AIMD affects composition and function of the gut microbiome

Twelve-week-old mice were given twice daily (i.e., every 12 h) oral gavage of ampicillin, vancomycin, neomycin, metronidazole, and amphotericin B (AIMD-treated group) or water (vehicle-treated group)23. Stool cultures prepared from vehicle-treated and AIMD-treated mice revealed far fewer colonies in the AIMD group (Fig. 1a and Supplementary Fig. 1A). The amount of stool-extracted DNA was nearly 20-fold lower in the AIMD mice compared to vehicle-treated mice (Supplementary Fig. 1B), suggesting depletion of all luminal organisms. Finally, AIMD resulted in a massive increase in cecal weight and size (Supplementary Fig. 1C, D), as well as increase in stool output (Supplementary Fig. 1E).

Fig. 1 AIMD depletes the gut microbiome. a Stool cultures from AIMD mice yielded fewer colonies, n = 5/group. For box plot, center is mean, box is 25th to 75th percentile, and whiskers are 5th to 95th percentile. Kruskal–Wallis test, *p < 0.05, **p < 0.01. b, c 16S results show that AIMD mice had a decrease in OTUs detected from the Firmicutes and Bacteroidetes phyla, and an increase in Proteobacteria phyla, n = 6–8/group, mean percent abundance (with SEM). These differences were significant as assessed by ANCOM24. d 16S results showing the shifts in microbiome in each treatment condition. e Principal coordinate analysis of the gut microbiome, n = 6–8/group. Fecal specimens were collected approximately 2 weeks after intervention Full size image

The pre-treatment microbiome of the mice was dominated by Firmicutes and Bacteroidetes species, as was the microbiome from the vehicle-treated mice (Fig. 1b, d, Supplementary Fig. 1F, and Supplementary Data 1). The microbiome of AIMD mice had far fewer sequences from the Firmicutes and Bacteroidetes phylum. Instead, the microbiome of AIMD mice had a compositional shift to Proteobacteria (Fig. 1c, d, Supplementary Fig. 1F, and Supplementary Data 1). These phylum level differences are significant based on an analysis of composition of microbiome (ANCOM) assessment, which uses log-ratio analysis to improve inferences from microbiome compositional survey data24. The pre-treatment and vehicle-treated mice shared a majority of their OTUs, whereas the AIMD mice, which had fewer OTUs, shared about a sixth of their OTUs with the other two conditions (Supplementary Fig. 1G)

Principal coordinate analysis of UniFrac distances25 showed the microbiomes of pre-treatment and vehicle-treated mice were similar to each other, but the microbiome of AIMD mice was quite distinct (Fig. 1e). Phylogenetic diversity (PD) whole-tree (a measure of α-diversity) assessment of the three different groups of microbiomes showed that the pre-treatment condition had the greatest diversity, with a slight decrease in the vehicle-treated mice and significant decrease in AIMD-treated mice (Supplementary Fig. 1H, I). Another measure of diversity, UniFrac distance, showed that within-group distances for AIMD was significantly greater than that of pre-treatment and vehicle-treated conditions (Supplementary Fig. 1J). Between-group UniFrac distances (β-diversity measure) showed that the AIMD condition was equally distant from the pre-treatment and vehicle conditions and the pre-treatment and vehicle conditions were nearly as distant from each other as they were from members of their own group (Supplementary Fig. 1J).

These compositional and diversity changes induced by AIMD have functional implications. We used PICRUSt, a bioinformatics tool designed to predict metagenomic functional content from 16S ribosomal RNA (rRNA) surveys using known microbial genomes as reference, to assess potential functional differences between the microbiomes of AIMD-treated and vehicle-treated mice26. Many of the predicted functional differences were in metabolic pathways (Supplementary Data 2). The lipid metabolism pathway, including secondary BA biosynthesis, was significantly different between vehicle-treated and AIMD-treated mice (Supplementary Fig. 1K). In summary, AIMD in mice consuming a normal-chow diet-induced microbiome depletion, especially of Firmicutes and Bacteroidetes species, decreased microbial diversity, and altered the potential functional capabilities of the microbiome.

