This post sort of grew from an exploration of valerate. It is worth our while to even bother?
- The 1st report comparing valerate with shorter short chain fatty acids, 
- SCFA in intestinal barrier function, relevant to so much, 
- Brief look at airway epithelial function and secretion of tissue plasminogen activator. 
The featured image was found in a review after the first three papers. G protein coupled receptors (GPCRs) GPR40, GPR43 and GPR41 were discovered based only by virtue of having a similar sequence to other GPCR are now named free fatty acid receptors FFA1, FFA2 and FFA3, respectively. FFAR1 is more a receptor for long chain fatty acids. Part of the featured image comes from the ProteinAtlas.org FFA2 site. Atlas Antibodies has found immuno reactivity to the basal ganglia. Staining is medium in neuronal cells yet low in glial cells. The ProteinAtlas.org FFA3 site provided FFAR3 data for the featured image.
Two orphan G Protein coupled Receptors find ligands
This study was published almost 20 years ago when we were just getting to know the concept of G protein coupled receptors and the fully sequenced human genome. Genes that were suspected of coding for G protein coupled receptors were cloned into yeast for a fishing expedition to figure out what ligands might activate of heterotrimeric G proteins docked to their receptors and subsequent exchange of GDP for GTP. The GTP bound Gα subunit causes it and the βγ subunits to leave the receptor. Hydrolysis of GTP will result in the trimer returning to the receptor. A non hydrolysable analog of GTP, GTPγS is used to measure activation of G protein coupled receptors. The S in GTPγS occupies the position of the third P of GTP and usually is the 35S radioactive form. .
Brown 2002 Fig1, acetic acid is a ligand
These G protein coupled receptors along with their heterotrimeric G proteins that bind to them are found in yeast. GPa1p is a yeast version of the mammalian Gα sub units. Various flavors of the human Gα sub unit were cloned into yeast using the yeast GPa1p promoter to drive expression. the G protein coupling specificity of hGPR43 was determined in a panel of yeast strains expressing the yeast wild-type G subunit Gpa1p or a range of different Gpa1p/G chimeras. Chimeras contained the five C-terminal amino acids of the indicated mammalian G subunits fused to the remainder of Gpa1p. FUS1-lacZ reporter gene induction due to the constitutive activity of hGPR43 was determined in acetate-free assay medium (white bars), and agonist-dependent activity was determined in the presence of 150 μM acetate (black bars). Results from single representative experiments are presented. Data shown are the means ± S.D.
Panel 1C is a good place to introduce pertussis toxic (PTX), a Whooping couch toxin that prevents Gαi from inhibiting the enzyme adenylate cyclase, and enzyme that produces cyclic AMP. YM254890 is an inhibitor of Gαq/11. The former activates pathways that involve cAMP and protein kinase A by inhibiting the inhibition of adenylate cyclase production of cAMP.
Brown 2002 Fig2, the three duties of G proteins
2A acetate increases the intracellular Ca2+, score one for Gαq for GPR43 .2B PTX does nothing. Gαi needs to be crossed off the GPR43 “friends” list.
Brown 2002 Fig 3, a survey of short chain fatty acids
When it come to only GTP binding, propionate is by far the most effective ligand, especially for GPR43. These may explain why propionate is considered to participate in the etiology of ASD while butyrate is beneficial. GPR41 seems to be less picky in that it also is activated by acetate and pentanoate/valerate.
Table 2 summarizes carboxylates, aka short chain fatty acids, from two to seven carbons
The FLIPR measures changes in intracellular calcium. The lower the EC50, the more active the short chain fatty acid. GPR seems to be exclusive for propionate. GPR41 seems to be promiscuous.
Brown 2002 Figs 4 and 5, not to be discussed..
This post will not discuss the making of mutant forms of GPR41 and its close relative GPR42 simply because it is not part of the focus as to whether propionate vs butyrate is relevant to autism based on short chain fatty acid receptors. Figure 5 discusses functions in cultured adipocytes that are also straying from out focus.
Intestinal barrier and SCFA 
Figure 1 of D’Souza 2017 examined a concentration gradient of the three main short chain fatty acids in the colon in CHO cells expressing GPR43. Two specific agonists were examined as positive controls.
Note that the IC50 is about 100x less for the specific agonists. Also note that there really is not a big difference between propionate and butyrate that are thought to be a huge difference between ASD and neurotypical. Note that agonists #44 and #58 are allosteric, meaning that the act on a receptor site other than the one that the natural ligand binds to, the orthosteric site.
Figure 2 GPR43 expression
Note that not all colonic and small intestine expression of GPR43 is explained by epithelial cells.
GPR43 can be in smooth muscle cells and neurons.
Figure 3 Improving cell culture leaky gut
Moving on to Figure 3 we see that the trans epithelial electrical resistance increases with all of the SCFA tested.  Note the increase is from about 900 Ohms to Over 2000 Ohms at 2.5 mM butryate. Permeability is measured with Lucifer Yellow. Some images have been added to the original figure. Note that the control value is 1.5 x10-4 cm/sec for migration of Lucifer Yellow across the bilayer of cells. At 2.5 mM butyrate the migration decreases to maybe 3 x 10-7 cm/sec. This is 500x slower.
Lucifer Yellow is not even as large as LPS. Even though the increase in electrical resistance is less than tripled, the decrease in permeability is much more dramatic.
Figure 4, preventing cell detachment
The rationale of this figure was not explained that well in the text. EDTA is a divalent metal ion chelator that is used to remove cultured cells from the surfaces of culture dishes. The resistance was measured by electrical impedance to sinusoidal currents. It is assumed they used differentiated C2BBe1, see Figure 2.
