Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 6;32(4):643-653.e4.
doi: 10.1016/j.cmet.2020.07.012. Epub 2020 Aug 11.

Microbial Imidazole Propionate Affects Responses to Metformin through p38γ-Dependent Inhibitory AMPK Phosphorylation

Ara Koh  1 , Louise Mannerås-Holm  2 , Na-Oh Yunn  3 , Peter M Nilsson  4 , Sung Ho Ryu  3 , Antonio Molinaro  2 , Rosie Perkins  2 , J Gustav Smith  5 , Fredrik Bäckhed  6
Affiliations

Microbial Imidazole Propionate Affects Responses to Metformin through p38γ-Dependent Inhibitory AMPK Phosphorylation

Ara Koh et al. Cell Metab. .

Abstract

Metformin is the first-line therapy for type 2 diabetes, but there are large inter-individual variations in responses to this drug. Its mechanism of action is not fully understood, but activation of AMP-activated protein kinase (AMPK) and changes in the gut microbiota appear to be important. The inhibitory role of microbial metabolites on metformin action has not previously been investigated. Here, we show that concentrations of the microbial metabolite imidazole propionate are higher in subjects with type 2 diabetes taking metformin who have high blood glucose. We also show that metformin-induced glucose lowering is not observed in mice pretreated with imidazole propionate. Furthermore, we demonstrate that imidazole propionate inhibits AMPK activity by inducing inhibitory AMPK phosphorylation, which is dependent on imidazole propionate-induced basal Akt activation. Finally, we identify imidazole propionate-activated p38γ as a novel kinase for Akt and demonstrate that p38γ kinase activity mediates the inhibitory action of imidazole propionate on metformin.

Keywords: AMPK; diabetes; imidazole propionate; individual variations; metformin; microbial metabolites; microbiota; p38γ.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests A.K. and F.B. are shareholders in Implexion Pharma AB.

