gut health, short chain fatty acids

FFAR1 and FFAR4

BL

The featured image was obtained from ProteinAtlas.org for GPR40/FFAR1 and GPR120/FFAR4. The preference of the number of carbons in the free fatty acid is from Kimura 2020. [1] Not in this image is GPR119 mRNA transcripts found in the pancreas and throughout the lower digestive tact.

An overview of the journey of FFAR1 and FFAR4 that included medium chain fatty acids

Background tidbits

This subject matter is turning out to be very confusing and tricky to relate to autism. Even more difficult is bringing in medium chain fatty acid triglycerides.

As FFAR1 relates to autism…

Aizawa and coworkers studied various behavior aspects in GPR40/FFAR1 knockout mice. [2] In some ways this was not a model of autism spectrum disorders.

images from sources as noted
  • These mice showed less anxiety in terms of entering and roaming about a strange enclosure of the open “field” test. [2]
  • No difference was seen between the wildtype mice and the GPR40 knockout mice in social interaction tests. [2]
  • The GPR40 knockout mice demonstrated a slight, but statistically significant, reduction in sucrose preference. [2]
  • These investigators measured 5-HT, noradrenaline, and dopamine in the hippocampus, midbrain, medulla, and hypothalamus.
  • NA was slightly increased in all four regions. [2]
  • All three neurotransmitters were statistically increased in the hippocampus, dopamine was more than doubled in the GPR40 knockout mice. [2]

Nice rat brain models of NA and dopamine pathways were not readily obtainable with a quick Internet search. It is worth noting that the olfactory bulb is the region of highest expression of FFAR1 and that these serotonergic neurons signal to other regions of the rat brain. It is debatable how much this study applies to humans as the olfactory bulb does not take up half human brains.

MCT oil “PKPD” [3]

IV injection studies (mouse, rat, monkey) of emulsions of medium chain triglycerides mixed with fish oil indicate accumulation in the liver followed by the visceral fat, skeletal muscle, and cardiac muscle have been performed. [3] These studies unfortunately bypassed mammalian lipases of the stomach and small intestine as well as bacterial metabolism in the colon.

Comparison of blood clearance, plasma lipids, and organ uptake of low-dose and high-dose MCT/FO 8:2 emulsions in male and female rats.Radiolabeled low- (0.016 mg TG/g BW) and high-dose (1.6 mg TG/g BW) MCT/FO emulsions were intravenously injected into male and female rats (n=6). (E) Organ uptake (F) were compared in male and female rats was measured 60 minutes after injection. The values represent the mean±SD. *p < 0.05 by Student’s t test. [3]

We don’t really know if the deposition is in the triglyceride form.

The distal small intestine and enteroendocrine cells [4]

This paper was not concerned about autism or back flux of short to medium chain fatty acids from the colon into the distal small intestine. The findings are relevant to our purposes.

The introduction of this paper had some enteroendocrine gems

  • K Cells, located in the upper small intestine, secrete GIP, The GIPR is the receptor and triggers insulin secretion.  Chronic high fat diet can stimulate GIP hyper secretion.
  • L Cells, located in the lower small intestine,  secrete GLP-1.  Pancreas cells are the target.

The K cells in this study were isolated from the mouse small intestine after feeding routines so that the mRNA could be isolated. An added nuance was that instead of secreting GIP the mice secreted a version of GIP that also contained green fluorescence protein from jelly fish (GFP). The investigators were able to sort those cells that contained GFP/GIP. Was this mRNA tied to mRNA coding for a particular FFAR?

Figure 3. Expressions of FFAR4/GPR120 mRNA and in K cells. Data are shown as relative expression to
that of peptidylprolyl isomerase A (PPIA) expression in parallel in the same samples (A, GPR40; B,
GPR120; C, GPR119; D, GPR41; E, GPR43) (n  12). Their mRNA levels in GAP-positive and GFP negative cells were expressed as each 2ˆ CT [CT (internal control) CT (target gene)]. GFP positive cells [GFP()] are represented by black bars and GAP-negative cells [GFP()] by white bars. *, P  .05, ***, P  .001; the upper small intestine vs the lower small intestine; #, P  <.05,###, P < .001; GAP-positive vs GAP-negative cells (A–E). ns, no significance.

PPIP is apparently a “housekeeping” gene expressed in supposedly equal amounts in all cells and is used for normalization. Except for GPR120/FFAR4, the distal small intestine seems to be the site of K cells that express FFFARs in a way that is linked to GIP production. GIP does have a central nervous system function that will be covered later.

