Chemogenetics defines a short-chain fatty acid receptor gut–brain axis

  1. Natasja Barki
  2. Daniele Bolognini
  3. Ulf Börjesson
  4. Laura Jenkins
  5. John Riddell
  6. David I Hughes
  7. Trond Ulven
  8. Brian D Hudson
  9. Elisabeth Rexen Ulven
  10. Niek Dekker
  11. Andrew B Tobin
  12. Graeme Milligan

  • Curated by eLife

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    Evaluation Summary:

    In this paper the authors study the Rolle of small chain fatty acids receptors FFA2 and FFA3 in the dorsal root ganglia with the goal to define molecularly a gut to brain axis. They identified MOMBA as a compound that binds to FFA2. This paper presents a powerful screening strategy to identify receptor agonists. The main concerns are the specificity of the model, and the functional purpose of this gut to brain axis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Volatile small molecules, including the short-chain fatty acids (SCFAs), acetate and propionate, released by the gut microbiota from the catabolism of nondigestible starches, can act in a hormone-like fashion via specific G-protein-coupled receptors (GPCRs). The primary GPCR targets for these SCFAs are FFA2 and FFA3. Using transgenic mice in which FFA2 was replaced by an altered form called a Designer Receptor Exclusively Activated by Designer Drugs (FFA2-DREADD), but in which FFA3 is unaltered, and a newly identified FFA2-DREADD agonist 4-methoxy-3-methyl-benzoic acid (MOMBA), we demonstrate how specific functions of FFA2 and FFA3 define a SCFA–gut–brain axis. Activation of both FFA2/3 in the lumen of the gut stimulates spinal cord activity and activation of gut FFA3 directly regulates sensory afferent neuronal firing. Moreover, we demonstrate that FFA2 and FFA3 are both functionally expressed in dorsal root- and nodose ganglia where they signal through different G proteins and mechanisms to regulate cellular calcium levels. We conclude that FFA2 and FFA3, acting at distinct levels, provide an axis by which SCFAs originating from the gut microbiota can regulate central activity.

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  1. Version published to 10.7554/elife.73777 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    Short chain fatty acids produced by gut microbiota interact with the short chain fatty acid receptors FFA2 and FFA3 (formerly GPR43 and GPR41). Barki and colleagues report the results of studies designed to define the roles of FFA2 and FFA3.

    Using a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) derived from human FFA2 modified to allow a BRET signal in the presence of agonists, 1210 compounds with structural similarity to the known agonist sorbic acid were screened in an appropriately validated assay. Reconfirmation screens identified sorbic acid and MOMBA. Assessment of 320 additional compounds identified chemicals related to MOMBA.

    MOMBA did not activate human FFA2 in interaction assays. However, interaction assays could not be performed with mouse FFA2, so Gi inhibition of cAMP assays were pursued. Neither sorbic acid nor MOMBA inhibited cAMP levels using human or mouse FFA2, and the same lack of effect was seen using human and mouse FFA3. MOMBA was shown to be an orthosteric agonist of hFFA2-DREADD.

    FFA2 and 3 were shown to be expressed in myenteric neurons. MOMBA increased GI transit time in hFFA2-DREADD-HA mice but not control mice. MOMBA also increased transit in animals expressing only FFA2 or only FFA3. MOMBA also increased GLP-1 release from colonic cells and tissues, an effect already reported by this group for sorbic acid. MOMBA also promoted release of PYY.

    Vagal afferents in the colon were stimulated by activation of FFA3 but not FFA2. Cells from the nodose ganglion were stimulated by C3 and to a lesser extent by MOMBA. FFA2 and FFA3 also appeared to be active and functional in DRG cells.

    Using wild type mice, C3 administered to the rectum activated spinal cord neurons. C3 and MOMBA activated c-Fos in hFFA2-DREADD mice. The authors conclude that there is a SCFA-gut-brain axis.

    1. This paper extends findings from this group published in a very strong paper in 2019 (Nat Chem Biol 15:489-498) using knockin receptor hFFA2-DREADD mice and showing that activation of FFA2 promotes GLP-1 release, accelerates gut transit, and promotes lipolysis in adipocytes. The GLP-1 observations are confirmed here, and a new agonist for FFA2 is identified, but it is difficult to appreciate how these studies "define" a short chain fatty acid receptor gut-brain axis.

    Answer: to address whether we have ‘defined’ a short chain fatty acid receptor-gut-brain axis, we have slightly altered the title to more correctly reflect the work performed. It is now ‘Chemogenetics defines the roles of short chain fatty acid receptors within the gut-brain axis’

    Why did the authors choose to pursue detailed studies of the myenteric neurons, nodose ganglion and DRG?

