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. 2011 May 4;13(5):517-26.
doi: 10.1016/j.cmet.2011.02.018.

The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon

Dallas R Donohoe  1 , Nikhil Garge, Xinxin Zhang, Wei Sun, Thomas M O'Connell, Maureen K Bunger, Scott J Bultman
Affiliations

The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon

Dallas R Donohoe et al. Cell Metab. .

Abstract

The microbiome is being characterized by large-scale sequencing efforts, yet it is not known whether it regulates host metabolism in a general versus tissue-specific manner or which bacterial metabolites are important. Here, we demonstrate that microbiota have a strong effect on energy homeostasis in the colon compared to other tissues. This tissue specificity is due to colonocytes utilizing bacterially produced butyrate as their primary energy source. Colonocytes from germfree mice are in an energy-deprived state and exhibit decreased expression of enzymes that catalyze key steps in intermediary metabolism including the TCA cycle. Consequently, there is a marked decrease in NADH/NAD(+), oxidative phosphorylation, and ATP levels, which results in AMPK activation, p27(kip1) phosphorylation, and autophagy. When butyrate is added to germfree colonocytes, it rescues their deficit in mitochondrial respiration and prevents them from undergoing autophagy. The mechanism is due to butyrate acting as an energy source rather than as an HDAC inhibitor.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Effects of Microbiota on Energy Homeostasis
Total NADH/NAD+ ratios (A) and ATP levels (B) in various tissues from GF and CONV-R mice. L, liver; H, heart; K, kidney; T, testis; C, colon. 6 GF and 6 CONV-R mice were analyzed, and results are mean ± SEM. Significant differences are indicated (**p <0.01).
Figure 2
Figure 2. Analysis of Microbiome Regulated Gene Products and Metabolites
Top KEGG pathways of transcriptome (A) and proteome (B) identified as differences between GF and CONV-R. Blue bars correspond to −log (p-values) shown on y-axis at left. Straight yellow lines represent threshold of significance for these −log (p-values). Yellow boxes represent the ratio, shown on y-axis at right, and indicate the number of genes or proteins affected divided by total number within category. 6 GF and 6 CONV-R mice were used for the transcriptome analysis. For the proteome analysis, 3 GF and 3 CONV-R mice were used.
Figure 3
Figure 3. Regulation of Colonocyte Oxidative Metabolism
(A) Percentage of 13C-butyrate metabolized to 13CO2 in CONV-R and GF colonocytes. (B–D) Measurement of intermediary metabolites. Levels of acetyl-CoA (B), malate (C), and oxaloacetate (D) in CONV-R and GF. (E, F) Levels of mitochondrial NADH and NAD+ in CONV-R (E) and GF (F) colonocytes. The different scales reflect different amounts of material loaded and detected by HPLC-LC-MS. (G) Oxidative metabolism indicated by MitoTracker Red CM-H2XRos (red fluorescence; top panel) and MitoTracker Green FM (green fluorescence; middle panel) in colonocytes from CONV-R, CONV-R with NaN3 (sodium azide, negative control), or GF. (H) Quantification of oxidative metabolism in different experimental groups. (I) Spectral counts from quantitative mass spectrometry of ATP synthase F1α and β. Results in A–D, G, and H are displayed as mean ± SE and significant differences are identified (*p <0.05, **p <0.01). For panels A–D, 4 GF and 4 CONV-R mice were used. In panels E–H, 3 mice were used per condition.
Figure 4
Figure 4. Microbial Regulation of Energy Metabolism and Autophagy
(A–C) Western blot analysis of phosho-AMPK and AMPK (A), phospho-p27 (B) and LC3-I and -II (C) with histone H3 or β-actin as loading controls in CONV-R and GF. (D) Transmission electron micrographs of CONV-R and GF colonic epithelium. Lysosomes (L) and autophagosomes (a) are indicated, and arrows show double membrane of autophagasome in inset. For A–C, western blots are representative of 4 experiments using 2 CONV-R and 2 GF mice with 2 technical replicates each.
Figure 5
Figure 5. Colonization and Butyrate Rescue Diminished Oxidative Metabolism
(A) Mitochondrial respiration indicated by MitoTracker Red CM-H2XRos (red fluorescence; top panel) and MitoTracker Green FM (green fluorescence; middle panel) in colonocytes from CONV-R, CONV-D, B. fibrisolvens, GF, GF with 10 mM butyrate, and GF with both 10 mM butyrate and 500 µM etomoxir. (B) Quantification of mitochondrial respiration in different experimental groups. (C–D) Western blot analysis of phospho-p27 (C) and LC3-I and -II (D) with and without 10 mM butyrate in CONV-R and GF with β-actin as loading controls. (E) Western blot analysis of LC3-I and –II of GF, GF with 10 mM butyrate, and GF with 10 mM butyrate and 500 µM etomoxir. For panels A and B, a total of 3 mice per condition were used, and results are displayed as mean ± SE, with significant differences indicated (**p <0.01). For panels C–E, western blots are representative of 4 experiments using 2 CONV-R and 2 GF mice with 2 technical replicates each.
Figure 6
Figure 6. Microbial Regulation of Colonocyte Metabolism
Schematic showing that dietary fiber is fermented by microbes into butyrate in the lumen of the colon, which is transported into the colonocyte. In the colonocyte, butyrate promotes oxidative metabolism and inhibits autophagy. Based on transcriptome and proteome experiments, enzymes regulated by microbes are boxed. In all cases, boxed enzymes that function in β-oxidation and the TCA cycle are downregulated in GF colonocytes, revealing that microbes positively regulate their expression. Diminished ATP results in phosphorylation of AMPK and p27, which culminates in autophagy.
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