AIMD improves glucose homeostasis

We then assessed the effects of AIMD on glucose homeostasis. AIMD induced a lower fasting blood glucose level compared to vehicle-treated mice (Fig. 2a) after a 4-h fast or a 16-h fast. Glucose clearance was much faster in AIMD mice, with glucose levels lower in the AIMD group compared to the vehicle-treated controls (Fig. 2b). The area under the curve (AUC) above baseline for AIMD mice was less than half of that of vehicle-treated mice (Fig. 2c). An insulin tolerance test (ITT) showed that AIMD mice had increased sensitivity to insulin compared to vehicle-treated controls (Fig. 2d). However, fasting insulin levels was not significantly different between AIMD-treated and vehicle-treated mice (Fig. 2e).

Fig. 2 AIMD improves glucose tolerance and insulin sensitivity. a Blood glucose levels after 4 or 16 h of fasting. b, c Blood glucose concentration after IP injection of a glucose bolus (1 g/kg BW) (GTT) (b) and area under the curve quantification (c). d Blood glucose levels after IP injection of insulin (0.75 U/kg BW) (ITT). e Fasting serum insulin levels. f Body weight before and after the start of antibiotics gavage. g Cumulative food consumption (kcal) after acclimation to gavage. h Body composition as a percentage of total body weight. f–h were replicated in a separate experiment (Supplementary Figure 2). GTT and glucose measures were performed 3 weeks after intervention, and ITT was performed 4 weeks after intervention. Unpaired Student’s t test, **p < 0.01, ***p < 0.001, ns nonsignificant. All error bars are SEM Full size image

AIMD did not affect the weight of the mice (Fig. 2f and Supplementary Fig. 2A). The cumulative food consumption was also indistinguishable between the two groups (Fig. 2g and Supplementary Fig. 2B). The body composition of AIMD mice was similar to vehicle-treated mice, with the same amount of fat and lean mass, but there was a change in tissue that could not be characterized as being lean or fat (Fig. 2h and Supplementary Fig. 2C, D). This difference is likely due to changes in cecal content. Hence, AIMD resulted in a drop in fasting blood glucose, increased glucose tolerance, and increased insulin sensitivity despite unchanged serum insulin levels in the setting of unchanged mouse body weight, food consumption, and adiposity.

AIMD alters luminal secondary metabolites

AIMD can affect luminal secondary metabolites and gut signaling, which can affect whole-body glucose homeostasis. AIMD led to a complete disappearance of butyrate from the feces (Fig. 3a). AIMD mice also had a significant decrease in the fecal concentration of propionic acid when compared to vehicle-treated mice (Fig. 3a). Hence, AIMD changes the SCFA pool, most notably by decreasing butyrate to undetectable levels.

Fig. 3 AIMD affects SCFA and BA profiles in the cecum. a Absolute quantification of the level of different SCFA in the cecal content. Insert: close-up of butyrate levels. Absolute quantification of primary (b) and secondary BAs (c) in the feces. CA cholic acid, aMCA α-muricholic acid, bMCA β-muricholic acid, TCA taurocholic acid, TaMCA tauro-α-muricholic acid, TbMCA tauro-β-muricholic acid, TCDCA taurochenodeoxycholic acid, DCA deoxycholic acid, HCA hyocholic acid, wMCA ω-muricholic acid, TDCA taurodeoxycholic acid, TUDCA tauroursodeoxycholic acid, TLCA taurolithocholic acid, THDCA taurohyodeoxycholic acid. Fecal samples were collected 2–3 weeks after intervention. Mann–Whitney U test, **p < 0.01, ***p < 0.001. All error bars are SEM Full size image

AIMD has substantial effects on the luminal BA pool. Fecal samples from AIMD mice had lower concentrations of many primary BAs (Fig. 3b and Supplementary Table 1) and secondary BAs (Fig. 3c and Supplementary Table 1). The ratio of primary to secondary fecal BAs is more than 20-fold higher in AIMD mice compared to vehicle-treated mice. Thus, AIMD is accompanied by significant changes in luminal SCFA and BAs. These changes confirm the functional predictions made by the PICRUSt analysis (Supplementary Fig. 1K).

