CoChat
Sign Up
Cited by 287Metabolism, Diabetes, and CancerBiochemical and Molecular ResearchPeroxisome Proliferator-Activated Receptors

The DOI matches its Crossref record; 52 of the 52 references it cites were found in a registry or an index.

Metformin activates AMP-activated protein kinase in primary human hepatocytes by decreasing cellular energy status

Open access
Stephenne, X., Foretz, M., Taleux, N., van der Zon, G. C., Sokal, E., Hue, L., Viollet, B., Guigas, B.·2011·Diabetologia
DOI 10.1007/s00125-011-2311-5PMC PMC3210354
Open this paper in CoChat — free accountOpen PDF

Abstract

AIM/HYPOTHESIS: The glucose-lowering drug metformin has been shown to activate hepatic AMP-activated protein kinase (AMPK), a master kinase regulating cellular energy homeostasis. However, the underlying mechanisms remain controversial and have never been investigated in primary human hepatocytes. METHODS: Hepatocytes isolated from rat, mouse and human livers were treated with various concentrations of metformin. Isoform-specific AMPKα abundance and activity, as well as intracellular adenine nucleotide levels and mitochondrial oxygen consumption rates were determined at different time points. RESULTS: Metformin dose- and time-dependently increased AMPK activity in rat and human hepatocytes, an effect associated with a significant rise in cellular AMP:ATP ratio. Surprisingly, we found that AMPKα2 activity was undetectable in human compared with rat hepatocytes, while AMPKα1 activities were comparable. Accordingly, metformin only increased AMPKα1 activity in human hepatocytes, although both AMPKα isoforms were activated in rat hepatocytes. Analysis of mRNA expression and protein levels confirmed that only AMPKα1 is present in human hepatocytes; it also showed that the distribution of β and γ regulatory subunits differed between species. Finally, we demonstrated that the increase in AMP:ATP ratio in hepatocytes from liver-specific Ampkα1/2 (also known as Prkaa1/2) knockout mice and humans is due to a similar and specific inhibition of the mitochondrial respiratory-chain complex 1 by metformin. CONCLUSIONS/INTERPRETATION: Activation of hepatic AMPK by metformin results from a decrease in cellular energy status owing to metformin's AMPK-independent inhibition of the mitochondrial respiratory-chain complex 1. The unique profile of AMPK subunits found in human hepatocytes should be considered when developing new pharmacological agents to target the kinase.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1007/s00125-011-2311-5

