Microbial dietary protein metabolism regulates GLP‐1 mediated intestinal transit

Depletion of gut microbiota is associated with inefficient energy extraction and reduced production of short‐chain fatty acids from dietary fibers, which regulates colonic proglucagon (Gcg) expression and small intestinal transit in mice. However, the mechanism by which the gut microbiota influences dietary protein metabolism and its corresponding effect on the host physiology is poorly understood. Enteropeptidase inhibitors block host protein digestion and reduce body weight gain in diet‐induced obese rats and mice, and therefore they constitute a new class of drugs for targeting metabolic diseases. Enteroendocrine cells (EECs) are dispersed throughout the gut and possess the ability to sense dietary proteins and protein‐derived metabolites. Despite this, it remains unclear if enteropeptidase inhibition affects EECs function. In this study, we fed conventional and antibiotic treated mice a western style diet (WSD) supplemented with an enteropeptidase inhibitor (WSD‐ETPi), analyzed the expression of gut hormones along the length of the intestine, and measured small intestinal transit under different conditions. The ETPi‐supplemented diet promoted higher Gcg expression in the colon and increased circulating Glucagon like peptide‐1 (GLP‐1) levels, but only in the microbiota‐depleted mice. The increase in GLP‐1 levels resulted in slower small intestinal transit, which was subsequently reversed by administration of GLP‐1 receptor antagonist. Interestingly, small intestinal transit was normalized when an amino acid‐derived microbial metabolite, p‐cresol, was supplemented along with WSD‐ETPi diet, primarily attributed to the reduction of colonic Gcg expression. Collectively, our data suggest that microbial dietary protein metabolism plays an important role in host physiology by regulating GLP‐1‐mediated intestinal transit.

are dispersed throughout the gut and possess the ability to sense dietary proteins and protein-derived metabolites.Despite this, it remains unclear if enteropeptidase inhibition affects EECs function.In this study, we fed conventional and antibiotic treated mice a western style diet (WSD) supplemented with an enteropeptidase inhibitor (WSD-ETPi), analyzed the expression of gut hormones along the length of the intestine, and measured small intestinal transit under different conditions.The ETPi-supplemented diet promoted higher Gcg expression in the colon and increased circulating Glucagon like peptide-1 (GLP-1) levels, but only in the microbiota-depleted mice.The increase in GLP-1 levels resulted in slower small intestinal transit, which was subsequently reversed by administration of GLP-1 receptor antagonist.Interestingly, small intestinal transit was normalized when an amino acid-derived microbial metabolite, p-cresol, was supplemented along with WSD-ETPi diet, primarily attributed to the reduction of colonic Gcg expression.Collectively, our data suggest that microbial dietary protein metabolism plays an important role in host physiology by regulating GLP-1-mediated intestinal transit.

| INTRODUCTION
The seminal discovery that adiposity can be phenocopied by transferring fecal microbiota from female twin donors discordant for obesity into germfree (GF) mice revealed that the gut microbiota adapts to the nutrient state in the gut lumen and contributes significantly to energy extraction from diet. 1,2In the absence of the gut microbiota, either in GF mouse models or through antibiotics, mice remain in a chronic energy-deficient state and eat significantly more to compensate for lower energy extraction. 3In response to lower energy extraction, GF mice exhibit higher basal expression of GLP-1, 4,5 which reduces small intestinal transit to allow increased absorption of energy from the diet. 4However, the focus so far has been on short-chain fatty acids (SCFAs), microbial fermentation products of dietary fibers, which contribute to ~10% of total daily energy requirements. 6he SCFA butyrate is the preferred energy substrate for colonocytes, 7 and therefore, when GF mice were provided diet supplemented with butyrate or energyrich high fat diet, Gcg expression and intestinal transit normalized. 4,8However, in contrast to dietary fibers, it is unclear how other macronutrients, such as dietary protein, interact with the gut microbiota to regulate gut hormones and related host physiology.
Dietary protein accounts for 10%-35% of total daily energy requirements in weight stable humans. 9Protein is efficiently digested in the upper digestive tract; however, residual peptides may reach the colon, where they undergo microbial degradation to produce a variety of amino acid derived metabolites. 10,11These metabolites serve as signaling molecules (kynurenine), 12 neurotransmitters (tryptamine, 4-aminobutyrate), 13 or intermediates in SCFA production. 14nteropeptidase inhibitors block host protein digestion and produce rapid reduction in body weight in diet-induced obese mouse models. 15,16However, gut microbiota-depleted mice resisted body weight loss when given an enteropeptidase inhibitor indicating the role of microbiota-dietary protein interaction in the maintenance of body weight. 16Gut hormone-producing enteroendocrine cells operate at the interface of host and microbiota, and are responsive to dietary proteins and protein-derived microbial metabolites.Therefore, we hypothesized that the gut microbiota-protein interaction regulates gut hormones and related physiological processes.
In this study, we used an enteropeptidase inhibitor to block host protein digestion in gut microbiota-depleted mouse model to demonstrate that microbial dietary protein metabolism reduces GLP-1-mediated small intestinal transit in mice.