AIMD alters gut metabolic signaling

Since changes in luminal secondary metabolites can alter intestinal homeostasis and gut signaling, we assessed gut-released hormones and other metabolically important hormones. First, we measured serum levels of glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), which are incretins that can modulate serum glucose levels. AIMD mice had a nearly 18-fold higher total GLP-1 (Fig. 4a). In addition, blood collection on Ddp4 inhibitor-coated tubes revealed a higher fasted (Fig. 4b) and fed (Fig. 4b) active GLP-1. Although fasting insulin levels were not different, insulin significantly increased after re-feeding AIMD mice (Supplementary Fig. 3A). This suggests that GLP-1 potentiated insulin response after feeding.

Fig. 4 AIMD increases GLP-1 and affects other gut hormones. a Fasting serum level of total GLP-1 (n = 9–10/group). b Serum level of active GLP-1 after 16 h of fasting (fasted) or 15 min after an oral bolus of glucose (1 g/kg BW; fed). Blood was collected on Ddp4 inhibitor-coated tubes (n = 5/group). c Quantification by RNA-sequencing of Ggc and Pcsk7 mRNA expression in the cecum (n = 7–8/group). Adjusted p value from negative binomial Wald test (corrected for multiple hypothesis testing with the Benjamini–Hochberg method), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d–g Serum endocrine panel—plasmatic levels of GIP (d), ghrelin (e), leptin (f), and glucagon (g) (n = 9–10/group). h Serum level of total GLP-1 in GF mice (n = 4/group). i, j AIMD or vehicle mice were IP injected Exendin-9 (250 nmol/kg) or saline 20 min prior to receiving an oral bolus of glucose (1 g/kg BW) and glucose concentration was monitored over 100 min (n = 8/group). Quantification of the area under the curve (AUC, j). Serum collection and oGTT were performed at 1 week after intervention. Unless otherwise stated, Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns nonsignificant. All error bars are SEM Full size image

In agreement with the observed increase in GLP-1, results from cecal transcriptome analysis (Fig. 4c) show that the expression level of the proglucagon gene (Gcg), which encodes GLP-1, was sevenfold higher in AIMD mice. In addition, the enzyme which converts preglucagon to GLP-1 and GLP-2, proprotein convertase subtilisin/kexin type 7 (Pcsk7) was elevated in AIMD mice. Dipeptidylpeptidase 4 (Dpp4), the enzyme that degrades GLP-1, was not different between AIMD-treated and vehicle-treated mice (Supplementary Fig. 3B). Finally, the expression of genes for other gut hormone that are co-secreted with GLP-1, including protein YY and cholecystokinin were also upregulated in AIMD mice (Supplementary Fig. 3B). GIP, on the other hand, was not affected by AIMD (Fig. 4d).

We also measured other metabolically important hormones. Ghrelin, a gut hormone released by the gut that affects appetite, was not significantly different between vehicle and AIMD mice (Fig. 4e). However, AIMD mice had significantly less leptin, another hormone that regulates appetite (Fig. 4f). Finally, AIMD mice had nearly twice the amount of glucagon in their serum (Fig. 4g).

