Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase

Abstract

Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5′-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P2), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet–fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Generation of the AMP-insensitive FBP1-G27P-KI mouse model.
Fig. 2: FBP1-G27P-KI mice display normal glucose homeostasis.
Fig. 3: FBP1-G27P-KI mice are resistant to the hypoglycemic action of an AMP-mimetic FBPase inhibitor.
Fig. 4: FBP1-G27P-KI mice are resistant to the hypoglycemic action of AICAR.
Fig. 5: FBP1-G27P-KI mice exhibit resistance to the acute glucose-lowering effect of metformin.
Fig. 6: FBP1-G27P-KI mice are resistant to the glucose lowering effects of metformin in an obesity-induced model of diabetes.

References

  1. 1.

    Rena, G., Pearson, E. R. & Sakamoto, K. Molecular mechanism of action of metformin: old or new insights? Diabetologia 56, 1898–1906 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Owen, M. R., Doran, E. & Halestrap, A. P. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem. J. 348, 607–614 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    El-Mir, M. Y. et al. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J. Biol. Chem. 275, 223–228 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Bridges, H. R., Jones, A. J., Pollak, M. N. & Hirst, J. Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria. Biochem. J. 462, 475–487 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Zhou, G. et al. Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108, 1167–1174 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Vogt, J., Traynor, R. & Sapkota, G. P. The specificities of small molecule inhibitors of the TGFß and BMP pathways. Cell. Signal. 23, 1831–1842 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. 7.

    Fullerton, M. D. et al. Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin. Nat. Med. 19, 1649–1654 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Foretz, M. et al. Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state. J. Clin. Invest. 120, 2355–2369 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Miller, R. A. et al. Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP. Nature 494, 256–260 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Madiraju, A. K. et al. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 510, 542–546 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Hasenour, C. M. et al. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) effect on glucose production, but not energy metabolism, is independent of hepatic AMPK in vivo. J. Biol. Chem. 289, 5950–5959 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Vincent, M. F., Marangos, P. J., Gruber, H. E. & Van den Berghe, G. Inhibition by AICA riboside of gluconeogenesis in isolated rat hepatocytes. Diabetes 40, 1259–1266 (1991).

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Pagliara, A. S., Karl, I. E., Keating, J. P., Brown, B. I. & Kipnis, D. M. Hepatic fructose-1,6-diphosphatase deficiency. A cause of lactic acidosis and hypoglycemia in infancy. J. Clin. Invest. 51, 2115–2123 (1972).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Bouskila, M. et al. Allosteric regulation of glycogen synthase controls glycogen synthesis in muscle. Cell Metab. 12, 456–466 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Ouyang, J., Parakhia, R. A. & Ochs, R. S. Metformin activates AMP kinase through inhibition of AMP deaminase. J. Biol. Chem. 286, 1–11 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. 16.

    Gidh-Jain, M. et al. The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure. J. Biol. Chem. 269, 27732–27738 (1994).

    CAS  PubMed  Google Scholar 

  17. 17.

    Zhang, Y. et al. Fructose-1,6-bisphosphatase regulates glucose-stimulated insulin secretion of mouse pancreatic beta-cells. Endocrinology 151, 4688–4695 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. 18.

    Faupel, R. P., Seitz, H. J., Tarnowski, W., Thiemann, V. & Weiss, C. The problem of tissue sampling from experimental animals with respect to freezing technique, anoxia, stress and narcosis. A new method for sampling rat liver tissue and the physiological values of glycolytic intermediates and related compounds. Arch. Biochem. Biophys. 148, 509–522 (1972).

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Erion, M. D. et al. MB06322 (CS-917): a potent and selective inhibitor of fructose 1,6-bisphosphatase for controlling gluconeogenesis in type 2diabetes. Proc. Natl. Acad. Sci. USA 102, 7970–7975 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    Vincent, M. F., Erion, M. D., Gruber, H. E. & Van den Berghe, G. Hypoglycaemic effect of AICAriboside in mice. Diabetologia 39, 1148–1155 (1996).

