The gut microbiota play important roles in lipid metabolism and absorption. However, the contribution of the small bowel microbiota of mammals to these diet-microbe interactions remains unclear. We determine that germ-free (GF) mice are resistant to diet-induced obesity and malabsorb fat with specifically impaired lipid digestion and absorption within the small intestine. Small bowel microbes are essential for host adaptation to dietary lipid changes by regulating gut epithelial processes involved in their digestion and absorption. In addition, GF mice conventionalized with high-fat diet-induced jejunal microbiota exhibit increased lipid absorption even when fed a low-fat diet. Conditioned media from specific bacterial strains directly upregulate lipid absorption genes in murine proximal small intestinal epithelial organoids. These findings indicate that proximal gut microbiota play key roles in host adaptability to dietary lipid variations through mechanisms involving both the digestive and absorptive phases and that these functions may contribute to conditions of over- and undernutrition.

Some evidence suggests that microbes are important for lipid absorption. For example, germ-free (GF) mice consuming a HF diet were shown to have elevated fecal lipid levels compared with conventional mice (). In addition, conventionalized GF zebrafish displayed increased long- and short-chain fatty acid uptake in the intestinal epithelium under fasted and fed conditions (). More recently, antibiotic-treated rats displayed reduced lymphatic lipids following fat challenge (). Despite these findings, diet-microbe interactions in the small intestine and mechanisms driving lipid digestion and absorption have not been thoroughly examined. To address this, we postulated that proximal small intestinal microbiota play an important role in regulating lipid digestion and absorption and are vulnerable to HF diet-induced alterations. Lastly, we hypothesized that these alterations promote lipid absorption, contributing to development of obesity. Thus, the goals of this study were to (1) determine whether microbes are required for proper digestion and absorption of dietary lipid using GF animals, (2) examine the impact of HF diets on small intestinal microbiota membership using 16s rRNA gene profiles, (3) determine the functional impact of HF diet-induced jejunal microbiota on fat absorption, and (4) test proof of concept that microbially derived metabolites or products from specific microbial strains directly affect lipid absorption. Here, we demonstrate that gut microbes act as indispensable signal transducers of dietary lipid, allowing the host to adapt fat absorption. Consumption of a HF diet profoundly alters the microbiota in the small intestine, and this community increases fat absorption in conventionalized animals. Lastly, we show that these effects may be promoted by specific bacterial strains through microbe-derived components or small molecules.

Numerous studies have shown that changes in cecal and stool microbiota are associated with dietary shifts, but it remains unclear whether they faithfully represent alterations of regional microbiota of the small intestine, which are more likely responsive to dietary perturbations and elicit a direct impact on host functions related to metabolism and fat absorption. To date, limited studies have evaluated small intestine microbiota structure under obese versus lean states or with dietary manipulation and whether these changes contribute to metabolic disease.characterized regional microbiota distal to the jejunum in rhesus macaques. It was concluded that stool microbiota structure was strongly correlated with colonic communities but only moderately with those in the small intestine; however, characterization of the duodenal microbiota was not performed. Obese and lean pigs were found to have increased abundance of Clostridium and SMB53 in jejunum and ileum; however, no clear differences between obese and lean states were evident ().demonstrated that western diets shifted gut microbiota structure along the length of the gut, including the small intestine; however, this region was not further investigated, nor was a comparison made between the different regions. Studies have examined microbiota changes or host responses in the duodenum (), jejunum, and ileum (), yet a comparative analysis between small intestinal regions in response to low-fat (LF) versus HF diets has not been performed.

The proximal small intestine serves as the major site of macronutrient digestion and absorption. Complex interactions between dietary cues, the gut endocrine response, bile release, exocrine function of the pancreas, and absorptive enterocyte function are needed for efficient absorption of lipids and other dietary nutrients. The small intestine harbors a complex microbial community, albeit with less diversity and abundance (≈10–10microbial cells per gram) than the colonic microbiota (≈10cells per gram;). This could be because microbial populations in the small intestine are routinely subjected to adverse host factors affecting assemblage, such as low pH, faster transit time, bile acids, and antimicrobial peptide exposure. Studies suggest that Firmicutes and Proteobacteria, which dominate the small intestine, are more tolerant of these factors (). Bacteroidetes are also bile tolerant but the abundance of this phylum is reduced in the small intestine and is increased in the large intestine ().

