Small Intestine Microbiota Regulate Host Digestive and Absorptive Adaptive Responses to Dietary Lipids

Abstract

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.

Keywords: bacteria; diacylglycerol O-acyltransferase; digestion; enteroendocrine; esterification; gut microbiota; high-fat diet; lipid absorption; lipid transport; small intestine.

Figure 1. GF mice are resistant to diet-induced obesity

SPF and GF mice were fed a low fat (LF) or high fat (HF) diet for 4 weeks (Table S1). A) Body weight was measured weekly and expressed as a percentage relative to baseline. B) Epididymal and mesenteric fat pad weights were collected at the end of the study and expressed as a percentage of total body weight. C) Triglycerides (TG), low density lipoprotein (LDL), and non-esterified fatty acid (NEFA) levels were measured in portal plasma. D) Fasting serum glucose levels. E) Markers of insulin resistance were measured in portal plasma. See also Figure S1. F) Stool TG, NEFA, bile, and total cholesterol levels were measured. Data were pooled across 2–3 independent experiments (each with 2–6 animals per group) and are shown as means +/− SEM (n=10–13 A; n=6–9 B; n=5–12 C; n=4–9 D; n=5–6 E; n=5–9 F). * p ≤ 0.05 (LF vs HF), # p ≤ 0.05 (SPF vs GF).

Figure 2. GF mice have impaired lipid absorption and transport compared to SPF mice

A) Schematic of experimental procedure is shown. Mice were treated for 10 minutes with (A–C) or without (E–G) tyloxapol followed by gavage with [³H]triolein and [¹⁴C]cholesterol. B) Radiolabeled lipid absorption was measured in SPF and GF mice that were fed a standard chow diet. C) Radiolabeled lipid absorption was measured in mice that were fed a low fat (LF) or high fat (HF) diet for 4 weeks. D) Schematic of experimental procedure without tyloxapol is shown. E) Radiolabeled lipid absorption was measured in SPF and GF mice fed a standard chow diet. F–G) Radiolabeled lipid was measured in intestinal epithelium or metabolic tissues. See also Figure S2. Data were pooled across 1–3 independent experiments and are shown as means +/− SEM (n= 7–9 B; n=5–11 C; n=5–6 E–G). B, E–G) * p ≤ 0.05 (SPF vs GF). C) * p ≤ 0.05 (LF vs HF), # p ≤ 0.05 (SPF vs GF).

Figure 3. GF mice exhibit impaired lipid digestive and absorptive function

A–E) SPF and GF mice were fasted for 4 hours and gavaged with corn oil (CO) or water (H₂O) for 2 hours. A) Gallbladder weights. B) Duodenum and jejunum lipase activity. C) Jejunal gene expression of cholecystokinin (Cck) and secretin (Sct). D) Plasma CCK and SCT levels were measured via commercially available EIA assays. E) Pancreatic gene expression of ccka receptor (Cckar), F) western blot of CCKaR, and densitometry of CCKaR. G) Pancreatic Cckar gene expression was determined in GF mice following gavage with vehicle (PBS) or heat-killed Bacteroidetes thetaiotamicron plus Lactobacillus rhamnosus gg. (H) Levels of [³H]oleic acid and of (I) [³H]glucose uptake in brush border membrane vesicles from jejunum and ileum of SPF vs GF mice. Data presented in A–F represent two independent experiments, with 3–4 animals each and are shown as means +/− SEM (n=4 A-D; n=3–4 E-G; n=4–5 H; n=3 I). A–F) * p ≤ 0.05 (LF vs HF), # p ≤ 0.05 (SPF vs GF). G–I) * p ≤ 0.05 (SPF vs GF).

Figure 4. HF diet-induced jejunal microbiota promotes lipid absorption

SPF mice were fed LF or HF diet for four weeks. Small intestinal mucosal scrapings and cecal contents were collected for 16S rRNA amplicon sequencing. A) PCoA plot showing Bray Curtis distances of microbial communities between LF and HF diet on a forced axis for intestinal region. B) Heat map displaying relative abundance of taxa in small intestine and cecal contents between LF and HF diets. C) Experimental design for conventionalization of jejunal microbiota D) Absorption of [³H]Triolein and [¹⁴C]Cholesterol is shown over time and expressed as dpms/µl plasma. See also Figure S3. Data are shown as means +/− SEM (n= 6 A–B; n=3–5 D). * p ≤ 0.05 (LF vs HF), # p ≤ 0.05 (SPF vs GF).

Figure 5. Clostridium bifermentans and Lactobacillus rhamnosus gg induces Dgat2 expression

A) Gene expression of esterification enzymes (monoacylglycerol O-acyltransferase (Mogat2), and diacylglcerol O-acyltransferase (Dgat1 and Dgat2), cholescystokinin (Cck), secretin (Sct), and fatty acid translocase (Cd36) following conditioned media (CM) treatment from Clostridium bifermentans (C. bif; green), Clostridium ramosum (C. ram; red), Lactobacillus rhamnosus gg (L. rham; purple) or reinforced clostridial media (RCM; blue) control in duodenal organoids. B) Uptake of [³H]oleic acid in CM-treated cultures of duodenal and jejunal organoids. C–G) Antibiotic-treated SPF mice were maintained on either a LF or HF diet and gavaged weekly for four weeks with vehicle control or 1×10⁹ CFUs Clostridium bifermentans (D–E), C. bifermentans CM (C), or L. rhamnosus gg (F–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 Figure S4 and S5. Data are shown as means +/− SEM (n=3 A; n=9–12 B; n=5 C–G). Data shown in panels A and B are representative of two independent experiments; * p ≤ 0.05.