Insight into AMPK regulation mechanism in vivo and in vitro: Responses to low temperatures in the olive flounder Paralichthys olivaceus

The olive flounder, Paralichthys olivaceus, is a commercially important maricultured fish in China, Japan, and Korea. Low winter temperatures influence its survival and growth and affect the output of the aquaculture industry. Energy metabolism is essential for fish survival, and the central energy-regulating factor – 5ʹ-AMP-activated protein kinase (AMPK) – plays an important role in responses to cold stress. However, the mechanism of AMPK pathway regulation in fish coping with cold stress remains poorly understood. In the present study, the expression of AMPK and its upstream (LKB1 and CaMKKβ) and downstream genes (SITR1, FOXO1A, and TFAM) in the brain, muscle, and heart was analyzed while the flounder was under cold stress (0.2 ± 0.2 °C). The results showed that low temperatures activated LKB1, CaMKKβ, and AMPK genes in the brain, and the activated AMPK induced expression of SITR1, FOXO1A, and TFAM. In the muscle tissue, the expression patterns of these genes presented a trend of initially decreasing and then increasing, and there was a delay in the response to low temperatures. At the cellular level, comparative analysis of the effects of the activator 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) and inhibitor compound C of the AMPK pathway demonstrated that cold stress was similar to AICAR, which activated the AMPK pathway with hysteresis. Thus, the regulation mechanism of AMPK under cold stress was preliminarily analyzed. In general, AMPK was involved not only in responses to low temperatures but also in energy regulation under cold stress.     

温度刺激对斑马鱼仔鱼基因转录表达的影响

许多优良鱼类养殖品种不耐低温或高温的特点给水产养殖业带来诸多限制和困难,这些鱼类在胚胎和仔鱼等早期阶段的抗寒和抗热能力比成体更差,育苗过程中很容易受到温度突然变化的影响。虽然目前利用基因芯片技术已研究了温度刺激对几种鱼类成体组织中基因表达的影响,但温度刺激对仔鱼基因转录表达的影响还未见报道。研究以斑马鱼受精后96h的出膜仔鱼为实验材料,分别在低温(16℃)和高温(34℃)条件下处理12h和24h,用基因芯片技术检测温度刺激对其基因表达的影响。与培养在28℃的对照相比,低温和高温处理后共有3633个基因发生差异表达,其中低温处理后差异表达基因数目多于高温处理,而且低温抑制基因数目多于诱导表达基因的数目。生物信息学分析结果表明,低温诱导基因主要参与RNA加工和核糖体生物发生等生物学过程,高温诱导基因则主要参与应激反应和未折叠蛋白结合。低温抑制基因主要参与蛋白质水解、视觉感知以及铁离子结合等生物学功能,高温抑制基因参与的生物学功能包括DNA复制、神经系统过程和类固醇激素生物合成等。除了已报道的温度刺激响应基因外,研究鉴定出了大量尚未报道与温度刺激相关的基因,如参与RNA加工的rnmtl1a和pus3基因,以及参与转录调控的twistnb和aebp2基因等。研究结果为进一步揭示鱼类冷或热适应的分子机理和培养耐寒或耐热的养殖新品种提供理论基础。     

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0°C and after long-term warming to 5°C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0°C for the polar and 6°C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.     

Early life history of the small yellow croaker (Larimichthys polyactis) in sandy ridges of the South Yellow Sea

The early life history of the small yellow croaker Larimichthys polyactis was studied using the otolith microchemistry of juvenile fish and a recruitment survey in the sandy ridges of the South Yellow Sea. The otolith Sr/Ca ratio regime shifted markedly at the age of 5–7 days (early April) and 37–41 days (around early May). Subsequently, the Sr/Ca ratio was almost constant until late June, indicating an aggregation of L. polyactis juveniles in the relatively stable habitat of the sandy ridges, which was supported by a recruitment survey that showed a rapid increase in the number of juveniles in the same area between early May and the end of June. Variations in the Sr/Ca ratio between early April and early May might reflect a habitat adaptive strategy of L. polyactis in the pelagic stage to hydrodynamic processes of the sandy ridges. Based on the aforementioned habitat shifts and recruitment phenology, it was hypothesised that L. polyactis juveniles disperse between early April and early May, inhabit sandy ridges to feed, and migrate offshore in late June.