Recent work highlights the role of the trillions of intestinal microbes in health and disease. Our microbial organ is highly sensitive to environmental factors; in particular, dietary stress. Several studies have revealed that high-fat (HF) diets profoundly (), rapidly, and sustainably alter microbial communities in as little as 24–48 hr in mice and humans (), resulting in the development of metabolic disturbances. Not only are changes in community membership evident (i.e., abundance of Firmicutes and Proteobacteria as well as decreasing diversity) but also microbial metabolic function is altered (i.e., decreasing short-chain fatty acid production) (). Many of these studies have relied on examining cecal or fecal microbiota. A critical, unanswered, question is whether diet-mediated changes in the small intestinal microbiome elicit a significant impact on the host in the proximal small intestine, a crucial site for macronutrient absorption and energy acquisition.

To determine whether other microbes had similar capabilities or worked through alternate mechanisms in vivo, we evaluated the effect of L. rhamnosus GG supplementation using the same experimental protocol and examined changes in weight gain, adiposity, and markers of lipid absorption. Mice fed a HF diet supplemented with L. rhamnosus GG gained significantly more weight compared with mice fed a LF diet with L. rhamnosus GG ( Figure S5 A). Supplementation of HF diet with L. rhamnosus GG does not further increase body weight significantly compared with HF diet alone ( Figure S5 A). L. rhamnosus GG significantly increased epididymal and mesenteric fat under HF diet conditions compared with LF-fed mice supplemented with L. rhamnosus GG ( Figure S5 B). Mice fed a HF diet and supplemented with L. rhamnosus GG displayed increased LDL and cholesterol levels compared with mice fed a LF diet and supplemented with L. rhamnosus GG ( Figure S5 C). L. rhamnosus GG induced Dgat1 under LF conditions compared with control in the duodenum and Dgat2 gene expression and protein levels in the jejunum, although not significantly ( Figures 5 F and 5G). Similar to C. bifermentans, L. rhamnosus GG did not have a significant impact on processes related to lipid digestion, including gallbladder weight, plasma CCK, or SCT levels in this study ( Figures S5 E–S5G). No differences were detected across groups in 16S rRNA gene abundance in cecal contents at the end of the study ( Figure S5 D). Although a significant increase in L. rhamnosus GG itself was not detected, taxonomic changes were apparent with L. rhamnosus GG supplementation compared with controls ( Figure S5 E). Collectively, our findings show that specific microbial strains affect expression of genes involved in re-esterification of TG, such as Dgat1 and Dgat2, but the exact mechanisms are yet to be explored.

To establish proof of concept that specific strains of bacteria affect lipid absorption pathways, a reference strain belonging to Clostridiaceae, Clostridium bifermentans, was tested in an in vitro model of small intestinal organoid cultures. This microbe was selected based on its fermentative activity and identification in human sewage and feces. C. bifermentans is a spore-forming gram-positive anaerobe that has been shown to produce a wide range of metabolites, including acetate, lactate, carbon dioxide, and hydrogen, that have potential in eliciting host responses (). However, a role of C. bifermentans in regulating host metabolism or small intestinal lipid absorption has not been previously described. Therefore, this bacterium was grown in anaerobic conditions after which conditioned media (CM), presumably containing bioactive metabolites, was collected and used to treat duodenal and jejunal cultures of enteroids derived from mice (). We also selected Clostridium ramosum due to previous findings that this bacterium was found to be associated with human obesity () and promoted diet-induced obesity in gnotobiotic mice (). L. rhamnosus GG CM was also selected as a comparison strain based on the finding from our previous experiment that this strain could affect Cckar expression in the pancreas, indicating an effect related to digestion but not absorption ( Figure 3 G). After 24 hr of treatment, C. bifermentans CM selectively induced the expression of critical esterification enzymes (i.e., monoacylglycerol O-acyltransferase, Mogat2, and diacylglycerol O-acyltransferases, Dgat1, Dgat2) involved in lipid transport but not Cck, Sct, or Cd36 ( Figure 5 A). In addition, C. bifermentans CM increased oleic acid uptake compared with C. ramosum CM in duodenal monolayers or reinforced clostridial media (RCM) control in jejunal monolayers ( Figure 5 B). Consistent with these findings, we noted that CM from C. bifermentans increased Dgat2 mRNA levels in the jejunum of LF-fed mice in vivo ( Figure 5 C). Based on these results, we further examined C. bifermentans in a separate in vivo study. SPF mice were treated with an antibiotic cocktail for 14 days followed by weekly gavage with C. bifermentans (1 × 10colony-forming units [CFUs]) under either LF or HF conditions for 4 weeks. Mice fed a HF diet supplemented with C. bifermentans gained significantly more weight compared with mice fed a LF diet with C. bifermentans ( Figure S4 A); however, supplementation of HF diet-fed mice with C. bifermentans does not further increase body weight significantly compared with HF diet alone ( Figure S4 A). C. bifermentans did not significantly increase body fat or plasma lipids compared with control groups (i.e., HF + C. bifermentans versus HF or LF + C. bifermentans versus LF) ( Figures S4 A–S4C). Supplementation with C. bifermentans significantly increased mRNA levels of Dgat2, but not Dgat1, in the duodenum and jejunum ( Figure 5 D) under LF conditions with a trend toward increased levels under HF conditions. No appreciable effects of C. bifermentans on DGAT2 protein levels were observed; however, this could be due to intra-group variation ( Figure 5 E). Gene expression of Fabp2 and Cd36 were significantly elevated by HF diet regardless of supplementation ( Figure 5 D). C. bifermentans did not significantly affect processes related to lipid digestion, including no change in gallbladder weight, plasma CCK, or SCT levels ( Figures S4 E–S4G). No differences were detected across groups in 16S rRNA gene abundance in cecal contents at the end of the study ( Figure S4 D). However, taxonomic changes were apparent with C. bifermentans supplementation compared with controls, including an increase in the abundance of Clostridiaceae under HF conditions ( Figure S4 E). While it remains unclear whether C. bifermentans elicits a direct increase in adiposity under HF conditions, both our in vitro and in vivo results suggest that this Clostridiaceae member increases oleic acid uptake and the expression of Dgat2 involved in TG synthesis, possibly through C. bifermentans-derived bioactive components or molecules.

