Introduction
Enteroendocrine cells release incretins in response to nutrient ingestion. The two main incretins—glucagon-like peptide (GLP) 1 and glucose-dependent insulinotropic peptide (GIP)—regulate the postprandial secretion of insulin [
1]. One limitation of GLP-1 and GIP activities is their short half-life, because they are rapidly cleaved and inactivated by dipeptidyl peptidase 4 (DPP-4). DPP-4 exists as a membrane-anchored cell surface peptidase and as a soluble form in the circulation [
2]. The expression and circulation levels of DPP-4 in people with type 2 diabetes are higher than in those without diabetes [
3]. Additionally, DPP-4 is considered an adipokine positively correlated to body weight, adipose tissue inflammation and insulin resistance in obese individuals [
4,
5].
Sitagliptin, anagliptin and vildagliptin are a new type of DPP-4 inhibitors which are administered orally and are well tolerated [
6]. Although first approved in 2006, their effects on essential intestinal functions remain poorly studied. In 2011, Waget et al described how inhibition of DPP-4 activity in the intestine was involved in regulating glycaemia [
7]. Recently, Mulvihill et al demonstrated that glucose tolerance depended on inhibition of DPP-4 activity in haematopoietic and endothelial cells, but there was no involvement of the DPP-4 expressed by enterocytes [
8]. DPP-4 activity modulates the functionality of more than 40 potential substrates, including cytokines, chemokines and growth factors, some of which are particularly relevant for gut homeostasis [
9]. For example, the preservation of GLP-2 with DPP-4 inhibitors (valine pyrrolidide) has been proposed to stimulate intestinal epithelial growth [
10,
11].
Besides cleaving gut peptides, DPP-4 plays a role in the immune response. Soluble DPP-4 increases the expression of Toll-like receptors (TLRs) and activates NFκB signalling, resulting in the secretion of proinflammatory cytokines [
12,
13]. These effects can be reduced by DPP-4 inhibitors [
14‐
16]. For instance, in macrophages vildagliptin counteracts the production of cytokines and expression of TLR-2 and TLR-4 induced by soluble DPP-4 and lipopolysaccharide [
13]. As TLRs are part of the innate immune response and play a crucial role in the maintenance of gut homeostasis, investigating the effect of vildagliptin in the context of intestinal health may produce particularly relevant results [
17].
Vildagliptin is principally absorbed in the small, and to a lesser extent in the large, intestine [
18]. During its passage through the intestinal tract, eukaryotic cells as well as the gut microbiota are exposed to vildagliptin. It has been widely described that intestinal microbes respond to environmental factors such as drugs and food, generating protective or detrimental effects [
19,
20]. Interestingly, some genera of bacteria in the gut microbiota, such as
Prevotella and
Lactobacillus, have been reported to exert DPP-4-like activity [
21‐
23]. Furthermore, some strains of
Bifidobacterium and
Lactobacillus can produce DPP-4 inhibitors [
24,
25]. To our knowledge, the DPP-4-like activity of the gut microbiota has never been studied. One study (without assessment of DPP-4 activity) reported that vildagliptin caused changes in the gut microbiota and that the changes were more significant in rats fed a high-fat diet than in those fed a control diet [
26]. Furthermore, a recent report showed that high-fat, high-sugar diets altered the activation of enteric neurons by GLP-1 regulating insulin secretion, an effect that seemed to be also dependent on the gut microbiota [
27]. We therefore assessed the effect of vildagliptin on the gut microbiota and intestinal functions of a murine model of obesity induced by a Western diet (WD), a model that induces gut dysfunction and related hepatic inflammation.
Methods
Animals and treatments
Two animal experiments were performed. In experiment 1, 27 male 9-week-old C57BL/6J mice (Janvier Labs, Saint-Berthevin, France) were purchased. Three mice were housed in one individually ventilated cage. In experiment 2, four male 12-week-old C57BL/6J mice (Janvier Labs) were housed in one individually ventilated cage. Mice were kept in a pathogen-free environment with a 12 h daylight cycle and free access to food and water. The acclimatisation period lasted 1 week on a standard diet (AIN-93M; ssniff, Soest, Germany). The experiments were approved by and performed in accordance with the guidelines of the local ethics committee of Université catholique de Louvain. Housing conditions were as specified by the Belgian Law of 29 May 2013 regarding the protection of laboratory animals (Agreement no LA 1230314).