C10 activates better than C8 in pancreatic β-cells. [5]

A company we’ve been talking to sells medium chain triglycerides, HNSstore.eu. HCA2, The hydroxycarboxylic acid receptor 2, is another G protein coupled receptor for niacin, β-hydroxybutyrate, and butyric acid.

Cell and Islet Culture

Rodent β-cell line INS1E were maintained in a common cell culture medium.The human islets were isolated from healthy donors that had been approved for research. Cells were treated with up to 500 µM MCFA.

Figure 3. from Pujol 20Expressions of FFAR mRNA and in K cells. Data are shown as relative expression to
that of peptidylprolyl isomerase A (PPIA) expression in parallel in the same samples (A, GPR40; B,
GPR120; C, GPR119; D, GPR41; E, GPR43) (n  12). Their mRNA levels in GFP-positive and GFP negative cells were expressed as each 2ˆ CT [CT (internal control) CT (target gene)]. GFP positive cells [GFP()] are represented by black bars and GFP-negative cells [GFP()] by white
bars. *, P  .05, ***, P  .001; the upper small intestine vs the lower small intestine; #, P  .05,
###
, P  .001; GFP-positive vs GFP-negative cells (A–E). ns, no significance.

IP3 production from PLC seems to be inferred by an intermediate degradation product IP1. Panels and b of Figure 2 from [5] are a bit confusing. The black traces are simply dose response curves of C8 and C10. Less of C10 is required to reach half maximal IP3 release. IP3 production from PLC seems to be inferred by an intermediate degradation product IP1 using a very complicated fluorescent reporter scheme that will not be covered in this post. . 

Figure 2. Activation of GPR40 by medium chain fatty acids, C8 and C10, in Chem1-GPR40 cells. (a) Dose–response curves of C8 in the presence of C10; dose–response of IP1 production with C8 activation in the presence of increasing concentrations of C10 and (b) dose–response curves of C10 in the presence of C8; C10 in the presence of increasing concentrations of C8. (c) Dose–response curves of C8 in GW1100; inhibitory effect of the GPR40 antagonist, GW1100, on the dose–response accumulation of IP1 by C8 or by (d) dose–response curves of C10 in GW1100; C10 in Chem1-GPR40 cells. Curves represent as means + SD of eight experimental replicates.

Panels 2c and 2d are simply dose response curves of C8 and C10 in the presence and absence of GPR40 antagonist GW1100 (50 nM).

INS1E cells grown to confluence in 6-well plates were treated with the

  • vehicle (DMSO)
  • GPR40 antagonist GW1100 (50 nM)
  • PLCβ  inhibitor U73122 (2 µM)
  • IP3 ↓ receptor inhibitor (-) Xestospongin C(3 µM)
  • L-type Ca2+ channel inhibitor Nifedipine (0.1 µM)

for 15 min prior to incubation with200 µM MCFA for 2 h in RPMI medium with 1 mM glucose. Analysis of 3-hydroxybutyric acid (BHB) was done with LC-MS was used as a down stream signalling endpoint. The lack of effect of the GW compound suggests that FFAR1 firing has no effect on acetyl CoA and the TCA cycle taking fatty acids to H2O and CO2. It is interesting ta hat C8 produces more BHB than C10

Figure 3. GPR40 signaling affects fatty acid β-oxidation in β-cells. (a) Ketone body β- hydroxybutyrate (BHB) production in INS1E β-cells in the presence of C8, C10 or C8:C10 40:60 mixture. (b) Inhibition of GPR40 signaling by the antagonist, GW1100 (GW), (c) the phospholipase C-β (PLCβ) inhibitor,U73122 (U7) and nifedipine (Nif) prevent BHB production from medium chain fatty acids (MCFA) in INS1E β-cells unlike the IP3 receptor inhibitor xantospongin (Xant). (d) Inhibition of GPR40 signaling by GW increased complete β-oxidation of palmitate. Data are presented as means + SEM of three independent experiments, * p < 0.05, ** p < 0.01 relative to control

Analysis of 3-hydroxybutyric acid (BHB) was done with LC-MS

A conclusion figure6 form Pujol 2018, [5] A TCA cartoon with the entry point of propionate and the exit of β-hydroxy butyrate are also shown,.