We further assessed whether these changes in GI hormones are specific to AIMD mice or were also observed in GF mice. Since GF mice used for these experiments were older, housed in a different room, with a different vendor for their normal-chow diet, and were not receiving twice daily gavage as an intervention, we used conventionally raised brethren as controls. Similar to AIMD mice, GF mice had elevated levels of GLP-1 compared to their controls (Fig. 4h). Also similar to AIMD mice, GF mice did not have significant differences in their fasting serum insulin (Supplementary Fig. 3C) or GIP (Supplementary Fig. 3D). However, unlike AIMD mice, there was no significant difference between GF mice and their controls in serum glucagon (Supplementary Fig. 3E) or serum leptin (Supplementary Fig. 3F). In addition, unlike AIMD mice, GF mice had lower production of ghrelin (Supplementary Fig. 3G).

We investigated whether the lower fasting blood glucose and the increased glucose clearance seen in GTT were caused by high levels of serum GLP-1. We used a high dose of Exendin-9 (Ex-9), a potent GLP-1 receptor antagonist to reverse the effects of GLP-1 in AIMD mice (Fig. 4i). If GLP-1 is the sole cause of hypoglycemia in AIMD mice, we would expect the GTT response to be similar between vehicle-treated and AIMD mice after Ex-9 injection. Although Ex-9 increased the glycemic surge in AIMD mice, it did not bring their oral glucose tolerate test (oGTT) response to the same level as that of vehicle-treated mice who also received the antagonist (Fig. 4j). In other words, the relative incretin effect of GLP-1 appears to be similar between AIMD-treated and vehicle-treated mice, which suggests that lower blood glucose in AIMD mice is not solely caused by excessive GLP-1.

AIMD remodels cecal enterocyte transcriptome

The effect of AIMD on luminal secondary metabolites and gut signaling suggests that this treatment is also affecting intestinal homeostasis. To further assess the effect of AIMD on the gut, we performed cecal transcriptome analysis. AIMD-treated and vehicle-treated mice cluster by treatment type in a principal component analysis analysis (Fig. 5a). The AIMD-treated and vehicle-treated groups had 6256 differentially expressed genes (Supplementary Fig. 4A and Supplementary Data 3). Ingenuity pathway analysis of these differentially expressed genes reveal that they are mainly involved in mitochondrial dysfunction and oxidative phosphorylation (Fig. 5b), which is consistent with the decrease in the main fuel of cecal enterocytes, butyrate (Fig. 3a). Furthermore, changes in cellular growth and proliferation, as well as cell death and survival pathways (Fig. 5c), suggest colonic remodeling, which is consistent with the larger cecum size observed in these mice (Supplementary Fig. 1C, D).

Fig. 5 AIMD induces major changes in the transcriptome of the cecum. a PCA analysis of the cecum RNA-sequencing data (n = 7/8/groups). b, c Functional annotation of differentially expressed genes using IPA. The top 15 canonical pathways (b) and the top 11 biological functions (c) are shown Full size image

AIMD affected gut inflammatory signaling by activating both pro-inflammatory and anti-inflammatory cascades. For example, some pro-inflammatory transcripts were significantly higher in AIMD mice (Tlr4, Tnfrs1a; Supplementary Fig. 4B). However, other pro-inflammatory transcripts (e.g., interleukin-18 (IL-18); Supplementary Fig. 4B) were significantly lower in AIMD mice, as was an inhibitor of the pro-inflammatory cytokine IL-1 (IL1rm; Supplementary Fig. 4B). Hence, it is difficult to determine how AIMD affects gut inflammatory pathways through transcriptomics alone. In sum, changes in cecal gene expression suggest that AIMD not only induces gut proliferation and alters inflammatory signaling but also affects cecal enterocyte metabolism.

