Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle.

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium

Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium.ISSN:1544-9173ISSN:1545-788

OligoMM12 mice have increased fat mass compared to GF mice and SPF C57B6/J mice.

(A) Schematic representation of isolator-based indirect calorimetry system, with a TSE PhenoMaster calorimeter connected to 2 flexible surgical isolators with 4 metabolic cages each. (B) Pictures of isolator-based indirect calorimetry system inside the facility. (C) Cecal mass (tissue including luminal content). (D) Total body mass at the end of the experiment and before cecum removal. (E) Total body mass after cecum removal. (F) Lean body mass acquired by EchoMRI before cecum removal (N of mice per group with EchoMRI and indirect calorimetry measurements: GF = 12, OligoMM12 = 8, SPF = 11). (G) Fat mass from iBAT, iWAT, and vWAT. Number of mice per group in all figures unless otherwise specified: GF = 16, OligoMM12 = 12, SPF = 11. p-values obtained by Tukey’s honest significance test. Data underlying this figure are supplied in S1 Data. GF, germ-free; iBAT, interscapular brown adipose tissue; iWAT, inguinal white adipose tissue; SPF, specific-opportunistic-pathogen-free; vWAT, visceral white adipose tissue.</p

Bacterial density in cecum content of OligoMM12 and SPF mice.

(A) Bacterial density in cecum content of OligoMM12 and SPF mice during the light and dark phase quantified by flow cytometry. Data underlying this figure are supplied in S1 Data. (TIF)</p

Energy metabolism in GF, OligoMM12, and SPF C57B6/J mice.

(A) Linear regression of energy expenditure and lean body mass based on EchoMRI during light and dark phase. Each colored vertical line represents energy expenditure measurements during the experiment for 1 mouse. (B) Energy expenditure during 24-h period or during the 12-h light or dark phase. Values represent area-under-curve normalized by regression-based analysis using lean body mass obtained by EchoMRI and dissected fat mass. (C) Average daily food intake per mouse. Mice represented in this figure include those that underwent long-term indirect calorimetry (Fig 3) and mice that only contribute to daily fecal pellet quantification/bomb calorimetry. (N of mice per group: GF = 24, OligoMM12 = 19, SPF = 10) (D) Dry fecal output per mouse collected during a 24-h period. (N of mice per group: GF = 12, OligoMM12 = 8, SPF = 4) (E) Energy content of dry fecal output by bomb calorimetry. (N of mice per group: GF = 21, OligoMM12 = 11, SPF = 11). (F-I) Estimation energy metabolism parameters. Number represented estimate mean value ± 1.96*combined standard uncertainty from measurements used for calculations. (F) Estimated daily energy input (food intake* 3.94 kcal/g). (G) Estimated daily energy excretion (daily fecal dry mass*fecal energy content). (H) Estimated daily energy extraction (daily energy input–daily energy excretion). (I) Estimated energy extraction from food as percentage of energy input ((daily energy input − daily energy excretion)/daily energy input*100). Note that calculations in F, G, and H are per mouse and are not normalized to body mass. Number of mice per group in all figures unless otherwise specified: GF = 9, OligoMM12 = 8, SPF = 10. p-values obtained by Tukey’s honest significance test. Data underlying this figure are supplied in S1 Data. GF, germ-free; SPF, specific-opportunistic-pathogen-free.</p

List of metabolites identified by targeted peak extraction in the UPLC/MS data.

Table indicates compound name, KEGG entry number, type of column was used for UPLC and if the peak ID matched the retention time and MS2 spectra identified with the chemical standard in liver and plasma samples. Data of all compounds in liver and plasma samples during the light phase (ZT 5) and dark phase (ZT 16) available in S1 Data. UPLC/MS, ultraperformance liquid chromatography coupled with mass spectrometry; ZT, Zeitgeber time. (DOCX)</p

Metabolic profile comparison of GF, OligoMM12, and SPF C57B6/J mice by UPLC/MS.

(A and B) Principal coordinate analysis using Canberra distances of metabolites identified by untargeted UPLC/MS in liver and plasma during the (A) light phase (Zeitgeber 5) and (B) dark phase (Zeitgeber 16). (C-F) Metabolic pathways identified in the KEGG PATHWAY database; red dots represent pathways containing compounds differentially enriched in OligoMM12 vs.GF and OligoMM12 vs. SPF comparisons and selected compounds obtained by targeted peak extraction from differentially expressed pathways. Samples obtained during the light phase (Zeitgeber 5) and dark phase (Zeitgeber 16) in (C and D) liver and (E and F) plasma. p-values obtained by Tukey’s honest significance test after log2 transformation of area value. Number of mice per group: Liver ZT5: GF = 4, OligoMM12 = 6, SPF = 7; ZT16: GF = 4, OligoMM12 = 6, SPF = 7 / Plasma ZT5: GF = 4, OligoMM12 = 7, SPF = 7; ZT16: GF = 5, OligoMM12 = 6, SPF = 6. Data underlying this figure are supplied in S1 Data. GF, germ-free; SPF, specific-opportunistic-pathogen-free; UPLC/MS, ultraperformance liquid chromatography coupled with mass spectrometry; ZT, Zeitgeber time.</p

Excel spreadsheet containing, in separate sheets, the underlying numerical data and statistical analysis for Figs 1C–1G, 2A–2I, 3A–3F, 4A–4F, S1A–S1D, S2A–S2K, S3A–S3E, S4, S5A–S5C, S6, S7, and S8.

(XLSX)</p

Metabolic profile comparison of GF, OligoMM12, and SPF C57B6/J mice by UPLC/MS in plasma.

Manually curated list of compounds obtained by targeted peak extraction from differentially expressed pathways in plasma samples during the light phase (ZT 5) and dark phase (ZT 16). p-values obtained by Tukey’s honest significance test after log2 transformation of area value. Number of mice per group: ZT5: GF = 4, OligoMM12 = 7, SPF = 7; ZT16: GF = 5, OligoMM12 = 6, SPF = 6. Data underlying this figure are supplied in S1 Data. GF, germ-free; SPF, specific-opportunistic-pathogen-free; UPLC/MS, ultraperformance liquid chromatography coupled with mass spectrometry; ZT, Zeitgeber time. (TIF)</p

Locomotor activity and total amount of cecal SCFAs.

(A-B) Locomotor activity in OligoMM12 and SPF mice (n = 9 per group): (A) Circadian variation in average breaks/minute per time point. (B) Average daily breaks/minute. (C) Estimation total amount of SCFAs and intermediate metabolites by multiplying measured concentration values by the cecal mass of the group. Number represented estimate mean value ± combined standard uncertainty from measurements used for calculations. Number of mice per group in all figures unless otherwise specified: GF = 13, OligoMM12 = 12, SPF = 10. p-values obtained by Tukey’s honest significance test. Data underlying this figure are supplied in S1 Data. GF, germ-free; SCFA, short-chain fatty acid; SPF, specific-opportunistic-pathogen-free. (TIF)</p