Figure 1.
Effects of microbiota on energy metabolism biomarkers in the GI tract and digestive tissues.
A, ATP levels in the stomach (S), small intestine (SI), proximal colon (PC), distal colon (DC), liver (L), and fat (F) (white adipose tissue) of CONV-R and GF mice. Five CONV-R and five GF mice were analyzed, and the results are mean ± standard error. Significant differences are indicated (*p<0.05). B, Western blot analysis of AMPKα phosphorylated at Thr172 (top panel) and total AMPKα (bottom panel) in different segments of the colon and small intestine from GF and CONV-R mice. Results are representative of 3 independent experiments.
Figure 2.
Microbial regulation of glucose uptake.
A, 2-deoxyglucose uptake observed as green fluorescence in CONV-R (left) and GF (right) colonocytes counterstained with DAPI (blue). B, Quantification of total 2-deoxyglucose fluorescence per cell in CONV-R and GF colonocytes. Results are based on 20 cells per colonocyte preparation from 3 independent mice and represent the mean ± SE with significant differences indicated (*p<0.01).
Figure 3.
Microbial regulation of glycolysis in colonocytes.
A–E, Levels of serum glucose (A), intracellular glucose (B), glucose-6-phosphate (Glu-6-P) (C), pyruvate (D), and lactate (E) in CONV-R and GF serum (A) or colonocytes (B–E). Results are mean ± standard error from 3 independent experiments with significant differences indicated (*p<0.05; **p<0.01).
Figure 4.
Microbes and butyrate stimulate oxidative metabolism.
A, Levels of free 13CO2 released from CONV-R and GF colonocytes following their incubation with uniformly labeled (13C(6)) or singly labeled (13C(1)) glucose. 13CO2 levels were detected by isotope-ratio mass spectrometry, and the results are presented as mean ± standard error from 3 independent experiments with significant differences indicated (*p<0.01). B, Oxidative metabolism indicated by MitoTracker Red CM-H2XRos (red fluorescence; second column) and total mitochondria indicated by the non-oxidizable MitoTracker Green FM (green fluorescence; first column) in colonocytes from CONV-R (top two rows) and GF (bottom three rows). The sodium azide treated cells in the second row serve as a negative control. The effect of glucose and butyrate on GF colonocytes in the two bottom rows is compared to untreated GF colonocytes in the row immediately above. C, Quantification of oxidative metabolism in the different experimental groups. Determined as the ratio of red fluorescence relative to green fluorescence (internal control). Results are based on 20 cells per condition from 3 independent experiments and represent the mean ± SE with significant differences indicated (*p<0.01). D, Spectral counts from quantitative mass spectrometry of pyruvate dehydrogenase subunits in CONV-R and GF colonocytes. E, Western blot analysis of the E1α and E1b subunits of PDF from CONV-R and GF colons with β–actin serving as a loading control. Results are representative of 3 independent experiments. F, Western blot analysis of phospho-PDH (E1α) and total PDH (E1α) from CONV-R and GF colons. Results are representative of 3 independent experiments.
Figure 5.
Microbes and butyrate promote cell-cycle progression at the G1-to-S phase transition.
A, Flow cytometry profiles of colonocytes from CONV-R mice (left), GF mice (center), and GF mice provided a tributyrin-fortified diet (right). PI levels are shown on the x-axis, and BrdU incorporation is shown on the y-axis. The percentage of cells in various stages of the cell cycle are indicated: P1, G1; P2, early S; P3, middle S; P4, late S; P5, G2. Results are representative of 3 independent experiments. B, Measurements of luminal butyrate (left) and tributyrin (right) levels from the colon of CONV-R mice and GF mice provided either a provided a control diet (GF) or a tributyrin-fortified diet (GF + Tributyrin). Results are mean ± standard error from 3 independent experiments with significant differences indicated (*p<0.01). N.D., not detectable.
Figure 6.
A model of microbial regulation of glucose metabolism in host colonocytes.
Microbes increase blood glucose levels and increase PDH levels and activity to facilitate oxidative metabolism of glucose in the mitochondria. In contrast, GF mice are hypoglycemic, and their colonocytes respond by increased expression of GLUTs. Although this leads to increased glucose uptake, Glu-6-P and pyruvate levels are diminished because of their conversion to lactate, which is increased in GF colonocytes. This increase in glycolysis can be attributed to decreased PDH expression/activity and decreased oxidative metabolism in the mitochondria. Not only are PDH levels decreased, but also enzymatic activity is inhibited via phosphorylation of the E1a regulatory subunit due to increased expression of PDK2 in GF colonocytes.