AIMD shifts cecal enterocyte metabolism

To better understand the altered glucose homeostasis associated with AIMD, we investigated the expression of genes involved in metabolic pathways. Most strikingly, AIMD leads to extensive reconfiguration of the cecal enterocytes’ metabolism. The decrease in luminal SCFAs, particularly of butyric acid (Fig. 3a) and BAs (Fig. 3b, c), suggests difficulty in the absorption of fatty acids, the primary source of nutrients for enterocytes. Accordingly, compared to vehicle-treated mice, AIMD mice down-regulate genes involved in fatty acid metabolism (Fig. 6a). A schematic of transcriptional changes in the cecum is illustrated in Fig. 6b. There is a downregulation of fatty acid receptors and transporters such as free fatty acid receptor 2 (Ffar2/Gpr41) and fatty acid-binding proteins (e.g., Fabp2). SCFAs are imported into the mitochondria by carnitine palmitoyltransferases (Cpt1a, Cpt2) and processed by acyl-CoA synthetases (Acss1), both of which are downregulated in AIMD mice. Similarly, all the enzymes of fatty acid β-oxidation are downregulated in AIMD mice, including acyl-CoA dehydrogenases (Acadl, Acad8, Acad12), enoyl CoA hydratases (Echdc2), 2,4-dienoyl CoA reductase (Decr1, Decr2), hydroxyacyl-coenzyme A dehydrogenase (Hadh, Hadhb), and acetyl CoA acyltransferase (Acaa1a, Acaa1b). A parsimonious explanation of the consistent downregulation of β-oxidation genes is that the enterocytes are not using SCFAs for cellular metabolism.

Fig. 6 Anaerobic glycolysis in the cecum is upregulated in AIMD mice. a Heatmap of differentially expressed genes involved in sugar and lipid metabolism in the cecum of vehicle and AIMD mice as assessed by RNA-sequencing analysis (n = 7–8/group). b Mapping of metabolic genes onto specific pathways, including gluconeogenesis and glycolysis, β-oxidation, ketone body metabolism, TCA cycle, fatty acid synthesis, maturation and storage. Adjusted p value from negative binomial Wald test (corrected for multiple hypothesis testing with the Benjamini–Hochberg method), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. c Quantification by qRT-PCR of Glut2, Gck, G6pc, Pcx, and Pgc1a mRNA expression in the liver (n = 8/group). Tissue collected 4 weeks after intervention. Mann–Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.001, ns nonsignificant. All error bars are SEM Full size image

Cecal enterocytes could be using SCFAs for energy storage through lipogenesis. However, cecal gene expression profiling again shows that this is not the case (Fig. 6a, b). AIMD mice have a downregulation of fatty acid synthase (Fasn) compared to vehicle-treated mice. There is also a downregulation of the stearoyl-CoA desaturase (Scd1) which elongates palmitate, the end product of fatty acid synthase complex, into oleate. Furthermore, the upregulation of 3-oxoacid CoA transferase (Oxct1) accompanied by a complementary downregulation in the genes involved in ketogenesis, including acyl-CoA thioesterase (Acot7) and HMG-CoA synthase (Hmgcs2), suggested increased utilization of ketone bodies as an energy source.

Cecal transcriptome analysis indicates that anaerobic glycolysis is upregulated in the enterocytes of AIMD mice (Fig. 6a, b). Out of the ten enzymes in anaerobic glycolysis, eight were upregulated in AIMD mice. AIMD have increased expression of hexokinase (Hk2), phosphofructokinase (Pfkp), aldolase B (Aldob), phosphoglyceratekinase (Pgk1), phosphoglycerate mutase (Pgam1), enolase (Eno1), pyruvate kinase (Pklr), and lactate dehydrogenase (Ldha). Appropriately, transcription for genes in the reverse pathway of glycolysis, gluconeogenesis (e.g., its rate-limiting enzyme phosphoenolpyruvate carboxykinase (Pck1) and its main transcriptional regulator, CREB, regulated transcription coactivator 2 (Crtc2)) are downregulated. Further analysis shows upregulation of genes transporting sugars to the glycolysis pathway (e.g., increased glycerol kinase (Gyk) and glycerol phosphate dehydrogenase (Gpd2)). These findings suggest that the primary source of energy for enterocytes is now glucose through anaerobic glycolysis, with less reliance on the mitochondria.

