Abstract
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
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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Acknowledgements
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.
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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.
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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.
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Miller, R., Shi, Y., Lu, W. et al. Targeting hepatic glutaminase activity to ameliorate hyperglycemia. Nat Med 24, 518–524 (2018). https://doi.org/10.1038/nm.4514
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DOI: https://doi.org/10.1038/nm.4514
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