Mitochondrial uncoupling proteins (UCPs) play central roles in vertebrate energy metabolism, with UCP1 specializing as a thermogenic effector in placental mammals. Comparative genomics demonstrated that UCP1, UCP2, and UCP3 originated prior to the emergence of endothermy, suggesting that their ancestral functions evolved in ectothermic vertebrates. Here, we investigated the genomic organization, conserved synteny, and mRNA expression of all three UCP paralogs and homoeologs in the allotetraploid amphibian Xenopus laevis. Comparative genome analyses revealed that all three UCP paralogs and their long (L) and short (S) homoeologs were retained following polyploidization, although local rearrangements were evident for neighboring genes at the UCP2/UCP3 locus. Quantitative expression analyses across adult frog tissues revealed pronounced tissue specificity, with predominant expression of UCP1 and UCP2 in kidney, UCP3 in muscle, and differences in UCP2 homoeolog expression levels. Together, these findings establish a framework for UCPs in an amphibian model organism, primarily linking them to energetically demanding tissues.

Uncoupling protein 1 (UCP1) is a crucial factor for mammalian metabolism, driving thermogenesis by uncoupling the respiratory chain from ATP production. In mammals, UCP1 is predominantly expressed in brown and beige adipose tissue. Interestingly, UCP1 is not exclusive to endothermic mammals; its orthologs are also found in ectotherms, such as amphibians and fish. The presence of UCP1 in these species raises the question of its ancient function. In contrast to mammals, fish UCP1 is localized to other metabolically active organs, primarily the liver and the brain. This tissue pattern is conserved among various fish species, including the common carp (Cyprinus carpio), killifish (Fundulus heteroclitus), and zebrafish (Danio rerio). A notable similarity between UCP1 in endotherms and ectotherms is the temperature-dependent expression. For instance, in C. carpio, ucp1 mRNA levels rise in the brain while decreasing in the liver when exposed to cold temperatures, demonstrating tissue specificity. Conversely, mammalian UCP1 levels consistently increase in response to cold temperatures. The specific function of UCP1 in ectotherms is unclear. In this thesis, I aim to contribute to our understanding of ectotherm UCP biology and its role in thermal physiology by examining ucp1-ablated D. rerio and establishing various respirometric analyses that provide insights into fish metabolism in response to temperature. Additionally, my research aims to clarify the poorly understood roles of UCP1 and its paralogs in amphibious ectotherms by examining syntenic regions of the genome and quantifying organ-specific gene expression in Xenopus laevis.

Manuscript I: We examined the ucp1 gene in D. rerio, investigating temperature-dependent gene expression. A novel zebrafish ucp1 knockout (KO) line (ucp1^uu4471) showed no major developmental or morphological defects. However, ucp1 KO mitochondria exhibited impaired complex I-driven respiration, and gene expression changes suggested the presence of compensatory mechanisms. My work establishes a new tool and fundamental data for deciphering UCP1s enigmatic role in teleost metabolism and acclimation.

Paper II: I adopted Seahorse XF96 respirometry to study the effects of temperature on zebrafish embryo bioenergetics. Embryos (28°C) were exposed to 18–37°C for 20 h before performing oxygen consumption rate (OCR) assays (at 18–45°C). At a temperature of 18°C, low basal OCR reflected reduced ATP-linked respiration. OCR rose with temperature, remaining stable up to 37°C, and pre-exposure to 37°C enhanced thermal tolerance up to 41°C. Proton leak increased above 28°C, reducing the efficiency of ATP synthesis. The heart rate (a metabolic indicator) peaked at 28°C, coherent with the OCR trends. This method enables high-throughput in situ analysis of whole-embryo responses to temperature acclimatization.

Manuscript III: I analyzed the evolutionary conservation and expression of UCPs in X. laevis, confirming that all three major UCP paralogs and their duplicated copies persisted post-polyploidization events. Bioenergetics assays in X. laevis kidney cells showed nominal responses to mitochondrial stress tests applied via Seahorse technology. However, unresponsiveness to canonical UCP1 activators suggested functional divergence. This study provides a foundation for probing the ancestral roles of UCPs in amphibians.

Oxygen consumption allows measuring the metabolic activity of organisms. Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (Danio rerio) in situ. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and penetration of various mitochondrial inhibitors, and confirm that sedation reduces the variability of OCR. At 48h post-fertilization, embryos (maintained at a routine temperature of 28°C) were exposed to different medium temperatures ranging from 18°C to 37°C for 20h prior OCR measurement. Measurement temperatures from 18°C to 45°C in the XF96 were achieved by lowering the room temperature and active in-built heating. At 18°C assay temperature, basal OCR was low due to decreased ATP-linked respiration, which was not limited by mitochondrial power, as seen in substantial spare respiratory capacity. Basal OCR of the embryos increased with assay temperature and were stable up to 37°C assay temperature, with pre-exposure of 37°C resulting in more thermo-resistant basal OCR measured at 41°C. Adverse effects of the mitochondrial inhibitor oligomycin were seen at 37°C and chemical uncouplers disrupted substrate oxidation gradually with increasing assay temperature. Proton leak respiration increased at assay temperatures above 28°C and compromised the efficiency of ATP production, calculated as coupling efficiency. Thus, temperature impacts mitochondrial respiration by reduced cellular ATP turnover at lower temperatures and by increased proton leak at higher temperatures. This conclusion is coherent with the assessment of heart rate, an independent indicator of systemic metabolic rate, which increased with exposure temperature, peaking at 28°C, and decreased at higher temperatures. Collectively, plate-based respirometry allows assessing distinct parts of mitochondrial energy transduction in zebrafish embryos and investigating the effect of temperature and temperature acclimation on mitochondrial bioenergetics in situ.