clays and binders, gut health, microbiomes, short chain fatty acids



Relevance to clinoptilolite

This post is about GPR109a and the response to LPS. It is a direct full circle to the post called Clinoptilolite that describes a decrease in the tight junction protein ZO-1 in the feces of human endurance atheletes. CLinoptilolite decreases the loss of barrier function. Clinoptilolite cont addresses a metabolomics study demonstrating changes in β-hydroxybutyrate and other metabolites that could be ligands for GPR109a in dairy cows.

The featured image: GPR109a structure and expression

The structure of GPRa is from Tunaru 2005 [1] with some modifications:

  • Ovals representing the Gα and Gβγ were added based on information from Protein Choice’s blogspot, This site also has a link to a YouTube video explaining how ligand binding to the receptor results in conformational changes in the receptor itself that facilitate exchange of GDP for GTP in the Gα subunit.
  • The 3D structures of niacin, butyrate, and 3-hydroxy butyrate were imparted from PubChem and placed next the ligand binding site structure from Tunaru 2005. [1]. The point being made here is that Serine 278 on the 2nd extracellular loop that connects trans membrane helices 3 and 4 has nothing to form hydrogen bond with on butyrate. The “OH” on the 3-carbon position of β-hydroxybutyrate depends on the Pka of the both this hydroxyl group and that of the Ser278 side chain. This post will examine evidence that butryate and β-hydroxybutyrate are agonists of GPR109a
  • The image of the man with organs with GPR109a expression sites is from Primary sites in the GI tract include the duodenum and he colon. The primary sites in the brain include the hippocampus, the pons, the cerebral cortex, and the basal ganglion.

Butyrate, leaky gut, and LPS [2]

This study by Macia and coauthors was performed on mice in which the GPR43/FFAR2 short chain fatty acid receptor and the GPR109a receptor that binds niacin, butyrate, and β-hydroxy butyrate had been knocked out. [2] The mice were fed low and high fiber diets as well as given acetate in their drinking water.   This post will not discuss any of the experiments but will present a summary figure. In really simple terms

  1. Lipopolysaccharide from bacteria binds to the TLR4 receptor resulting in production of precursors to cytokines and the inflammasome.
  2. Short chain fatty acids from bacteria bind to GPR43 and GPR109a. This causes an increase in intracellular Ca2+ and subsequent release of cytokine IL-18 into the blood.
Figure 9: A Model of inflammasome activation in gut epithelium, and the role of fibre, gut microbiota, SCFAs and metabolite-sensing GPCRs.(a) Under high-fibre feeding, a contact between the healthy microbiota under injury conditions such as DSS colitis ensures a proper priming of the gut epithelium through products such as TLR ligands. SCFA are highly abundant as a result of fermentation of dietary fibre, which through GPR109A and GPR43 will activate NLRP3 through mechanisms that involve Ca2+i mobilization and membrane hyperpolarization. Activated caspase 1 will cleave IL-18, which will be released promoting epithelial repair and protection from colitis development. (b) On the other hand, dysbiosis linked to consumption of a zero-fibre diet will not promote an optimal priming of the inflammasome and the low level of SCFA and thus decrease signalling of GPR43 and GPR109A will altogether not contribute to proper inflammasome activation. In this situation, IL-18 levels are not properly raised that contribute to exacerbated colitis development.

Gβγ activation of K+ channels was not really addressed and may not be important in this system. The general assumption is that GPR109a fires exclusively through Gαq.  Since increased intracellular Ca2+ is usually a part of neurotransmitter release, the priming event in the brain could be anything that triggers synthesis of packaging of neurotransmitters in synaptic vesicles. 

β-hydroxy butyrate, Parkinson’s Disease, and LPS [3]

The second paper turned out to be more like the first than anticipated.  The Macia paper did not specify LPS as one of the specific microbial products for priming. [2] A role of the inflammasome processing of IL-18 was suggested. [2] The Fu study went directly to LPS as a way of producing inflammation in the substantia nigra par compacta (SNpc) of the midbrain, a region in which dopaminergic neurons are lost in Parkinson’s Disease. [3] TLR4, part of the assemble that binds LPS is expressed widely throughout the brain. Protein Atlas TLR4. Methamphetamine also affects dopaminergic neurons by binding to teh TLR4 receptor of astrocytes taht are caretakers of said neurons. [4] NLRP3 is a a subunit of the inflammasome that produces proinflammatory cytokines IL-18 and IL-1β. [4]

TLR4 expression data from ProteinAtlas. The astrocyte/glia image was adapted from Du 2017. [4] “Methamphetamine” binding to the TLR4 receptor in astrocytes nuring dopaminergic neurons with “LPS” Note that NFκB requires phosphorylation to act as a transcription factor.

This particular scenario may apply to all regions of the brain wherever TLR4 is expressed and may apply to cases of leaky gut. Fu and coauthors injected the DHBA subcutaneously and the LPS diredtly into the substantia nigra. [3]

Fu Fig 1 [3] Effects of β-hydroxybutyric acid (BHBA) on lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons in mesencephalic neuron-glia cultures. Cultures were pretreated for 30 min with vehicle or indicated concentrations of BHBA before treatment with 10 ng/ml LPS. Seven days later, The immunohisotchemistry is not shown,. (B),TH-ir neuron count (C) [3H]DA uptake. The scale bar indicates 250 μm. The results are expressed as a percentage of the vehicle-treated control cultures and presented as the mean ± SD from three independent experiments performed in triplicate. **P <0.01 compared with the LPS-treated cultures; and ## P <0.01 compared with the vehicle-treated cultures.