In experiment 1, mice were randomised based on body composition assessed by NMR (LF50 minispec; Bruker, Rheinstetten, Germany) to minimise differences (initial mean body weight ± SEM was 24.46 ± 0.23 g). No blinding procedure was followed. The groups (
n = 9) were: (1) group-fed a control diet (D12450K; Research Diets, New Brunswick, NJ, USA) containing 10% kJ fat; (2) group-fed a WD (D12451; Research Diets) containing 45% kJ fat and 17% kJ sucrose; and (3) group-fed a WD plus vildagliptin (Cayman, Ann Arbor, MI, USA [supplied by Sanbio, Uden, the Netherlands]) in the drinking water (0.6 mg/ml according to previous studies [
13], corresponding to approximately 50 mg kg
−1 day
−1). To discriminate the effect of vildagliptin from the effect of the WD on DPP-4 activity, we pretreated with vildagliptin for 2 weeks. In the third week, the WD was introduced. A scheme of the experimental design is shown in electronic supplementary material (ESM) Fig.
1.
After 8 weeks and 6 h of fasting, glycaemia was measured using a glucometer (Roche Diagnostics, Basel, Switzerland) on blood from the tail. Mice were anaesthetised with isoflurane gas (Abbot, Lake Bluff, IL, USA). Portal blood was collected. Mice were necropsied after cervical dislocation. Liver, adipose tissue and caecal content and tissue were weighed. Blood, liver, caecal content and tissue, ileum and colon were collected, frozen in liquid nitrogen and stored at −80°C until analysed.
Details of experiment 2 are given below in the section Precision-cut liver slices.
Discussion
Inhibition of DPP-4 activity to tackle metabolic disorders through the preservation of incretins has been widely described. However, the additional effects of DPP-4 inhibitors on the intestinal ecosystem remain poorly studied. In the present study, we evaluated whether the DPP-4 inhibitor vildagliptin could impact intestinal homeostasis—including the composition and activity of gut microbiota—using in vivo, ex vivo and in vitro approaches. Our work is the first to consider the DPP-4-like activity of the gut microbiota as a potential target of DPP-4 inhibitors and evaluate, in an animal model of diet-induced obesity, the effect of vildagliptin on gut barrier function, innate immune response and liver. Overall, our results show that vildagliptin improved gastrointestinal function judged by restoration of the expression of AMPs and the depth of the crypts in the ileum. In the liver, vildagliptin was associated with a reduction of cytokine expression. However, the ex vivo experiment with PCLS showed that reductions were not due to a direct effect of the drug, reinforcing the idea that reduced hepatic inflammatory tone might be related to changes at the intestinal level. Furthermore, vildagliptin was associated with changes in beta diversity indexes, reductions in
Oscillibacter spp. and increases in
Lactobacillus spp. and caecal propionate levels. Among the 67 OTUs changed by vildagliptin, we can pinpoint some unclassified Lachnospiraceae and Ruminococcaceae, as well as
P. goldsteinii, as potential contributors to the increase in propionate levels [
35], even if no statistically significant correlation backed up this fact. DPP-4 is a multifunctional protein and therefore it is plausible that its inhibition by vildagliptin has induced the broad changes that we observed through several mechanisms. In this regard, we demonstrated three effects of vildagliptin on the intestine, namely: (1) restoration of the active form of gut peptides such as GLP-1; (2) modulation of the innate immune response; and (3) modulation of the gut microbiota. There could be a link between these effects, as illustrated for instance in a study performed in obese prebiotic-fed mice which reported that the gut microbiota influenced the release of substrates of DPP-4 such as GLP-1 and GLP-2 [
36]. Furthermore, a high-fat diet can impair enteroendocrine cell function and the release of GLP-1, as confirmed here [
37]. We propose that the increase in active GLP-1 in mice treated with vildagliptin can also be partly explained by the increases in propionate in the caecal content [
38]. The commutative relationship between microbiota and host is also supported by the fact that AMPs shape the composition of the gut microbiota, and, conversely, the gut microbiota influences host immunity [
39].
Since we know that some bacteria exert DPP-4-like activity, one of our outcome measures was to assess whether vildagliptin targets DPP-4 activity of the gut microbiota [
21‐
23]. We have shown for the first time that vildagliptin completely abolished DPP-4 activity in the caecal content and faeces. Analyses of the composition of the gut microbiota showed that
Oscillibacter spp. was the most striking target of vildagliptin, but its reduction was not explained by DPP-4 inhibition. Only one study has considered the impact of the DPP-4 inhibitors saxagliptin, vildagliptin and sitagliptin on bacterial DPP-4-like activity [
40]. Inhibition of the DPP-4-like activity of
Streptococcus mutans found in the oral microbiota impaired its biofilm formation [
40]. None of the DPP-4 inhibitors tested affected the growth of
S.
