Progress in Fishery Sciences (Dec 2023)
Role of the Dissostichus mawsoni LeptinB Gene in Cellular Cold Resistance
Abstract
Variation in fish temperature is determined by changes in the external aquatic environment, and temperature serves as a crucial regulatory factor for the behavior, physiology, and molecular processes of fish. A rapid decline in water temperature can elicit a strong stress response in fish, potentially inhibiting growth and even leading to mortality. Low temperature stress significantly impacts various life processes of organisms, including growth and development, energy metabolism, and reproductive development. In recent years, there has been increasing research on the effects of low-temperature stress on organisms, with a greater focus at the molecular level. Low temperatures induce a decrease in cellular activity and even growth arrest, resulting in apoptosis. Apoptosis is a form of programmed cell death ubiquitous in the biological world, and it plays a vital role in maintaining the normal physiological function of an organism. Once apoptosis occurs, it is irreversible and cannot be stopped. Temperature regulation is crucial for fish, and current knowledge of the role of leptin in fish primarily focuses on the regulation of feeding, lipid energy metabolism, and reproduction, with limited reports on its role in temperature regulation.Leptin, a protein hormone produced by adipocytes, has long been recognized as a product of the obese gene, which regulates organismal metabolism, neuroendocrine function, and other physiological processes. In animals, it exerts diverse physiological functions related to cell growth, proliferation, apoptosis, and metabolism. The homology of the leptin gene in fish is relatively low compared to that in mammals, and multiple leptin proteins encoded by multiple leptin genes can be produced due to genomic duplication events. Regarding the role of leptin in temperature regulation, more research has been conducted on mammals, mainly related to the regulation of energy metabolism and the influence on temperature adaptation evolution. There has been limited research on the regulatory mechanism of leptin in fish during cold stress.Dissostichus mawsoni belongs to the group of Antarctic vertebrates and has lived in the cold and isolated environment of the Southern Ocean at a temperature of –1.9 ℃ for ~30 million years. It has adapted to the extremely low temperatures of the surrounding Antarctic waters and is an excellent material and living fossil for studying temperature adaptation mechanisms in extreme environments. Compared with other fish species at the same depth, it has a large amount of lipid deposition in its subcutaneous and muscle tissues, mainly triglycerides (TGs). Predictions based on 3D structural models suggest that the partial absence of the lepB structure in D. mawsoni leads to only three α-helices, and lepA has four α-helices and a short and twisted E-helix as well as several irregular turns, forming a hollow barrel-like structure, which differs from the protein structure of all other known leptins. Moreover, previous studies have found a close correlation between lepA and temperature evolution, leading to speculation that Antarctic fish lepB may play an essential role in low-temperature adaptation.In this study, a eukaryotic expression vector of the Antarctic toothfish lepb gene was constructed and transfected into ZFL cells to establish a model of overexpression of the D. mawsoni lepb (DMLB) gene in the ZFL cells. After conducting a cell culture temperature experiment, 10 ℃ was selected as the significant difference temperature. Following 2 weeks of cold stimulation, the DMLB experimental group cells remained viable, whereas the control group cells died. The results indicated that the overexpression of the DMLB gene provided strong resistance to low-temperature conditions. By detecting changes in cell proliferation, apoptosis, reactive oxygen species (ROS) level, and ATP content under low-temperature stress, it was discovered that DMLB maintained cell growth and proliferation, reduced ROS production, and inhibited cell apoptosis. DMLB effectively maintained ATP levels in cells under low-temperature stress, which helps maintain the mitochondrial status and reduce the effects of apoptosis and necrosis caused by low-temperature stress, thereby protecting cells from cold stress. Additionally, the results of Oil red O staining and TG detection suggested that the DMLB gene may slow down the depletion of cell lipids under low-temperature stress, perhaps by lowering lipid metabolism to preserve lipids to cope with low-temperature damage. Consequently, the DMLB gene may be functional in the cold resistance of D. mawsoni by protecting cells from damage at low temperatures, but its specific molecular regulatory mechanism needs to be further explored. In this study, 10 ℃ was used as the key temperature, providing a basis for understanding the regulation of energy homeostasis by the lepb gene in D. mawsoni under low-temperature stress. This study explored the role of the lepB gene in the adaptation of D. mawsoni to extremely low-temperature environments and provided fundamental data for further study of the evolution of similar species in the Antarctic region. Furthermore, this study enriched the basic data related to low-temperature tolerance and provided reference materials for further investigation of the low-temperature tolerance mechanism of leptin in Notothenioids, as well as laid a foundation for further study of the mechanism of leptin in temperature regulation.
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