Figure 2 of Fu 2017 examined the BHBA induced improvement of behavior aspects of this LPS induced Parkinson’s Disease model. [2] Figure 3 examined the influence of DHBA pretreatment on the influence of LPS injection unilaterally into the substantia nigra on dopamine and its metabolite in the striatum. DHBA, 0.4 to 1.6 mmol/kg/day helped normalize these levels. [3] Figure 4 of Fu 2017 documented improved survival of dopaminergic neurons and tyrosine hydroxylase protein expression in response to the same BHBA pretreatment after unilateral LPS injection. [3]

Figure 5 of Fu 2017 show results of tests of the ability of BHBA to prevent micoglia activation. OX-42, aka CD11c, is a microglia surface marker found on activated peritoneal macrophages, Kupffer cells, around 35% of alveolar macrophages, dendritic cells, granulocytes and microglial cells in the brain. Fu and coworkers measured protein levels of this marker and normalized to the “house keeping” protein actin. [3] The figures were not particularly clear in the original publication. Liberty (in magenta) has been taken to relabel the graphs. These experiments were performed in mice. The immunohistochemistry staining and the Western blot for OX-42 are not shown in this post.

β-hydroxybutyric acid (BHBA) treatment inhibits microglial activation and downregulates mRNA expression of pro-inflammatory mediators in the substantia nigra (SN) of lipopolysaccharide (LPS)-induced Parkinson’s disease (PD) model rats. (A) The morphological changes of the microglia in the SN as shown by IBA-1 immunostaining. Representative photomicrographs of the SN area are shown. The scale bar indicates 100 μm. (B) Western blot assay of O-X42 expression. The experiments were repeated three times. A representative immunoblot is shown. (C-G) Real-time RT-PCR analysis of pro-inflammatory enzyme (iNOS and COX-2) and pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) expression in the SN of LPS-induced PD model rats. The data are expressed as fold changes relative to the sham-operated control rats. The results are expressed as the mean ± SD. *P <0.05 and **P <0.01 compared with the LPS-treated rats; and ## P <0.01 compared with the sham-operated control rats.

Experiments in primary microglia cells

  • Figure 6 Exposure to LPS causes an increase in GPR109a mRNA in dose and time dependent manners.
  • Figure 7 BHBA reduces LPS induced iNOS and Cox-2 pro-inflammatory enzyme production in cultured microglia. Use of silencing RNA to inhibit GPR109a mRNA translation prevents this reduction.
  • Figure 8 BHBA reduces LPS induced pro-inflammatory cytokine production in cultured microglia. Use of silencing RNA to inhibit GPR109a mRNA translation prevents this reduction.
  • Figure 9 GPR109a and wildtype primary microglia cultures were treated with LPS ±BHBA. BHBA prevented the phosphorylation of the transcription factor NFκB inhibitor subunit Iκ B. [3]

More on NFκB

A 2018 study by Chen and coauthors was discovered after the majority of this post was written. The anti-inflammatory activity of sodium butyrate (SB) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mice was produced in order to better understand how sodium butyrate might alleviate symptoms. [5] A knock out GPR109a−/− and wild-type (WT) mice were given sodium butyrate (5 g/L) in their drinking water for 6 weeks. The authors performed a side series of experiments with LPS and various varieties of macrophages. Sodium butyrate inhibited the LPS-induced phosphorylation of the NF-κB p65 and AKT signaling pathways, but failed to inhibit the phosphorylation of the MAPK signaling pathway. [5]

Parting comments

Further searching on the molecular mechanisms of how butyrate binding to GPR109a inhibts AKt phophorylation has been suspended.


  1. Tunaru S, Lättig J, Kero J, Krause G, Offermanns S. Characterization of determinants of ligand binding to the nicotinic acid receptor GPR109A (HM74A/PUMA-G). Mol Pharmacol. 2005 Nov;68(5):1271-80.
  2. Macia L, Tan J, Vieira AT, Leach K, Stanley D, Luong S, Maruya M, Ian McKenzie C, Hijikata A, Wong C, Binge L, Thorburn AN, Chevalier N, Ang C, Marino E, Robert R, Offermanns S, Teixeira MM, Moore RJ, Flavell RA, Fagarasan S, Mackay CR. (2015) Metabolite-sensing receptors GPR43 and GPR109A facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome. Nat Commun. 2015 Apr 1;6:6734. free paper
  3. Fu SP, Wang JF, Xue WJ, Liu HM, Liu BR, Zeng YL, Li SN, Huang BX, Lv QK, Wang W, Liu JX. (2015) Anti-inflammatory effects of BHBA in both in vivo and in vitro Parkinson’s disease models are mediated by GPR109A-dependent mechanisms. J Neuroinflammation. 2015 Jan 17;12:9. PMC free article
  4. Du SH, Qiao DF, Chen CX, Chen S, Liu C, Lin Z, Wang H, Xie WB. (2017) Toll-Like Receptor 4 Mediates Methamphetamine-Induced Neuroinflammation through Caspase-11 Signaling Pathway in Astrocytes. Front Mol Neurosci. 2017 Dec 12;10:409. PMC free article
  5. Chen G, Ran X, Li B, Li Y, He D, Huang B, Fu S, Liu J, Wang W. (2018) Sodium Butyrate Inhibits Inflammation and Maintains Epithelium Barrier Integrity in a TNBS-induced Inflammatory Bowel Disease Mice Model. EBioMedicine. 2018 Apr;30:317-325 PMC free article
  6. Li Z, Li X, Lin S, Chen Y, Ma S, Fu Y, Wei C, Xu W. Nicotinic Acid Receptor GPR109A Exerts Anti-Inflammatory Effects Through Inhibiting the Akt/mTOR Signaling Pathway in MIN6 Pancreatic β cells. Ann Clin Lab Sci. 2017 Nov;47(6):729-737.

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