Metabolites 2021, 11, 755. https://doi.org/10.3390/metabo11110755 www.mdpi.com/journal/metabolites Article Glutamine Homeostasis and Its Role in the Adaptive Strategies of the Blind Mole Rat, Spalax Dmitry Miskevich 1, *, Anastasia Chaban 1 , Maria Dronina 2 , Ifat Abramovich 3 , Eyal Gottlieb 3 and Imad Shams 1,2, * 1 Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel; anastasia@chaban.su 2 Institute of Evolution, University of Haifa, Haifa 3498838, Israel; dronina.maria@gmail.com 3 Technion Faculty of Medicine, Haifa 3525433, Israel; ifat.a.g@gmail.com (I.A.); e.gottlieb@technion.ac.il (E.G.) * Correspondence: miskevichd@gmail.com (D.M.); imad.shams@univ.haifa.ac.il (I.S.) Abstract: Oxidative metabolism is fine-tuned machinery that combines two tightly coupled fluxes of glucose and glutamine-derived carbons. Hypoxia interrupts the coordination between the me- tabolism of these two nutrients and leads to a decrease of the system efficacy and may eventually cause cell death. The subterranean blind mole rat, Spalax, is an underexplored, underground, hy- poxia-tolerant mammalian group which spends its life under sharply fluctuating oxygen levels. Primary Spalax cells are an exceptional model to study the metabolic strategies that have evolved in mammals inhabiting low-oxygen niches. In this study we explored the metabolic frame of gluta- mine (Gln) homeostasis in Spalax skin cells under normoxic and hypoxic conditions and their im- pacts on the metabolism of rat cells. Targeted metabolomics employing liquid chromatography and mass spectrometry (LC-MS) was used to track the fate of heavy glutamine carbons ( 13 C5 Gln) after 24 h under normoxia or hypoxia (1% O2). Our results indicated that large amounts of gluta- mine-originated carbons were detected as proline (Pro) and hydroxyproline (HPro) in normoxic Spalax cells with a further increase under hypoxia, suggesting a strategy for reduced Gln carbons storage in proteins. The intensity of the flux and the presence of HPro suggests collagen as a can- didate protein that is most abundant in animals, and as the primary source of HPro. An increased conversion of αKG to 2 HG that was indicated in hypoxic Spalax cells prevents the degradation of hypoxia-inducible factor 1α (HIF-1α) and, consequently, maintains cytosolic and mitochondrial carbons fluxes that were uncoupled via inhibition of the pyruvate dehydrogenase complex. A strong antioxidant defense in Spalax cells can be attributed, at least in part, to the massive usage of glutamine-derived glutamate for glutathione (GSH) production. The present study uncovers addi- tional strategies that have evolved in this unique mammal to support its hypoxia tolerance, and probably contribute to its cancer resistance, longevity, and healthy aging. Keywords: glutamine; metabolome; hypoxia; proline cycle; GSH; adaptation; bioenergetics 1. Introduction Glucose (Glc) and glutamine (Gln) are two central metabolic nutrients that maintain cellular metabolism. Gln has a more pleiotropic role in cellular metabolism than Glc. It incorporates directly into proteins [1] and participates in the proteostasis regulation [2]; provides carbons and nitrogen for the biosynthesis of amino acids, nucleotides, and hexosamines [1,3,4]; replenishes the tricarboxylic acid (TCA) cycle with carbons [5]; and plays an important role in the gluconeogenesis (GNG) [6]. Hypoxia upregulates the level of hypoxia-inducible factor 1α (HIF-1α) which switches cellular bioenergetics to anaerobic mode [7]. The anaerobic mode includes re- duced pyruvate metabolism, uncoupling of the glycolysis and the TCA cycle, and the subsequent shortage of glucose-derived acetyl-CoA (Ac-CoA) [8,9]. In turn, the deficit of ac-CoA could result in a decreasing citrate (Citr) level that is essential for multiple bio- Citation: Miskevich, D.; Chaban, A.; Dronina, M.; Abramovich, I.; Gottlieb, E.; Shams, I. Glutamine Homeostasis and Its Role in the Adaptive Strategies of the Blind Mole Rat, Spalax. Metabolites 2021, 11, 755. https://doi.org/10.3390/ metabo11110755 Academic Editors: Matthew Pa- menter and Ken Storey Received: 9 October 2021 Accepted: 30 October 2021 Published: 31 October 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and insti- tutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses /by/4.0/).