Metabolic Regulation Research Group

Our current focus

The Metabolic Regulation Research Group bridges two highly relevant and interconnected areas of biomedical science: therapeutic hypothermia and reductive stress. Both have major implications for improving outcomes in conditions such as stroke, cardiac arrest, organ transplantation, and hypoxic-ischemic encephalopathy, as well as in disorders linked to mitochondrial dysfunction and reactive oxygen species accumulation.

Therapeutic hypothermia (targeted temperature management) - cooling body to around 32-36°C - has long been used clinically, but its cellular mechanisms remain poorly understood. Our group has shown that mild hypothermia is not simply suppressive, but rather triggers protective signaling programs, including Nrf2-driven antioxidant pathways. We aim to decipher the temperature-sensitive transcriptional responses.

Reductive stress is a redox imbalance caused by the excessive buildup of NADH and NADPH, leading to a highly reduced cellular environment. This disrupts mitochondrial respiration, impairs energy production, and makes cells more vulnerable to damage—especially during reoxygenation after hypoxia or ischemia. While oxidative stress has long dominated redox research, reductive stress is now recognized as an equally important driver of metabolic dysfunction. Our research focuses on understanding and alleviating reductive stress, with the goal of maintaining mitochondrial integrity and improving cell survival in metabolically challenging environments. We explore strategies such as modulating nutrient availability and applying redox-active compounds.

We combine classical physiology with advanced molecular and biochemical techniques, including gene and protein profiling, redox assays, genetic models, ChIP, and multi-omics. A cornerstone of our research is the use of a custom gas flux measurement system that allows simultaneous, real-time measurement of both oxygen consumption (O₂) and carbon dioxide production (CO₂) in live mammalian cells. The system was originally developed by plant physiologists for photosynthetic studies. In close collaboration with them, the system was adapted and optimized for use in eukaryotic (animal) cells—resulting in a one-of-a-kind tool. Unlike commercial systems such as Seahorse XF (which measures extracellular acidification) or Oroboros O₂k (which measures only O₂), our adapted system offers continuous, high-resolution monitoring of both gases. This enables us to map dynamic metabolic shifts under conditions such as hypoxia, hypothermia, nutrient deprivation, and anoxia with exceptional detail.

Ongoing projects

Hypothermia-Responsive Gene Regulation
We are investigating how mild hypothermia (approximately 32°C) activates protective transcriptional programs in cells. Specifically, we are exploring the role of HIF1B as a potential hypothermia-responsive transcription factor, distinct from the classical HIF1A/2A pathways known in hypoxia. This project involves multi-omics approaches, chromatin immunoprecipitation (ChIP), and in vitro hypothermia models to identify HIF1B target genes. The long-term goal is to develop pharmacological strategies that replicate the protective effects of hypothermia without the need for physical cooling.

Reductive Stress Modulation in Hypoxia
We are studying how cells accumulate excess reducing equivalents (NADH/NADPH) under low-oxygen conditions, leading to reductive stress. Our research focuses on identifying metabolic interventions—such as pyruvate supplementation, glucose withdrawal, and the use of redox-reactive agents like resazurin—that can relieve reductive stress and restore mitochondrial respiration. This work is highly relevant to ischemia-reperfusion injury and cancer metabolism.

Cellular Metabolism During Anoxia
Using our custom dual O₂/CO₂ gas flux measurement system, we are examining how cells produce CO₂ in the absence of oxygen. We aim to uncover which metabolic pathways remain active under anoxia and how these processes help maintain redox balance and survival. We also explore how factors like temperature and substrate availability influence these alternative pathways, particularly in relation to mitochondrial function and reductive stress.

Mechanisms of Resazurin Reduction
Traditionally used as a cell viability dye, resazurin is now being studied in our lab as a biologically active compound that participates in redox modulation. We are investigating its dual reaction mechanisms—fluorescent and reversible—and how it interacts with key metabolic intermediates. Our work suggests that resazurin can act as an electron sink, alleviating reductive stress and improving mitochondrial respiration under hypoxic or nutrient-stressed conditions.

