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The role of autophagy and lipid catabolism in enhancing cold stress resistance in zebrafish

The Journal of Physiology2019Vol. 597(8), pp. 2123–2125

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Charles B. Trelford, Stephanie Wojtowicz, Caroline A. Banas

Abstract

Temperature has been a key selection factor for evolution. Natural selection favours organisms that develop biological and behavioural mechanisms to adapt to acute decreases in temperature, which is a known environmental stressor. Homeothermic animals maintain body temperature by increasing food intake whereas poikilothermic animals, such as fish, have evolved to adapt to cold temperatures by decreasing food intake (Lu et al., 2019). Although the physiological mechanism to explain this phenomenon remains unknown, it is hypothesized that fasting attenuates cellular damage mediated by stressors. It has been proven that when an organism is in a fasted state, the mechanistic target of rapamycin (mTOR) intracellular signalling pathway is inhibited, which increases macroautophagy (Kaur and Debnath, 2015). Macroautophagy, hereafter referred to as autophagy, is a catabolic process where a cell recycles its macromolecules and organelles via lysosome-dependent degradation. Autophagy has the potential to eliminate cellular damage induced by cold stress while providing cells with sufficient energy to survive the fasting state for a short period (Kaur and Debnath, 2015). Fatty acids, a key component of the lipids of biological membranes, provide the most energy per macromolecule when degraded. Lipophagy, autophagy-dependent lipid droplet breakdown and other forms of lipid catabolism are up-regulated during cell stress responses to meet the energy demands of the cell (Zechner et al., 2017). Therefore, autophagy and lipid catabolism are important for maintaining cellular homeostasis. As such, deregulating either process may influence how cells adapt to damage and render them more susceptible to cold stress (Zechner et al., 2017). In a recent issue of The Journal of Physiology, Lu et al. (2019) investigated how fasting induces cold resistance and characterized the underlying protective mechanisms of fasting. Specifically, the group wanted to determine if the metabolic adjustments initiated by fasting can protect female zebrafish from cold stress. In order to achieve this, the group used wild-type, autophagy-related protein 12 knockout (ATG12−/−) and carnitine palmitoyltransferase-1b knockout (CPT-1b−/−) zebrafish. ATG12−/− impaired autophagy whereas CPT-1b−/− decreased fatty acid β-oxidation, which is required for lipid catabolism. These experimental groups were subjected to fasting and fed conditions prior to being exposed to cold water to induce cold stress. The article assessed viability, autophagy and fatty acid β-oxidation efficiency as experimental outcomes. Several in vitro and in vivo studies were employed to determine if enhancing or dampening lipid catabolism or autophagy via fasting or knockout mutants, respectively, could improve zebrafish survival rate to acute cold stress. In the presence of cold water, fasting led to a significant increase in zebrafish survival rate, suggesting an enhanced resistance to acute cold stress. When assessing mRNA expression of genes related to apoptosis and endoplasmic reticulum stress, the fasted cold stressed zebrafish had significantly lower expression compared to the fed cold stressed zebrafish. In addition, transmission electron microscopy of zebrafish liver tissue samples showed significant impairment of endoplasmic reticulum structures and damaged mitochondria in fed cold-stressed zebrafish but fasted cold-stressed zebrafish showed reversal of organelle impairments. This suggests that fasting in a cold environment is protective against apoptosis, endoplasmic reticulum stress and mitochondrial damage. When zebrafish were fed an activator of fatty acid β-oxidation, fenofibrate, the survival rate of zebrafish significantly increased when exposed to cold stress, but when zebrafish were fed an inhibitor, mildronate, the survival rate decreased significantly with respect to the control zebrafish. When a key enzyme in fatty acid β-oxidation, CPT-1b, was knocked out (CPT1b−/−), cold stressed zebrafish had a lower survival rate. This suggests that fatty acid β-oxidation is necessary for cold stress resistance in fasted zebrafish. Finally, when zebrafish were treated with chloroquine and rapamycin, an inhibitor and activator of autophagy, respectively, rapamycin increased, and chloroquine decreased survival rate. When ATG12, a protein necessary for autophagy, was knocked out (ATG12−/−), the cold-stressed zebrafish had a significantly lower survival rate compared to wild-type zebrafish. Together, this suggests that autophagy plays an important role in fasting to reduce cold stress toxicity in zebrafish. Although it has been reported previously that fasting increases cold stress resistance using Drosophila melanogaster (Isabelle and Bourg, 2015), the mechanism of this phenomenon remained unknown. However, the work completed by Lu et al. (2019) provides evidence that the processes of both autophagy and lipid catabolism could be responsible for this phenomenon