The starvation caused a significant weight loss in 96 h treatment, which was in agreement with reports in rainbow trout (Salmo gairdneri) and gibel carp (Carassius auratus gibelio) (Reinitz 1983; He et al. 2022). Meanwhile, VSI and HSI were both largely reduced (Table 1), these decreasing was also presented in rainbow trout (Salmo gairdneri Richardson) (Storebakken and Austreng 1987), Atlantic salmon (Salmo salar) (Einen et al. 1998) and red sea bream (Pagrus major) (Mohapatra et al. 2015). Particularly, the decline of HSI was more pronounced than the change of VSI, which might because liver was considered the most active organ for nutrient metabolism (Bradbury 2006), and the loss of liver weight was confirmed the energy consumption caused by starvation.
It is widely accepted that largemouth bass had limited glucose utilization (Ma et al. 2019; Li et al. 2020). Starvation can trigger the mobilization of energy stores in fish, and fish could show a clear and distinct sequences of energy mobilization during starvation (Black and Love 1986; Bychek et al. 2005; Li et al. 2018). These above confirmed the feasibility of investigating whether largemouth bass could effectively utilize glycogen through the background of starvation. In the present study, the liver glycogen, crude protein and crude lipid contents of largemouth bass were significantly reduced immediately after starvation, and this reduction was also been reported in catfish (Rhamdia hilarii) (Machado et al. 1988); red sea bream (Pagrus major) (Mohapatra et al. 2015); jundiá (Rhamdia quelen) (Barcellos et al. 2010) and gibel carp (Carassius auratus gibelio) (He et al. 2022). It indicated that both glycogen, protein and lipid were mobilized as energy resources during starvation. Additionally, the proportion of crude protein in hepatic was presented up-regulated trend in this study, as previously confirmed in channel catfish (Ictalurus punctatus) (Luo et al. 2009), which might account for the mobilization of glycogen and lipids in the liver. Indeed, in the present study, the hepatic glycogen contents (mg) showed a greater decline trend than the hepatic crude protein and crude lipid, it confirmed that the consumption of hepatic glycogen is higher than lipid and protein. Previous studies have revealed hepatic glycogen is usually the first mobilized store during starvation in sea bass (Dicentrarchus labrax) (Alliot et al. 1984), golden perch (Macquaria ambigua) (Collins and Anderson 1995) and traíra (Hoplias malabaricus) (Rios et al. 2006). Thus, these results of above mentioned revealed that largemouth bass is preferentially use glycogen as the energy source during starvation.
Target of rapamycin (TOR) and amino acid response (AAR) are two complementary nutrient-sensing signaling pathways that act to regulate protein synthesis and downstream metabolism (Kimball and Jefferson 2002; Wullschleger et al. 2006; Guo and Cavener 2007). In this regard, our results showed that the down-regulation of TOR pathway in largemouth bass liver was reflected by the decreasing activity of TOR and S6, which was consistent with some previous studies (Li et al. 2017; Davis and Gaylord 2011; Sheng et al. 2023). IGF-1 regulates the protein accretion (Stitt et al. 2004), and it plays an important role in promoting anabolism and growth, cell differentiation, reproduction and osmoregulation (Dubois and Callard 1993). Moreover, S6 is a phosphorylated target of S6K1, which is a downstream protein of TORC1, and linked to translation. Therefore, the down-regulation of IGF-1 and S6 expression induced by starvation further confirmed the inhibition of TOR pathway and protein synthesis the present study. Additionally, AAR signaling pathway usually acts in opposition to the TOR signaling pathway, and it could be activated by the amino acid deficiency to inhibit protein synthesis (Kilberg et al. 2005; Song et al. 2016). REDD1 is considered to be a negative regulator of the TOR signaling pathway in mammals (Jin et al. 2009), and eIF2α could be also phosphorylated by the scarce or imbalanced amino acids to promote the translation of ATF4 and REDD1, and reduced protein synthesis (Kilberg et al. 2009; Whitney et al. 2009; Castilho et al. 2014). It could be observed in this study that starvation could up-regulate the expression of eIF2α and REDD1, which indicated the activation of the AAR pathway in some extent. However, the expression of ATF4 was decreased significantly within 192 h, which seemed contradictory with previous studies in Mandarin Fish (Siniperca chuatsi) (Zou et al. 2022) and Gibel Carp (Carassius gibelio) (Xu et al. 2020). The potential underlying mechanism for this could be that starvation mobilized stores energy and reduced the glycogen accumulation in the liver of largemouth bass, which down-regulated the gene expression of ATF4 to inhibiting the AAR pathway to some extent. The negative impact of glycogen accumulation was alleviated in some extent. The underlying mechanism for the regulation of starvation on growth should be further explored. In summary, the inhibition of the TOR pathway and the activation of the AAR pathway could confirm that starvation for 192 h had negative effect on the protein metabolism.