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig. 1 Time- and dose-dependent effects of metformin on AMPK activity and intracellular adenine nucleotide levels in primary rat hepatocytes. Primary cultured rat hepatocytes were incubated for 0, 0.5, 3, 6, 9, 18 and 24 h in M199 medium with different concentrations of metformin (white triangle, 0.5 mmol/l; white square, 1 mmol/l; white diamond, 5 mmol/l) or vehicle (black dots). a AMPK activity was measured after polyethylene glycol fractionation. b Intracellular ATP and (c) AMP concentrations were determined by HPLC, and the AMP:ATP ratios (d) calculated. Results are expressed as means ± SEM; n = 3–4; *p < 0.05 compared with control-vehicle
Fig. 1 Time- and dose-dependent effects of metformin on AMPK activity and intracellular adenine nucleotide levels in primary rat hepatocytes. Primary cultured rat hepatocytes were incubated for 0, 0.5, 3, 6, 9, 18 and 24 h in M199 medium with different concentrations of metformin (white triangle, 0.5 mmol/l; white square, 1 mmol/l; white diamond, 5 mmol/l) or vehicle (black dots). a AMPK activity was measured after polyethylene glycol fractionation. b Intracellular ATP and (c) AMP concentrations were determined by HPLC, and the AMP:ATP ratios (d) calculated. Results are expressed as means ± SEM; n = 3–4; *p < 0.05 compared with control-vehicleCC BY-NC · PubMed Central
Fig. 2 Dose-dependent effects of metformin on AMPK activity and the AMP:ATP ratio in primary rat and human hepatocytes. a–c Primary cultured hepatocytes isolated from rats or (d–f) human liver segments were incubated for 24 h in M199 medium with increasing concentrations of metformin (white bars) or vehicle (black bars). a, d AMPK activity was measured after polyethylene glycol fractionation and the AMP:ATP ratios (b, e) were calculated after determination of intracellular adenine nucleotides by HPLC. c, f The phosphorylation states (p) of Thr172-AMPK and Ser79-ACC, and the abundance of AMPKα were assessed by western blot. Results (a, b, d, e) are expressed as means ± SEM; n = 3–4; *p < 0.05 compared with control-vehicle
Fig. 2 Dose-dependent effects of metformin on AMPK activity and the AMP:ATP ratio in primary rat and human hepatocytes. a–c Primary cultured hepatocytes isolated from rats or (d–f) human liver segments were incubated for 24 h in M199 medium with increasing concentrations of metformin (white bars) or vehicle (black bars). a, d AMPK activity was measured after polyethylene glycol fractionation and the AMP:ATP ratios (b, e) were calculated after determination of intracellular adenine nucleotides by HPLC. c, f The phosphorylation states (p) of Thr172-AMPK and Ser79-ACC, and the abundance of AMPKα were assessed by western blot. Results (a, b, d, e) are expressed as means ± SEM; n = 3–4; *p < 0.05 compared with control-vehicleCC BY-NC · PubMed Central
Fig. 3 Effects of metformin on AMPKα1 and α2 activities. a Primary cultured hepatocytes isolated from rats or (b) human liver segments were incubated for 24 h in M199 medium in the presence of 500 μmol/l metformin (white bars) or vehicle (black bars). AMPKα1 and α2 activity was measured after immunoprecipitation with isoform-specific antibodies. Results are expressed as means ± SEM; n = 3–4; *p < 0.05 compared with control-vehicle
Fig. 3 Effects of metformin on AMPKα1 and α2 activities. a Primary cultured hepatocytes isolated from rats or (b) human liver segments were incubated for 24 h in M199 medium in the presence of 500 μmol/l metformin (white bars) or vehicle (black bars). AMPKα1 and α2 activity was measured after immunoprecipitation with isoform-specific antibodies. Results are expressed as means ± SEM; n = 3–4; *p < 0.05 compared with control-vehicleCC BY-NC · PubMed Central
Fig. 4 a Protein levels of AMPK subunits and (b) the corresponding mRNA gene expression were determined in hepatocytes isolated from human (black bars), rat (hatched bars) or mouse (white bars) livers by western blot and RT quantitative PCR, respectively. Western blot analysis (a) is representative of three separate experiments and shows that AMPK subunits migrated to their expected molecular mass (α1, 62 kDa; α2, 62 kDa; β1, 38 kDa; β2, 30 kDa; γ1, 38 kDa; γ2full-length, ∼75 kDa; γ2truncated, ∼55 kDa; γ3, 55 kDa). The western blots for AMPKα2 were performed using three different primary antibodies cross-reacting with human, rat and mouse forms (see ESM Table 1). b The results from RT quantitative PCR were corrected for GAPDH and are expressed as means ± SEM (n = 3). a.u., arbitrary units
Fig. 4 a Protein levels of AMPK subunits and (b) the corresponding mRNA gene expression were determined in hepatocytes isolated from human (black bars), rat (hatched bars) or mouse (white bars) livers by western blot and RT quantitative PCR, respectively. Western blot analysis (a) is representative of three separate experiments and shows that AMPK subunits migrated to their expected molecular mass (α1, 62 kDa; α2, 62 kDa; β1, 38 kDa; β2, 30 kDa; γ1, 38 kDa; γ2full-length, ∼75 kDa; γ2truncated, ∼55 kDa; γ3, 55 kDa). The western blots for AMPKα2 were performed using three different primary antibodies cross-reacting with human, rat and mouse forms (see ESM Table 1). b The results from RT quantitative PCR were corrected for GAPDH and are expressed as means ± SEM (n = 3). a.u., arbitrary unitsCC BY-NC · PubMed Central
Fig. 5 Effects of metformin on AMPK activity, AMP:ATP ratio, and cellular and mitochondrial JO2 in intact or permeabilised hepatocytes from wild-type and liver-specific Ampkα1/2 −/− mice. Hepatocytes from wild-type or Ampkα1/2 −/− mice were incubated at 37°C in a Krebs/bicarbonate medium containing lactate/pyruvate/octanoate (20, 2, 4 mmol/l) in the presence of vehicle (black bars) or 5 mmol/l metformin (white bars). After 30 min incubation, AMPK activity in U/g of dry cells (a), isoform-specific AMPKα abundance and Ser79-ACC phosphorylation (p) state (b), and the AMP:ATP ratio (c) were determined. d–i Oligomycin-sensitive JO2 was measured after the successive addition of 6 μg/ml oligomycin (basal) and 100 μmol/l DNP in separate experiments, after hepatocytes from wild-type (d, f, h) and liver-specific Ampkα1/2 −/− (e, g, i) mice had been incubated as described above and permeabilised in a KCl medium containing 200 μg/ml digitonine. JO2 was measured in the presence of glutamate/malate (5 mmol/l; 2.5 mmol/l) (f, g) or succinate/malate/rotenone (5 mmol/l; 0.5 mmol/l; 1.25 μmol/l) (h, i) after the successive addition of 1 mmol/l ADP (state 3), 6 μg/ml oligomycin (state 4) and 75 μmol/l DNP. All JO2 results are expressed as percentage of the maximum activity of cytochrome oxidase (cyt.ox) determined after the final addition of 0.15 μg/ml antimycin and TMPD/ascorbate (1 mmol/l, 5 mmol/l). Results are expressed as means ± SEM; n = 3; *p < 0.05 compared with control-vehicle; † p < 0.05 compared with wild-type mice
Fig. 5 Effects of metformin on AMPK activity, AMP:ATP ratio, and cellular and mitochondrial JO2 in intact or permeabilised hepatocytes from wild-type and liver-specific Ampkα1/2 −/− mice. Hepatocytes from wild-type or Ampkα1/2 −/− mice were incubated at 37°C in a Krebs/bicarbonate medium containing lactate/pyruvate/octanoate (20, 2, 4 mmol/l) in the presence of vehicle (black bars) or 5 mmol/l metformin (white bars). After 30 min incubation, AMPK activity in U/g of dry cells (a), isoform-specific AMPKα abundance and Ser79-ACC phosphorylation (p) state (b), and the AMP:ATP ratio (c) were determined. d–i Oligomycin-sensitive JO2 was measured after the successive addition of 6 μg/ml oligomycin (basal) and 100 μmol/l DNP in separate experiments, after hepatocytes from wild-type (d, f, h) and liver-specific Ampkα1/2 −/− (e, g, i) mice had been incubated as described above and permeabilised in a KCl medium containing 200 μg/ml digitonine. JO2 was measured in the presence of glutamate/malate (5 mmol/l; 2.5 mmol/l) (f, g) or succinate/malate/rotenone (5 mmol/l; 0.5 mmol/l; 1.25 μmol/l) (h, i) after the successive addition of 1 mmol/l ADP (state 3), 6 μg/ml oligomycin (state 4) and 75 μmol/l DNP. All JO2 results are expressed as percentage of the maximum activity of cytochrome oxidase (cyt.ox) determined after the final addition of 0.15 μg/ml antimycin and TMPD/ascorbate (1 mmol/l, 5 mmol/l). Results are expressed as means ± SEM; n = 3; *p < 0.05 compared with control-vehicle; † p < 0.05 compared with wild-type miceCC BY-NC · PubMed Central
Fig. 6 Effects of metformin on JO2 in permeabilised human hepatocytes. Hepatocytes isolated from human liver segments were incubated in the presence of vehicle (black bars) or 5 mmol/l metformin (white bars), and the JO2 was measured in the presence of (a) glutamate/malate (5 mmol/l, 2.5 mmol/l) or (b) succinate/malate/rotenone (5 mmol/l; 0.5 mmol/l; 1.25 μmol/l) as described (Fig. 5). The results are expressed as means ± SEM; n = 3; *p < 0.05 compared with control-vehicle
Fig. 6 Effects of metformin on JO2 in permeabilised human hepatocytes. Hepatocytes isolated from human liver segments were incubated in the presence of vehicle (black bars) or 5 mmol/l metformin (white bars), and the JO2 was measured in the presence of (a) glutamate/malate (5 mmol/l, 2.5 mmol/l) or (b) succinate/malate/rotenone (5 mmol/l; 0.5 mmol/l; 1.25 μmol/l) as described (Fig. 5). The results are expressed as means ± SEM; n = 3; *p < 0.05 compared with control-vehicleCC BY-NC · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 52 references

