Dallas R. Donohoe

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

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.

The Warburg Effect Dictates the Mechanism of Butyrate-Mediated Histone Acetylation and Cell Proliferation

Widespread changes in gene expression drive tumorigenesis, yet our knowledge of how aberrant epigenomic and transcriptome profiles arise in cancer cells is poorly understood. Here, we demonstrate that metabolic transformation plays an important role. Butyrate is the primary energy source of normal colonocytes and is metabolized to acetyl-CoA, which was shown to be important not only for energetics but also for HAT activity. Due to the Warburg effect, cancerous colonocytes rely on glucose as their primary energy source, so butyrate accumulated and functioned as an HDAC inhibitor. Although both mechanisms increased histone acetylation, different target genes were upregulated. Consequently, butyrate stimulated the proliferation of normal colonocytes and cancerous colonocytes when the Warburg effect was prevented from occurring, whereas it inhibited the proliferation of cancerous colonocytes undergoing the Warburg effect. These findings link a common metabolite to epigenetic mechanisms that are differentially utilized by normal and cancerous cells because of their inherent metabolic differences.

A Gnotobiotic Mouse Model Demonstrates that Dietary Fiber Protects Against Colorectal Tumorigenesis in a Microbiota- and Butyrate-Dependent Manner

UNLABELLED Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues. SIGNIFICANCE These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy.

Metaboloepigenetics: Interrelationships between energy metabolism and epigenetic control of gene expression†

Diet and energy metabolism affect gene expression, which influences human health and disease. Here, we discuss the role of epigenetics as a mechanistic link between energy metabolism and control of gene expression. A number of key energy metabolites including SAM, acetyl‐CoA, NAD+, and ATP serve as essential co‐factors for many, perhaps most, epigenetic enzymes that regulate DNA methylation, posttranslational histone modifications, and nucleosome position. The relative abundance of these energy metabolites allows a cell to sense its energetic state. And as co‐factors, energy metabolites act as rheostats to modulate the activity of epigenetic enzymes and upregulate/downregulate transcription as appropriate to maintain homeostasis. J. Cell. Physiol. 227: 3169–3177, 2012.

Microbial Regulation of Glucose Metabolism and Cell-Cycle Progression in Mammalian Colonocytes

A prodigious number of microbes inhabit the human body, especially in the lumen of the gastrointestinal (GI) tract, yet our knowledge of how they regulate metabolic pathways within our cells is rather limited. To investigate the role of microbiota in host energy metabolism, we analyzed ATP levels and AMPK phosphorylation in tissues isolated from germfree and conventionally-raised C57BL/6 mice. These experiments demonstrated that microbiota are required for energy homeostasis in the proximal colon to a greater extent than other segments of the GI tract that also harbor high densities of bacteria. This tissue-specific effect is consistent with colonocytes utilizing bacterially-produced butyrate as their primary energy source, whereas most other cell types utilize glucose. However, it was surprising that glucose did not compensate for butyrate deficiency. We measured a 3.5-fold increase in glucose uptake in germfree colonocytes. However, 13C-glucose metabolic-flux experiments and biochemical assays demonstrated that they shifted their glucose metabolism away from mitochondrial oxidation/CO2 production and toward increased glycolysis/lactate production, which does not yield enough ATPs to compensate. The mechanism responsible for this metabolic shift is diminished pyruvate dehydrogenase (PDH) levels and activity. Consistent with perturbed PDH function, the addition of butyrate, but not glucose, to germfree colonocytes ex vivo stimulated oxidative metabolism. As a result of this energetic defect, germfree colonocytes exhibited a partial block in the G1-to-S-phase transition that was rescued by a butyrate-fortified diet. These data reveal a mechanism by which microbiota regulate glucose utilization to influence energy homeostasis and cell-cycle progression of mammalian host cells.

