiota, which has recently been associated to weight loss and the metabolic outcomes following bariatric surgery.Previous research in mouse models of RYGB support this hypothesis suggesting that the metabolic benefits of this type of surgery may be directly linked to alterations in the gut microbiota [111].Moreover, studies involving fecal microbiota transplantation from humans post-RYGB surgery into GF mice also indicate that the shift of gut microbiota after surgery can result in reduced adiposity and metabolic improvements in the recipient mice [112][113][114].In humans, bariatric surgery increases gut bacterial diversity and richness in several studies (Table 2).At phylum level, most of the studies show an increase in Proteobacteria following bariatric surgery [115][116][117][118][119][120][121][122][123][124].Bariatric surgery leads to anatomical and physiological alterations in the gastrointestinal tract, resulting in decreased gastric acid secretion and a shift in overall pH levels [125].These changes increase the availability of oxygen, facilitating the expansion of facultative anaerobes belonging to the Proteobacteria phylum.Several studies also observed an increase of Akkermansia and its species A. muciniphila after bariatric surgery [116,118,122,[126][127][128][129]. A. muciniphila utilizes mucus as a carbon and nitrogen source to produce SCFAs and it has been increasingly tested as a therapeutic tool for obesity treatment.In mice with genetic or diet-induced obesity, the levels of A. muciniphila are reduced compared to lean mice while the oral administration of the bacterium improved mice body weight and composition [130].In humans, the administration of A. muciniphila has several beneficial effects such as the improvement of insulin sensitivity, reduction of insulinemia, and body weight [58].However, a recent study observed that the increase of A. muciniphila following RYGB was not correlated with glucose homeostasis and other clinical variables [131].The question of whether A. muciniphila or other bacteria could effectively treat obesity is still under investigation.Additionally, it is also unclear whether gut microbiota shifts induced by bariatric surgery are durable and whether they can be reversed over time.There is a limited number of studies evaluating this issue, and more systematic longitudinal studies are , Bacteroides vulgatus Farin et al. [118] SG (n=89) RYGB (n=108) needed to provide further insight.Ground-breaking in this field was a study evaluating changes induced by RYGB and vertical banded gastroplasty (VBG) 9.4 years after surgery.This study suggests that both surgical procedures yield comparable and long-lasting changes, with an increased presence of Proteobacteria (Escherichia, Klebsiella and Pseudomonas) and a reduction in some species from the Firmicutes phylum (Clostridium difficile, Clostridium hiaronis and Gemella sanguinis) [113].A more recent study assessing changes induced by RYGB, 12 years after surgery, found that β-diversity was significantly different compared to non-surgery patients, showing that Verrucomicrobiaceae and Streptococcaceae were significantly higher in patients who underwent RYGB, while Bacteroidiaceae was significantly lower [132].
Another important matter to consider is whether the composition of the gut microbiota before surgery can predict the amount of weight loss.There are relatively few studies that have analyzed the role of gut microbiota in determining weight loss trajectory.A recent study based on lifestyle intervention highlight the importance of baseline gut microbiota as a predictor of weight loss trajectory, outperforming factor such as caloric intake, physical activity, or body weight [133].Another study that analyzed baseline gut microbiota in relation to weight loss achievement after SG [134] observed that the pre-operative gut microbiota of individuals who did not respond to surgery in terms of weight loss was enriched in Rikenellaceae, Lachnospiraceae, Thermomonisporaceae and Enterobacteraceae families, and also in Hungatella, Alistipes, Brassicibacter and Siccibacter genera.They also observed that patients who responds successfully to surgery had an enrichment of Fibrobacteraceae, Peptoniphilaceae, Campylobacteraceae, the genera Gordonibacter, Anaerofustis, Finegoldia, Hathewaya and Campylobacter.
While the findings of these studies are promising, they had a limited  sample size and short follow-up.More research is needed to explore whether distinct patterns in gut microbiota could influence the trajectory of weight loss following bariatric surgery.

Obesity medications and gut microbiota
Incretin-based treatments involving GLP-1 receptor agonists (GLP1-RA), like Liraglutide, have become a widely accepted approach for managing obesity [135].The mechanism of action of Liraglutide is linked to reduced food intake, resulting from the inhibition of appetite and gastric emptying induced by GLP-1 [20,136].However, some studies have shown that Liraglutide could induce more weight loss than by simply restricting food intake alone, suggesting the presence of other mechanisms contributing to weight-loss [137,138].Recently, there has been a growing focus on the potential mechanistic role of gut microbiota in the metabolic effects of GRAs.In preclinical studies, these beneficial effects have been postulated to be mediated in part by medication-induced changes in the intestinal microbiome.In hyperglycemic and obese mice, Liraglutide was shown to increase microbial diversity and the presence of beneficial bacteria such as Lactobacillus and Turicibacter.However, it was not investigated whether this was a direct effect of Liraglutide administration or simply an effect linked to weight loss and reduced caloric intake.Another study found that SCFAs producing bacteria (Bacteroides, Lachnospiraceae and Bifidobacterium) were increased in liraglutide-treated diabetic male rats [139].Accordingly, other studies performed in both obese and diabetic obese rats reported that liraglutide can reduce obesity-associated bacteria (Erysipolotrichaceae, Marvincryantia, Roseburia, Candidatus, Arthromitus, Parabacteroides, Romboutsia and Ruminiclostridium) and increase bacteria associated with a lean phenotype (Prevotella, Blautia and Coprococcus).Finally, a study on subjects with T2DM who received liraglutide or metformin showed that patients treated with liraglutide had a higher abundance of Akkermansia compared to those treated with metformin [140].However, it remains unclear whether the weight-loss effect of liraglutide is related to structural modulation of gut microbiota in obese individuals.

Conclusion
Several potential connections between gut microbes and the host have been identified.Investigating whether targeting the gut microbiota could be a promising approach to enhance endogenous production of incretins requires further investigations.Although preliminary studies in humans have demonstrated that the modulation of gut microbiota trought bariatric surgery or anti-obesity medications can boost incretin secretion, the actual impact on glucose homeostasis, appetite, and fat mass needs clarification through extensive large-scale studies.Furthermore, the intricate mechanisms linking the gut microbiota, the metabolites produced by specific microbes, and host metabolism are still the subject of ongoing research.Consequently, the forthcoming studies should define the mechanisms involved in the intricate interplay between gut microbes and the host.Determining whether specific microbes or metabolites could serve as therapeutic strategies to regulate obesity is a key focus for the future.
Non-digestible, fermentable polysaccharides supplementation enhance gut microbiota production of SCFAs that binds to GPR43 increasing GLP-1 secretion from intestinal L-cells.Bile acids stimulate GLP-1 release by crossing the epithelium to access G protein-coupled bile acid receptor 1 (GPBAR1) located basolaterally on L-cells.Indole, produced by bacterial breakdown of dietary tryptophan, has been found to have opposite effects on GLP-1 release, a stimulatory action through blockage of voltage-gated K+ channels

Table 1
Gut microbiota composition in subjects with obesity and lean subjects.

Table 2
Gut microbiota composition following bariatric surgery.