    Article  CAS  PubMed  Google Scholar 

  21. 21.

    Guigas, B. et al. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside and metformin inhibit hepatic glucose phosphorylation by an AMP-activated protein kinase-independent effect on glucokinase translocation. Diabetes 55, 865–874 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Vincent, M. F., Bontemps, F. & Van den Berghe, G. Substrate cycling between 5-amino-4-imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes. Biochem. Pharmacol. 52, 999–1006 (1996).

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Hunter, R. W. et al. Mechanism of action of compound-13: an α1-selective small molecule activator of AMPK. Chem. Biol. 21, 866–879 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Bailey, C. J., Wilcock, C. & Scarpello, J. H. Metformin and the intestine. Diabetologia 51, 1552–1553 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Yoshida, T. et al. Metformin primarily decreases plasma glucose not by gluconeogenesis suppression but by activating glucose utilization in a non-obese type 2 diabetes Goto-Kakizaki rats. Eur. J. Pharmacol. 623, 141–147 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Takashima, M. et al. Role of KLF15 in regulation of hepatic gluconeogenesis and metformin action. Diabetes 59, 1608–1615 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Stepensky, D., Friedman, M., Raz, I. & Hoffman, A. Pharmacokinetic–pharmacodynamic analysis of the glucose-lowering effect of metformin in diabetic rats reveals first-pass pharmacodynamic effect. Drug Metab. Dispos. 30, 861–868 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. 28.

    Duca, F. A. et al. Metformin activates a duodenal Ampk-dependent pathway to lower hepatic glucose production in rats. Nat. Med. 21, 506–511 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Wu, H. et al. Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug. Nat. Med. 23, 850–858 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M. & Koepsell, H. Drug excretion mediated by a new prototype of polyspecific transporter. Nature 372, 549–552 (1994).

    Article  PubMed  Google Scholar 

  31. 31.

    Kjøbsted, R. et al. Prior AICAR stimulation increases insulin sensitivity in mouse skeletal muscle in an AMPK-dependent manner. Diabetes 64, 2042–2055 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Shaw, R. J. et al. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 310, 1642–1646 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Patel, K. et al. The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver. Nat. Commun. 5, 4535 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Samuel, V. T. et al. Fasting hyperglycemia is not associated with increased expression of PEPCK or G6Pc in patients with type 2 diabetes. Proc. Natl. Acad. Sci. USA 106, 12121–12126 (2009).

  35. 35.

    Cool, B. et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 3, 403–416 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Graham, G. G. et al. Clinical pharmacokinetics of metformin. Clin. Pharmacokinet. 50, 81–98 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. 37.

    Christensen, M. M. et al. The pharmacogenetics of metformin and its impact on plasma metformin steady-state levels and glycosylated hemoglobin A1c. Pharmacogenet. Genomics 21, 837–850 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. 38.

    Lalau, J. D., Lemaire-Hurtel, A. S. & Lacroix, C. Establishment of a database of metformin plasma concentrations and erythrocyte levels in normal and emergency situations. Clin. Drug Investig. 31, 435–438 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. 39.

    Bleasby, K. et al. Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical species: a resource for investigations into drug disposition. Xenobiotica 36, 963–988 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. 40.

    Terada, T. et al. Molecular cloning, functional characterization and tissue distribution of rat H + /organic cation antiporter MATE1. Pharm. Res. 23, 1696–1701 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. 41.

    Gormsen, L. C. et al. In vivo imaging of human 11c-metformin in peripheral organs: dosimetry, biodistribution, and kinetic analyses. J. Nucl. Med. 57, 1920–1926 (2016).

    Article  CAS  PubMed  Google Scholar 

  42. 42.

    Jensen, J. B. et al. [11C]-labeled metformin distribution in the liver and small intestine using dynamic positron emission tomography in mice demonstrates tissue-specific transporter dependency. Diabetes 65, 1724–1730 (2016).