The BRG1 Chromatin Remodeler Regulates Widespread Changes in Gene Expression and Cell Proliferation During B Cell Activation

Widespread changes in gene expression underlie B cell development and activation, yet our knowledge of which chromatin‐remodeling factors are essential is limited. Here, we demonstrate that the BRG1 catalytic subunit of SWI/SNF complexes was dispensable for murine B cell development but played an important, albeit selective, role during activation. Although BRG1 was dispensable for CD69 induction and differentiation into plasma cells based on the ability of mutant B cells to undergo hypertrophy and secrete IgM antibodies, it was required for robust cell proliferation in response to activation. Accordingly, BRG1 was required for only ∼100 genes to be expressed at normal levels in naïve B cells but >1,000 genes during their activation. BRG1 upregulated fivefold more genes than it downregulated, and the toll‐like receptor pathway and JAK/STAT cytokine‐signaling pathways were particularly dependent on BRG1. The importance of BRG1 in B cell activation was underscored by the occurrence of opportunistic Pasteurella infections in conditionally mutant mice. B cell activation has long served as a model of inducible gene expression, and the results presented here identify BRG1 as a chromatin‐remodeling factor that upregulates the transcriptome of B cells during their activation to promote rapid cell proliferation and to mount an effective immune response. J. Cell. Physiol. 229: 44–52, 2014.

Butyrate decreases its own oxidation in colorectal cancer cells through inhibition of histone deacetylases

Colorectal cancer is characterized by an increase in the utilization of glucose and a diminishment in the oxidation of butyrate, which is a short chain fatty acid. In colorectal cancer cells, butyrate inhibits histone deacetylases to increase the expression of genes that slow the cell cycle and induce apoptosis. Understanding the mechanisms that contribute to the metabolic shift away from butyrate oxidation in cancer cells is important in in understanding the beneficial effects of the molecule toward colorectal cancer. Here, we demonstrate that butyrate decreased its own oxidation in cancerous colonocytes. Butyrate lowered the expression of short chain acyl-CoA dehydrogenase, an enzyme that mediates the oxidation of short-chain fatty acids. Butyrate does not alter short chain acyl-CoA dehydrogenase levels in non-cancerous colonocytes. Trichostatin A, a structurally unrelated inhibitor of histone deacetylases, and propionate also decreased the level of short chain acyl-CoA dehydrogenase, which alluded to inhibition of histone deacetylases as a part of the mechanism. Knockdown of histone deacetylase isoform 1, but not isoform 2 or 3, inhibited the ability of butyrate to decrease short chain acyl-CoA dehydrogenase expression. This work identifies a mechanism by which butyrate selective targets colorectal cancer cells to reduce its own metabolism.

Abstract PL03-01: A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner

It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energentic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We colonized BALB/c mice with wild-type or mutant strains of a butyrate-producing bacterium in a gnotobiotic facility, provided them with high- or low-fiber diets that were otherwise identical and isocaloric, and used azoxymethane (AOM) to induce colorectal tumors. Analysis of these gnotobiotic mouse models demonstrated that fiber conferred a significant tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. To confirm that butyrate is a causal factor, the anticancer chemoprotective effect was recapitulated in mice without any butyrate-producing bacteria when they were provided a tributyrin-fortified diet. Our data support a general mechanism that includes microbial fermentation of fiber rather than fiber exclusively speeding colonic transit to minimize the exposure of colonocytes to ingested carcinogens. Our data also support a molecular mechanism that is metaboloepigenetic. Normal colonocytes utilize butyrate as their primary energy source, whereas cancerous colonocytes rely on glucose because of the Warburg effect. Due to this metabolic difference, butyrate accumulated in tumors and functioned as an HDAC inhibitor to increase histone acetylation levels globally and at pro-apototic (Fas) and cell-cycle (p21 and p27) target genes, which culminated in increased apoptosis and decreased cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone acetylation levels are elevated in colorectal adenocarcinomas compared to normal colonic tissues. These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy. Citation Format: Dallas Donohoe, Darcy Holley, Leonard Collins, Stephanie Montgomery, Alan Whitmore, Kaitlin Curry, Sarah Renner, Alicia Greenwalt, Elizabeth Ryan, Virginia Godfrey, Deborah Threadgill, James Swenberg, David Threadgill, Scott Bultman. A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PL03-01.