as summarized in Fig. 1. In order to achieve this, the group assessed cellular, structural and molecular changes induced by acute cold stress in fed and fasted experimental conditions. Autophagy and fatty acid β-oxidation were attenuated using knockout zebrafish models and pharmacological agents such as rapamycin and chloroquine modulated autophagy whereas fenofibrate and mildronate altered fatty acid β-oxidation. Summary of the cold stress response model in zebrafish The model proposes that by fasting in cold water, zebrafish increase autophagy and lipid catabolic pathways to produce energy and alleviate damage induced by cold stress. Pharmacological agents that induced fatty acid β-oxidation and autophagy increased zebrafish survival rate whereas knockout zebrafish and inhibitors of these pathways decreased survival rate. Rapamycin increases autophagy because it inhibits mTOR, which is responsible for suppressing the formation of the phagophore (Zechner et al., 2017). On the other hand, chloroquine inhibits autophagy by decreasing lysosomal pH, which prevents autolysosome formation and hydrolase-dependent degradation (Kaur and Debnath, 2015). Since ATG12 is essential for phagophore extension, the assembly of autophagosomes in ATG12−/− zebrafish was impaired (Zechner et al., 2017). As illustrated by Lu et al. (2019), experimental conditions that increased or decreased autophagy were associated with an increase or decrease in zebrafish survival to cold stress, respectively. Therefore, this work provided evidence that highlighted autophagy as necessary for eliminating damage mediated by cold stress. When the rate of fatty acid β-oxidation was decreased using mildronate or CPT-1b−/− zebrafish, the number of surviving zebrafish decreased. This is likely due to the fact that after carbohydrate stores were depleted, zebrafish could not utilize their lipid stores and thus had no energy to reverse the structural and molecular damage mediated by cold stress. However, zebrafish fed with fenofibrate were able to break down the lipid stores to produce sufficient energy to maintain the cellular processes vital for survival. This evidence suggests that lipid catabolic pathways are essential in the cold stress response to supply zebrafish with energy to survive in a prolonged fasted state. Unlike homeothermic animals that rely on a calorie rich diet to sustain body temperature (Lu et al., 2019), it is hypothesized that zebrafish have adapted to manipulate mTOR signalling and lipid catabolism to survive cold climates. Therefore, it is possible that poikilothermic animals have a selective advantage versus homeothermic animals under fasted conditions because their cells are primed to digest themselves and lipid stores rather than rely on external sustenance. While this study contributed to uncovering the underlying metabolic pathway that protects poikilothermic animals from cold stress and emphasized autophagy and lipid catabolic functions, it did have some limitations. Since metabolic pathways and body fat composition vary between sexes in some species, investigating sex differences in autophagy and lipid catabolism in zebrafish would have been beneficial. Furthermore, the pharmacological agents used to modulate autophagy, rapamycin and chloroquine, are relatively non-specific. For instance, mTOR is involved with many molecular pathways that are independent of autophagy, and chloroquine impacts the pH of endosomes, which are necessary for receptor trafficking and several intracellular signalling pathways. It is also worth noting that LC3 protein levels are proportional to the number of autophagosomes, which is not a reliable measure of autophagic activity. However, these are more idealistic circumstances, as both chloroquine and rapamycin have been proven to decrease and increase the rate of autophagy, respectively (Kaur and Debnath, 2015). Future studies could include experiments to determine whether the rate of autophagic degradation changes for a given experimental condition and whether the proteasome plays a role in cold stress resistance in zebrafish. The rationale for studying the proteasome is that the two primary cellular protein degradation pathways are autophagy and the ubiquitin–proteasome system, which play a compensatory role with one another (Ding et al., 2007). Conducting this study could verify whether autophagy is the primary mechanism in which zebrafish eliminate damaged macromolecules and organelles or a combination of both degradation pathways. Fasting in cold environments has been proven to be crucial for survival of poikilotherms. The current study confirmed this by showing that fasted zebrafish had a higher survival rate when exposed to cold stress. When metabolic processes such as autophagy and fatty acid β-oxidation were inhibited by specific gene knockouts and pharmacological inhibitors, the survival rate of the fasted cold stressed zebrafish decreased, providing evidence that these two processes are key players in cold stress resistance in zebrafish. None declared. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None. The authors would like to thank Dr Gianni Di Guglielmo for his assistance in preparation of this review article.

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