Earlier studies have successfully shown the limited use of glucose in largemouth bass, where an excessive intake of carbohydrates had a detrimental impact on their growth and overall health [26, 3, 27]. In the present investigation, largemouth bass subjected to excessive carbohydrate intake exhibited a notable decline in WG, survival, and SGR. Recent research has increasingly revealed the multifaceted benefits of astaxanthin in aquatic animals, including growth promotion, antioxidation, stress reduction, immune enhancement, and inflammation alleviation. In this study, the supplementation of astaxanthin led to an improvement of growth performance in largemouth bass fed HC diet. The inclusion of astaxanthin with a concentration of 0.01% had notable beneficial effect on the growth of Trachinotus ovatus when fed a high-fat diet [28]. Nevertheless, there was no notable disparity in the developmental progress of Oncorhynchus mykiss when exposed to a 0.05% ASX concentration [29]. A prior investigation has demonstrated that astaxanthin alleviated the obesity caused by a high-fat diet in mice [30]. CF and VSI were employed as indicators of fish fatness [31, 32, 33], and our findings showed that the inclusion of astaxanthin effectively reduced the increase in CF and VSI in largemouth bass fed with HC diet.
Generally speaking, the elevation of serum insulin level was the primary physiological response to a rise in plasma glucose levels during the GTT test [34]. This study confirmed the phenomenon of glucose intolerance in largemouth bass, characterized by higher levels of serum glucose during a GTT, which consistent with the discoveries of prior research [8, 35]. In the present study, HC diet leads to increased serum glucose levels and decreased plasma insulin levels, which indicated a reduced sensitivity to insulin, which may be caused by insufficient insulin secretion or insulin resistance. RNA-seq analysis of largemouth bass indicated that both HC and HCA diets caused significant impacts on glycolysis/gluconeogenesis pathways. This suggests that astaxanthin plays an essential role in the modulation of glucose homeostasis in response to treatment with HC diet. Similar to other vertebrates, insulin plays a crucial role in the regulation of glycolysis and gluconeogenesis processes in fish [36, 22]. Our study demonstrated that astaxanthin enhances systemic glucose tolerance and reduces serum insulin levels, but, it did not alter expression of insulin resistance genes in the liver during the GTT. Arguably, astaxanthin is likely to be different in different organs, or perhaps at different time in the same organ.
Insulin primarily exerts its metabolic effects in the liver via the PI3K/Akt signaling pathway. Insulin resistance may occur when there is a dysfunction in this communication pathway within liver tissues. Previous research has demonstrated that AST has the ability to mitigate growth, decrease oxidative stress, enhance insulin sensitivity, and activate the IRS/PI3K/Akt signaling pathway in mice fed a high-fat and high-fructose diet [9, 10]. Ezzat et al. [37] have identified PTP1B as a possible treatment target for diabetes, functioning as a suppressor of the insulin signaling pathway. In the current study, we present provide evidence for the initial instance to indicate that astaxanthin upregulated pi3kr1 mRNA expression and downregulated PTP1B protein expression level. Notably, astaxanthin exhibited an enhancement in Akt phosphorylation. Presumably, astaxanthin could potentially assume a crucial role in HC fed largemouth bass by reinstating insulin secretion and insulin sensitivity. Moreover, this research offers the preliminary validation that astaxanthin efficiently controls the PTP1B/PI3K/Akt signaling cascade in the liver. Although insulin promotes glycogen synthesis by suppression of GSK3 kinase, the role of this regulatory pathway is very limited [38]. Li et al. found a GSK3-independent insulin-stimulated glycogen synthesis pathway in mice [39]. However, more research is needed to determine the specific mechanism.
