Glucagon levels increase under homeostatic, fasting conditions, promoting the release of glucose from the liver by accelerating the breakdown of glycogen (also known as glycogenolysis). Glucagon also enhances gluconeogenic flux, including from an increase in the hepatic consumption of amino acids1. In type 2 diabetes, dysregulated glucagon signaling contributes to the elevated hepatic glucose output and fasting hyperglycemia that occur in this condition. Yet, the mechanism by which glucagon stimulates gluconeogenesis remains incompletely understood. Contrary to the prevailing belief that glucagon acts primarily on cytoplasmic and nuclear targets, we find glucagon-dependent stimulation of mitochondrial anaplerotic flux from glutamine that increases the contribution of this amino acid to the carbons of glucose generated during gluconeogenesis. This enhanced glucose production is dependent on protein kinase A (PKA) and is associated with glucagon-stimulated calcium release from the endoplasmic reticulum, activation of mitochondrial α-ketoglutarate dehydrogenase, and increased glutaminolysis. Mice with reduced levels of hepatic glutaminase 2 (GLS2), the enzyme that catalyzes the first step in glutamine metabolism, show lower glucagon-stimulated glutamine-to-glucose flux in vivo, and GLS2 knockout results in higher fasting plasma glucagon and glutamine levels with lower fasting blood glucose levels in insulin-resistant conditions. As found in genome-wide association studies (GWAS), human genetic variation in the region of GLS2 is associated with higher fasting plasma glucose2,3; here we show in human cryopreserved primary hepatocytes in vitro that these natural gain-of-function missense mutations in GLS2 result in higher glutaminolysis and glucose production. These data emphasize the importance of gluconeogenesis from glutamine, particularly in pathological states of increased glucagon signaling, while suggesting a possible new therapeutic avenue to treat hyperglycemia.

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Change history

  • Corrected online 12 June 2018

    In the version of this article initially published, the "[13C2]α-ketoglutarate" label on Fig. 1g is incorrect. It should be "[13C5]α-ketoglutarate". Additionally, in Fig. 3b, the "AAV-GFP" group is missing a notation for significance, and in Fig. 3c, the "AAV-GLS2-sh" group is missing a notation for significance. There should be a double asterisk notating significance in both panels. Finally, in the Fig. 4g legend, "[13C6]UDP-glucose" should be "[13C3]UDP-glucose", and in the Fig. 4h legend, "[13C6]hexose" should be "[13C3]hexose". The errors have been corrected in the HTML and PDF versions of this article.


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All data generated and analyzed within this study are presented in the article and supplementary procedures. The stable-isotope-based metabolomics work was supported by US NIH grant CA211437 to W.L. This work was also supported by FONDECYT grant 1160332 to C.C. and CONICYT/FONDAP 15150012 to C.C.

Author information

Author notes

    • Russell A Miller
    • , Yuji Shi
    •  & Wenyun Lu

    These authors contributed equally to this work.


  1. Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Russell A Miller
    •  & Morris J Birnbaum
  2. Pfizer Internal Medicine Research Units, Cambridge, Massachusetts, USA.

    • Russell A Miller
    • , Yuji Shi
    • , Aditi Jatkar
    • , Min Wan
    •  & Morris J Birnbaum
  3. Chemistry and Integrative Genomics, Princeton University, Princeton, New Jersey, USA.

    • Wenyun Lu
    • , Junyoung O Park
    •  & Joshua D Rabinowitz
  4. Pfizer Worldwide Research and Development, Groton, Connecticut, USA.

    • David A Pirman
    • , Matthew Blatnik
    •  & Hong Wu
  5. Anatomy and Developmental Biology Program, Institute of Biomedical Sciences, University of Chile, Santiago, Chile.

    • César Cárdenas
  6. Geroscience Center for Brain Health and Metabolism, Santiago, Chile.

    • César Cárdenas
  7. Buck Institute for Research on Aging, Novato, California, USA.

    • César Cárdenas
  8. Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, California, USA.

    • César Cárdenas
  9. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • J Kevin Foskett
  10. Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • J Kevin Foskett
    •  & Morris J Birnbaum
  11. Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, USA.

    • Junyoung O Park
  12. Touchstone Diabetes Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

    • Yiyi Zhang
    •  & William L Holland


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R.A.M. designed and performed all experiments and drafted and edited the manuscript. Y.S. designed and performed experiments related to GLS2 knockout and in vivo infusion and drafted and edited the manuscript. W.L. performed mass spectrometry experiments in rodent cells and tissues and edited the manuscript. D.A.P. performed mass spectrometry experiments in human hepatocytes and edited the manuscript. A.J. performed experiments on human hepatocytes and edited the manuscript. M.B. performed mass spectrometry experiments in human hepatocytes and edited the manuscript. H.W. performed experiments on human hepatocytes and edited the manuscript. C.C. performed mouse hepatocyte calcium experiments and edited the manuscript. M.W. performed experiments related to GLS2 knockout and in vivo infusions and edited the manuscript. J.K.F. edited the manuscript. J.O.P. performed flux modeling experiments and edited the manuscript. Y.Z. performed pancreas histology experiments and edited the manuscript. W.L.H. designed pancreas histology experiments and edited the manuscript. J.D.R. designed experiments and drafted and edited the manuscript. M.J.B. designed experiments and drafted and edited the manuscript.

Competing interests

R.A.M., Y.S., D.A.P., A.J., M.B., H.W., M.W., and M.J.B. were employed by Pfizer during the reported studies.

Corresponding authors

Correspondence to Russell A Miller or Morris J Birnbaum.

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