| Mouse studies
The mouse experiments were performed at the University of Copenhagen, Denmark, in accordance with bioethical guidelines fully compliant with institutionally accepted principles for the care and use of laboratory animals approved by Animal Experiments Inspectorate under the Danish Ministry of Food, Agriculture and Fisheries.Swiss Webster male mice (age 8-12 weeks, n = 5-8) were group housed in individual ventilated cages in temperature-and humidity-controlled room maintained at 22°C on a 12:12 light dark cycle and were randomly allocated into four groups.Three days prior to start of dietary treatments, the drinking water of two groups of mice was supplemented with antibiotics: Ampicillin sodium salt (1 g/L, Sigma, A9518) and Neomycin trisulfate salt hydrate (0.5 g/L, Sigma, N1876). 17Both non-antibiotic (non-Abx) and antibiotic treated (Abx) mice were then fed either western style diet (WSD, 58% kcal fat and sucrose) (Formula D12331, Research Diets, Table S1), or WSD mixed with enteropeptidase inhibitor, compound 6b 15 (WSD-ETPi, 0.535 g/kg of WSD) ad libitum for 9 days.The Abx groups received fresh antibiotic supplemented water, and their body weight and food intake were monitored three times a week.At the end of the experiment, blood samples were collected by puncturing retro-orbital sinus, and plasma was separated by centrifugation at 10 000g for 5 min.Weights of epididymal fat and cecum were recorded.The entire small intestine was divided into 8 segments (SI 1-8), and 2-cm segments of full-thickness intestine were collected from SI1(duodenum), SI5 (jejunum), and SI8 (ileum).Additionally, a 2-cm section of the proximal colon (after cecum) was collected and all intestinal segments were snap frozen in liquid nitrogen.

| Small intestinal transit studies
Two independent cohorts of Abx-treated Swiss Webster mice on WSD or WSD-ETPi diets were orally gavaged 200 μL of 6% carmine red (Sigma, C1022) solution prepared in 0.5% methylcellulose (Sigma, M0512) after a 4 h fast.After 45 min, the entire small intestinal tract was dissected and percentage of small intestine traveled by carmine red was quantified to determine small intestinal transit, as previously described. 4nother cohort of (n = 3-4) 4 h fasted Abx-treated Swiss Webster mice on a WSD-ETPi diet were either injected intraperitoneally with saline or the GLP-1 receptor antagonist Exendin (9-39) (Avexitide, HY-P0264, MedChemExpress) twice, 2 h and 30 min, prior to carmine red gavage and their small intestine transit was recorded.
Next, a cohort of 10-weeks-old C57/bl6J mice (n = 4-5) were procured from Janvier, Denmark, and the mice were acclimatized for one week on the chow diet.The mice were then given Abx-treated water 3 days prior to start of WSD and daily oral gavage of ETPi (40 mg/kg body weight) suspended in 0.5% methylcellulose for 7 days.On the day of experiment, 4-h-fasted mice were similarly injected with saline or Exendin (9-39) twice, 2 h and 30 min, prior to carmine red gavage, and their small intestine transit was recorded after 40 min.
In an independent cohort (n = 4), the drinking water of Abx-treated WSD-ETPi fed Swiss Webster mice was additionally supplemented with 0.25 g/L p-cresol 18 (Sigma, W233706,) for 9 days, small intestinal transit was determined, and section of the proximal colon was collected as described before.

| GLP-1 measurement
Total GLP-1 was analyzed in the plasma samples using V-plex GLP-1 Total kit on Mesoscale Discovery platform.