Long versus medium free fatty acids on FFAR1 and FFAR4 [6]

This study is nice in that the authors came up with a model that suggests medium chain fatty acids act as inhibitors.

Basic CCK functions

These comments are from a Medical Forum for physicians and medical students.

Cholecystokinin (CCK)

  • Source: – I cells (duodenum, jejunum)
  • Action: – increase pancreatic secretion; gallbladder contraction; Oddi sphincter relaxation – decreases gastric emptying
  • Regulation: – Inc: fatty acids, AAs
  • Notes: – CCK acts on neural muscarinic pathways to cause pancreatic secretion

GIP

  • Source: – K cells (duodenum, jejunum)
  • Action: exocrine: Decrease gastric H+ secretion endocrine: Increase insulin release Regulation: – Incresaes in FA, AA, oral glucose
  • Notes: – Glucose-dependent insulinotropic peptide aka gastric inhibitory peptide.
These images are from Figures 2 and 3 of Murata 2021 [6 CCK has three actions examined were examined. It is assumed that the CCK agonist is some other ligand that binds to and activates teh CCK receptor in the absence to CCK not secreted due to inhibition by MCFA. Left MCT mixed with the olive oil inhibited emptying of the gal bladder unless the CCK agonist was added. Right: Lipase activity. Figure 3 data were in fasted mice…

MCTs inhibit GPR120 but not GPR40 CCK secretion

Modeling fatty acids binding to GPR120

In silico molecular docking was based on active and inactive structures of bovine rhodopsin. For some reason Table1 focuses on Hydrogen bonding energy in arbitrary units. [6] This post will not go into hydrophobicity based interactions. The more negative the energy is, the more “downhill” the reaction is. Note that many of the fatty acids in Table1 are present in olive oil.

Agonist: When the energy of active form of the receptor is lower than that of inactive form of the receptor. C8 Caprylic acid binds with more negative energy to the inactive model of GPR120 supporting other results that it is an inhibitor.

Antagonist when the energy of inactive form is lower than that of in active form. C8 caprylic acid binds to the inactive state more strongly than C18 oleic acid.

Table 1 from Murata 2021 [6]

References

  1. Kimura I, Ichimura A, Ohue-Kitano R, Igarashi M. Free Fatty Acid Receptors in Health and Disease. Physiol Rev. 2020 Jan 1;100(1):171-210. free paper
  2. Aizawa F, Nishinaka T, Yamashita T, Nakamoto K, Kurihara T, Hirasawa A, Kasuya F, Miyata A, Tokuyama S. GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior. J Pharmacol Sci. 2016 Dec;132(4):249-254. PMC free article
  3. Hu C, Ding H, Zhuang Q, Llanos P, Pillay T, Hernandez C, Carpentier YA, Deckelbaum RJ, Chang CL. Blood clearance kinetics and organ delivery of medium-chain triglyceride and fish oil-containing lipid emulsions: Comparing different animal species. Clin Nutr. 2021 Mar;40(3):987-996. PMC free article
  4. Iwasaki K, Harada N, Sasaki K, Yamane S, Iida K, Suzuki K, Hamasaki A, Nasteska D, Shibue K, Joo E, Harada T, Hashimoto T, Asakawa Y, Hirasawa A, Inagaki N. (2015) Free fatty acid receptor GPR120 is highly expressed in enteroendocrine K cells of the upper small intestine and has a critical role in GIP secretion after fat ingestion. Endocrinology. 2015 Mar;156(3):837-46. 6. PMC free article
  5. Pujol JB, Christinat N, Ratinaud Y, Savoia C, Mitchell SE, Dioum EHM. (2018) Coordination of GPR40 and Ketogenesis Signaling by Medium Chain Fatty Acids Regulates Beta Cell Function. Nutrients. 2018 Apr 12;10(4):473. PMC free article
  6. Murata Y, Harada N, Kishino S, Iwasaki K, Ikeguchi-Ogura E, Yamane S, Kato T, Kanemaru Y, Sankoda A, Hatoko T, Kiyobayashi S, Ogawa J, Hirasawa A, Inagaki N. (2021) Medium-chain triglycerides inhibit long-chain triglyceride-induced GIP secretion through GPR120-dependent inhibition of CCK. iScience. 2021 Aug 9;24(9):102963 free article
  7. Grundmann M, Bender E, Schamberger J, Eitner F. Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators. Int J Mol Sci. 2021 Feb 10;22(4):1763. PMC free article

Published by BL

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