Grants

• Estonian Research Council: HIF1B as a Novel Hypothermia Response Factor with Therapeutic Benefits (Associate Professor Kattri-Liis Eskla, GMVBS24069PS, 01.01.2024−31.12.2028)

Current collaborations

• Assoc Prof Darja Lavõgina, Institute of Chemistry, University of Tartu, Estonia
• Assoc Prof Kalle Kilk, Department of Biochemistry, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia
• Dr Indrek Reile, National Institute of Chemical Physics and Biophysics, Tallinn, Eesti
• Pro Vallo Volke, Department of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia
• Prof Daniel Tennant, University of Birmingham, UK
• Prof Roberta Foresti & Prof. Roberto Motterlini, University Paris-Est Créteil, France

The most important publications

• Vera-Siguenza E, Escribano-Gonzalez C, Serrano-Gonzalo I, Eskla KL, Spill F, Tennant D. Mathematical reconstruction of the metabolic network in an in-vitro multiple myeloma model. PLoS Comput Biol. 2023 Sep 15;19(9):e1011374. doi: 10.1371/journal.pcbi.1011374. PMID: 37713666; PMCID: PMC10503963.
• Vellama H, Eskla KL, Eichelmann H, Hüva A, Tennant DA, Thakker A, Roberts J, Jagomäe T, Porosk R, Laisk A, Oja V, Rämma H, Volke V, Vasar E, Luuk H. VHL-deficiency leads to reductive stress in renal cells. Free Radic Biol Med. 2023 Nov 1;208:1-12. doi: 10.1016/j.freeradbiomed.2023.07.029. Epub 2023 Jul 26. PMID: 37506952; PMCID: PMC10602395.
• Lavogina D, Lust H, Tahk MJ, Laasfeld T, Vellama H, Nasirova N, Vardja M, Eskla KL, Salumets A, Rinken A, Jaal J. Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon. Biosensors (Basel). 2022 Mar 25;12(4):196. doi: 10.3390/bios12040196. PMID: 35448256; PMCID: PMC9032648.
• Eskla KL, Vellama H, Tarve L, Eichelmann H, Jagomäe T, Porosk R, Oja V, Rämma H, Peet N, Laisk A, Volke V, Vasar E, Luuk H. Hypothermia Alleviates Reductive Stress, a Root Cause of Ischemia Reperfusion Injury. Int J Mol Sci. 2022 Sep 3;23(17):10108. doi: 10.3390/ijms231710108. PMID: 36077504; PMCID: PMC9456258.
• Westbrook RL, Bridges E, Roberts J, Escribano-Gonzalez C, Eales KL, Vettore LA, Waler PD, Vera-Siguenza E, Cuozzo F, Eskla KL, Vellama H, Shaaban A, Nixon C, Luuk H, Lavery GG, Hodson D, Harris A, Tennant DA.Proline Synthesis Through PYCR1 is Required to Support Cancer Cell Proliferation and Survival in Oxygen-Limiting Conditions. Cell Reports. 2021.
• Eskla KL, Porosk R, Reimets R, Visnapuu T, Vasar E, Hundahl CA, Luuk H. Hypothermia augments stress response in mammalian cells. Free Radic Biol Med. 2018 Jun;121:157-168. doi: 10.1016/j.freeradbiomed.2018.04.571. Epub 2018 Apr 25. PMID: 29704622.
• Ilmjärv S, Hundahl CA, Reimets R, Niitsoo M, Kolde R, Vilo J, Vasar E, Luuk H. Estimating differential expression from multiple indicators. Nucleic Acids Res. 2014 Apr;42(8):e72. doi: 10.1093/nar/gku158. Epub 2014 Feb 27. PMID: 24586062; PMCID: PMC4005682.