(C–G) Antibiotic-treated SPF mice were maintained on either a LF or HF diet and gavaged weekly for 4 weeks with vehicle control or 1 × 10CFUs C. bifermentans (D and E), C. bifermentans CM (C), or L. rhamnosus GG (F and G). Expression of esterification enzymes (Dgat2 and Dgat1) and fat transport genes (fatty acid binding protein, Fabp2 and Cd36) were measured in the duodenum and jejunum via qRT-PCR in the C. bifermentans study (C) and the L. rhamnosus GG study (E). DGAT2 protein levels were measured in the C. bifermentans study (D) and the L. rhamnosus GG study (F). See also Figures S4 and S5

To directly compare the impact of jejunal versus cecal microbiota on lipid absorption, GF mice were colonized with microbiota from these regions under chow-fed conditions in both the donor and recipient mice. Conventionalization with chow-fed microbiota did not restore lipid absorption compared with GF mice from either region ( Figure S2 E). Interestingly, this lack of restoration was similar to findings with purified LF jejunal microbiota shown in Figure 4 D. Thus, HF diet-induced microbiota may be necessary to elicit this host response. Future studies are needed to further interrogate this theory using HF jejunal microbiota versus HF cecal microbiota for conventionalization of GF mice.

We next determined whether conventionalization of GF animals with HF jejunal microbiota increased lipid absorption compared with conventionalization with LF jejunal microbiota and whether these changes required the selective pressure of the donor diet ( Figure 4 C). GF mice were conventionalized with HF diet jejunal microbiota and maintained on a HF diet (ConvD HF⇒HF), conventionalized with HF jejunal microbiota, and maintained on a LF diet (ConvD HF⇒LF), or conventionalized with LF jejunal microbiota and maintained on a LF diet (ConvD LF⇒LF) for 3 weeks ( Figure 4 C). Prior to conventionalization, GF mice were acclimated to their respective diets for 1 week. HF jejunal microbes increased lipid absorption to the same degree, regardless of whether they were maintained on LF or HF diets. Both groups that received HF microbes exhibited increased lipid absorption compared with mice receiving LF microbes (ConvD LF⇒LF) ( Figure 4 D). Notably, 16S rRNA gene abundance did not differ between groups at any time point following conventionalization from day 3 (d3) post conventionalization through day 21 (d21) or at the completion of the study ( Figure S2 D). We calculated transplantation efficiency by determining the percentage of donor oligotypes represented in recipient stool at d3 and d21. At d3, ∼79% donor oligotypes were represented in the recipients and, at d21, 50%–60% donor oligotypes were represented in the recipients ( Figure S3 E). Despite this, microbial community structure remained similar between groups receiving HF microbes (i.e., ConvD HF⇒HF and ConvD HF⇒LF groups) until d21, where community structure became more similar between ConvD HF⇒LF and ConvD LF⇒LF based on Bray-Curtis beta diversity analyses as indicated in the PCoA plot shown in Figure S2 C. Thus, even though HF⇒LF community structure was shifted toward a LF⇒LF community by d21, the HF microbes influenced the level of lipid absorption in these mice, perhaps through differential reprograming of the GF mouse small intestine. These results suggest that small bowel microbes from HF diet conditions increase lipid absorption compared with LF-derived microbes independent of post-conventionalization dietary pressure.

