Pendulum Probiotics

This post summarizes a Pendulum Biosciences clinical trial that tested the hypothesis the hypothesis that their three to five strain probiotic mix could improve glucose handing parameters in type 2 diabetes participants.

T2D in this adult population was  defined as a fasting glucose ≥126 mg/dL or glycated hemoglobin (A1c) of ≥6.8%.  Subjects were being treated with diet and exercise alone, or in combination with metformin with or without a sulfonylurea.  The body mass index of the participants was between 25 and 45 kg/m² were eligible to be considered eligible. This trial lasted for a total of 12 weeks.  Some of the plasma samples were reanalyzed for other markers after the results of the initial study were published.  Each subject received 12 bottles containing 45 size 0 capsules plus three more.  It is assumed that each bottle is per week and the participants were taking six capsules a day, presumably two with each meal.  Titanium dioxide and inulin were the inactive ingredients. Plasma was the source of most of the outcome measures.

Table Figure 2 ref [1], all about butyrate

The Pendulum Bio authors like to report their results as change. In Panel 2A they are reporting change in plasma short chain fatty acids. The new members Akkermansia muciniphila and Anaerobutyricum hallii resulted in a significant increase in butyrate compared to the placebo (p<0.05) and compared to the starting value (p=0.007).

The other short chain fatty acid plasma concentrations have not changed. Only in the WBF-011 group does the fecal butyrate content correlate with plasma butyrate content suggesting that the latter is the source of the former.

Does the change in the plasma butyrate correlate with the change in glycosylated hemoglobin, otherwise known as A1c? The answer is yes! As the plasma butyrate increases, the A1c decreases.

These results and other T2D markers are summarized in Table 2.

Table 2 ref [1]

Table 2 documents the MEAN changes from the baseline to the end of the study. 

In glucose AUC, area under the curve, time is on the X-axis and blood glucose conentration is on the Y-axis. The area is under the curve. The incremental AUC is usually associated with calculating the glycemic index of foods. The increment is blocks of time. White bread and pure glucose will result in a larger AUC for the first time increment than a food that is slower to break down. A1c is simply glycated hemoglobin. HOMA IR is a method to quantitate insulin resistance. The concentration of insulin and glucose are multiplied together and divided by a constant to give units of amount of glucose per unit volume.

Followup data analysis [2] sought to establish a mechanism

Probiotic dosing stopped at week 12. By week 14 only Clostridium beijerinckii was detected in in the feces of over 1/8th of the subjects.

  Since plasma and fecal butyrate were well correlated, this improvement may have been due to the effect of butyrate directly on the intestine or other targets once it was absorbed.  Urodeoxycholic acid and its glucoronidated conjugate was the only bile acid assayed for that increased in the plasma.  UDCA was shown to be a metabolite of Clostridium butyricum.  Only CDCA was able to support the production of UDCA by C butyricum in a mono culture.

The conversion of CDCA to UDCA is generally considered to be catalyzed by two enzymes:  7ɑ- and 7β-hydroxysteroid dehydrogenase (7ɑβ-HSDH).  The authors found evidence of these enzymes only in the genome of C butyricum and not the other probiotics.

Previous studies referenced in this report [2] showed that Akkermansia increased the antagonism of UDCA of the FXR receptor on enteroendocrine L cells.  The other bile acids are agonists of this receptor that inhibits the production of GLP-1.  Butyrate also stimulates release of GLP-1 from L-cells according to studies referenced in this report.

The Model

• The GPR41/43 post on this site reviews short chain fatty acid specificity of both receptors. GPR43 seems to “prefer” propionate. GPR41 seems to be less “picky.”
• TGR5 is primarily expressed in “glandular cells of the GI tract.
• Wiipedia authors have written a good page on GLP-1. Parts of the image on the Wikipedia site were melded with the Greiner and Bäckhed to fit into the Pedulum Bio model of how their probiotic might be working.
• Bile acids, produced by the liver and secreted into the duodenum, signal through distinct receptors such as TGR5 and the nuclear receptor farnesoid X receptor (FXR), both of which are expressed by L-cells.[3]
• FRX5 is a teterotrimeric G protein coupled receptor that increases the levels of cAMP, and therefor downstream Porotein Kinase A pathways.
• Farnesoid X Receptor, FXR, is a transcription factor. Chenodoxycholic acid (CDCA) is one of the activators. Activation of this transcription factor results in the INHIBITION of transcription of the gene for GLP-1 [4] FXR activation in L-cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. [4]
• The tricky point is that UDCA activates GLP-1 transcription by competing with the inhibitor CDCA.

• The GPR41&43 post discusses work from Brown 2002. A multitude of short chain fatty may activate increases in intracellular calcium levels that may play a role in releaseof GLP-1 from secretory vesicles. Note that propionate is a better activator of GPR41 and GPR43 than butyrate. And that there are other scfa activators….

Conclusions

1. Clostridium butyricum is a star in the GLP-1 show for converting CDCA to UDCA and releaving inhibition of GLP-1 secretion by L-cells
2. Akkermansia muncinophila, one of the new additions that makes the probiotic work, may be doing its thing by making propionate… that is the better activator of GRP41 and GPR43. The one issue is that propionate was not increased in the blood plasma. Perhaps this is a good thing.

References

1. Perraudeau F, McMurdie P, Bullard J, Cheng A, Cutcliffe C, Deo A, Eid J, Gines J, Iyer M, Justice N, Loo WT, Nemchek M, Schicklberger M, Souza M, Stoneburner B, Tyagi S, Kolterman O. (2020) Improvements to postprandial glucose control in subjects with type 2 diabetes: a multicenter, double blind, randomized placebo-controlled trial of a novel probiotic formulation. BMJ Open Diabetes Res Care. 2020 Jul;8(1):e001319 PMC free article
2. McMurdie PJ, Stoeva MK, Justice N, Nemchek M, Sieber CMK, Tyagi S, Gines J, Skennerton CT, Souza M, Kolterman O, Eid J. (2022) Increased circulating butyrate and ursodeoxycholate during probiotic intervention in humans with type 2 diabetes. BMC Microbiol. 2022 Jan 8;22(1):19. PMC free article
3. Greiner TU, Bäckhed F. (2016) Microbial regulation of GLP-1 and L-cell biology. Mol Metab. 2016 May 28;5(9):753-8. PMC free paper
4. Trabelsi MS, Daoudi M, Prawitt J, Ducastel S, Touche V, Sayin SI, Perino A, Brighton CA, Sebti Y, Kluza J, Briand O, Dehondt H, Vallez E, Dorchies E, Baud G, Spinelli V, Hennuyer N, Caron S, Bantubungi K, Caiazzo R, Reimann F, Marchetti P, Lefebvre P, Bäckhed F, Gribble FM, Schoonjans K, Pattou F, Tailleux A, Staels B, Lestavel S. (2015) Farnesoid X receptor inhibits glucagon-like peptide-1 production by enteroendocrine L cells. Nat Commun. 2015 Jul 2;6:7629. PMC free paper