Cited by

Sorted by citations

Items 201–285 of 285

Related

See all

Metadata

Authors
Stephenne, X., Foretz, M., Taleux, N., van der Zon, G. C., Sokal, E., Hue, L., Viollet, B., & Guigas, B.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • Inserm (FR)
  • Cliniques Universitaires Saint-Luc (BE)
  • de Duve Institute (BE)
  • Leiden University Medical Center (NL)
Corresponding author
Guigas, B.
ORCID
Venue
Diabetologia
Publisher
Springer Science and Business Media LLC
Published
2011-09-23
Volume
54
Issue
12
Pages
3101-3110
ISSN
  • 0012-186X (ISSN-L)
  • 1432-0428
DOI
10.1007/s00125-011-2311-5
PMC
PMC3210354
OpenAlex
W2042871909
Cited by
287
References
52
Citations by year
2026: 14 · 2025: 12 · 2024: 20 · 2023: 10 · 2022: 22 · 2021: 14 · 2020: 24 · 2019: 30 · 2018: 25 · 2017: 26 · 2016: 19 · 2015: 16 · 2014: 22 · 2013: 19 · 2012: 12
FWCI
6.97
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Diabetologia (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
  • Leiden Repository (Leiden University) (submittedVersion)
  • DIAL (Catholic University of Leuven) (submittedVersion)
Funders
  • European Commission (LSHMCT-2004-005272)
  • Agence Nationale de la Recherche (ANR-10-BLAN-1123, ANR-10)
  • Fonds De La Recherche Scientifique - FNRS
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Peroxisome Proliferator-Activated Receptors
Keywords
metformin, AMPK, AMPKα1, AMPKα2, human hepatocytes, cellular energy status, AMP/ATP ratio, mitochondrial complex I inhibition
MeSH terms
Adenine Nucleotides, Animals, Cells, Cultured, Humans, Hypoglycemic Agents, Isoenzymes, Male, Metformin, Mitochondria, Liver, Oxygen Consumption, Mice, Knockout, Hepatocytes, Mice, Rats, AMP-Activated Protein Kinases

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review