Understanding small intestinal microbiota composition is integral to identifying key host-microbe interactions that drive fat absorption. Thus, we characterized the microbial community structure of the small intestine in SPF mice under LF and HF conditions. Sequencing of 16S rRNA gene amplicons was performed in mucosal scrapings collected from the duodenum, jejunum, and ileum, as well as cecal contents of mice fed either a LF or HF diet for 4 weeks. Differences between LF and HF microbiota were apparent in the jejunum and ileum based on Bray-Curtis beta diversity analyses as indicated in the principal coordinates analysis (PCoA) plot expressed on a forced axis for regional site ( Figure 4 A) and in PCoA plots for individual intestinal regions ( Figure S3 A). Adonis and analysis of similarities (ANOSIM) tests were performed for Bray-Curtis and Canberra diversity metrics to examine differences between LF and HF diet groups in each intestinal region ( Table 1 ). HF diet increased the relative abundance of the family Clostridiaceae compared with LF diet in all regions, especially in the jejunum and ileum ( Figure 4 B). Similar results were found in a pilot study conducted early in the development of this project (data not shown). HF diet also decreased abundance of the families Bifidobacteriaceae and Bacteroidaceae in all intestinal regions ( Figure 4 B). Analysis between minimum entropy decomposition (MED) oligotypes from LF- or HF-fed mice revealed significant differences in the jejunum ( Table 2 ) and duodenum in which the abundance of the family Peptostreptococcaceae was significantly increased (Bonferroni p value = 0.0472, data not shown). Significant differences in MED oligotypes were not found between LF and HF diets in the ileum and cecum. MED oligotypes found to be significantly increased by HF diet in the jejunum belonged to the Clostridium and Turicibacter genera and Peptostreptococcaceae family, whereas oligotypes that were significantly decreased by HF diet include those mapping to Bifidobacterium, Allobaculum, and Bacteroidales ( Table 2 ). Abundance of the 16S rRNA gene increased along the length of the proximal intestine but did not significantly differ in HF versus LF conditions ( Figure S2 B). Altogether, microbiota structure differs along the length of the small intestine and HF diet has a dramatic impact on microbial structure, particularly in the small intestine.

(C and D) Experimental design for conventionalization of jejunal microbiota (C). Absorption of [H]triolein and [C]cholesterol is shown over time and expressed as disintegrations per minute (dpm) per microliter of plasma (D). See also Figure S3

To examine whether reduced lipid transport was occurring at the site of absorptive enterocytes (e.g., apart from the enteroendocrine signaling required for digestion), brush border membrane vesicles were isolated from the jejunum and ileum of GF and SPF mice, followed by incubation with [H]oleic acid. Brush border membrane vesicles from GF mice exhibited decreased [H]oleic acid ( Figure 3 H) as well as [H]glucose uptake compared with those from SPF counterparts ( Figure 3 I), implicating a role for microbes in regulating processes related to membrane transport. Gene expression of fatty acid transporters and esterification enzymes ( Figure S1 D) was measured in the duodenum and jejunum of SPF and GF mice under LF and HF conditions. The fatty acid translocase Cd36 was upregulated under HF versus LF diet conditions and in GF versus SPF mice in the jejunum. Dgat1 was upregulated in GF versus SPF mice under LF conditions in the duodenum and jejunum ( Figure S1 D). Genes related to lipogenesis and fat oxidation were also measured in the duodenum and jejunum ( Figure S1 E). The transcriptional regulator Pparα, involved in fat oxidation, was upregulated in GF versus SPF mice under LF and HF conditions in the jejunum ( Figure S1 E). Intriguingly, in the duodenum, Dgat2 was induced by HF diet in SPF mice but not GF mice, suggesting that the regulation of Dgat2 may involve diet-microbe interactions. To better elucidate mechanisms behind potential host-microbe interactions, we next sought to identify the role that HF diet-induced gut microbes play in lipid absorption.

