Heat stress reduces the health and activity of animals as well as agricultural productivity. Although many research studies on the physiological reactions of animals to heat have been conducted, there are no direct countermeasures to offset the negative effects and any such measure is limited to general environmental temperature reduction and feed additives. The use of early heat exposure for reducing the thermal stress of broilers has emerged as an economically viable method compared with other countermeasures. Although strategies for reducing heat stress have increased the heat resistance of animals, the productivity of the animals in the hot summer season has yet to be fully normalized. Therefore, a detailed understanding of the biological responses to heat stress is necessary to aid the development of techniques for normalizing the heat-related physiological and metabolic responses of animals. Among the livestock affected by high temperatures, poultry was selected as it is the most susceptible to heat and therefore the most suitable for studying the metabolic response during heat exposure.
Given that protein metabolism in animals can be altered by heat, researchers have studied the heat-induced proteomic responses in various animals, such as cattle, pigs, and birds. For example, {Victoria Sanz Fernandez, 2015 #241} reported that heat stress affects carbohydrate metabolism in pigs. Heat stress decreases ATP production through oxidative phosphorylation and increases energy production through aerobic sugar degradation, thereby significantly altering intracellular energy (11). This is similar to the Warburg effect that is used in tumor cells to gain energy (12). Our differentially expressed proteins were consistent with the results of these reports, in that proteins in pathways of carbohydrate metabolism—such as those in glycolysis/gluconeogenesis (acetyl-CoA synthetase 2-like, mitochondrial isoform X1 (ACSS1L), glucose 6-phosphate isomerase (GPI), hexokinase domain-containing 1 (HKDC1)), the citrate cycle (OGDHL), starch and sucrose metabolism (GPI, HKDC1, UGT2A1), and propanoate metabolism (ACSS1L, propionyl-CoA carboxylase subunit alpha (PCCA))—were upregulated in the liver tissue of the CH chickens compared with that of the CC and HH chickens. The overall metabolic process is shown in Fig. 5.
In glycolysis, HKDC1 (a member of the hexokinase family) plays an important role in regulating glucose metabolism by catalyzing the ATP-dependent phosphorylation of glucose-6-phosphate at the first step of the glycolytic pathway (13). Although the specific biological functions of HKDC1 are still unclear, it has been proposed to play an especially greater role in glucose metabolism when the fetus needs to be supplied with sufficient nutrients during pregnancy (14). In a study of the sugar degradation pathways of various hyperthermophilic archaea, the extremophiles were found to degrade glucose, maltose, cellobiose, and starch through modified Embden-Meyerhof pathways (15). Additionally, it has been found that enzyme activity increases exponentially at high temperatures (above 55℃) (16). GPI, which catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate, plays a role in glycolysis and gluconeogenesis in the cytoplasm and is also involved in the pentose phosphate pathway (17). This protein has been studied as a neurotrophic factor for promoting the survival of skeletal and sensory neurons and inducing immunoglobulin secretion, and as a tumor-secreting cytokine that plays a role in tumor angiogenesis and metastasis and cell migration, proliferation, and apoptosis (18, 19). GPI deficiency induces hemolytic anemia, whereas its overexpression is related to carcinogenesis and its elevated serum level is used as a prognostic biomarker of colon, rectal, breast, lung, and kidney cancers (20). Therefore, the upregulation of both HKDC1 and GPI by chronic heat stress would promote glycolysis and activate pathways to obtain energy from glucose in the body. However, such overexpression may induce cell damage as well. Sorbitol dehydrogenase (SORD), which converts sorbitol to fructose in the polyol pathway, is closely related to various diabetic complications (viz., neuropathy, retinopathy, cataracts, and nephropathy) (21). The decreased expression of SORD causes an excessive accumulation of sorbitol, leading to osmotic damage to the retinal endothelial cells and pericytes through oxidative-nitridation stress and activation of the protein kinase C pathways, with resultant inflammation and growth factor imbalance (22). Our results showed that the expression of SORD was reduced by chronic heat stress, which is coincident with the finding that SORD is inactivated with increasing temperature (23). However, the SORD reduction in the CH group was recovered in the HH group, indicating that early heat exposure plays a protective role against heat stress in the liver cells.
In the tricarboxylic acid (TCA) cycle, the main precursor acetyl-CoA is essential for energy generation toward the mitochondria (24). During a lack of carbohydrate intake or utilization, acetate is used as an essential source for producing acetyl-CoA under ketogenic conditions and is involved in heat generation (25). In broilers, ACSS1L catalyzes the synthesis of acetyl-CoA for use in glycolysis. The feed intake of the CH broilers was significantly decreased compared with that of the CC broilers, and thus the expression of ACSS1L was increased in that group owing to the insufficient nutrients for metabolism. Citrate synthase (CS) catalyzes the condensation reaction to form citrate from oxaloacetate and acetyl-CoA, which are the first steps in the TCA cycle. It is also used as an enzymatic marker of intact mitochondria (26). CS activity has been found to be decreased by mitochondrial dysfunction and inhibited by oxidative stress (27). Another study confirmed the protective function of CS in thermally stressed yeast cells (28), where deletion of the CS-coding gene citl resulted in temperature-sensitive ROS accumulation, nuclear and DNA fragmentation, and phosphatidylserine translocation, all of which are hallmarks of cytological apoptosis. From the results of our study, we can postulate that heat stress causes damage to the mitochondria as a result of the downregulation of CS, but early heat exposure can regulate the CS expression level. OGDHL, which is located in the mitochondria, is also a TCA cycle-related enzyme and indirectly responsible for the induction of apoptosis. The increased expression of OGDHL contributes to the acceleration of the TCA cycle (29). It was found in a previous study that OGDHL overexpression significantly induced ROS production and lipid peroxidation in the mitochondria of cervical cancer cells and resulted in apoptosis by inducing caspase-3 activity and PARP protein expression, whereas OGDHL suppression significantly inhibited these processes and promoted cell proliferation (30). These results suggest that overexpressed OGDHL plays an important role in inducing ROS-mediated apoptosis. In our study, chronic heat stress induced OGDHL expression, which suggests that it may induce ROS production followed by cell damage. Thus, the increase in carbohydrate metabolism observed in chronically heat-stressed broilers may be the result of a lack of energy in the body.
In general, heat stress increases energy consumption for panting to dissipate heat, maintaining homeostasis, and protecting the cells (31). In a study on the fatty acid levels in broilers under heat stress, the plasma concentration of non-esterified fatty acids was reduced in the heat-stressed animals, and better absorption and storage of triglycerides in the intestine or liver were observed (32). This is because the heat emitted from fat metabolism is higher than that generated through carbohydrate metabolism. Similarly, in heat-stressed dairy cows, energy was preferentially supplied by the carbohydrates to reduce the body’s own heat generation (33). However, in our study, chronic heat stress, which increases the requirement of a considerable amount of energy, increased the expression of enzymes of the acyl-CoA dehydrogenase (ACAD) family (i.e., ACAD11 and ACAD9) to obtain energy through fat. This confirms that a considerable amount of energy is required, along with a reduction in feed intake, in response to chronic heat stress. Although heat stress significantly reduced the nutrient (and thus potential energy) intake by the broilers, a large amount of energy was needed from metabolism to cope with the stress. Thus, the expression of carbohydrate metabolism-related proteins changed.