    Article  CAS  PubMed  Google Scholar 

  43. 43.

    Chen, L. et al. OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin. Proc. Natl. Acad. Sci. USA 111, 9983–9988 (2014).

    Article  CAS  PubMed  Google Scholar 

  44. 44.

    Hawley, S. A., Gadalla, A. E., Olsen, G. S. & Hardie, D. G. The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes 51, 2420–2425 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. 45.

    Hawley, S. A. et al. Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation. Cell Metab. 11, 554–565 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Argaud, D., Roth, H., Wiernsperger, N. & Leverve, X. M. Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes. Eur. J Biochem. 213, 1341–1348 (1993).

    Article  CAS  PubMed  Google Scholar 

  47. 47.

    McCarty, M. F. A proposal for the locus of metformin’s clinical action: potentiation of the activation of pyruvate kinase by fructose-1,6-diphosphate. Med. Hypotheses 52, 89–93 (1999).

    Article  CAS  PubMed  Google Scholar 

  48. 48.

    van Poelje, P. D., Dang, Q. & Erion, M. D. Discovery of fructose-1,6-bisphosphatase inhibitors for the treatment of type 2 diabetes. Curr. Opin. Drug Discov. Devel. 10, 430–437 (2007).

    PubMed  Google Scholar 

  49. 49.

    Tao, H., Zhang, Y., Zeng, X., Shulman, G. I. & Jin, S. Niclosamide ethanolamine–induced mild mitochondrial uncoupling improves diabetic symptoms in mice. Nat. Med. 20, 1263–1269 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Dang, Q. et al. Discovery of potent and specific fructose-1,6-bisphosphatase inhibitors and a series of orally-bioavailable phosphoramidase-sensitive prodrugs for the treatment of type 2 diabetes. J. Am. Chem. Soc. 129, 15491–15502 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. 51.

    Giroux, E., Williams, M. K. & Kantrowitz, E. R. Shared active sites of fructose-1,6-bisphosphatase. Arginine 243 mediates substrate binding and fructose 2,6-bisphosphate inhibition. J. Biol. Chem. 269, 31404–31409 (1994).

    CAS  PubMed  Google Scholar 

  52. 52.

    Tashima, Y., Mizunuma, H. & Hasegawa, M. Purification and properties of mouse liver fructose 1,6-bisphosphatase. J. Biochem. 86, 1089–1099 (1979).

  53. 53.

    Smiley, K. L. Jr., Berry, A. J. & Suelter, C. H. An improved purification, crystallization, and some properties of rabbit muscle 5′-adenylic acid deaminase. J. Biol. Chem. 242, 2502–2506 (1967).

    CAS  PubMed  Google Scholar 

  54. 54.

    Han, P., Han, G., McBay, H. & Johnson, J. Adenosine 5′-monophosphate-removing system in fructose-1,6-bisphosphatase assay mixture: a new approach. Anal. Biochem. 122, 269–273 (1982).

    Article  CAS  PubMed  Google Scholar 

  55. 55.

    Nelson, S. W., Choe, J. Y., Honzatko, R. B. & Fromm, H. J. Mutations in the hinge of a dynamic loop broadly influence functional properties of fructose-1,6-bisphosphatase. J. Biol. Chem. 275, 29986–29992 (2000).

    Article  CAS  PubMed  Google Scholar 

  56. 56.

    Chomczynski, P. & Rymaszewski, M. Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. Biotechniques 40, 454 (2006). 456, 458.

    Article  CAS  PubMed  Google Scholar 

  57. 57.

    Zarghi, A., Foroutan, S. M., Shafaati, A. & Khoddam, A. Rapid determination of metformin in human plasma using ion-pair HPLC. J. Pharm. Biomed. Anal. 31, 197–200 (2003).

    Article  CAS  PubMed  Google Scholar 

  58. 58.

    Nakamura, K., Maeda, H. & Kawaguchi, H. Enzymatic assay of hemoglobin in tissue homogenates with chlorpromazine. Anal. Biochem. 165, 28–32 (1987).