To better understand the mechanisms behind decreased fat absorption in GF mice, fat digestion and transport was assessed in GF versus SPF mice. Fat digestion is largely facilitated by the enteroendocrine hormones cholecystokinin (CCK) and secretin (SCT) (). These peptide hormones are produced in I and S endocrine cells, respectively, of the epithelium lining the proximal small intestine and are released upon feeding. CCK stimulates the release of bile from the gallbladder for emulsification of fat as well as pancreatic secretion of lipase. Similarly, SCT stimulates pancreatic secretion of digestive enzymes and bicarbonate for neutralization of chyme and proper enzymatic function (). Notably, enteroendocrine cell number is not altered in the duodenum or jejunum of GF compared with SPF animals (), but we expected that a breakdown in enteroendocrine signals may explain reduced lipid absorption observed in GF mice. To evaluate the involvement of impaired enteroendocrine signaling in GF mice, GF and SPF mice were fasted for 4 hr and challenged with a lipid bolus of corn oil (CO) or HO control for 2 hr (). Gallbladder weights were significantly higher in GF versus SPF mice under control conditions but not following CO gavage ( Figure 3 A). Gene expression of the receptor for CCK (Cckar) was not different in SPF versus GF mice (data not shown). Interestingly, CO significantly increased lipase activity in the duodenum of SPF but not GF mice, implicating an impairment in CCK signaling to the pancreas in GF mice ( Figure 3 B). A similar trend was seen in the jejunum. GF mice exhibited reduced gene expression of Cck and Sct in the jejunum compared with SPF mice following CO gavage ( Figure 3 C). Unexpectedly, reduced lipase activity in GF mice could not be explained by circulating CCK levels as plasma CCK levels were elevated in GF mice ( Figure 3 D). This compelled us to investigate whether pancreatic expression of Cckar was reduced in GF mice. Indeed, GF mice had significantly lower mRNA of Cckar in the pancreas compared with SPF mice ( Figure 3 E). Western blot analysis revealed that SPF mice gavaged with CO exhibited elevated CCKaR protein levels compared with HO control, whereas GF mice did not display an elevated CCKaR response to CO challenge relative to HO control ( Figure 3 F). However, this effect was not significant based on densitometry analysis. Circulating SCT levels were not significantly different in GF versus SPF mice ( Figure 3 D), nor were expression levels of secretin receptor (Sctr) in the pancreas (data not shown). Next, GF mice were gavaged with or without a combination of heat-killed gram-negative (Bacteroides thetaiotamicron) and gram-positive (Lactobacillus rhamnosus GG) bacteria and pancreatic Cckar expression was measured. Exposure to both gram-negative and gram-positive heat-killed bacteria increased expression of Cckar in the pancreas compared with GF controls ( Figure 3 G). Taken together, these results suggest that reduced lipid absorption in GF mice involves a breakdown in CCKaR-mediated stimulation of the pancreas.

It should be noted that, in these experiments, the SPF and GF mice were not maintained in identical bedding conditions, as SPF mice were maintained in corn cob bedding and GF mice were housed in pine shavings. To address this issue, we repeated the radiolabeled lipid absorption assay using tyloxapol in SPF versus GF mice raised in pine shavings. Consistent with our previous results, we found that GF mice had significantly reduced [H]triolein and [H]cholesterol absorption compared with SPF mice ( Figure S2 D).

To examine the level of peripheral [H]triolein and [C]cholesterol uptake, an identical experiment was conducted in chow-fed SPF and GF mice but without tyloxapol pretreatment ( Figure 2 D). Consistent with previous results, GF mice had significantly lower plasma levels of [H]triolein and [C]cholesterol compared with SPF mice at 5 and 7 hr ( Figure 2 E). Notably, SPF mice had lower [H]triolein and [C]cholesterol levels without tyloxapol compared with when tyloxapol was used ( Figure S2 C), but levels in GF animals were rather similar between the two conditions ( Figure S2 C), suggesting that there is more rapid peripheral uptake in SPF mice compared with GF mice. In addition, we observed that GF mice have significantly lower levels of radiolabeled lipid in the duodenal and jejunal epithelium ( Figure 2 F) and in the liver ( Figure 2 G) compared with SPF mice.

To determine whether lipid absorption could be restored by HF diet feeding, radiolabeled lipid absorption was examined in SPF and GF mice fed a purified LF or HF diet ( Table S1 ). HF-fed SPF mice had significantly higher plasma levels of [H]triolein and [C]cholesterol compared with GF mice fed a HF diet at 5 and 7 hr ( Figure 2 C). SPF mice fed a HF diet displayed higher [C]cholesterol levels compared with SPF mice fed a LF diet at 7 hr ( Figure 2 C). There was no increase in [H]triolein and [C]cholesterol absorption in GF animals fed HF versus LF diets ( Figure 2 C). Thus, HF diet alone was not sufficient to restore lipid absorption in GF mice, indicating that the presence of microbes is necessary for adequate intestinal lipid absorption.