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 10.0.1 and presented as mean ± Standard Error of the Mean (SEM).Normal distribution of the data was checked using Kolmogorov-Smirnov analysis and outliers were identified using ROUT method available in Graph-Pad Prism 10.0.1.To assess the effect of diet, microbiota, and their interaction, two-way ANOVA was used, followed by Tukey's post hoc analysis.The nonparametric Mann-Whitney U test was used to determine significance between the two groups.

| WSD-ETPi diet promotes gut microbiota-dependent body weight loss
To investigate how the gut microbiota interacts with dietary protein, we supplemented WSD diet with ETPi compound 6b and observed no effect on cumulative food intake in either non-Abx or Abx-treated groups (Figure 1A).However, WSD-ETPi promoted body weight loss compared with control WSD, but only in the non-Abx groups of mice (Figure 1B).While the percentage epididymal adipose tissue did not differ between the dietary treatments in either non-Abx or Abx-treated groups (Figure 1C), percentage cecum weight, as a proxy of gut microbiota depletion, was significantly higher in Abx-treated groups compared with non-Abx groups of mice (Figure 1D).Within the Abx-treated mice, cecum weight was significantly higher in WSD-ETPi mice compared with WSD control (Figure 1D).To account for cecum enlargement in body weight measurements, cecum weight was subtracted from body weight at day 9 in both non-Abx and Abx-treated groups.The WSD-ETPi mice in non-Abx group still weighed less than the WSD control mice, but no difference was observed between dietary groups in Abx-treated mice (Figure S1).Overall, these results indicate that inhibition of host dietary protein digestion promotes microbiota dependent body weight loss and increases cecum size in antibiotics mediated microbiota-depleted mice.

| WSD-ETPi diet interacts with the gut microbiota to regulate colonic gut hormone expression
Energy status in the gut lumen regulates proglucagon (Gcg) expression and GLP-1 levels 4 ; therefore, we investigated whether the inhibition of host protein metabolism affects gut hormone expression and levels in our dietary model.In the non-Abx groups, ETPi supplementation did not affect expression of gut hormones, such as Gcg, peptide YY (Pyy), and cholecystokinin (Cck) (Figure 2A-C) in duodenum, jejunum, ileum, or colon.In agreement, fasting plasma GLP-1 levels did not differ between the non-Abx WSD and WSD-ETPi groups (Figure 2D).Among the Abx groups, no difference in expression of gut hormones was observed in duodenum and jejunum due to ETPi supplementation in the diet.However, colonic expression of all hormones, that is, Gcg, Pyy, and Cck, was significantly upregulated in WSD-ETPi mice compared with the WSD control in Abx-treated groups (Figure 3A-C).In addition, the fasting plasma GLP-1 level was significantly elevated in Abx-treated WSD-ETPi group compared with WSD controls (Figure 3D), indicating that colonic transcription levels of gut hormones and plasma GLP-1 levels are responsive to interaction between gut microbiota-dietary protein.

GLP-1R-mediated small intestinal transit in microbiota-depleted mice
Next, we measured small intestinal transit in Abx-treated WSD control and WSD-ETPi mice.Small intestinal transit was significantly slower in WSD-ETPi mice compared with WSD controls (Figure 4A).Since previous studies suggest that colonic Gcg regulates small intestinal transit, 4,19 Abx-treated WSD-ETPi mice were injected with GLP-1 receptor antagonist, Exendin (9-39) to block GLP-1 receptors.We found that Exendin (9-39) treatment reversed the phenotype by promoting faster small intestinal transit in the Abx-treated WSD-ETPi group compared with the vehicle group (p = .06,Figure 4B).We also repeated this experiment in another mouse strain and found a similar faster small intestinal transit when GLP-1 receptor was blocked in the Abx-treated WSD-ETPi group (Figure S2).Thus, this indicates that gut microbiota-dietary protein interaction regulates GLP-1 receptor-mediated small intestinal transit in mice.

| p-Cresol reverses WSD-ETPi diet-mediated effects on GLP-1 and intestinal transit
We anticipated that the inhibition of host dietary protein digestion increases protein levels in the distal intestine, leading to enhanced microbial protein degradation and the production of a variety of amino acid-derived microbial metabolites.Due to gut microbiota depletion in Abx-treated mice, dietary protein would remain intact in the colonic lumen, resulting in limited production of microbial metabolites.1][22] Therefore, we hypothesized that administration of p-cresol would reverse WSD-ETPimediated effects in microbiota-depleted mice.To test this hypothesis, p-cresol was supplemented along with antibiotics in the drinking water of mice fed WSD-ETPi diet.We found that administration of p-cresol reversed the effects of WSD-ETPi diet, with reduction in the colonic Gcg expression (Figure 4C) and faster small intestinal transit compared with Abx WSD-ETPi group of mice (Figure 4D).