    Answer: That there is connectivity between gut and brain and therefore a ‘gut-brain axis’ is now well established. Many products of both anabolic and catabolic metabolism by the gut microbiota have been suggested to play roles in this connection. Chief amongst these are the short chain fatty acids (SCFAs). The hypothesis explored herein is that SCFAs derived from the microbiota will impact on the gut-brain axis at many levels, hence regulating physiology and function. The FFA2 and FFA3 receptors are major targets for the SCFAs and the animal models and novel ligands we have developed allow us to define explicitly whether function at key points in this axis involve activation of FFA2, FFA3, both receptors or neither, and this is the first large scale study to have undertaken such mapping at multiple different levels. This is why, among other tissues and cells types we have explored myenteric neurons, nodose ganglion (NG) and DRG. Each of these tissues showed expression of FFA2 and/or FFA3. This naturally lead to the question of whether afferents from the gut innervating DRG/NG were activated by FFA2/3 stimulation – this was the case. It was then entirely rational to assess whether FFA2/3 were functionally expressed in the DRG and NG – this would give information regarding the possibility that there is receptor expression in nerve fibers innervating the gut from the DRG/NG. Through this investigation we present a previously unappreciated profile of FFA2/3 expression and function in neurons associated with the gut.

    Other mediators of this purported axis could also be involved. What is the purpose of this axis? GI motility?

    Answer: Other mediators certainly will play roles and co-ordinate with the actions of the SCFAs. We had shown previously that FFA2 in the gut controls aspects of gut motility and we now show that FFA3 also plays a role. We hypothesised that the roles of SCFAs might also invove control of sensory function. The results provided herein
    confirm this and dissect the specific roles of each of FFA2 and FFA3.

    If that is the focus, there are technical issues that limit the conclusions that can be drawn.

    Answer: As noted above, the work and the conclusions are not restricted to gut motility but include actions from the gut to the spinal cord.

    1. A strength of the paper is the elegant and rigorous screening strategy and validation of receptor agonists.

    Thank you for this comment: it was indeed a substantial task and component of the work because without rigorous assessment of the selective actions of MOMBA and TUG1907 it would have been impossible to fully dissect the specific contributions of FFA2
    and FFA3.

    1. A weakness is that expression of hFFA2-DREADD is induced by use of a whole-body Cre mouse. Given the broad distribution of FFA2, it is likely that this receptor is being activated in multiple tissues when MOMBA is administered. How can the authors be sure that observed effects after administration of agonist in drinking water are due to local expression as opposed to an effect mediated at a distant site removed from the myentery?

    Answer: We can understand where the concern of the reviewer comes from but our aim was to understand the pathway in as close as possible to whole animal and physiologically normal conditions. Hence, we wanted to maintain all elements of FFA2/3 expression whilst investigating the effects of introducing ligands into the gut, which is where the endogenous SCFAs largely originate. So the question we wanted to ask was by introducing FFA2/3 ligands into the gut (thereby mimicking the release of SCFA by the microbiota) what were the physiological consequences of this at levels of the gut, enteric neurons, DRG/NG and spinal cord.

    Reviewer #2 (Public Review):

    Strengths. Barki et al. report extensive and rigorous studies which convincingly establish that FFA2 and FFA3 are functionally expressed in dorsal root ganglia and nodose ganglia where they signal through different G proteins and mechanisms that regulate intracellular calcium concentrations. The authors further demonstrate that activation of both FFA2 and FFA3 within the gut lumen stimulates spinal cord activity and that activation of gut FFA3 directly regulates sensory afferent neuronal firing. These data support the authors' contention that their investigations define a SCFA-gut-brain axis.

    The authors have employed a number of complimentary pharmacological, genetic, cell culture, and ex vivo approaches to obtain their data. The use of these diverse methodological approaches is a key strength of the work. They have employed transgenic mice where FFA2 was replaced by an altered form of FFA2 referred to as FFA2-DREADD (Designer Receptor Exclusively Activated by Designer Drugs), which can be activated by novel ligands but not SCFAs. They have further identified through screens of chemical libraries a novel FFA2-DREADD agonist, referred to as MOMBA, for use in their investigations. Using the FFA-DREADD mice, which are also HA tagged to allow for immunologic detection, and other related transgenic lines, they have been able to establish and identify distinct roles for FFA2 and FFA3 in signaling SCFA production and presence in the gut (specifically the colon) to neuronal pathways that communicate directly with the brain. Using isolated cells, the authors further establish roles for different G proteins and mechanisms that affect cellular calcium levels. Collectively, the data obtained using these diverse experimental approaches support the existence of a SCFA-gut-brain axis.