Transport of dietary lipids from the gut lumen into the systemic circulation requires both digestive and absorptive functions of the gastrointestinal (GI ) tract (). To determine whether GF mice exhibit impaired lipid digestion and absorption, GF and SPF mice maintained on a standard chow diet ( Table S1 ) were gavaged with [H]triolein (triglyceride consisting of oleic acid) and [C]cholesterol, and the amount of radiolabeled lipid accumulating in the plasma was measured over 7 hr. Prior to gavage, mice were injected retro-orbitally with tyloxapol, a peripheral lipoprotein lipase (LPL) inhibitor that blocks peripheral uptake of lipid, thereby allowing for the detection of accumulating radiolabeled lipid in the blood over time ( Figure 2 A,). GF mice displayed a dramatically decreased rate of [H]triolein and [C]cholesterol absorption compared with SPF mice ( Figure 2 B). To examine whether this was due to slower small intestinal transit time, SPF and GF mice were gavaged with corn oil plus activated charcoal for 2 hr to track the distance traveled. There was no significant difference in small intestinal transit time between SPF and GF animals ( Figures S2 A and S2B).

To establish whether GF mice display signs of fat malabsorption as previously reported, specific-pathogen free (SPF) and GF mice were fed a purified LF or HF diet ( Table S1 ) for 4 weeks. Metabolic indices and stool lipid content were measured. Consistent with previous reports, GF mice fed a HF diet were resistant to weight gain ( Figure 1 A;) and exhibited reduced epididymal and mesenteric fat pad mass ( Figure 1 B). Food consumption was not significantly different between groups on purified diets ( Figure 1 A). Markers of insulin resistance, gut peptide hormones that regulate insulin signaling (i.e., GLP-1), and circulating lipids were measured in portal and peripheral plasma. GF mice exhibited profoundly reduced levels of portal plasma triglyceride (TG) compared with SPF mice on a LF diet and reduced low-density lipoprotein (LDL) levels compared with SPF mice on both LF and HF diets ( Figure 1 C). Non-esterified fatty acids (NEFA) were elevated in GF mice on LF but not HF diets ( Figure 1 C). Similar trends in lipid profiles were found in peripheral plasma (data not shown). In addition, GF mice fed an HF diet displayed reduced levels of fasting blood glucose ( Figure 1 D), insulin, leptin, and plasminogen activator inhibitor-1 (PAI-1) compared with SPF mice ( Figure 1 E). Gut peptide hormones ghrelin and GLP-1 were higher in GF versus SPF mice under LF conditions, which are indicative of a state of energy deficit and improved insulin signaling (), respectively ( Figure S1 A). Concurrently, stool triglyceride levels tended to be elevated in GF compared with SPF mice fed an HF diet ( Figure 1 F). Levels of NEFA tended to be lower in GF mice fed a HF diet compared with SPF mice. Stool bile acids and total cholesterol were significantly increased following HF diets in both SPF and GF mice ( Figure 1 F). We noted that GF mice fed a HF diet excreted significantly more stool (grams of dry weight) over 24 hr compared with HF-fed SPF mice ( Figures S1 B and S1C). Based on these observations, we speculated that GF mice fed a HF diet have impaired fat absorption and digestive capacity, thereby conferring protection from HF diet-induced adiposity, elevated plasma lipids, and markers of insulin resistance. Therefore, we sought to further examine whether GF mice display an active impairment in lipid absorption and to identify the mechanisms involved.

Discussion

Most studies examining the relationship between gut microbiota and host metabolism have primarily relied on fecal or colonic luminal samples. In contrast, few studies have considered the roles of small bowel microbiota, particularly for nutrient digestion and absorption, which largely occur in the small intestine. This study finds that small bowel microbiota associated with the intestinal mucosa are highly sensitive to dietary cues and play an important role in nutrient assimilation, particularly in the physiological and possibly pathophysiological regulation of host lipid digestive and absorptive pathways. Even though the small bowel microbiota has fewer members and is less diverse than its colonic microbiota counterpart, it is most proximate in timing and exposure to nutrient-rich dietary cues, which may dramatically affect its composition and function relative to downstream microbiota and nearest to tissues most important for digestion and absorption (most affected by and affective to diet and metabolism). Indeed, we show that HF diet feeding dramatically affects the small bowel microbiota. Due to the highly unstable environment of the duodenum, with an influx of bile and pancreatic secretions, microbes could elicit a less influential role on host responses in this region. Downstream of this in the jejunum, host absorption is still highly active, yet the environment may allow for important host-microbe interactions. Therefore, we propose that, along with dietary residuals, host-driven cues in this region may have a great impact on shaping the microbiota.