Figures

None
Graphical abstract
Figure 1
Figure 1
Imidazole Propionate Is Associated with Poor Glucose Control on Metformin (A) Plasma levels of imidazole propionate (ImP) or urocanate in metformin (Met)-treated subjects with type 2 diabetes divided according to glucose control. Poor glucose control is defined as glucose levels ≥7.8 mM, approximately 6.5% HbA1c. (B–G) Effects of ImP in metformin-treated mice models. Mice were injected intraperitoneally with ImP (100 μg) or vehicle (Veh, 1% DMSO) followed 1 h later by oral administration of Met (200 mg/kg) or water. (B) Percent change in fasting blood glucose levels in chow-fed mice 45 min after Met (or water) versus start of experiment. Veh+Water (n = 7), ImP+Water (n = 8), Veh+Met (n = 7), and ImP+Met (n = 7). (C) Intraperitoneal glucose tolerance tests in chow-fed mice treated with Veh+Water (n = 9) or ImP+Water (n = 10) (left) or Veh+Met (n = 9) or ImP+Met (n = 9) (right). (D) Serum insulin levels during intraperitoneal glucose tolerance tests shown in (C). (E) Percent changes in fasting blood glucose levels in western diet-fed mice 45 min after Met (or water) versus start of experiment. Veh+Water (n = 4), ImP+Water (n = 6), Veh+Met (n = 7), or ImP+Met (n = 6). (F) Intraperitoneal glucose tolerance tests in western diet-fed mice treated with Veh+Water (n = 4) or ImP+Water (n = 6) (left) or Veh+Met (n = 7) or ImP+Met (n = 6) (right). (G) Intraperitoneal glucose tolerance tests in diabetic (db/db) mice treated with Veh+Met (n = 7) or ImP+Met (n = 7). Data in (A) are presented as box plots showing minimum, 25% quartile, median, 75% quartile, maximum, and mean (marked as +). Other data are mean ± SEM.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. p values were determined by Wilcoxon rank-sum test (A), unpaired two-tailed Student’s t tests (C, F, and G), one-way ANOVA with Tukey’s multiple comparisons test (B and E), and two-way ANOVA with Tukey’s multiple comparisons test (D).
Figure 2
Figure 2
Imidazole Propionate Induces Inhibitory AMPK S485 Phosphorylation (A) Effects of imidazole propionate (ImP) on metformin (Met)-induced AMPK T172 phosphorylation in the liver. Mice were injected intraperitoneally with vehicle (1% DMSO in water) or ImP (100 μg) and after 1 h Met (200 mg/kg) was orally administered; liver was collected 45 min after Met administration (n = 3 mice per group). (B) Immunoblot (and quantification) of liver lysates taken from mice 105 min after one intraperitoneal injection of ImP. Vehicle (n = 3) and ImP (n = 4). (C) Immunoblot (and quantification) of liver lysates from mice (taken after an intraperitoneal glucose tolerance test) showing effects of oral administration of Met preceded by an intraperitoneal injection of vehicle or ImP. Vehicle (n = 3), Met (n = 4), and ImP+Met (n = 3). (D) Time-dependent effects of ImP (100 μM) on AMPK S485 phosphorylation in serum-starved HEK293 cells (n = 3). (E) Concentration-dependent effects of ImP on AMPK S485 phosphorylation in serum- and amino acid-deprived HEK293 cells (representative of n = 2). (F) Comparison of effects of ImP (100 μM) or amino acids (a.a) on AMPK S485 and S6K1 phosphorylation in serum- and amino acid-deprived HEK293 cells (representative of n = 2). (G) Role of AMPK S485 phosphorylation on inhibitory action of ImP on Met-induced AMPK T172 phosphorylation. HEK293 cells were transfected with HA AMPK wild-type (WT), HA AMPK S485A, or HA AMPK T479A mutant and incubated with 0.5 mM Met with or without ImP (100 μM) for 6 h (n = 3). Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. One-way ANOVA with Dunnett’s multiple comparisons test (A, D, and G), unpaired two-tailed Student’s t tests (B and C).
Figure 3
Figure 3
Imidazole Propionate-Induced Basal Akt Activation Is Responsible for Inhibitory AMPK S485 Phosphorylation (A) Effect of 2 h treatment with imidazole propionate (ImP) (at the indicated concentrations) on Akt S473 phosphorylation in amino acid-deprived HEK293 cells (n = 3). (B) Time-dependent effects of ImP (100 μM) on Akt phosphorylation in serum-starved HEK293 cells (n = 3). (C) Effects of mTORC1 inhibition (by 200 nM Rapamycin, Rap) or Akt inhibition (by 200 nM MK2206, MK) on ImP-induced Akt or mTORC1 activation. HEK293 cells preincubated with Rap or MK for 30 min were stimulated with 100 μM ImP for 1 h in the absence of amino acids (n = 4). (D and E) Effects of mTORC1 inhibition by rapamycin (20 nM Rap) or the Akt inhibitor MK (200 nM) on ImP-induced Akt or mTORC1 activation. Serum-starved HEK293 cells were co-incubated with 100 μM ImP and Rap or MK for 6 h (D) (n = 3) and for 8 h (E) (representative of n = 2). (F) mTORC1- and Akt-dependent inhibitory AMPK S485 phosphorylation by ImP. Serum-starved HEK293 cells were co-incubated with ImP and Rap or MK for 6 h (n = 3). Data are mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001. One-way ANOVA with Dunnett’s multiple comparisons test (A–D), one-way ANOVA with Tukey’s multiple comparisons test compared to ImP-treated groups (F).
Figure 4
Figure 4
Imidazole Propionate-Activated p38γ Is a Direct Kinase for Akt, Responsible for Mediating Inhibitory AMPK S485 Phosphorylation (A) In vitro kinase assay (n = 4). p38γ and inactive Akt1 were preincubated and the kinase reaction was started by adding ATP in the absence or presence of ImP at the indicated concentrations. (B) Effects of p38γ depletion on ImP (100 μM)-induced AMPK and Akt phosphorylation in serum-starved HEK293 cells (n = 3). (C) Effects of the p38γ inhibitor pirfenidone (Pirf) on ImP-induced inhibitory action on metformin (Met). Serum-starved HEK293 cells were co-incubated with 0.5 mM Met, 100 μM ImP, and Pirf at the indicated concentrations for 6 h (n = 3). (D and E) Effects of Pirf on ImP-induced inhibition of response to Met. Mice were injected intraperitoneally with vehicle (Veh, 1% DMSO), ImP (100 μg), Pirf (700 μg), or ImP with Pirf followed 1 h later by oral administration of Met (200 mg/kg) or water. (D) Immunoblot (and quantification) of liver lysates taken from chow-fed mice 45 min after Met (n = 4 mice per group). (E) Percent change in fasting blood glucose levels in chow-fed mice 45 min after Met (or water) versus start of experiment: Veh+Water (n = 7), Pirf+Water (n = 6), and Pirf+Met (n = 7) (left); Veh+Met (n = 4), ImP+Met (n = 4), and ImP+Met+Pirf (n = 4) (right). (F) Schematic depiction of imidazole propionate signaling. Interaction between Met and the ImP/p38γ/Akt/AMPK axis investigated in this study was shown together with previously reported ImP/alternative p38/p62/mTORC1 axis (Koh et al., 2018). Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. One-way ANOVA followed by Dunnett’s multiple comparisons test (A and B), one-way ANOVA followed by Tukey’s multiple comparisons test (C–E).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aeder S.E., Martin P.M., Soh J.W., Hussaini I.M. PKC-eta mediates glioblastoma cell proliferation through the Akt and mTOR signaling pathways. Oncogene. 2004;23:9062–9069. - PubMed
    1. Alessi D.R., Andjelkovic M., Caudwell B., Cron P., Morrice N., Cohen P., Hemmings B.A. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996;15:6541–6551. - PMC - PubMed
    1. American Diabetes Association 6. Glycemic targets: standards of medical care in diabetes-2019. Diabetes Care. 2019;42(Suppl 1):S61–S70. - PubMed
    1. Bachrach W.H. Sulfasalazine: I. An historical perspective. Am. J. Gastroenterol. 1988;83:487–496. - PubMed
    1. Bentzinger C.F., Romanino K., Cloëtta D., Lin S., Mascarenhas J.B., Oliveri F., Xia J., Casanova E., Costa C.F., Brink M. Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell Metab. 2008;8:411–424. - PubMed

Publication types

MeSH terms