Histopathological examinations revealed that astaxanthin supplementation effectively ameliorated liver vacuolization, excessive accumulation of liver glycogen and induced by the HC diet, which indicated that inflammation was induced by a high carbohydrate feed in largemouth bass. Additionally, the activities of serum AST and ALT were notably reduced, indicating that astaxanthin indeed alleviated liver injury in largemouth bass subjected to the HC diet. Astaxanthin has been widely studied and acclaimed as a powerful antioxidant and anti-inflammatory agent under certain pathological conditions [40]. When the liver becomes damaged, hepatocytes secrete excessive amounts of inflammatory factors, such as tnf-α, il-1β, il-6 and il-8 etc. [41]. In the present study, tnf-α, il-6 and il-8 increased, and il-10 expression decreased in HC diet, whereas, astaxanthin has the ability to improve the high-carbohydrate induced hepatic inflammation. On the other hand, HC diet finally induced a reduction on mRNA levels cat and sod1 in the liver of largemouth bass, which indicated that HC diet would breakdown the antioxidant system, resulting in weak antioxidant capacity of the liver. Similarly, astaxanthin dietary supplementation recovery the liver redox state.
In this study, GO analysis showed that astaxanthin plays an important role in regulating the signaling pathways of apoptosis and programmed death. Programmed cell death, also referred to as apoptosis, consists of two primary routes: the intrinsic pathway, which engages the mitochondria, and the extrinsic pathway, which involves death receptors [42]. Activation of executioner caspase-3 occurs in both pathways [43]. Upon activation of the intrinsic pathway, the pro-apoptotic proteins Bax and Bad in the Bcl-2 family were increased, while the anti-apoptotic proteins Bcl-2 was decreased, resulting in an imbalance in the Bax to Bcl-2 ratio [44]. The utilization of RT-PCR analysis revealed that the stimulation of HC diet caused the increase of caspase-3, caspase-9 and bad expressions, and concurrently reduced the expression of bcl-2. However, the administration of astaxanthin effectively restored the expression levels of these genes to their normal levels. These findings unequivocally demonstrate that astaxanthin serves as a protective agent for largemouth bass against HC diet. The existing scholarly investigations pertaining to the impact of excessive carbohydrate consumption on largemouth bass primarily concentrate on transcript levels, enzyme activity, and metabolites at the individual level [14, 45]. However, there is a scarcity of literature regarding the examination at the cellular level. Afterwards, we utilized primary hepatocytes cultured in a high glucose setting as a representation to evaluate the changes caused by astaxanthin on cell growth and cell death. Our findings indicate that astaxanthin exhibited a significant ability to enhance cell survival and reduce the rate of apoptosis in a dose-dependent manner. Under typical cellular circumstances, the production and removal of ROS maintain in a balanced and ever-changing state. However, when the body is stimulated by specific factors, an overproduction of ROS can occur [46]. The overabundance of ROS has the potential to initiate a series of successive responses, which may involve the initiation of the caspase signaling pathway, ultimately leading to cellular apoptosis [47]. Through the utilization of primary hepatocytes, our study demonstrated that exposure to high glucose levels induced a substantial rise in ROS generation, which may be the reason why high glucose induces apoptosis.
It has been demonstrated that MAPK pathway activation is crucial to a wide variety of cellular processes such as cell proliferation, differentiation, and apoptosis [48]. In the present investigation, the involvement of MAPK signaling in the development of metabolic liver diseases in largemouth bass was examined, and it was found that the phosphorylation of ERK1/2, JNK1/2, and p38MAPK was significantly increased. As a super antioxidant, astaxanthin has been shown to exhibit efficacy in the treatment of diabetic mellitus by activating the NF-κB pathway, suppressing anti-apoptotic activity via modulation of MAPKs and PI3K/Akt pathways [49]. Our observation that astaxanthin significantly inhibits phosphorylation of p38MAPK, but not ERK1/2 and JNK1/2. This result indicates that the mechanism of astaxanthin-inhibited apoptosis might differ from previous studies. In response to various stressors, p38MAPK plays a vital role in triggering apoptosis. To further explore and confirm the pivotal role of p38MAPK in HG-induced primary hepatocytes apoptosis, a p38MAPK inhibitor, SB203580, was utilized. SB203580 did inhibit the protein expression of CAS3 and affect the gene expression of bcl-2, bax and caspase-3, underlining the key role of p38MAPK in promoting cell apoptosis. Moreover, the present study also demonstrated that astaxanthin may hinder apoptosis induced by high glucose by targeting p38MAPK/bcl-2/caspase-3 signaling pathway.