The influence of altered community membership in the small intestine on host outcomes has not been previously examined using microbiota transplant specific to the jejunum under HF versus LF conditions. We show that conventionalization of GF mice with HF diet-induced, mucosa-associated, jejunal microbiota increased radiolabeled lipid absorption compared with LF diet-induced, mucosa-associated, jejunal microbiota, independent of the diet that mice were consuming ( Figure 4 D). In other words, maintaining the GF mice on an LF diet was not protective against the lipid absorption-promoting effects of the HF microbiota. We speculate that microbial communities derived from HF conditions differentially program the GF small intestine, promoting increased lipid absorption upon lipid challenge, compared with LF microbes. Given the decreased lipid absorption found in GF mice maintained on a HF diet ( Figure 2 C), we conclude that microbes can play an essential role in regulating host lipid absorption. Therefore, changes in the small bowel microbiota could have significant consequences for the functional role of the small intestine in regulating host macronutrient digestion and absorption. This has important implications in malnutrition and obesity, as a specific restructuring of the small bowel microbiota may be required to either increase or decrease lipid transport, respectively.

Rabot et al., 2010 Rabot S.

Membrez M.

Bruneau A.

Gerard P.

Harach T.

Moser M.

Raymond F.

Mansourian R.

Chou C.J. Germ-free C57BL/6J mice are resistant to high-fat-diet-induced insulin resistance and have altered cholesterol metabolism. Sato et al., 2016 Sato H.

Zhang L.S.

Martinez K.

Chang E.B.

Yang Q.

Wang F.

Howles P.N.

Hokari R.

Miura S.

Tso P. Antibiotics suppress activation of intestinal mucosal mast cells and reduce dietary lipid absorption in Sprague-Dawley rats. Duca et al. (2013) Duca F.A.

Sakar Y.

Covasa M. The modulatory role of high fat feeding on gastrointestinal signals in obesity. Duca et al., 2013 Duca F.A.

Sakar Y.

Covasa M. The modulatory role of high fat feeding on gastrointestinal signals in obesity. Our findings are similar to previous reports demonstrating elevated TG in the stool of GF compared with SPF mice () and reduced lymphatic transport of lipid in antibiotic-treated rats (). We further demonstrate an active impairment of lipid absorption in GF mice using radiolabeled lipid absorption assays ( Figure 2 ). We showed that impaired CCK signaling may be partly responsible for these effects. Whiledemonstrated decreased CCK protein levels in the proximal intestine, we found that GF mice have elevated circulating CCK levels. However, we found that the defect in CCK signaling may be due to reduced expression of Cckar in the pancreas as opposed to changes in the amount of CCK protein produced, a finding that has not been previously reported ( Figure 3 E). These differences might be explained by an overall improper development of the GF mouse. However, it was previously demonstrated that enteroendocrine cell number was not altered in the duodenum or jejunum of GF animals () and our results show that heat-killed bacteria directly regulate the pancreatic expression of Cckar ( Figure 3 F). In addition to altered digestive function, we demonstrate that GF animals have reduced brush border membrane transport of free oleic acid ( Figure 3 G). Taken together, these findings indicate that gut microbes regulate fat uptake locally in the gut and affect extra-intestinal organs such as the pancreas.

El Aidy et al. (2013) El Aidy S.

Merrifield C.A.

Derrien M.

van Baarlen P.

Hooiveld G.

Levenez F.

Doré J.

Dekker J.

Holmes E.

Claus S.P.

et al. The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation. El Aidy et al., 2013 El Aidy S.

Merrifield C.A.

Derrien M.

van Baarlen P.

Hooiveld G.

Levenez F.

Doré J.

Dekker J.

Holmes E.

Claus S.P.

et al. The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation. Bäckhed et al. (2007) Bäckhed F.

Manchester J.K.

Semenkovich C.F.