    The authors' new findings significantly extent understanding of the molecular actions of SCFA's produced in the colon through bacterial fermentation of dietary fiber. Multiple publications have identified altered SCFA levels in the gut as a significant contributor to dysregulated metabolism and metabolic disease. Often such studies fail to provide insight into the molecular basis for the observed linkages between SCFAs and altered metabolic states, only reporting the association. The new data being reported by Barki et al. provide new possibilities for understanding these associations.

    Answer: We thank the reviewer for these very positive comments.

    Weaknesses. The perceived weaknesses of the work are minor compared to the strengths. Although the authors provide a description of the general characteristics of the chemical libraries they screened to identify MOMBA, sparse other information is provided. This is especially true for the second screen of 320 compounds where the data provided indicate a number of compounds may be equally potent agonists. What similarities were there in these structures?

    Answer: We state specifically in the text that compound 132 was 4-methoxy-3-chlorobenzoic acid and compound 235 4-methoxy-3-hydroxy-benzoic acid. These are indeed closely related to MOMBA. We did not test the other hits from the secondary screen for their broader selectivity as none of them were substantially more potent than the MOMBA-related compounds and the others did not provide the close structure-activity relationship of MOMBA, compound 132 and compound 235, so there is no useful information we can provide.

    Were any of these compounds naturally occurring or resemble naturally occurring molecules?

    Answer: No, they were not, although as small molecule carboxylates they can of course be considered to ‘resemble’ certain naturally occurring compounds. For example they ‘resemble’ short chain fatty acids.

    The issue here is one of trying to understand whether endogenous substances exist that might have influenced experimental outcomes and/or data interpretation. This is far from being transparent.

    Answer: We have no evidence to suggest that an endogenous FFA2-DREADD activator exists – in fact our data supports the opposite, that the FFA2-DREADD is a true DREADD receptor i.e. is only activated by a synthetic ligand.

    In the authors' first report of the FFA2-DREADD mice (reference 15), they establish that sorbic acid is an agonist for FFA2-DREADD. Many of the studies being reported in the present manuscript are repetitive of this earlier work but using MOMBA in place of sorbic acid. The authors provide some justification for this, but this reviewer does not find these very compelling. Seemingly, sorbic acid stimulated FFA2-DREADD signaling could have been studied without the need to screen libraries and characterize MOMBA actions. This issue detracts from the overall positive feelings towards the work. Why was the identification of MOMBA needed prior to undertaking the present studies? The authors note greater activity of MOMBA over sorbic acid, but why was this important for establishing FFA2 presence and actions in signaling to the brain?

    Answer: Although this is a good point one of the important outcomes of this study is that we have generated a novel FFA2-DREADD agonist, and in so doing further validated the FFA2-DREADD model – i.e. it is not dependent on just one ligand:receptor pairing but in fact we are now able to employ two chemically distinct ligands. Interestingly,
    reviewer 2 found the identification of a novel FFA2-DREADD ligand an important component of the paper.

  3. eLife

    Evaluation Summary:

    In this paper the authors study the Rolle of small chain fatty acids receptors FFA2 and FFA3 in the dorsal root ganglia with the goal to define molecularly a gut to brain axis. They identified MOMBA as a compound that binds to FFA2. This paper presents a powerful screening strategy to identify receptor agonists. The main concerns are the specificity of the model, and the functional purpose of this gut to brain axis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Short chain fatty acids produced by gut microbiota interact with the short chain fatty acid receptors FFA2 and FFA3 (formerly GPR43 and GPR41). Barki and colleagues report the results of studies designed to define the roles of FFA2 and FFA3.

    Using a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) derived from human FFA2 modified to allow a BRET signal in the presence of agonists, 1210 compounds with structural similarity to the known agonist sorbic acid were screened in an appropriately validated assay. Reconfirmation screens identified sorbic acid and MOMBA. Assessment of 320 additional compounds identified chemicals related to MOMBA.

    MOMBA did not activate human FFA2 in interaction assays. However, interaction assays could not be performed with mouse FFA2, so Gi inhibition of cAMP assays were pursued. Neither sorbic acid nor MOMBA inhibited cAMP levels using human or mouse FFA2, and the same lack of effect was seen using human and mouse FFA3. MOMBA was shown to be an orthosteric agonist of hFFA2-DREADD.

    FFA2 and 3 were shown to be expressed in myenteric neurons. MOMBA increased GI transit time in hFFA2-DREADD-HA mice but not control mice. MOMBA also increased transit in animals expressing only FFA2 or only FFA3. MOMBA also increased GLP-1 release from colonic cells and tissues, an effect already reported by this group for sorbic acid. MOMBA also promoted release of PYY.

    Vagal afferents in the colon were stimulated by activation of FFA3 but not FFA2. Cells from the nodose ganglion were stimulated by C3 and to a lesser extent by MOMBA. FFA2 and FFA3 also appeared to be active and functional in DRG cells.