In general, lipid content is related to lipid synthesis, decomposition and oxidation. As reported in some fish species, such as rainbow trout (Oncorhynchus mykiss) (Salem et al. 2007) and Atlantic salmon (Salmo salar) (Martin et al. 2010). In the present results, FAS, ACC1 and ACC2, which are involved in lipogenesis, exhibited significant decline in gene expressions of starved largemouth bass, and the inhibition of lipogenesis caused by starvation was also observed in zebrafish (Lu et al. 2019) and gibel carp (Carassius auratus gibelio var. CAS III) (Li et al. 2018). In addition, triglyceride was considered the most accessible lipid stored form during the lipolysis caused by starvation (Navarro and Gutierrez 1995). In this study, starvation up-regulated the expression of lipolysis related gene HSL in largemouth bass. which was also observed in previous studies (Li et al. 2018; Dar et al. 2018). These above results confirmed that the stored lipids were breakdown to providing energy. Generally, free fatty acid produced by the stored lipid (triglyceride formed as lipid droplets) could oxidize to provide energy for the body when the energy intake is insufficient. In the present study, the expression of CPT1 in the hepatic of largemouth bass was up-regulated under starvation, which was in line with results in grass carp (Ctenopharyngodon idellus) (Gong et al. 2017). Interestingly, we found that the β-oxidation gene ACO was significant down-regulated in starved for 192 h, which was consistent with the result in the 70 days starvation of zebrafish (Danio rerio) (Drew et al. 2008), and contradictory with gibel carp (Carassius auratus gibelio var. CAS III) (Li et al. 2018). ACO is considered to catalyze the first rate-limiting step in peroxisomal β-oxidation (Morais et al. 2007). Above all, the data confirmed that starvation could mobilize lipids as energy sources by activating fatty acid β-oxidation and suppressing lipogenesis.
Glycolysis and gluconeogenesis are opposing metabolic pathways involved in the degradation and synthesis of carbohydrates to maintain glucose homeostasis. PFKL was observed increasing in 12 h and 24 h treatments in this study, and PK was presented the same trend in the 12 h treatment. It reflected starvation encourages glycogen to participate in energy supply, which was contrary to the conclusions in barred sand bass (Paralabrax nebulifer) (Lowery et al. 1987) and gilthead seabream (Sparus aurata) (Metón et al. 2003). This might be explained by the fact that the short-term starvation induced hepatic glycogenolysis in largemouth bass to provide energy. Then, the expressions of PFKL and PK was decreased significant in 48 h, 96 h and 192 h, which might be related to the decreasing of hepatic glycogen stored, limiting the glycogenolysis and reducing the efficiency of energy supply. Meanwhile, the expression of gluconeogenesis related genes, FBP and PEPCK, was significantly down-regulated in present study, which was contradictory with previous studies in many fish species, such as rohu fingerlings (Labeo rohita) (Dar et al. 2018), gilthead sea bream (Sparus aurata) (Caseras et al. 2002) and Siberian sturgeon (Acipenser baerii) (Liang et al. 2017). Liver gluconeogenesis could maintain the glycemic level changes caused by starvation (Soengas et al. 1996; De Pedro et al. 2003). It might be related to the starvation period in this study was insufficient to completely consume the “excess” glycogen in largemouth bass, and the inhibition of glycolysis and low protein consumption resulted in the lack of precursor substances required for gluconeogenesis. The delayed activation of the gluconeogenic has also been observed in rainbow trout (Salmo gairdneri) (Morata et al. 1982) and Atlantic salmon (Salmo salar) (Soengas et al. 1996), and the regulation mechanism between food deprivation period and gluconeogenesis of largemouth bass should be further explored.
In conclusion, the results obtained in this study suggested that during starvation, glycogen could participate in the energy mobilization by activating the glycolysis and maintaining gluconeogenesis at a low expression level, which was an indispensable substance in largemouth bass energy regulation. Although starvation also activated lipolysis, largemouth bass mainly used liver glycogen during short-term starvation, which also resulted in a protein-saving effect. Therefore, the hepatic glycogen depletion and the inhibition hepatic gluconeogenesis during starvation could consume the excess stores of glycogen, contributing to relieve the damage of previous high carbohydrate in feed. These findings provided a more appropriate understanding in the carbohydrate utilization in carnivorous fish and supplied theoretical basis to the solution of high carbohydrate diet induced liver glycogen accumulation in aquaculture industry.