Abstract SY08-03: Metaboloepigenetic effects of microbial-produced butyrate in cancer prevention.

It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. These studies have been complicated by the participants9 genetic heterogeneity and differences in the composition of microbiota within their gastrointestinal tracts. To eliminate these confounding variables, we utilized a gnotobiotic mouse model of colorectal cancer. Our experiments were designed to investigate the function of butyrate because it is a short-chain fatty acid produced by bacterial fermentation of fiber in the colon at high (mM) levels and has potent energetic and epigenetic properties in host colonocytes. Here, we report that fiber did, in fact, have a chemoprotective effect but in a microbiota- and butyrate-dependent manner. The incidence, number, size, and histopathologic progression of AOM/DSS-induced colorectal tumors were significantly diminished when BALB/c mice were provided a high-fiber diet only if they were colonized with defined microbiota that included a butyrate-producing bacteria. This chemoprotective effect was attenuated when mice were colonized with the same microbiota except that the wild-type butyrate producer was replaced by a mutant strain with a 0.8-kb deletion in the butyryl-CoA synthesis operon. To confirm that butyrate was a causal factor, the chemoprotective effect was also observed in mice without any butyrate-producing bacteria if their diet was fortified with a butyrate derivative. Our data support a general mechanism that includes microbial fermentation of fiber rather than fiber exclusively speeding colonic transit to minimize the exposure of colonocytes to ingested carcinogens. Our data also support a molecular mechanism that is metaboloepigenetic. Normal coloncytes utilize butyrate as their preferred energy source, whereas cancerous colonocytes rely on glucose because of the Warburg effect. Due to this metabolic difference, butyrate accumulated in tumors (as measured by LC-MS/MS) and functioned as an HDAC inhibitor to increase global histone acetylation levels and apoptosis. To support the applicability of this model to human cancer, we demonstrate that butyrate also accumulates at higher levels in human colorectal tumors than in normal colonic tissue, and this is associated with higher levels of histone acetylation in tumors. These results link diet and microbiota to a common metabolite that influences epigenetics and cancer predisposition. To investigate the metaboloepigenetic mechanism in more detail, we evaluated the effect of butyrate in colorectal cancer cell lines in the presence of the Warburg effect and when it was prevented from occurring by growing the tumor cells in low glucose or depleting lactate dehydrogenase levels (siLDHA). Low doses of butyrate (0.5-1 mM) inhibited cell proliferation in the presence of the Warburg effect by acting as an epigenetic factor (by inducing histone acetylation) but stimulated proliferation in the absence of the Warburg effect by acting as an energy source. Low doses of butyrate also stimulated the proliferation of non-cancerous colonocytes, which do not undergo the Warburg effect without any experimental manipulation. Higher doses of butyrate (2-5 mM), which exceed the metabolic capacity of the cell to oxidize butyrate (but are still physiologically relevant), induced histone acetylation and apoptosis regardless of the Warburg effect. At the lower doses, where butyrate was metabolized, it was converted to acetyl-CoA, and this was important not only for energetics but also for epigenetics because it served as a HAT co-factor to stimulate histone acetylation. Although the acetyl-CoA/HAT and HDAC inhibition mechanisms both stimulate histone acetylation, they were differentially utilized and upregulated different target genes. The acetyl-Co-A/HAT mechanism was predominant in normal cells and at low butyrate doses regardless of the Warbug effect and upregulated cell proliferation genes, whereas the HDAC inhibition mechanism was predominant in cancerous colonocytes and at high butyrate doses regardless of the Warburg effect and upregulated pro-apoptotic genes. These data have important implications in vivo. Because mucus produced by goblet cells within the crypts flows upward into the lumen, an endogenous butyrate gradient is believed to exist with lower concentrations at the base of crypts ( Citation Format: Dallas Donohoe, Stephanie Montgomery, Leonard Collins, Darcy Holley, Virgina Godfrey, James Swenberg, Scott Bultman. Metaboloepigenetic effects of microbial-produced butyrate in cancer prevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY08-03. doi:10.1158/1538-7445.AM2013-SY08-03