Diabetes Volume 67, October 2018 1949 Liver Glutamate Dehydrogenase Controls Whole-Body Energy Partitioning Through Amino Acid–Derived Gluconeogenesis and Ammonia Homeostasis Melis Karaca,1,2 Juliette Martin-Levilain,1,2 Mariagrazia Grimaldi,1,2 Lingzi Li,1,2 Eva Dizin,1 Yalin Emre,3 and Pierre Maechler1,2 Diabetes 2018;67:1949–1961 | https://doi.org/10.2337/db17-1561 fi + Ammonia detoxi cation and gluconeogenesis are major further accumulation of the neurotoxic ammonium (NH4 ). hepatic functions mutually connected through amino The liver is the central organ of ammonia metabolism, acid metabolism. The liver is rich in glutamate dehy- andthemusclesandkidneysplayaroleintheinterorgan drogenase (GDH) that catalyzes the reversible oxidative exchange and final ammonia disposal (1,2). The liver has deamination of glutamate to a-ketoglutarate and ammo- a remarkable capacity to remove ammonia via the high- nia, thus bridging amino acid–to–glucose pathways. Here capacity/low-affinity urea cycle as well as low-capacity/ we generated inducible liver-specific GDH-knockout high-affinity glutamine synthetase (GS) (3). Both of these METABOLISM Glud12/2 mice (Hep ) to explore the role of hepatic GDH ammonia-removal systems engage glutamate as an in- on metabolic homeostasis. Investigation of nitrogen termediary metabolite. metabolism revealed altered ammonia homeostasis in Glutamine serves, along with alanine, as a major non- HepGlud12/2 mice characterized by increased circu- toxic interorgan ammonia shuttle in the body (2,4). Glu- lating ammonia associated with reduced detoxification tamine is deamidated to glutamate by glutaminase (GLS), process into urea. The abrogation of hepatic GDH also modified energy homeostasis. In the fasting state, thereby generating ammonia. Alanine aminotransferase HepGlud12/2 mice could barely produce glucose in re- (ALAT) catalyzes the transfer of an amino group from a a sponse to alanine due to impaired liver gluconeogenesis. the donor alanine to the acceptor -ketoglutarate ( -KG), Compared with control mice, lipid consumption in thereby producing pyruvate and glutamate. Then, gluta- 2/2 a HepGlud1 mice was favored over carbohydrates mate is deaminated to -KG through glutamate dehydro- as a compensatory energy fuel. The changes in energy genase (GDH), releasing NH3 for urea synthesis. GDH is partitioning induced by the lack of liver GDH modified abundant in the liver and is the only mammalian enzyme the circadian rhythm of food intake. Overall, this study that catalyzes the deamination of an amino acid. Despite demonstrates the central role of hepatic GDH as a major its specificity for glutamate, GDH, via the coupling to regulator for the maintenance of ammonia and whole- aminotransferase reactions, plays a key role in the de- body energy homeostasis. amination of most amino acids. Beside its role in nitrogen metabolism, the liver is also the major site for gluconeogenesis, which is a fundamental The turnover of amino acids is characterized by the re- process under metabolic-challenging conditions, such as spective metabolisms of nitrogen and carbon skeletons. fasting or sustained physical exercise (5–9). Primary car- The crucial steps for nitrogen metabolism are first, the bon skeletons used for gluconeogenesis may derive from removal of the amino group in the form of ammonia pyruvate, lactate, glycerol, and amino acids. During fasting, (NH3), and second, its detoxification into urea to prevent glutamine and alanine account for 60–80% of the amino 1Department of Cell Physiology and Metabolism, University of Geneva Medical Received 21 December 2017 and accepted 1 July 2018. School, Geneva, Switzerland This article contains Supplementary Data online at http://diabetes 2 Faculty Diabetes Center, University of Geneva Medical School, Geneva, Switzer- .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1561/-/DC1. land © 2018 by the American Diabetes Association. Readers may use this article as 3Department of Pathology and Immunology, University of Geneva Medical School, long as the work is properly cited, the use is educational and not for profit, and Geneva, Switzerland the work is not altered. More information is available at http://www.diabetesjournals Corresponding authors: Melis Karaca, [email protected], and Pierre .org/content/license. Maechler, [email protected]. 