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Colonic Gene Expression in Conventional and Germ-Free Mice with a Focus on the Butyrate Receptor GPR109A and the Butyrate Transporter SLC5A8

  • 2009 SSAT Plenary Presentation
  • Published:
Journal of Gastrointestinal Surgery Aims and scope

Abstract

Introduction

Butyrate is a bacterial fermentation product that produces its beneficial effects on colon through GPR109A, a butyrate receptor, and SLC5A8, a butyrate transporter. In this study, we compared the expression of GPR109A and SLC5A8 between conventional mice and germ-free mice to test the hypothesis that the expression of these two proteins will be decreased in germ-free mice compared to conventional mice because of the absence of bacterial fermentation products and that colonization of germ-free mouse colon with conventional bacteria will reverse these changes.

Methods

RNA was prepared from the ileum and colon of conventional mice and germ-free mice and used for RT-PCR to determine mRNA levels. Tissue sections were used for immunohistochemical analysis to monitor the expression of GPR109A and SLC5A8 at the protein level. cDNA microarray was used to determine the differential expression of the genes in the colon between conventional mice and germ-free mice.

Results

In conventional mice with normal bacterial colonization of the intestinal tract, GPR109A and SLC5A8 are expressed on the apical membrane of epithelial cells lining the ileum and colon. In germ-free mice, the expression of GPR109A and SLC5A8 is reduced markedly in the ileum and colon. The expression returns to normal levels when the intestinal tract of germ-free mice is colonized with bacteria. The expression of the Na+-coupled glucose transporter, SGLT1, follows a similar pattern. Microarray analysis identifies ∼700 genes whose expression is altered more than twofold in germ-free mice compared to conventional mice. Among these genes are the chloride/bicarbonate exchanger SLC26A3 and the water channel aquaporin 4. The expression of SLC26A3 and AQP4 in ileum and colon is reduced in germ-free mice, but the levels return to normal upon bacterial colonization.

Conclusion

Gut bacteria play an active role in the control of gene expression in the host intestinal tract, promoting the expression of the genes that are obligatory for the biological actions of the bacterial fermentation product butyrate and also the genes that are related to electrolyte and water absorption.

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Acknowledgments

We thank Krishnan Dhandapani, PhD (Department of Neurosurgery, Medical College of Georgia) for the monoclonal antibody specific for AQP4. This work was supported by the Scientist Training Program from the Medical College of College of Georgia Research Institute, Inc.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, 30912, USA

    Muthusamy Thangaraju, Kebin Liu & Vadivel Ganapathy

  2. Department of Surgery, Medical College of Georgia, Augusta, GA, 30912, USA

    Gail A. Cresci & John D. Mellinger

Authors
  1. Gail A. Cresci
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  2. Muthusamy Thangaraju
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  3. John D. Mellinger
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  4. Kebin Liu
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  5. Vadivel Ganapathy
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Corresponding author

Correspondence to Vadivel Ganapathy.

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Discussant

DR. ROBERT MARTINDALE (Portland, OR): Thank you, Gail, for this very interesting work. This shows us that we truly are mutualistic with our bacteria as this paper and many others at this meeting have shown us.

There are several things you have very nicely shown through microarray and immunohistofluorescence data.

The question I have for you today is regarding the mechanism. Is it the bacteria themselves or some product of the bacteria? Can you speculate on the mechanism?

Discussant

GAIL CRESCI: Thank you for your very interesting question. We were interested in that question as well. We’ve done some preliminary work, and I don’t have the data to show here, but because we saw down-regulation of the two genes that we are studying, SLC5A8 and GPR109A, and that we have previously shown that they are silenced in colon cancer by DNA methylation, we sought out to see if this might be the possible cause with germ-free animals.

Our preliminary work has actually shown that, in fact, for the silencing of these two genes, SLC5A8 and GPR109A, DNA methylation is definitely involved and DNMT1 seems to be the main isoform involved.

We also know butyrate alters gene expression as shown in other people’s work. One may predict there to be an absence of butyrate in a germ-free mouse intestine as the production of butyrate results from bacterial fermentation of undigested polysaccharides. Thus with the absence of this molecule, gene expression may be altered.

I have attended many lectures here at this conference and have read many papers that lead to the thought that perhaps the bacteria itself may be influencing gene expression by secreting proteins or altering the lumen pH. So, without the presence of the gut microbiota, these proteins and other alterations would not exist. That is future work for us. Thank you.

Discussant

DR. ROBERT MARTINDALE (Portland, OR): Do you think having two mechanisms, including the receptor you’ve shown, as well as a transporter, shows the importance of butyrate? Thus, if one is knocked out, you still have another alternate way for butyrate to elicit its biologic effects on the cell?

Discussant

GAIL CRESCI: Yes. I think that’s very important. We are very excited to see that both have a relationship with butyrate. We actually now have knockout mice for GPR109A as well as SLC5A8. So we are, in fact, going to start some studies looking at the potential mechanism there.

Discussant

DR. TEREZ SHEA-DONOHUE (Baltimore): I was interested that you have SGLT1 expression in the colon and one doesn’t normally think of the colon as having a nutrient transporter like that. Can you comment on what you think the role of the transporter is there?

Closing discussant

GAIL CRESCI: We were surprised by that as well. Just speculating as I really wouldn’t expect to find glucose in the colon. I’ve been to other presentations this weekend showing that some of these different micobiota rely on different nutrient sources. So perhaps the presence of glucose in the colon is for that or to help with sodium and water absorption as well.

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Cresci, G.A., Thangaraju, M., Mellinger, J.D. et al. Colonic Gene Expression in Conventional and Germ-Free Mice with a Focus on the Butyrate Receptor GPR109A and the Butyrate Transporter SLC5A8. J Gastrointest Surg 14, 449–461 (2010). https://doi.org/10.1007/s11605-009-1045-x

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  • DOI: https://doi.org/10.1007/s11605-009-1045-x

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