Increased oral detection, but decreased intestinal signaling for fats in mice lacking gut microbiota

Abstract

Germ-free (GF) mice lacking intestinal microbiota are significantly leaner than normal (NORM) control mice despite consuming more calories. The contribution of microbiota on the recognition and intake of fats is not known. Thus, we investigated the preference for, and acceptance of, fat emulsions in GF and NORM mice, and associated changes in lingual and intestinal fatty acid receptors, intestinal peptide content, and plasma levels of gut peptides. GF and NORM C57Bl/6J mice were given 48-h two-bottle access to water and increasing concentrations of intralipid emulsions. Gene expression of the lingual fatty acid translocase CD36 and protein expression of intestinal satiety peptides and fatty-acid receptors from isolated intestinal epithelial cells were determined. Differences in intestinal enteroendocrine cells along the length of the GI tract were quantified. Circulating plasma satiety peptides reflecting adiposity and biochemical parameters of fat metabolism were also examined. GF mice had an increased preference and intake of intralipid relative to NORM mice. This was associated with increased lingual CD36 (P<0.05) and decreased intestinal expression of fatty acid receptors GPR40 (P<0.0001), GPR41 (P<0.0001), GPR43 (P<0.05), and GPR120 (P<0.0001) and satiety peptides CCK (P<0.0001), PYY (P<0.001), and GLP-1 (P<0.001). GF mice had fewer enteroendocrine cells in the ileum (P<0.05), and more in the colon (P<0.05), relative to NORM controls. Finally, GF mice had lower levels of circulating leptin and ghrelin (P<0.001), and altered plasma lipid metabolic markers indicative of energy deficits. Increased preference and caloric intake from fats in GF mice are associated with increased oral receptors for fats coupled with broad and marked decreases in expression of intestinal satiety peptides and fatty-acid receptors.

Figure 1. Preference (A), raw intake (B), and calorie intake from intralipid emulsions (C) in GF and NORM C57B6/J mice during 48-h two-bottle intralipid vs. water tests.

(A) GF mice preferred the lowest concentration (0.156% oil) of intralipid emulsion test more than NORM mice. (B) Intake of intralipid emulsions was similar at each concentration tested, but increased overall in GF mice relative to NORM controls. (C) GF mice consumed more energy from the highest concentration (1.25% oil) of intralipid emulsion tested. Data are expressed as means±SEM. *P<0.05 compared to NORM.

Figure 2. Gene expression of (A) lingual CD36, and protein expression of (B) intestinal CD36, and (C) intestinal FIAF.

(A) GF mice exhibited 3-fold up-regulation of lingual CD36 mRNA in the posterior lingual epithelium relative to NORM mice during fasting. GF mice displayed a slight, but non-significant, increase in lingual CD36 expression following intralipid exposure. (B) Intestinal CD36 was significantly down-regulated in GF compared to NORM mice. (C) Intestinal FIAF expression was increased over 3-fold in GF relative to NORM mice Data are expressed as means±SEM. *P<0.05, **P<0.01, ***P<0.001, compared to NORM.

Figure 3. Intestinal epithelial protein expression of fatty-acid responsive receptors in GF and NORM mice.

GF mice displayed down-regulation of proximal intestinal GPR40, 41, 43, and 120 relative to NORM mice. Data are expressed as means±SEM. *P<0.05, ***P<0.001, compared to NORM mice.

Figure 4. Intestinal epithelial protein expression of satiety peptide in GF and NORM mice.

GF mice exhibited down-regulation of proximal intestinal CCK, GLP-1 and PYY relative to NORM mice. Data are expressed as means±SEM. **P<0.01, ***P<0.001, compared to NORM mice.

Figure 5. (A) Cell counts of enteroendocrine cells expressing chromogrannin-A and (B) representative microphotographs of ileum and colon sections at 100× magnification.

GF mice had significantly less enteroendocrine cells in the ileum, but more in the colon, compared to NORM controls. Data are expressed as means±SEM. *P<0.05, compared to NORM mice.

Figure 6. Plasma levels of (A) leptin, (B) ghrelin, and (C) PYY in GF and NORM mice following an overnight fast or re-feeding with 1-ml of 20% intralipid.

(A) Plasma leptin was lower in GF mice relative to NORM controls. Re-feeding elevated plasma leptin in both groups. (B) GF mice displayed lower levels of plasma PYY compared to NORM mice while re-feeding increased plasma PYY in both groups. (C) GF mice exhibited lower circulating of ghrelin in both feeding conditions while re-feeding increased plasma ghrelin in NORM, but not GF mice. Data are expressed as means±SEM. **P<0.01 ***P<0.001, compared to NORM mice. ^††P<0.01, ^††† P<0.001, compared to fasting condition within group.

Figure 7. Plasma levels of (A) glucose, (B) total triglycerides, (C) cholesterol, and (D) HDL in GF and NORM mice following an overnight fast or re-feeding with 1-ml of 20% intralipid.

(A) Plasma glucose levels of GF mice were lower than NORM controls and re-feeding increased glucose levels in GF, but not NORM mice. (B) Plasma triglycerides were similar between both groups and feeding conditions. (C) Plasma total cholesterol was increased in GF mice relative to NORM controls and re-feeding increased cholesterol levels in both NORM and GF mice. (D) Total plasma HDL was increased in GF mice relative to NORM mice with re-feeding increasing total HDL in both groups. Data are expressed as means±SEM. **P<0.01 mice, ***P<0.001 compared to NORM. ^†P<0.05, ^†† P<0.01, compared to fasting condition within group.