There are significant changes in the role of mitochondria in the metabolism of enterocytes. The proportion of total reads that map to mitochondrial chromosome M are reduced in AIMD mice, suggesting a reduced number of mitochondria (Supplementary Fig. 5A). Furthermore, mitochondrial cytochrome b and some subunits of cytochrome c oxidase such as Cox6a1 (complex IV) expression are significantly reduced in AIMD mice (Supplementary Fig. 5B). In agreement with increased utilization of glucose for anaerobic glycolysis as the main source of energy for enterocytes, expression of the sodium/potassium-transporting ATPase is upregulated (Atp1b1) (Fig. 6a, b). In addition, the sugar efflux transporter (Slc50a1) is downregulated, suggesting that the enterocytes are preventing glucose export.

These changes in intestinal glucose regulation can be responsible for the observed differences in whole-body glucose homeostasis in AIMD mice. Since the liver is the main regulator in glucose homeostasis, we characterized the hepatic expression of key glucose metabolic pathways using quantitative reverse transcription PCR (qRT-PCR). Unlike enterocytes, hepatic gluconeogenesis is upregulated with an increase in pyruvate carboxylase (Pcx) and its activator peroxisome proliferator-activated receptor gamma coactivator 1α (Pgc1a), as well as glucose-6-phosphatase (G6pc; Fig. 6c). Conversely, glucokinase, an enzyme in the glycolysis/reverse pathway, is downregulated. These results suggest that the liver in AIMD mice is actively producing more glucose. This is further confirmed by the upregulation of hepatic glucose transporter 2 (Glut2/Slc2a2), which is a facilitated transporter of glucose from the liver into the blood. There was no difference in hepatic histology (Supplementary Fig. 5C) or hepatic triglyceride load (Supplementary Fig. 5D) between AIMD-treated and vehicle-treated mice. Transcripts involved in lipid metabolism, such as Pparα (Supplementary Fig. 5E) and Rev-erbα (Supplementary Fig. 5E), are not affected by AIMD or secondary luminal metabolites.

In summary, examination of cecal transcriptomes reveals that proliferative enterocytes shift their metabolic nutritional needs away from SCFAs toward the utilization of glucose. The low amount of luminal SCFAs likely leads to a downregulation of fatty acid uptake, β-oxidation, and lipogenesis. Instead, there is an upregulation of anaerobic glycolysis and downregulation of gluconeogenesis and function of mitochondria. The upregulation of anaerobic glycolysis is similar to a Warburg effect in the setting of upregulated proliferation that correlates with high levels of GLP-1 growth factors. Furthermore, hepatic gene expression suggests that the liver responds to AIMD as though it is in starvation, upregulating the expression of genes in gluconeogenesis, while downregulating glycolysis genes.

AIMD alters BA metabolism and potentiates GLP-1 response

Since the luminal BA pool was quite different in AIMD compared to vehicle-treated mice, we examined the cecal enterocytes sequencing results for genes involved in BA processing (Fig. 7a). The expression of the primary transporter for intestinal BA uptake, Slc10a2/Ibat (also known as Asbt), is increased 16-fold in AIMD mice compared to vehicle-treated controls. In addition, organic solute transporter β (Slc51b/Ostb), a subunit of the Ost basolateral transporter complex responsible for exporting BA from enterocytes to the enterohepatic circulation system, is increased 2.5-fold. Both transporters are activated by FXR, and the luminal BA pool contains both FXR agonists (e.g., taurocholic acid (TCA), cholic acid (CA)) and antagonists (e.g., TaMCA, TbMCA). FXR transcription is significantly increased in AIMD mice. In addition, FXR forms an obligate heterodimer with the retinoid X receptor (RXRa) which is also upregulated in AIMD mice. These changes suggest that FXR signaling in the gut is actively increasing the uptake of BAs from the lumen.