1950 Liver GDH and Energy Homeostasis Diabetes Volume 67, October 2018 acids released from the skeletal muscles and subsequently porin (ab34726; Abcam), G6Pase (developed by Dr. taken up by the liver (10). GDH represents an essential link G. Mithieux’s laboratory, INSERM U855, Lyon, France), between amino acids and carbohydrates, channeling the and PEPCK-c (ab70358; Abcam) were used for immu- carbon skeletons of several amino acids into the tricar- noblotting experiments on whole-tissue extracts. For boxylic acid cycle for anaplerosis and the provision of immunofluorescence, tissue sections were stained using gluconeogenic substrates (2,11,12). The importance of antibodies against GDH and glutamine synthetase GDH activity is witnessed by the severity of disorders (MAB302; Millipore) and bodipy dye (D3922; Molecular where GDH function is altered (gain of function muta- Probes) for lipid droplets. Primary hepatocytes were tions), such as hyperinsulinism/hyperammonemia syn- labeled with MitoTracker Orange CMTMRos (M-7510; drome (13,14). Molecular Probes) to visualize mitochondria. Nuclei Although our current knowledge points to liver GDH were stained with DAPI or Hoechst (Molecular Probes). as a key player in nitrogen metabolism and amino acid– Detailed protocols are described in the Supplementary derived gluconeogenesis, no study has challenged yet such Experimental Procedures. an assumption by in vivo abrogation of GDH in hepato- cytes. Here, we generated inducible liver-specificGDH- GDH Enzymatic Activity knockout mice to investigate the role of liver GDH in GDH enzymatic activity was measured in tissues collected metabolic homeostasis under basal and energy-challenging in ice-cold saline before homogenization in 50 mmol/L conditions. We found that hepatic GDH was critical for Tris/HCl (pH 7.4), 0.5 mmol/L EDTA, and 1 mmol/L b maintenance of ammonia and urea homeostasis, playing a -mercaptoethanol in glass potter. After 40-min centrifu- central role in the circadian rhythm of food intake and en- gation at 8,000g, supernatants were assayed for enzymatic ergy partitioning through alanine-induced gluconeogenesis. activity as previously described (17). Determination of Metabolic Parameters RESEARCH DESIGN AND METHODS Blood, urine, and tissue parameters were determined on Mice samples collected from animals fed or fasted overnight lox/lox tm1.1Pma Glud1 floxed (Glud1 )mice(Glud1 , MGI:3835667 (O/N) for 18 h (from 4 P.M. to 10 A.M.). Specific measure- [15]) were crossed with mice carrying the tamoxifen- ments are described in detail in the Supplementary Ex- dependent Cre-ERT2 recombinase coding sequence pre- perimental Procedures. ceded by an internal ribosomal entry site inserted in the 39-untranslated region of the serum albumin gene (16). In Vivo Experiments 2 2 The in vivo deletion of GDH in hepatocytes (HepGlud1 / Body composition was assessed by an EchoMRI-700 quan- mice) was induced at 8 weeks of age by subcutaneous titative nuclear magnetic resonance analyzer (Echo Med- implantation of tamoxifen pellets (Tamoxifen free base, ical Systems, Houston, TX). Metabolic parameters and 25 mg/pellet, 21-day release, E-361; Innovative Research physical activity were measured using a LabMaster sys- of America) in male Glud1lox/lox mice carrying the tem (TSE Systems GmbH, Bad Homburg, Germany) at albumin–Cre-ERT2 construct and used for analyses 4 weeks the Small Animal Phenotyping Core Facility (University later at 12 weeks of age, unless otherwise specified. Animals of Geneva, Geneva, Switzerland) under controlled temper- 6 – were maintained on a mixed (C57BL/6J 3 129/Sv) genetic ature (22 1°C) and lighting (light on: 0700 1900 h). background to avoid inbred strain-specificphenotypes. Energy expenditure and respiratory exchange ratio (RER) As control mice, we used Glud1lox/lox littermates to opti- were determined by indirect calorimetry, locomotor activ- mize standardization of the genetic background between ity was recorded by an infrared frame, and food and water the two groups. To rule out the possibility that the intake were measured by highly sensitive feeding and albumin-Cre transgene used in the current study may drinking sensors. These parameters were measured in have independently contributed to the metabolic pheno- mice housed individually in LabMaster metabolic cages type, we studied in parallel cohorts of mice carrying the (TSE Systems GmbH). albumin-Cre transgene without floxed Glud1 littermates Glucose and insulin tolerance tests were performed to investigate some metabolic parameters, revealing upon i.p. injection of D-glucose (2 g/kg; Sigma-Aldrich) no effects of Albumin-Cre per se. Mice were maintained after an O/N fast or insulin (0.75 units/kg; NovoRapid, in our certified animal facility (12-h 3 12-h light/ Novo Nordisk) on fed mice. Glycerol, pyruvate, alanine, and glutamine challenge tests were performed upon i.p. dark cycle with 7 A.M. on and 7 P.M. off) according to procedures that were approved by the animal care and injection of glycerol (2 g/kg; Sigma-Aldrich), sodium py- experimentation authorities of the Canton of Geneva ruvate (2 g/kg; Sigma-Aldrich), L-arginine (2 g/kg; Sigma- (#1034-3615-1R). Aldrich), or L-glutamine (2 g/kg;