mutans in the concentration range considered (4–2048 μg/ml) [
40]. In our study, however, vildagliptin (600 μg/ml) inhibited the growth of
O.
valericigenes, even though this bacterium does not exhibit DPP-4 activity. Taken together these results demonstrate the impact of vildagliptin on different members of the microbiota (intestinal and oral) via inhibition of DPP-4-like activity and through a mechanism still unknown. A recent study reported changes in the composition of the gut microbiota by vildagliptin in Sprague–Dawley rat models of diabetes [
26]. In agreement with our results, vildagliptin caused a reduction in
Oscillibacter that was interpreted as a beneficial effect [
26]. Furthermore, other animal studies have associated increases in
Oscillibacter with obese and diabetic phenotypes [
41‐
43], and with increases in intestinal permeability, a condition involved in the development of metabolic disorders [
41]. Consequently, vildagliptin induces beneficial changes in the composition of the intestinal microbiota that might become a therapeutic strategy beyond the preservation of incretins.
The gut is populated with multiple types of immune and epithelial cells that work together to maintain tolerance to the gut microbiota and food [
17]. The interaction between microbiota and host cells is mediated by TLRs, which are sensors involved in the innate immune response. In addition, a chemical barrier consisting of the secretions of AMPs and other cell products (cytokines and immunoglobulins) is part of the first line of defence [
44]. Here, on the one hand, we have shown that vildagliptin reduces levels of TLR-2 and TLR-4 agonists in the caecal content. Previously, it has been reported that vildagliptin suppresses TLR-2 and TLR-4 content in macrophages stimulated with soluble DPP-4 and lipopolysaccharide [
13]. This observation is in agreement with the reduction in TLR-4 agonist activity (such as lipopolysaccharide) in the caecal content of mice treated with vildagliptin and demonstrates that vildagliptin impacts the crosstalk between the gut microbiota and the host. On the other hand, vildagliptin also influenced the innate immune response via restoration of AMP expression in the ileum. In agreement with previous studies carried out by our group, a fat-enriched diet caused a reduction in AMPs [
34,
45]. Vildagliptin completely counteracted this reduction. We hypothesise that this effect might be linked to GLP-2. As GLP-2 is inactivated by DPP-4, vildagliptin could have preserved its functionality [
10,
11]. The role of GLP-2 in inducing antimicrobial products has been shown in GLP-2 receptor knockout mice which had reduced AMPs and impaired mucosal bactericidal activity [
46]. Also, the restoration of crypt depth in mice that received vildagliptin could be explained, at least in part, by GLP-2 stimulation of crypt cell proliferation and inhibition of apoptosis [
47]. Unfortunately, we could not confirm experimentally this hypothesis due to the lack of commercially available methods to measure active GLP-2.
Finally, we investigated whether the changes observed at the intestinal level could also have impacted the inflammatory tone in the liver. Mice treated with vildagliptin showed a reduction in the expression of lymphocytes (
Cd3g), macrophage activation (
Cd11c) and proinflammatory markers. The ex vivo experiment in which PCLS were exposed to vildagliptin showed no changes in any of the inflammatory markers analysed. Even if there is a bias between the in vivo and ex vivo approaches, our observations indicate that the changes induced by vildagliptin at the intestinal level might have also impacted hepatic homeostasis. Specifically, the reinforcement of gut immunity induced by vildagliptin could result in a lower exposure of the liver to microbial stimuli. It would be useful to evaluate how vildagliptin influences gut microbial activity and the gut–liver axis, since microbial metabolites that are prone to reach the liver through the portal vein, such as bile acids (via the farnesoid X receptor) or SCFA (like butyrate), modulate hepatic inflammation and thereby the occurrence of non-alcoholic fatty liver disease [
48,
49].
For our study, we selected vildagliptin for its potential effect on inflammation (reduction of TLR expression) [
14]. The pharmacokinetics of vildagliptin is different from that of the other DPP-4 inhibitors (being more lipophilic). For instance, the percentage of vildagliptin excreted in the faeces is 4.5%, whereas for sitagliptin and saxagliptin it is reported to be 13% and 22%, respectively [
50]. Therefore, the effects of other gliptins on the microbiota—and related metabolic effects—could be relevant, since they are found in higher quantities in the lower intestine. Human data may confirm this hypothesis.
In conclusion, we show that in WD-fed mice presenting gut disorders, vildagliptin affected the composition of the gut microbiota and improved intestinal homeostasis. Due to the multifunctional roles of DPP-4, further studies are needed to decipher the precise molecular mechanisms. If the beneficial effect of vildagliptin on the intestinal tract is confirmed in human studies, it might represent a novel mechanism of vildagliptin to improve human health beyond its standard use.