Fig. 7 AIMD alters BA metabolism which can potentiate GLP-1 response. a Quantification by RNA-sequencing of Slc10a2, Fxr, Rxra, and Slc51b mRNA expression in the cecum (n = 7–8/group). Adjusted p value from negative binomial Wald test (corrected for multiple hypothesis testing with the Benjamini–Hochberg method), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. b, c Absolute quantification of primary (b) and secondary BAs (c) in the serum. Mann–Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Quantification by qRT-PCR of Slc10a1, Fxr, Shp, and Cyp7a1 mRNA expression in the liver (n = 8/group). Mann–Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant. e Serum GLP-1 level after 4 days of bi-daily TCA gavage (400 mg/kg) (n = 4/group). Tissue and serum samples from AIMD studies collected at 4 weeks after intervention. Serum from TCA study was collected after 1 week intervention. Student’s t test, *p < 0.05. All error bars are SEM Full size image

Since cecal transcriptome analysis indicates that there is an upregulation of BA reabsorption from the cecum, we assessed whether the altered luminal BA pool resulted in differences in the serum BA pool. Serum samples from AIMD mice have significantly higher amounts of common primary BAs (Fig. 7b and Supplementary Table 2). Serum secondary BAs remain higher in samples collected from vehicle-treated mice compared to those from AIMD mice (Fig. 7c and Supplementary Table 2). The ratio of primary to secondary serum BAs is more than tenfold higher in AIMD compared to vehicle-treated mice (Supplementary Fig. 6A). Consistent with observations from fecal BAs, CA and bMCA were significantly decreased in AIMD mice (Fig. 7b and Supplementary Table 2). Furthermore, there was nearly a twofold increase in TCA, a threefold increase in TbMCA, and a threefold increase in TCDCA (Fig. 7b and Supplementary Table 2). Despite altered serum and luminal BA pool in AIMD mice, there was no significant difference in serum cholesterol and triglyceride levels (Supplementary Fig. 6B, C).

Altered serum BA pool suggests altered BA metabolism in hepatocytes. We assessed the expression level of key enzymes by qRT-PCR in whole liver extracts of AIMD-treated and vehicle-treated mice (Fig. 7d). AIMD-induced serum BA pool changes coincide with increased expression of the primary basolateral membrane BA transporter, liver BA transporter (Slc10a1/Lbat). The primary BA increase did not affect the expression of FXR in hepatocytes, but did affect the expression of its main target and metabolic mediator, small heterodimer partner (Nr0b2/Shp). Shp regulates BA synthesis by inhibiting cholesterol 7 α-hydoxylase (Cyp7a1), the rate-limiting enzyme in de novo synthesis of BAs from cholesterol. As expected with downregulation of Shp, there is a nearly threefold increase in the expression of Cyp7a1 in hepatocytes.

Finally, we investigated whether the altered luminal BA pool contributed to the altered glucose homeostasis and increased GLP-1 observed in the AIMD mice. To assess this, we gavaged 12-week-old male wild-type C57BL/6 with TCA twice daily (i.e., every 12 h) for 4 days. TCA was selected since it was elevated in AIMD condition, whereas other BAs were not. TCA-treated mice had a significant increase in GLP-1 compared to vehicle-treated controls (Fig. 7E) and a decrease in fasting blood glucose level that trended toward significance (Supplementary Fig. 6D). Compared to vehicle-gavaged controls, glucagon, insulin, leptin, ghrelin, and GIP levels were unchanged in TCA-gavaged mice (Supplementary Fig. 6E–I).

In summary, in response to the lower luminal BA concentration, cecal enterocytes in AIMD mice increase the expression of FXR and upregulate BA transporters likely to conserve as much BAs as possible. The transport of primary/conjugated BAs that are FXR antagonists (e.g., TbMCA) into the serum in turn suppresses hepatic FXR targets, such as Shp, which allows Cyp7a1 to upregulate further BA synthesis. Finally, increasing luminal primary BAs by oral gavage with TCA elevates serum GLP-1, suggesting a potential mechanism by which altered luminal BAs in AIMD mice could lead to altered glucose homeostasis.