Figure 5, silencing cytokines
This figure required some background work. Peripheral blood mononuclear cells, PBMC, are lymphocytes (T cells, B cells, NK cells) and monocytes. According to ProteinAtlas, monocytes are the only PBMC that express PPAR2/GPR43 mRNA. Human PBMC were incubated with mM concentrations of butyrate or μM concenttions of the GPR43 allosteric agonists for 45 minutes and then challenged with 1 μg/mL LPS. Three representative cytokines were measured in the cell culture medium. None of the agonists influenced the viability of the cells. 
The question here is if the concentration of butyrate can reach 10mM.
Valeric acid lessons from airway epithelial cells 
This study out of Japan was interested in chronic rhinosinusitis (CRS) that arises from nasal nolyps NPs in which there is excessive fibrin deposition.  t-PA is expressed in epithelial cells that are accessible to topical agents. A list of short chain fatty acids was explored for the purpose of synthesis and/or release of t-PA from airway epithelial cells. Figures 1 and 2 demonstrated GPR41 and GPR43 in nasal polyps more so than in normal tissue. This paper gets very confusing because the authors switch between measuring tPA secretion and messenger RNA that could be under Gαq/ Ca2+ control…. Ca2+ is a common 2nd messenger for neurotransmitter vesicle release…Gαi tends to turn off cAMP, that leads to protein kinase A activation, that phosphorylates transcription factors that make blue prints for more proteins. I’ve copied and pasted the figure legends, which are quite good, and high lighted what they are really looking at.
3 SCFA making tPA messages versus secreting protein
4. Inhibiting SCFA production of tPA messages
One thing that needs to be clarified is that Imoto and coworkers are looking at tPA mRNA transcription rather than synthesis or release into the culture medium. Note that propionate (3 carbons) to valerate (5 carbons ) cause an increase of tPA. These results are consistent with FFAR3/GPR43. Note that Imoto and coworkers are looking at tPA synthesis inside a cultured cell line, not release of this enzyme.
5 With cultured cells, Imoto could just get rid of the SCFA creptors
Pharmacologically, both receptors appear to be involved in tPA release. The next strategy was to knock down the expression of one, the other, or both using silencing RNA.
In yet another review on the subject, Grundmann and coauthors cite studies showing that the FFAR3 protomer functions as a positive allosteric modulator of FFAR2 signaling (Ca2+ increase downstream of FFAR2) and FFAR2 allosterically modulating FFAR3 function vice versa (β-arrestin 2 recruitment to FFAR3). The FFAR2-FFAR3 dimer lacked Gi/o-mediated cAMP decrease.  It is an interesting concept that a FFAR2-FFAR3 heterodimer might increase the carbon range of SCFA. An even more interesting concept is the possible ability of a SCFA triglyceride to bind to both members of the hetero or homodimer and elicit an even greater action.
6. Primary cultured airway epithelial cells, the real deal
This is all well and good GPR41 and GPR43 are involved in the synthesis of tPA in a cultured cell line. Some times functions get lost with splitting, regrowing, freezing back, and so one. What about primary cultures of real human cells that are closer to the real thing?
- The 2003 Brown  study demonstrated that GPR43/FFAR2 prefers propionate to other SCFA while GPR41/FFAR3 responds well to all short chain fatty acids. This means we have to look closely as to where GPR43 is expressed and if this can explain as to why propionate is toxic to kids with ASD and butyrate is a good thing
- The D’Sourza study never really looked at valeric acid.  The interesting aspect of this study is the importance of SCFA and GPR41/43 in maintaining GI barrier function.
- The insightful thing about the Imoto airway epithelial cell study  is that it made the point that SCFA can control mRNA transcription, protein synthesis, and release. I did not go to deep into this one because there is no obvious relationship to ASD. A nice followup would be hormones that enteroendocrine cells release by propionate, butyrate, valerate….
The next assignment is MCT, medium chain triglyceride, oil. GPR40/FFAR1 is the receptor for the medium chain free fatty acids.
- Brown AJ, Goldsworthy SM, Barnes AA, Eilert MM, Tcheang L, Daniels D, Muir AI, Wigglesworth MJ, Kinghorn I, Fraser NJ, Pike NB, Strum JC, Steplewski KM, Murdock PR, Holder JC, Marshall FH, Szekeres PG, Wilson S, Ignar DM, Foord SM, Wise A, Dowell SJ. (2003) The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J Biol Chem. 2003 Mar 28;278(13):11312-9. Free article
- D’Souza WN, Douangpanya J, Mu S, Jaeckel P, Zhang M, Maxwell JR, Rottman JB, Labitzke K, Willee A, Beckmann H, Wang Y, Li Y, Schwandner R, Johnston JA, Towne JE, Hsu H. (2017) Differing roles for short chain fatty acids and GPR43 agonism in the regulation of intestinal barrier function and immune responses. PLoS One. 2017 Jul 20;12(7):e0180190. PMC free article
- Imoto Y, Kato A, Takabayashi T, Sakashita M, Norton JE, Suh LA, Carter RG, Weibman AR, Hulse KE, Stevens W, Harris KE, Peters AT, Grammer LC, Tan BK, Welch K, Conley DB, Kern RC, Fujieda S, Schleimer RP. (2018) Short-chain fatty acids induce tissue plasminogen activator in airway epithelial cells via GPR41&43. Clin Exp Allergy. 2018 May;48(5):544-554. PMC free article
- Milligan G, Stoddart LA, Smith NJ. Agonism and allosterism: the pharmacology of the free fatty acid receptors FFA2 and FFA3. Br J Pharmacol. 2009 Sep;158(1):146-53. PMC free article
- Grundmann M, Bender E, Schamberger J, Eitner F. (2021) Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators. Int J Mol Sci. 2021 Feb 10;22(4):1763. PMC free article