    Using wild type mice, C3 administered to the rectum activated spinal cord neurons. C3 and MOMBA activated c-Fos in hFFA2-DREADD mice. The authors conclude that there is a SCFA-gut-brain axis.

    1. This paper extends findings from this group published in a very strong paper in 2019 (Nat Chem Biol 15:489-498) using knockin receptor hFFA2-DREADD mice and showing that activation of FFA2 promotes GLP-1 release, accelerates gut transit, and promotes lipolysis in adipocytes. The GLP-1 observations are confirmed here, and a new agonist for FFA2 is identified, but it is difficult to appreciate how these studies "define" a short chain fatty acid receptor gut-brain axis. Why did the authors choose to pursue detailed studies of the myenteric neurons, nodose ganglion and DRG? Other mediators of this purported axis could also be involved. What is the purpose of this axis? GI motility? If that is the focus, there are technical issues that limit the conclusions that can be drawn.

    2. A strength of the paper is the elegant and rigorous screening strategy and validation of receptor agonists.

    3. A weakness is that expression of hFFA2-DREADD is induced by use of a whole-body Cre mouse. Given the broad distribution of FFA2, it is likely that this receptor is being activated in multiple tissues when MOMBA is administered. How can the authors be sure that observed effects after administration of agonist in drinking water are due to local expression as opposed to an effect mediated at a distant site removed from the myentery?

  5. eLife

    Reviewer #2 (Public Review):

    Strengths. Barki et al. report extensive and rigorous studies which convincingly establish that FFA2 and FFA3 are functionally expressed in dorsal root ganglia and nodose ganglia where they signal through different G proteins and mechanisms that regulate intracellular calcium concentrations. The authors further demonstrate that activation of both FFA2 and FFA3 within the gut lumen stimulates spinal cord activity and that activation of gut FFA3 directly regulates sensory afferent neuronal firing. These data support the authors' contention that their investigations define a SCFA-gut-brain axis.

    The authors have employed a number of complimentary pharmacological, genetic, cell culture, and ex vivo approaches to obtain their data. The use of these diverse methodological approaches is a key strength of the work. They have employed transgenic mice where FFA2 was replaced by an altered form of FFA2 referred to as FFA2-DREADD (Designer Receptor Exclusively Activated by Designer Drugs), which can be activated by novel ligands but not SCFAs. They have further identified through screens of chemical libraries a novel FFA2-DREADD agonist, referred to as MOMBA, for use in their investigations. Using the FFA-DREADD mice, which are also HA tagged to allow for immunologic detection, and other related transgenic lines, they have been able to establish and identify distinct roles for FFA2 and FFA3 in signaling SCFA production and presence in the gut (specifically the colon) to neuronal pathways that communicate directly with the brain. Using isolated cells, the authors further establish roles for different G proteins and mechanisms that affect cellular calcium levels. Collectively, the data obtained using these diverse experimental approaches support the existence of a SCFA-gut-brain axis.

    The authors' new findings significantly extent understanding of the molecular actions of SCFA's produced in the colon through bacterial fermentation of dietary fiber. Multiple publications have identified altered SCFA levels in the gut as a significant contributor to dysregulated metabolism and metabolic disease. Often such studies fail to provide insight into the molecular basis for the observed linkages between SCFAs and altered metabolic states, only reporting the association. The new data being reported by Barki et al. provide new possibilities for understanding these associations.

    Weaknesses. The perceived weaknesses of the work are minor compared to the strengths. Although the authors provide a description of the general characteristics of the chemical libraries they screened to identify MOMBA, sparse other information is provided. This is especially true for the second screen of 320 compounds where the data provided indicate a number of compounds may be equally potent agonists. What similarities were there in these structures? Were any of these compounds naturally occurring or resemble naturally occurring molecules? The issue here is one of trying to understand whether endogenous substances exist that might have influenced experimental outcomes and/or data interpretation. This is far from being transparent.

    In the authors' first report of the FFA2-DREADD mice (reference 15), they establish that sorbic acid is an agonist for FFA2-DREADD. Many of the studies being reported in the present manuscript are repetitive of this earlier work but using MOMBA in place of sorbic acid. The authors provide some justification for this, but this reviewer does not find these very compelling. Seemingly, sorbic acid stimulated FFA2-DREADD signaling could have been studied without the need to screen libraries and characterize MOMBA actions. This issue detracts from the overall positive feelings towards the work. Why was the identification of MOMBA needed prior to undertaking the present studies? The authors note greater activity of MOMBA over sorbic acid, but why was this important for establishing FFA2 presence and actions in signaling to the brain?

  7. Version published to 10.1101/2020.01.11.902726v1 on bioRxiv