    Article  CAS  PubMed  Google Scholar 

  59. 59.

    Bosselaar, M., Smits, P., van Loon, L. J. & Tack, C. J. Intravenous AICAR during hyperinsulinemia induces systemic hemodynamic changes but has no local metabolic effect. J. Clin. Pharmacol. 51, 1449–1458 (2011).

    Article  CAS  PubMed  Google Scholar 

  60. 60.

    Ayala, J.E. et al. Hyperinsulinemic–euglycemic clamps in conscious, unrestrained mice. J. Vis. Exp. (57), e3188 (2011).

  61. 61.

    Hasenour, C. M. et al. Mass spectrometry–based microassay of 2H and 13C plasma glucose labeling to quantify liver metabolic fluxes in vivo. Am. J. Physiol. Endocrinol. Metab. 309, E191–E203 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Antoniewicz, M. R., Kelleher, J. K. & Stephanopoulos, G. Measuring deuterium enrichment of glucose hydrogen atoms by gas chromatography/mass spectrometry. Anal. Chem. 83, 3211–3216 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Young, J. D. INCA: a computational platform for isotopically non-stationary metabolic flux analysis. Bioinformatics 30, 1333–1335 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Antoniewicz, M. R., Kelleher, J. K. & Stephanopoulos, G. Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab. Eng. 8, 324–337 (2006).

    Article  CAS  PubMed  Google Scholar 

  65. 65.

    Landau, B. R. et al. Contributions of gluconeogenesis to glucose production in the fasted state. J. Clin. Invest. 98, 378–385 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Satapati, S. et al. Elevated TCA cycle function in the pathology of diet-induced hepatic insulin resistance and fatty liver. J. Lipid Res. 53, 1080–1092 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Jakobsen, S. et al. A PET tracer for renal organic cation transporters, 11C-metformin: radiosynthesis and preclinical proof-of-concept studies. J. Nucl. Med. 57, 615–621 (2016).

  68. 68.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Keppler, D. & Decker, K. Glycogen determination with amyloglucosidase. in Methods of Enzymatic Analysis, Vol. 3 (ed. Bergmeyer, H.U.) 1127–1131 (Verlag Chemie, Weinheim, Germany, 1974).

  70. 70.

    Ryll, T. & Wagner, R. Improved ion-pair high-performance liquid chromatographic method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells. J. Chromatogr. 570, 77–88 (1991).

    Article  CAS  PubMed  Google Scholar 

  71. 71.

    Noll, F. Methods of Enzymatic Analysis 3rd edn, Vol. 6 (ed. Bergmeyer, H. U.) 582–588 (Verlag Chemie, Weinheim, Germany, 1984).

  72. 72.

    Passonneau, J. V. & Lowry, O. H. Enzymatic analysis. A practical guide. (Humana Press, New York, 1993).

    Google Scholar 

  73. 73.

    Racker, E. Methods of enzymatic analysis 1st edn (ed. Bergmeyer, H. U.) 160–163 (Verlag Chemie, Weinheim, Germany, 1965).

  74. 74.

    Van Schaftingen, E., Lederer, B., Bartrons, R. & Hers, H. G. A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate. Eur. J. Biochem. 129, 191–195 (1982).

    Article  PubMed  Google Scholar 

  75. 75.

    Davidson, A. L. & Arion, W. J. Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity. Arch. Biochem. Biophys. 253, 156–167 (1987).

    Article  CAS  PubMed  Google Scholar 

  76. 76.

    Castaño, J. G., Nieto, A. & Felíu, J. E. Inactivation of phosphofructokinase by glucagon in rat hepatocytes. J. Biol. Chem. 254, 5576–5579 (1979).

    PubMed  Google Scholar 

  77. 77.

    Blair, J. B., Cimbala, M. A., Foster, J. L. & Morgan, R. A. Hepatic pyruvate kinase. Regulation by glucagon, cyclic adenosine 3′-5′-monophosphate, and insulin in the perfused rat liver. J. Biol. Chem. 251, 3756–3762 (1976).