| DISCUSSION
In this study, using a mouse model of host protein deficiency combined with gut microbiota depletion, we demonstrate that microbial dietary protein metabolism regulates colonic gut hormone expression and GLP-1mediated small intestinal transit, potentially reversible by supplementation with the tyrosine-derived microbial metabolite p-cresol.
Enteropeptidase is a membrane-bound protease on the brush border of the duodenum that activates trypsin, initiating a cascade of enzymatic reactions involved in dietary protein digestion.By using a dietary model that blocks host protein digestion through an enteropeptidase inhibitor, we observed a significant drop in body weight gain of mice, independent of food intake.However, this effect was evident only in the presence of the gut microbiota.These findings are consistent with a previous report suggesting that oral administration of compound 6b reduced body weight gain in diet-induced obese rats. 15Sugama et al. 16 also observed that enteropeptidase inhibition induced microbiota-dependent effects on body weight loss and they additionally observed reduced food intake in diet-induced obese mice, changes in metabolome, and shifts in the gut microbiota composition accompanied by a significant increase in Akkermansia muciniphila, a bacterium recognized for its anti-obesity potential. 23Another report suggested that body weight loss due to chronic protein deficiency was not observed in GF mice, mediated by fibroblast growth factor 21. 24 Thus, the prevention of body weight loss in chronic and acute protein deficiency in microbiota-deficient mice may be attributed to the absence of microbiota-regulated circulating factors.
Partially digested protein hydrolysates such as peptones, 25,26 oligopeptides, 27 and amino acids 28,29 increase the expression and secretion of gut hormones, particularly GLP-1.Therefore, we hypothesized that increased protein levels in the intestinal lumen due to enteropeptidase inhibition would modulate expression patterns of the gut hormones.Although we did not observe any marked in colonic expression of gut hormones the conventional mice with intact microbiota when treated with ETPi, enteropeptidase inhibition in the absence of the gut microbiota resulted in a substantial increase in the expression of gut hormones in the colon, including GLP-1.This is likely due to increased load of undigested proteins in the distal gut where density of L-cells is higher, which may induce Gcg expression.An alternate hypothesis is that the absence of protein-derived microbial metabolites might regulate Gcg expression in the colon. 22An earlier report also showed that chow fed GF mice exhibited higher colonic Gcg expression, 4 which could be reversed by SCFAs administration. 8LP-1 secreting L-cells are scattered along the length of the intestine.However, GLP-1 from the small intestine appears to be more important for glucose regulation, whereas GLP-1 from the colon may have paracrine roles, such as the regulation of the intestinal motility. 19Given the profound increase in colonic Gcg expression, we focused here on small intestinal transit and observed significantly slower transit mediated by GLP-1 receptor in the microbiota-depleted mice fed WSD-ETPi.Slower intestinal transit promotes efficient energy extraction from the diet, which may also contribute to resistance to body weight loss in microbiota-depleted mice fed WSD-ETPi.
It has been shown previously that gut microbiota regulated metabolites, such as SCFAs, modulate GLP-1 levels, either as energy substrates 4 or as FFAR2/3 agonists. 30,31ince the absence of gut microbiota also reduces the production of amino acid-derived microbial metabolites (e.g., indole and p-cresol), which are reported to modulate GLP-1 expression and secretion, and gut motility, 22,32 we investigated here whether p-cresol might be responsible for the changes in GLP-1 and small intestinal transit.Our study demonstrated that supplementing mice with the tyrosine-derived microbial metabolite p-cresol reversed the enteropeptidase inhibition mediated effects on Gcg expression and small intestinal transit.It is important to note that we used ETPi in combination with WSD, as demonstrated in other metabolic studies 15,16 and therefore, if ETPi supplementation would produce similar effects in the mice fed a chow diet needs to be further studied.
In conclusion, our data demonstrate a link between microbial dietary protein metabolism and the host physiology.As protein malnutrition is associated with slower intestinal motility, 33 our study highlights an important role of the gut microbiota in this process, and the potential to reverse effects on gut transit by dietary proteinderived microbial metabolite.Further investigations into gut microbiota-dietary protein interactions may provide novel mechanisms to improve metabolic health and gut motility.