Gordon J.I. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. An alternative explanation for the resistance of GF mice to HF diet-induced obesity is increased fat oxidation in the gut and peripheral tissues such as liver and muscle.demonstrated that fecal transplantation in GF mice resulted in significant changes in lipid metabolic pathways of peripheral tissues occurring as early as 1 day post transplantation. Genes involved in fatty acid oxidation were downregulated (i.e., Pparα) in the jejunum, while genes involved in glycolysis were upregulated, suggesting that conventionalization shifted energy utilization (). We also show that GF mice display an upregulation of genes involved in fat oxidation, such as Cd36 and Pparα, compared with SPF mice ( Figure S1 ). Thus, increased oxidation of any absorbed fats in the intestine would prevent their incorporation into chylomicrons and delivery to the periphery. Earlier work byshowed that GF mice exhibit increased phosphorylated AMPK levels in the muscle and liver, as well as carnitine-palmitoyltransferase, involved in fatty acid oxidation. Our results expand on this finding by showing that intestinal absorption as well as peripheral uptake of lipid in the liver and adipose tissue is reduced in GF animals ( Figure S2 C). Altogether, GF mice may be resistant to diet-induced obesity due to a combination of impaired fat digestion and absorption, as well as increased fat oxidation in the gut and other metabolically active tissues.

Yen et al., 2008 Yen C.-L.E.

Stone S.J.

Koliwad S.

Harris C.

Farese R.V. Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. Hung et al., 2017 Hung Y.H.

Carreiro A.L.

Buhman K.K. Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat. Stone, 2004 Stone S.J. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice. Yen et al., 2008 Yen C.-L.E.

Stone S.J.

Koliwad S.

Harris C.

Farese R.V. Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. −/− mice only survive a few hours after birth and the carcasses have ∼90% less TG than WT counterparts ( Stone, 2004 Stone S.J. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice. Yen et al., 2008 Yen C.-L.E.

Stone S.J.

Koliwad S.

Harris C.

Farese R.V. Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. Ferré, 2004 Ferré P. The biology of peroxisome proliferator-activated receptors. To examine a direct interaction between microbial cues and lipid absorption, we examined the capacity of the reference strains, C. bifermentans and L. rhamnosus GG, to affect lipid absorption in vitro and in vivo. Our in vitro studies suggest that different strains have specific actions and targets in duodenal organoid cultures. For example, soluble mediators or components from C. bifermentans have a selective impact on Dgat2 expression ( Figure 5 A). Notably, DGAT2 is an enzyme critical for lipid TG synthesis and storage (). Overexpression of DGAT2 increases the number and total area of cytoplasmic lipid droplets compared with wild-type (WT) mice in the jejunum (). DGAT2 deficiency leads to severely reduced plasma and liver TG levels (); DGAT2mice only survive a few hours after birth and the carcasses have ∼90% less TG than WT counterparts (), demonstrating the importance of this enzyme for lipid homeostasis and life. Thus, control of Dgat2 expression by microbe-derived components or molecules may have significant implications for host physiology. The specific microbe-derived components or molecules driving host fat absorption also remain elusive. Future studies will focus on interrogating these bioactive bacterial components or molecules using classic digestion, filtration, and biochemical fractionation approaches. At this time, our study establishes proof of concept that microbes play a role in dietary lipid processing facilitated, in part, through regulation of DGAT2. It should be noted, however, that this is only one of many collective mechanisms necessary for fully functional lipid digestion and absorption. The use of these candidate strains does not recapitulate the entire host response to the microbiota, where a complete restoration of lipid absorption likely involves the complex integration of diverse microbial taxa and microbial signals. Thus, the entire community of microbes and their corresponding gene function and metabolite production is likely required to influence other processes underlying the complexities of lipid digestion and transport. In addition, the interactive effects of diet, particularly those high in fat, or the direct effect of diet likely influences the degree of fat absorption. Notably, HF diets directly affect host responses. For instance, long-chain fatty acids can mediate activation of nuclear hormone receptors, such as PPARγ (), that lead to the upregulation of fat transport genes like Cd36. Further study is warranted to understand how different microbial taxa, alone or in concert, versus dietary cues differentially or additively affect host lipid digestion and absorption pathways.

Limitations to this study include bedding conditions between SPF and GF mice. In most cases, SPF mice were maintained in corn cob bedding and GF mice were maintained in pine shavings. We addressed this caveat by demonstrating that GF mice displayed a significant reduction in lipid absorption compared with SPF mice when maintained in identical bedding conditions. Another limitation to the study was the use of antibiotics in the experiments conducted to address the impact of specific microbial strains on lipid absorption ( Figure 5 ), as antibiotics may elicit direct effects on host pathways and may prove difficult to replicate in future studies. Notably, all mice in these experiments were treated with the same antibiotic regimen prior to placement on LF or HF diet and supplementation of the microbial strains, and thus the differences observed should reflect the dietary effects (LF versus HF) and the presence/absence of the bacterial strains used.