    CAS  PubMed  Google Scholar 

  78. 78.

    Petrescu, I. et al. Determination of phosphoenolpyruvate carboxykinase activity with deoxyguanosine 5′-diphosphate as nucleotide substrate. Anal. Biochem. 96, 279–281 (1979).

    Article  CAS  PubMed  Google Scholar 

  79. 79.

    Saheki, S., Takeda, A. & Shimazu, T. Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity. Anal. Biochem. 148, 277–281 (1985).

    Article  CAS  PubMed  Google Scholar 

  80. 80.

    Srere, P. A. Citrate synthase. Methods Enzymol. 13, 3–11 (1969).

    Article  CAS  Google Scholar 

  81. 81.

    Thomas, J. A., Schlender, K. K. & Larner, J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal. Biochem. 25, 486–499 (1968).

    Article  CAS  PubMed  Google Scholar 

  82. 82.

    Gilboe, D. P., Larson, K. L. & Nuttall, F. Q. Radioactive method for the assay of glycogen phosphorylases. Anal. Biochem. 47, 20–27 (1972).

    Article  CAS  PubMed  Google Scholar 

  83. 83.

    Stalmans, W. & Hers, H. G. The stimulation of liver phosphorylase b by AMP, fluoride and sulfate. A technical note on the specific determination of the a and b forms of liver glycogen phosphorylase. Eur. J. Biochem. 54, 341–350 (1975).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Deak for molecular biology assistance and S. Jakobsen and J. Frøkiær for support in method development of the [11C]metformin-uptake study. We also thank E. Heikkilä for performing islet isolation, S. Ducommun for performing the pTBC1D1 blot, and S. Cotting for constructing Wollenberger tongs.GLUT2 antibody was provided by B. Thorens, GCK/HXK4 antibody was provided by M. Magnuson, GCKR antibody was provided by M. Shiota, G6PC antibody was provided by G. Mithieux, PFKFB1 antibody was provided by S. Baltrusch, pS33 PFKFB1 antibody was provided by J. Xie and pS8 GYS2 antibody was provided by J. Guinovart. This study was supported by Vanderbilt Mouse Metabolic Phenotyping Center Grant DK059637 (D.H.W.) and R37 DK050277 (D.H.W.), a Foundation Grant (FND 143277) from the Canadian Institutes of Health Research (F.S.), the Danish Council for Independent Research DFF—4183-00384 (N.J.) and the Novo Nordisk Foundation NNF13OC0003882 (N.J.). E.Z. was supported by a Sir Henry Wellcome postdoctoral fellowship. C.C.H. was supported by a Canadian Diabetes Association postdoctoral fellowship.

Author information

Affiliations

Authors

Contributions

R.W.H. and K.S. designed the study. R.W.H. performed all biochemical assays and the majority of in vivo experiments, assisted by K.S. Analysis of FBP1 structure and design of the mutants were performed by E.Z. and F.S. M.P. performed molecular cloning and mutagenesis of FBP1. N.J. and E.I.S. performed the [11C]metformin-uptake kinetics study and analyzed the data. C.C.H. and L.L. performed the metformin euglycemic clamp and analyzed the data. D.H.W. supervised C.C.H. and L.L. and contributed to interpretation of data from the clamp study. R.W.H and K.S. wrote the manuscript. All authors reviewed, edited and approved the manuscript.

Corresponding author

Correspondence to Kei Sakamoto.

Ethics declarations

Competing interests

K.S. is a full-time employee of the Nestlé Institute of Health Sciences S.A., Switzerland.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–9 and Supplementary Tables 1–7

Reporting Summary

Combined Source Data

Uncropped gels for Figs. 2h, 4k 5d, and Supplementary Fig. 6a

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hunter, R.W., Hughey, C.C., Lantier, L. et al. Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase. Nat Med 24, 1395–1406 (2018). https://doi.org/10.1038/s41591-018-0159-7

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing