High Starch Induces Hematological Variations, Metabolic Changes, Oxidative Stress, Inflammatory Responses, and Histopathological Lesions in Largemouth Bass (Micropterus salmoides)

This study evaluated effects of high starch (20%) on hematological variations, glucose and lipid metabolism, antioxidant ability, inflammatory responses, and histopathological lesions in largemouth bass. Results showed hepatic crude lipid and triacylglycerol (TAG) contents were notably increased in fish fed high starch. High starch could increase counts of neutrophils, lymphocytes, monocytes, eosinophils, and basophils and serum contents of TAG, TBA, BUN, and LEP (p < 0.05). There were increasing trends in levels of GLUT2, glycolysis, gluconeogenesis, and LDH in fish fed high starch through the AKT/PI3K signal pathway. Meanwhile, high starch not only triggered TAG and cholesterol synthesis, but mediated cholesterol accumulation by reducing ABCG5, ABCG8, and NPC1L1. Significant increases in lipid droplets and vacuolization were also shown in hepatocytes of D3–D7 groups fed high starch. In addition, high starch could decrease levels of mitochondrial Trx2, TrxR2, and Prx3, while increasing ROS contents. Moreover, high starch could notably increase amounts of inflammatory factors (IL-1β, TNF-α, etc.) by activating NLRP3 inflammasome key molecules (GSDME, caspase 1, etc.). In conclusion, high starch could not only induce metabolic disorders via gluconeogenesis and accumulation of glycogen, TAG, and cholesterol, but could disturb redox homeostasis and cause inflammatory responses by activating the NLRP3 inflammasome in largemouth bass.


Introduction
As the cheapest and widest energy source or ingredient, starch is also a good binder for animal feeds [1,2].In most cases, adequate amount of dietary starch could enhance growth and feed efficiency, but it can also save protein by redirecting amino acids away from the oxidative process in humans, terrestrial animals, and fish species [3,4].Meanwhile, optimal contents of dietary starch could improve metabolism and enhance antioxidant capacity and immunity in animals [5][6][7].Nevertheless, carnivorous fish exhibit reduced capacity to digest dietary starch compared to omnivorous and herbivorous fish, as indicated by various research studies involving grass carp (Ctenopharyngodon idellus), Chinese longsnout catfish (Leiocassis longirostris) [8], jundiá catfish (Rhamdia quelen), Nile tilapia (Oreochromis niloticus) [9], and largemouth bass (Micropterus salmoides) [10].Several studies have proved that long-term intake of excessive dietary starch could induce metabolic dysfunction [6,11]

Diets, Fish, and Feeding Trials
Two isonitrogenous and isolipidic (crude protein: 46.72%, crude lipid: 9.86%) trial diets were produced with two levels of dietary starch (0% and 20%), respectively (Table 1).All used ingredients were firstly filtrated with 60-mesh screens, mixed, produced into pellets with 2.5 mm diameter, air-dried, and then stored following methods outlined by Wu et al. [39].Juvenile largemouth bass were acquired from a nearby fish breeder and introduced to recirculating tanks (1000 L) with a starch-free diet for two weeks.Following the acclimation period, 720 healthy fish (with an average starting weight of 7.43 ± 0.15 g) were chosen and distributed at random among 24 recirculating tanks (each with a capacity of 500 L and containing 30 fish).After the distribution, all fish were still fed a diet without starch for one month to maintain physiological consistency.Then, control groups with 3 tanks were still fed with a control diet without starch; these experimental groups (21 tanks) were fed with a high-starch diet (20%) and randomly distributed into seven groups (D1-D7) for 7 days.During the culturing time, all fish were fed with 3% of the total fish weight per day at 08:00 and 17:00.The recirculated water was filtered, with a temperature range of 26.3 to 28.4 degrees Celsius, pH range of 6.8 to 7.1, dissolved oxygen levels above 5.8 mg/L, low nitrite levels less than 0.02 mg/L, low ammonia nitrogen levels less than 0.05 mg/L, and exposed to natural sunlight.

Fish Tissue Samples' Preparation
After the experiment, all fish in these trial groups were first fasted for about 24 h, anesthetized with MS-222 (Sigma, St. Louis, MO, USA), and weighed to evaluate fish body indices.Twenty fish in each tank were randomly selected, weighed, and used for blood sampling for hematological assays.All the serum and liver samples were gathered with methods supplied by Wu et al. [39].These tested samples were firstly frozen using liquid nitrogen and subsequently kept at −80 • C for additional measurements.Frozen liver tissue samples were powderized in liquid nitrogen, suspended with physiological saline, and centrifuged with methods supplied by Wu et al. [39].All the supernatants were gathered, divided into Eppendorf tubes, and then frozen at −80 • C for further experimental tests.

Measurement of Antioxidant and Inflammatory Parameters in the Liver
Commercial diagnostic kits from Jiancheng Biotech (Nanjing, China) were used to measure levels of total superoxide dismutase (T-SOD), glutathione S-transferase (GST), CAT, malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), and total antioxidant capacity (T-AOC).ELISA kits from Jiangsu Meimian Industrial Co., Ltd.(Hangzhou, China) were used to evaluate the contents of ROS in the liver.Levels of TNF-α, IL-1β, IL-6, IFN-γ, IL-8, IL-12, and IL-17 in these fish liver samples were also assessed with ELISA kits purchased from Shanghai Hengyuan Biotech (Shanghai, China) following their recommended protocols.All measurements were performed with 9 replicates.

Measurement of Gene Expression in the Liver
RNA extraction, RNA integrity examination, quantification, and RNA reverse transcription were performed with methods supplied by Wu et al. [13].All the cDNA samples were treated with ethanol (95%) and then frozen at −80 • C for further analysis.All the primers were designed according to relative gene sequences from largemouth bass in the GenBank (Table 2).The CFX96 machine from Bio-Rad in Hercules, CA, USA was utilized for conducting real-time qPCR analysis, with expression variations analyzed using the 2 −∆∆CT method as outlined by Yang et al. [37].All measurements were performed with 9 replicates for further analyses.

Measurement of Histomorphometrical Parameters of the Liver
Six fish were selected at random from each category, and liver tissue samples measuring 0.5 cm × 0.5 cm × 0.5 cm were rinsed with 0.6% saline and then immersed in a 4% paraformaldehyde solution for 48 h.The liver tissue samples were then fixed, dehydrated, embedded, and stained with hematoxylin and eosin (HE), periodic acid Schiff (PAS), or oil red O (ORO) before being examined under light microscopy (DM500, Leica, Leica Microsystems (Schweiz) AG, Heerbrugg, Switzerland).Liver micrographs were captured at 20 × and 40 × magnification and recorded using a digital camera following the procedures outlined in our laboratory's previous publication [39].K-Viewer (https://kv.kintoneapp.com/en/user/,accessed 31 March 2023) 1.0 software (1.0.4) (Konfoong Bioinf Tech Co., Ltd, Ningbo, China) and Slide Viewer (accessed 21 April 2023) 2.5 software (2.5.0) (3DHISTECH Ltd., Budapest, Hungary) were utilized for image analysis conducted by Wu et al. [39].Each analysis was conducted with 3 duplicates.

Data Analysis
All results were expressed as mean ± SD (standard deviation).Groups were compared using SPSS 25.0 (IBM, Chicago, IL, USA) through a one-way analysis of variance (ANOVA).Tukey's multiple interval test was used for multiple comparisons between different dietary treatments.Furthermore, orthogonal polynomial contrasts about linear and/or quadratic effects were used with the methods supplied by Wu et al. [39].A significant difference was indicated when p was less than 0.05.

Body Indices and Lipid Contents in the Liver
The viscerosomatic index (VSI), hepatosomatic index (HSI), and intestinal somatic index (ISI) of the fish showed a tendency to rise as the number of days on a high starch diet increased in largemouth bass juveniles, reaching their peak in the D7 trial groups (p < 0.05) (Figure 1A-C).Furthermore, no significant variances were seen in ISI among the D3, D5 and D7 trial groups (p > 0.05).The liver's crude lipid contents increased as the high starch diet was cultured for more days, reaching its peak in the D7 groups (p < 0.05), although there were no notable differences between the D1 and D3 groups (p > 0.05).

Biochemical and Hormone Indicators in the Serum
High dietary starch significantly increased contents or levels of GLU, HDL-C LDL-C in the serum of D1-D7 groups when compared with that in the D0 groups fed a no-starch diet (p < 0.05).The peaked levels of HDL-C, and LDL-C were all shown D7 groups (p < 0.05), with no notable differences among D1, D3, D5, and D7 group

Hematological Parameters
With the increase in the consumption of a high-starch diet by largemouth bass over several days, there was first a rise and then a decrease in the trends on RBC counts in the D1-D7 groups (p > 0.05), while there were no notable differences in RBC counts between the D0 and D7 groups (p > 0.05).Furthermore, HGB levels rose in the D1, D3, and D5 groups and reached their peak in the D5 trial groups before declining in the D7 treated groups (p < 0.05).Additionally, there was a notable increase in the counts of WBC and LYM (p < 0.05), with WBC peaking in the D7 groups (p > 0.05), and LYM peaking in the D5 groups (p > 0.05).In the meantime, all NEU, MON, and EOS levels showed marked increases when compared to the D0 group, peaking in the D7 groups (p < 0.05).There were no notable differences in MON and EOS counts between the D3 and D5 groups (p > 0.05).Conversely, there was a marked inverse correlation between PLT counts and the duration of consuming a high-starch diet in comparison to the D0 groups (p < 0.05).However, no significant differences were presented between the D5 and D7 groups (p > 0.05) (Table 3).

Biochemical and Hormone Indicators in the Serum
High dietary starch significantly increased contents or levels of GLU, HDL-C, and LDL-C in the serum of D1-D7 groups when compared with that in the D0 groups fed with a no-starch diet (p < 0.05).The peaked levels of HDL-C, and LDL-C were all shown in the D7 groups (p < 0.05), with no notable differences among D1, D3, D5, and D7 groups (p > 0.05).TG and TC contents were notably heightened and peaked in the D5 and D7 groups (p < 0.05), respectively.AST activities were firstly heightened and then maintained stable trends in D1, D3, D5, and D7 groups (p > 0.05).Similarly, ALT activities and TBA contents were also markedly heightened to the maximal levels in the D5 and D3 groups, respectively, with no significant differences between the D3 and D5 groups (p > 0.05).In addition, amounts of ALP, ALB, LDH, and BUN were all first increased in the D1 and D3 groups when compared with that in the D0 groups (p < 0.05) and then reduced in the D5 and/or D7 groups.However, amounts of ALP, ALB, LDH, and BUN in the D7 groups were still significantly higher than those in the D0 groups (p < 0.05).In addition, the contents of INS and ADPN first rose to their peaks in the D3 groups when compared to those in the D0 groups (p < 0.05) and then decreased to the lower levels in the D5 and D7 groups when compared to that in the D3 groups (p < 0.05).However, GC amounts were increased and peaked in the D7 groups when compared to that in the D0 groups.The levels of LEP were also firstly heightened and reached maximal values in the D5 groups and then decreased in the D7 groups, although LEP levels in the D7 groups were markedly higher than those in the D0 groups (p < 0.05) (Table 4).Note: a-e Values in the same row with different letters indicate significant differences (p < 0.05).A = mmol/L, B = U/L, C = umol/L, D = g/L, E = mU/L, F = pg/ml, G = µg/L.

Activities or Contents of Glucose and Lipid Metabolism in Fish Liver
Compared to the D0 groups, amounts and activities of GLUT2, GCK, PFK, PK, LDH, PEPC, and ACC were all notably heightened in the livers of D3, D5, and D7 groups fed with high-starch diet (p < 0.05) and reached maximal levels in D5 or D7 groups, respectively.The contents of PA and LA were also notably heightened in the livers of D1-D7 groups fed with a high-starch diet (p < 0.05), although decreased in the D7 groups when compared with their peak in D5 and D3 groups (p < 0.05), respectively.In addition, activities of FBP, G6Pase, GCS, GBE, and FAS were first increased in D3 or D5 treated groups (p < 0.05) and then decreased in D7 groups.However, activities of GDE were significantly reduced in the D1-D7 groups when compared with D0 groups (p < 0.05), although there were no differences between the D1-D3, and the D5-D7 groups.While, LAG contents were increased in D1-D7 groups and reached the maximal level in the D7 groups (p < 0.05).Although there was no significant variation among the D0, D1, and D3 groups (p > 0.05), Ac-CoA contents were increased in D5 and D7 groups (p < 0.05).Moreover, there were similar variable patterns in the contents of Mal-CoA and LTAG.Compared to the D0 groups, the maximal contents of Mal-CoA and LTAG were both shown in the D5 groups, although no significant variation could be observed between the D5 and D7 groups (p > 0.05) (Table 5).Note: a-e Values in the same row with different letters indicate significant differences (p < 0.05).A = ng/mg prot, B = U/mg, C = U/g prot, D = IU/L, E = U/mg prot, F = nmol/min/mg prot, G = IU/g prot, H = mg/g, I = mmol/g prot, J = µmol/mg prot, K = mmol/h/mg prot, L = U/L.

Gene Expression Variations of FA and TAG Metabolism in Fish Liver
The high-starch diet significantly upregulated the expression of genes involved in fatty acid synthesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), Furthermore, there were rising trends in the levels of insulin receptor (ISR), insulin receptor substrate 2 (ISR2b), serine/threonine kinase 1 (AKT1), and sterol regulatory element binding protein 1c (SREBP1c) observed in the livers of subjects on high-starch diets (p < 0.05).In addition, the levels of PIK3c were significantly higher in the D5 groups compared to the D0 groups (p < 0.05), with no statistical differences observed among the latter (p < 0.05).The highest levels of SREBP1c were shown in the D5 groups compared to those in the other four groups, although there were no statistical differences observed among D1, D3, and D7 groups (p > 0.05) (Figure 4).Furthermore, there were rising trends in the levels of insulin receptor (ISR), insulin receptor substrate 2 (ISR2b), serine/threonine kinase 1 (AKT1), and sterol regulatory element binding protein 1c (SREBP1c) observed in the livers of subjects on high-starch diets (p < 0.05).In addition, the levels of PIK3c were significantly higher in the D5 groups compared to the D0 groups (p < 0.05), with no statistical differences observed among the latter (p < 0.05).The highest levels of SREBP1c were shown in the D5 groups compared to those in the other four groups, although there were no statistical differences observed among D1, D3, and D7 groups (p > 0.05) (Figure 4).

Gene Expression Variations of FA and TAG Metabolism in Fish Liver
The high-starch diet significantly upregulated the expression of genes involved in fatty acid synthesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), acyl-CoA desaturase (SCD), and NADP-dependent malic enzyme 1 (ME1), with the highest levels observed in the D5 groups compared to those in D0 groups (p < 0.05).Levels of

Gene Expression Variations of FA and TAG Metabolism in Fish Liver
The high-starch diet significantly upregulated the expression of genes involved in fatty acid synthesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), acyl-CoA desaturase (SCD), and NADP-dependent malic enzyme 1 (ME1), with the highest levels observed in the D5 groups compared to those in D0 groups (p < 0.05).Levels of mRNA for fatty acid-binding protein liver type (FABP-L) and fatty acid transport protein 4 (FATP4) were significantly increased to their highest levels in D7 or D5 groups (p < 0.05), respectively.Additionally, high starch was found to notably enhance the expression of glycerol kinase (GK), glycerol-3-phosphate acyltransferase 3 (GPAT3), GPAT4, and diacylglycerol O-acyltransferase 2 (DGAT2) related to triglyceride production in the livers of fish fed with high-starch diet (p < 0.05).Similarly, the levels of perilipin-2 (Plin2) were notably increased to the highest point in the D5 groups and then decreased in the D7 groups (p < 0.05), with no significant variations between the D3 and D7 groups (p > 0.05).Moreover, the levels of the triglyceride transporter apolipoprotein B-100 (APOB100) were significantly increased to the highest point in the D5 groups, then decreased in the D7 groups (p < 0.05), with no significant difference between the D1 and D3 groups (p > 0.05) (Figure 5).

Cholesterol Metabolism and Bile Acid Synthesis of Gene Expression
In the hepatic cells of fish fed a high-starch diet, the expression levels of genes in volved in cholesterol metabolism, including sterol regulatory element-binding protein (SREBP2), acetoacetyl-CoA synthetase (AACS), 3-hydroxy-3-methylglutaryl-Coenzyme reductase a (HMGCRa), hydroxymethylglutaryl-CoA synthase (HMGCS), and lanoster 14-alpha demethylase (CYP51), were significantly elevated in the D3 treated groups com pared to the D0 control groups (p > 0.05) and then decreased in the D7 groups compare to the D3 treated groups (p < 0.05).Both HMGCRa and HMGCS showed no significan differences not just between the D1 and D3 groups, but also between the D5 and D groups (p > 0.05).SREBP2 and AACS were not significantly different between D0, D5, an D7 groups (p > 0.05), and CYP51 was not significantly different between D7 and D0 group (p > 0.05).Expression levels of sterol O-acyltransferase 2 (SOAT2) consistently rose in high starch groups as feeding time increased, peaking in the D7 groups (p < 0.05).Conversel the expression of ATP-binding cassette sub-family G member 5 (ABCG5) and ABCG8 wer reduced in the D3, D5, and D7 treated groups compared with the D0 control groups (p 0.05).Additionally, there was first an increase trend on the levels of Niemann-Pick C1-lik protein 1 (NPC1L1) in the D1 and D3 groups and then, they reduced to the original leve in D5 and D7 groups when compared with the D0 groups (Figure 6).

Cholesterol Metabolism and Bile Acid Synthesis of Gene Expression
In the hepatic cells of fish fed a high-starch diet, the expression levels of genes involved in cholesterol metabolism, including sterol regulatory element-binding protein 2 (SREBP2), acetoacetyl-CoA synthetase (AACS), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase a (HMGCRa), hydroxymethylglutaryl-CoA synthase (HMGCS), and lanosterol 14-alpha demethylase (CYP51), were significantly elevated in the D3 treated groups compared to the D0 control groups (p > 0.05) and then decreased in the D7 groups compared to the D3 treated groups (p < 0.05).Both HMGCRa and HMGCS showed no significant differences not just between the D1 and D3 groups, but also between the D5 and D7 groups (p > 0.05).SREBP2 and AACS were not significantly different between D0, D5, and D7 groups (p > 0.05), and CYP51 was not significantly different between D7 and D0 groups (p > 0.05).Expression levels of sterol O-acyltransferase 2 (SOAT2) consistently rose in high-starch groups as feeding time increased, peaking in the D7 groups (p < 0.05).Conversely, the expression of ATP-binding cassette sub-family G member 5 (ABCG5) and ABCG8 were reduced in the D3, D5, and D7 treated groups compared with the D0 control groups (p < 0.05).Additionally, there was first an increase trend on the levels of Niemann-Pick C1-like protein 1 (NPC1L1) in the D1 and D3 groups and then, they reduced to the original levels in D5 and D7 groups when compared with the D0 groups (Figure 6).
starch groups as feeding time increased, peaking in the D7 groups (p < 0.05).Convers the expression of ATP-binding cassette sub-family G member 5 (ABCG5) and ABCG8 w reduced in the D3, D5, and D7 treated groups compared with the D0 control groups 0.05).Additionally, there was first an increase trend on the levels of Niemann-Pick C1protein 1 (NPC1L1) in the D1 and D3 groups and then, they reduced to the original le in D5 and D7 groups when compared with the D0 groups (Figure 6).In addition, the mRNA transcription levels of bile acid processing, such as CYP7A1 and CYP27A1, were significantly increased in the D1 and D3 groups (p < 0.05) and then reduced in the D7 groups in comparison to the D0 groups.The expression of the bile salt export pump (BSEP) only decreased significantly in the D7 groups compared to D0 (p < 0.05), with no significant differences observed among D0, D1, D3, and D5 groups.The transcription levels of sodium/taurocholate cotransporting polypeptide (NTCP) were significantly increased in the D3 and D5 treated groups (p < 0.05) and then reduced in the D7 treated groups in comparison to the D0 groups.Amounts of solute carrier organic anion transporter family member 1 (OATP1) were constantly up-regulated and reached the peak in the D7 treated groups (p < 0.05).Moreover, organic solute transporter subunit alpha (OST-α) was notably up-regulated in D1-D7 groups and obtained the maximal levels in D3 treated groups compared with D0 control groups (p < 0.05).However, no significant variations were observed in the transcription of canalicular multispecific organic anion transporter 2 (MRP3) (p > 0.05) (Figure 7).
Metabolites 2024, 14, x FOR PEER REVIEW 13 In addition, the mRNA transcription levels of bile acid processing, such as CYP and CYP27A1, were significantly increased in the D1 and D3 groups (p < 0.05) and reduced in the D7 groups in comparison to the D0 groups.The expression of the bile export pump (BSEP) only decreased significantly in the D7 groups compared to D0 0.05), with no significant differences observed among D0, D1, D3, and D5 groups.transcription levels of sodium/taurocholate cotransporting polypeptide (NTCP) were nificantly increased in the D3 and D5 treated groups (p < 0.05) and then reduced in th treated groups in comparison to the D0 groups.Amounts of solute carrier organic a transporter family member 1 (OATP1) were constantly up-regulated and reached the p in the D7 treated groups (p < 0.05).Moreover, organic solute transporter subunit a (OST-α) was notably up-regulated in D1-D7 groups and obtained the maximal leve D3 treated groups compared with D0 control groups (p < 0.05).However, no signifi variations were observed in the transcription of canalicular multispecific organic a transporter 2 (MRP3) (p > 0.05) (Figure 7).

Variation of Antioxidant and Oxidant Parameters
The liver's T-SOD and CAT activities were notably reduced in D1-D7 groups fed the high-starch diet compared to the D0 groups (p < 0.05).There were no significant ferences in T-SOD levels among the D1, D3, and D5 groups (p > 0.05), nor in CAT le between the D1 and D3 groups and among the D3, D5, and D7 groups (p > 0.05).ROS H2O2 levels were both elevated and peaked in the D5 groups (p < 0.05), with no signifi variation observed between the D3 and D7 groups in terms of ROS (p > 0.05).Amoun GST and MDA were peaked in the D7 groups (p < 0.05), with no significant differe observed among D0, D1, and D3 for GST (p > 0.05) and among D1, D3, and D5 tre groups for MDA levels (p > 0.05).T-AOC levels were lower in the D5 and D7 groups c pared to the D0 groups (p > 0.05), with no significant differences observed among the D1, and D3 groups, as well as between the D5 and D7 groups (p > 0.05) (Table 6).Table 6.Effects of dietary days on the antioxidative and oxidative indices of liver in largem bass fed high starch (mean ± SD, n = 9).

Variation of Antioxidant and Oxidant Parameters
The liver's T-SOD and CAT activities were notably reduced in D1-D7 groups fed with the high-starch diet compared to the D0 groups (p < 0.05).There were no significant differences in T-SOD levels among the D1, D3, and D5 groups (p > 0.05), nor in CAT levels between the D1 and D3 groups and among the D3, D5, and D7 groups (p > 0.05).ROS and H 2 O 2 levels were both elevated and peaked in the D5 groups (p < 0.05), with no significant variation observed between the D3 and D7 groups in terms of ROS (p > 0.05).Amounts of GST and MDA were peaked in the D7 groups (p < 0.05), with no significant differences observed among D0, D1, and D3 for GST (p > 0.05) and among D1, D3, and D5 treated groups for MDA levels (p > 0.05).T-AOC levels were lower in the D5 and D7 groups compared to the D0 groups (p > 0.05), with no significant differences observed among the D0, D1, and D3 groups, as well as between the D5 and D7 groups (p > 0.05) (Table 6).The expressions of Cu/Zn superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) were initially reduced to a minimum in the D1 and D3 groups compared to the D0 control groups (p < 0.05), respectively, both followed by an increase in the D5 groups (p < 0.05) and then were reduced in the D7 groups.There were no significant differences in the transcription levels of CAT in the D0-D7 groups (p > 0.05).Furthermore, GST1 transcriptions were notably elevated in the D3 and D5 groups compared to the D0 group, reaching its peak in the D3 and D5 groups (p < 0.05), with no significant differences between these two groups (p > 0.05).In contrast, the expressions of thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2), and peroxiredoxin 3 (Prx3) were decreased as high-starch feeding increased compared to the D0 groups (p < 0.05) (Figure 8A).The livers of largemouth bass fed a high-starch diet showed increased transcription variations of cytochrome c oxidase 4 (COX4), NADPH oxidase 1 (NOX1), NADPH oxidase activator 1 (NOXA1), and heme oxygenase 1 (HO1), reaching their peaks in the D5 and/or D7 groups compared to those in the D0 groups (p < 0.05).Furthermore, there were no significant variations in COX4 levels among D0, D1, and D7 groups.There were no notable variations in HO1 transcription levels between the D5 and D7 treated groups (p > 0.05) (Figure 8B).The expressions of Cu/Zn superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) were initially reduced to a minimum in the D1 and D3 groups compared to the D0 control groups (p < 0.05), respectively, both followed by an increase in the D5 groups (p < 0.05) and then were reduced in the D7 groups.There were no significant differences in the transcription levels of CAT in the D0-D7 groups (p > 0.05).Furthermore, GST1 transcriptions were notably elevated in the D3 and D5 groups compared to the D0 group, reaching its peak in the D3 and D5 groups (p < 0.05), with no significant differences between these two groups (p > 0.05).In contrast, the expressions of thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2), and peroxiredoxin 3 (Prx3) were decreased as high-starch feeding increased compared to the D0 groups (p < 0.05) (Figure 8A).The livers of largemouth bass fed a high-starch diet showed increased transcription variations of cytochrome c oxidase 4 (COX4), NADPH oxidase 1 (NOX1), NADPH oxidase activator 1 (NOXA1), and heme oxygenase 1 (HO1), reaching their peaks in the D5 and/or D7 groups compared to those in the D0 groups (p < 0.05).Furthermore, there were no significant variations in COX4 levels among D0, D1, and D7 groups.There were no notable variations in HO1 transcription levels between the D5 and D7 treated groups (p > 0.05) (Figure 8B).

Variation of Molecules in Inflammatory Responses in Fish Liver
In the livers of largemouth bass fed high starch, TNF-α contents were constantly heightened and peaked in the D7 treated groups compared to the D0 control groups (p < 0.05).There were similar variation trends in the levels of IL-6, IL-8, IL-17, and IL-1β in the D1-D7 groups and were notably higher than those in the D0 control groups (p < 0.05).IL-6 contents were firstly heightened and peaked in the D3 treated groups compared to the D0 groups (p < 0.05).Similarly, contents of IL-8, IL-17, and IL-1β were also firstly heightened and all reached the maximum in the D5 treated groups compared to those in the D0 groups (p < 0.05).Contents of IL-12 in high-starch groups were notably higher than that in the D0 groups, although there were no differences among the D1-D7 treated groups (p > 0.05).IFN-γ levels were notably increased in the D5 and D7 groups compared with those in D0, D1, and D3 treated groups (p < 0.05), while there were no significant differences among the D0, D1, D3 groups and between the D5, D7 groups (Table 7).Expression levels of IL-1β, IL-8, and TNF-α were first increased and reached the maximum in the D3 and/or D5 groups compared to those in the D0 groups (p < 0.05) and then reduced in D7 groups.Although there were no differences in the D0, D1, and D3 groups (p >0.05), expression levels of IL-12 and IFN-γ were notably increased in the D5 and D7 groups compared to those in the D0 groups (p < 0.05), with no significant differences observed among D5 and D7 (p > 0.05).In addition, transcription levels of apoptosis genes (caspase 1 and caspase 3) were increased in the D5 and D7 groups, and both peaked in the D7 trial groups (p > 0.05).And the transcription levels of caspase 6, caspase 8, and caspase 9 also first increased and reached peaks in the D5 and/or D3 groups compared to those in the D0 groups (p < 0.05) and then all decreased in D7 groups, with notable differences between D7 and D0 groups (p > 0.05) (Figure 9A).Moreover, the expression levels of NLRP3 inflammasome key molecules, including nucleotide-binding oligomerization domain-containing protein 1 (NOD1), NOD2, and gasdermin-E (GSDME), were notably elevated after consuming a high-starch diet, peaked in the fish liver of D7 or D5 groups compared to that of D0 groups (p < 0.05).Although there were no significant differences in the D0-D5 groups, apoptosis-associated speck-like protein-containing CARD (ASC) levels were notably elevated in the D7 groups (p < 0.05) (Figure 9B).

Fish Hepatic Histopathological Lesions
The liver samples of juvenile largemouth bass in the high-starch group (B-E) and the control group (A) were observed by photographing, hematoxylin-eosin staining (HE), periodic acid-Schiff staining (PAS), and Oil Red O staining (ORO) (Figure 10).Within the D0 category (A1-A3), the livers of the largemouth bass displayed a reddish hue and no apparent abnormal characteristics, featuring well-formed hepatocytes with distinct nuclei.Additionally, the glycogen granules and lipid droplets in the liver cells of the largemouth bass were both smaller and of a lighter shade.Within the D1 groups (B1-B3), there were instances of pale livers displaying a dense nuclear phenotype and indistinct liver cords, often indicating the early stages of liver fibrosis in clinical settings.Additionally, there was an increase in glycogen granules and lipid droplets.In D3 (C1-C3), D5 (D1-D3), and D7 (E1-E3) groups, the white areas in the livers of largemouth bass increased and the shape became larger, and glycogen granules and lipid droplets were increased with culturing times with the high-starch diet.

Fish Hepatic Histopathological Lesions
The liver samples of juvenile largemouth bass in the high-starch group (B-E) and the control group (A) were observed by photographing, hematoxylin-eosin staining (HE), periodic acid-Schiff staining (PAS), and Oil Red O staining (ORO) (Figure 10).Within the D0 category (A1-A3), the livers of the largemouth bass displayed a reddish hue and no apparent abnormal characteristics, featuring well-formed hepatocytes with distinct nuclei.Additionally, the glycogen granules and lipid droplets in the liver cells of the largemouth bass were both smaller and of a lighter shade.Within the D1 groups (B1-B3), there were instances of pale livers displaying a dense nuclear phenotype and indistinct liver cords, often indicating the early stages of liver fibrosis in clinical settings.Additionally, there was an increase in glycogen granules and lipid droplets.In D3 (C1-C3), D5 (D1-D3), and D7 (E1-E3) groups, the white areas in the livers of largemouth bass increased and the shape became larger, and glycogen granules and lipid droplets were increased with culturing times with the high-starch diet.

Discussion
Previous studies have found dietary starch levels could modulate body indices (VSI, HSI, and ISI), which can also influence animal body compositions [41,42].The current re-

Discussion
Previous studies have found dietary starch levels could modulate body indices (VSI, HSI, and ISI), which can also influence animal body compositions [41,42].The current research found that high-starch diet (20%) led to a notable increase in VSI, HSI, and ISI in largemouth bass as the culturing days increased, similar with previous studies on giant gourami (Osphronemus goramy) [7], blunt snout bream (Megalobrama amblycephala) [43], and black carp (Mylopharyngodon piceus) [13].Additionally, our findings indicated a significant rise in hepatic lipid levels over time due to high starch exposure, aligning with earlier studies on largemouth bass [44,45] and GIFT tilapia (Oreochromis niloticus) [5].Higher dietary starch, when combined with these findings and our results, was found to induce hepatic lipid synthesis and accumulation in largemouth bass.
Hematological parameters have been important physiological assays for analyzing pathological variations in human and fish species [31,46,47].Our results showed that high starch increased RBC counts and HGB levels, especially in the D1, D3 and D5 groups, which was in line with former results in Siberian sturgeon (Acipenser baerii) [48].It is well-known that RBC and HGB undertake key roles in increasing oxygen-carrying, metabolic capacity and redox regulation in humans and animals [49][50][51].Therefore, combining these findings and our results, it could be inferred that the largemouth bass needs more oxygen to carry out various physiological activities during the absorbing and metabolic processes of higher dietary starch.In addition, our results found high starch significantly increased counts of WBC, NEU, LYM, MON, EOS, and BAS with increasing culturing times, which agrees with previous results in Labeo rohita [52] and gilthead sea bream (Sparus aurata) [53].It is well-known that adequate counts of WBC, NEU, LYM, MON, EOS, and BAS could improve immunity and enhance the defense effects when the animal is infected by some kinds of pathogens or fed with adequate nutrients [54].However, excessive counts of WBC, NEU, MON, EOS, and BAS could induce inflammation and/or inflammatory responses in dairy goat [55], rat [56], and humans with type 2 diabetes mellitus [57].The combination of these discoveries and our study outcomes indicates that increased consumption of dietary starch may lead to inflammation or inflammatory reactions in largemouth bass.
Serum biochemical indices are widely recognized as crucial indicators of health and metabolic status in both humans and animals.ALT and AST variations reflect hepatic metabolism and environmental abnormalities in animals [58].Increased serum ALT and AST levels in the D3, D5, and D7 groups indicated that hepatic metabolism might be disturbed or damaged mediated by high dietary starch in largemouth bass [17].Meanwhile, serum BUN variations could correlate with nitrogen utilization and reflect the homeostasis of exogenous amino acids [59].So, higher BUN contents presented in D1-D7 groups indicated high dietary starch might impair exogenous nitrogen utilization in largemouth bass.Although ALP could act as an immune defense biomarker [60,61], excessive levels of ALP could couple degeneration, necrosis, and destruction of the liver due to cellular damage [62].Moreover, higher levels of serum ALP are also correlated with the bile ducts obstructed in metabolic liver diseases [60].Combined with higher serum ALP activities in fish fed high-starch diets in this study, it is suggested high dietary starch could cause cellular damage in largemouth bass.In addition to being an important immune index, serum ALB also plays a key role in the antioxidant function [63].Considering the lower levels of serum ALB in D7 groups, it indicated that high dietary starch could impair immunity and increase the danger of oxidative stress in largemouth bass.Previous studies have found higher levels of serum TBA could be correlated with early-stage metabolic liver disease mediated by higher contents of cholesterol in animals [37,64].Therefore, elevated levels of serum TBA suggest that increased dietary starch not only worsens liver damage but also triggers cholestatic disease in largemouth bass [65,66].In typical physiological conditions, HDL-C moves total cholesterol from peripheral tissues to the liver for additional processing and bile acid production, whereas LDL-C transports hepatic cholesterol to other tissues [67].Our findings showed contrasting trends in the HDL-C and LDL-C levels in D7 groups when compared with those in D0 groups (Table 4), which is consistent with previous findings in mice [68] and juvenile blunt snout bream [43].Moreover, some researches have shown the level of TG and TC is positively proportional to the severity of fatty liver [67,69].Combined with higher contents of serum TAG and TC, it is suggested that high dietary starch could not only prevent TC and TAG from being absorbed and utilized by the body, but also induce their accumulation in the body and then result in dyslipidemia and metabolic fatty liver [70].
Carnivorous fish are recognized as having weak capabilities for processing glucose and often present persistent hyperglycemia after feeding on a diet with high starch [2].INS, ADPN, and GC play a crucial role as endocrine hormones in maintaining the glucose balance by managing fluctuations in serum glucose levels in both mammals and animals, as highlighted in studies by del sol Novoa et al. [71], Xu et al. [72], and Zhong et al. [17].Elevated levels of serum glucose can trigger INS and ADPN release or decrease glucagon production to alleviate stress from hyperglycemia and maintain the glucose balance [73].Our research discovered contrasting trends in the levels of serum INS, ADPN, and GC, despite a consistent increase in serum glucose levels in largemouth bass fed a high-starch diet.Likewise, earlier research has demonstrated that a 20% wheat starch diet led to a notable increase in blood glucose and INS levels in largemouth bass [6] and over a 4-week period in rainbow trout [74].Conversely, it was observed that a high-starch diet resulted in significantly higher blood glucose levels after 3 h of feeding, but had no impact on INS levels in largemouth bass fed a diet with high starch (13.56%) [18].As for these differences on the INS contents, it might be due to the different sample time in these studies.Furthermore, the levels of INS and ADPN were both decreased in the D7 groups, indicating that inadequate secretion of these hormones was the primary cause of glucose intolerance in carnivorous fish species [2].In addition to reducing appetite signal, LEP also plays a crucial role in controlling fat or lipid reserves in both humans and animals [75][76][77].Considering these findings and higher contents of serum LEP in this study, it indicated that higher starch could induce LEP secretion, which could then initiate the self-regulatory mechanism in response to higher serum glucose in largemouth bass [78].Moreover, higher LEP levels were also presented in these obese individuals with a higher percentage of body fat, which was mainly mediated by typical LEP resistance in obesity [79].The combination of these discoveries and our own results indicates that elevated starch levels may lead to LEP resistance, ultimately disrupting the regulation of lipid metabolism in largemouth bass, although further research about this resistance mechanism is needed to fully understand this.
As a major metabolic organ, the liver tissue plays important roles regulating glucose and lipid metabolic processes, include glycolysis, gluconeogenesis, glycogen synthesis, lipogenesis and lipolysis [10].The important role of GLUT2 in regulating glucose transfer between the liver and serum through the PI3K/AKT signaling pathway has been widely recognized [80,81].Our research found that elevated levels of dietary starch significantly increased GLUT2 levels at both transcription and protein levels, consistent with findings in rainbow trout (Oncorhynchus mykiss) [82], blunt snout bream [83], and Gibel carp (Carassius gibelio) [84].This indicates that high starch intake may enhance the transport of plasma glucose into hepatic cells by stimulating GLUT2 in largemouth bass.Numerous past research has indicated that elevated levels of starch can trigger glycolysis pathways through the INS signal pathway in mice [85], blunt snout bream [86], common carp (Cyprinus carpio) [87], and largemouth bass [45].Levels of GCK, PFK, PK, ISRa, ISRS2b, PI3Kc, and AKT1 were significantly increased in the D5-D7 groups that were fed the high-starch diet, as well as with higher PA contents in our findings.Our results, along with these discoveries, suggest that elevated levels of starch may enhance glucose metabolism by stimulating glycolysis through the activation of the typical PI3K/AKT signaling pathway in largemouth bass.Additionally, gluconeogenesis was also stimulated with elevated levels of FBP and G6Pase in the D5-D7 groups that were given high-starch diet, which is similar with former results in largemouth bass fed 13.56% dietary starch [18], suggesting there was a competition between dietary glucose and endogenous productive glucose.Based on these findings, it was suggested that high levels of starch could lead to glucose metabolic disorders and hinder glucose utilization in largemouth bass.
Furthermore, the hepatic production of glycogen in animals may be controlled by the PI3K/AKT signaling pathway, which acts on its downstream proteins and enzymes (GYG2, GCS, GBE, and GDE) as indicated by Yang et al. [37] and Zhang et al. [88].Typically, increased levels of GYG2, GCS, and GBE may play important functions in enhancing glycogen production, whereas elevated GDE levels could facilitate the breakdown of hepatic glycogen [2].Past research has shown that the amounts of glycogen in the liver increased as the levels of starch in the diet rose for red spotted grouper (Epinephelus akaara) [89] and blunt snout bream [90].Furthermore, by utilizing [U-14C] glucose as a marker, it showed a significant rise in glycogen synthesis from glucose in largemouth bass when the dietary starch level was raised from 5% to 15% [91].Combined with these findings, it indicated that high starch may induce hepatic glycogen synthesis in largemouth bass through the PI3K/AKT signal transduction pathway, as evidenced by the variations of these key molecules, LAG contents, and PAS-stained histological sections in our results.Nevertheless, levels of hepatic LA and LDH activities in serum and liver showed significant increases in the D1-D7 groups, mirroring findings in goats [92] and cattle [93] that were given a high-starch diet.Past research has shown that increased LA accumulation can lead to risks such as lactic acidosis, nonalcoholic fatty liver disease, and tumors in humans and animals [94][95][96][97].Based on these discoveries and our findings, it appears that excessive LA levels could lead to lactic acidosis and metabolic disorders in largemouth bass fed a diet high in starch.
Typically, Ac-CoA is converted by ACC1 into Mal-CoA, which is then transformed by FAS into fatty acids (FAs) within the liver cells [18,98].The citrate-pyruvate pathway, facilitated by ME, and the pentose-citrate pathway, facilitated by G6PD, supply the necessary energy for this process [37].Consistent with prior findings in largemouth bass that were given a high-starch diet [18], we also observed elevated levels of ACC, FAS, Mal-CoA, SCD, ME1, G6PD, and PGD in fish fed a high-starch diet.This suggests that high starch intake can stimulate fatty acid production by up-regulating the expression of these genes associated with lipid synthesis in the liver of largemouth bass.Moreover, FATP1 can facilitate the transfer of FA to the endoplasmic reticulum within liver cells for the production of LTAG, as demonstrated by Ipsen et al. [99] and López [100].And GK, GPAT3, GPAT4, and DGAT2 are the rate-limiting enzymes during LTAG synthesizing processes [101].Then, these synthesized LTAG could bind apolipoprotein B100 (APOB100) and enter the bloodstream by free diffusion [102].Meanwhile, Plin2, as a key lipid droplet coated protein, is tightly related with lipid droplet biogenesis and storage in tissues [103].Elevated levels of Plin2 are often associated with various metabolic disorders, including insulin resistance and type 2 diabetes in human and animals [103,104].The study found that FABP10, GPAT3, GPAT4, DGAT2, APOB100, and Plin2 levels were notably higher in groups consuming high-starch diets, consistent with earlier findings in largemouth bass [18] and gilthead sea bream [105].Previous research has shown that the AKT/SREBP1c signaling pathway can stimulate LTG synthesis in both animals and fish, as documented by Wang et al. [106] and Ferre et al. [107].In addition to increased levels of these important molecules, this study also showed that high starch in largemouth bass can activate the AKT/SREBP1c signal pathway, leading to enhanced FA and LTAG synthesis, as well as lipid droplet formation, as demonstrated by LTAG levels and histological sections stained with HE and ORO in this study.SREBP2, a crucial transcription factor, has the ability to regulate the metabolism and balance of cholesterol in the liver by influencing the transcription of genes that are regulated by sterols, such as AACS, HMGCS, HMGCRa, and CYP51 [18,19].AACS has the ability to use ketone bodies for the creation of cholesterol and fatty acids, converting acetoacetate into its CoA ester to generate acetoacetyl-CoA in the cytosol [108].This process is facilitated by HMGCS, which transforms acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) [109,110].Subsequently, HMGCRa is able to facilitate the transformation of HMG-CoA into mevalonic acid in order to generate cholesterol [111].CYP51 plays an essential role in the sterols' production and is a significant focus of cholesterol-lowering medications according to recent studies [112][113][114].Our results showed that a diet with high-starch significantly increased the levels of SREBP2 and its target genes related to cholesterol synthesis (AACS, HMGCS, HMGCRa, and CYP51) in groups D1, D3, and D5.This effect was consistent with findings in largemouth bass [18] and male C57BL/6J mice [115], suggesting that high starch intake may stimulate cholesterol production through the SREBP2 signaling pathway in the hepatic cells of largemouth bass.The expression of these genes was significantly reduced in the D7 groups compared to that in the D3 groups, which might be due to the negative feedback regulatory mechanism mediated by higher contents of TC in the liver of largemouth bass [19,20], although further research is needed to fully understand this regulatory process.In addition, SOAT2 plays an important function in lipid droplet biogenesis and storage by converting cholesterol to cholesteryl ester in cells [104,116].Prior research has shown that an increase in lipid droplets can be triggered by elevated glucose levels in normal colon cells and colorectal cancer stem cells [117], as well as in mice with type 2 diabetes [118].Based on these results, it is proposed that increased levels of SOAT2 may be responsible for the accumulation of lipid droplets in largemouth bass fed a highstarch diet.Meanwhile, ABCG5 and ABCG8 act as key proteins transporting endogenous cholesterol out of the liver into other tissues, while NPC1L1 plays key roles in up-taking intestine cholesterol into hepatic cells [119,120].Combined with these findings and lower levels of ABCG5, ABCG8, and NPC1L1 in this study, it indicated these three key reduced transporters might mediate the hepatic cholesterol decomposition in largemouth bass [116].
In general, bile acid synthesis is mainly regulated by two key rate-limiting enzymes (CYP7A1 and CYP27A1) mediating the oxidation of cholesterol in hepatocytes [120,121].Higher CYP7A1 and CYP27A1 levels in D1-D3 groups indicated high starch could induce bile acid synthesis via the oxidation of cholesterol in hepatocytes of largemouth bass.The reduced levels of CYP7A1 and CYP27A1 in D5-D7 groups might be caused by the negative feedback regulatory mechanism mediated by higher TBA contents in hepatocytes [121,122].Furthermore, liver bile acids may be moved into the bile canaliculus using the bile salt transporter known as the canalicular bile salt export pump (BSEP) [123,124].Although there were no marked differences among D0-D5 groups, lower BSEP levels in D7 groups indicated high starch might block the transportation of hepatic bile acids into bile canaliculus and then result in possible cholestasis [120,121].Two important transporting polypeptides, NTCP and OATP1, facilitate the absorption of bile acids from the portal vein into hepatocytes [125].Higher NTCP and OATP1 levels in D1-D5 groups indicated that bile acids could be taken up into hepatocytes induced by high starch, which might further aggravate cholestasis in largemouth bass.In humans and rodents, studies have found higher levels of hepatic OST-α could help bile acid excretion into the circulating system [122,126].Combined with higher OST-α mRNA levels and serum TBA contents, it indicated that high starch could drive bile acid excretion into the circulating system via a heightening expression of hepatic OST-α in largemouth bass, although this driving mechanism needed to be studied in fish species [127].
It is well known that cellular ROS is mainly generated by NADPH oxidases (NOX) and the respiratory electron transport chain (ETC) of mitochondria during glucose metabolic processes in animals [28,128,129].Our study revealed that the expression of certain genes, such as G6PD, NOX1, NADPH oxidase activator 1 (NOXA1), and cytochrome c oxidase 4 (COX4), along with the levels of H 2 O 2 significantly increased with high-starch diets.This is consistent with findings in humans with diabetes [23,25,130], suggesting that high starch intake may enhance oxidative phosphorylation, leading to elevated ROS levels in largemouth bass liver.In general, SOD and CAT in antioxidant systems could catalyze ROS and reduce oxidative stress in animals [5,21].Meanwhile, increased GST and MDA amounts can symbolize physiological biomarkers to oxidative or inflammatory stress in cultured animals [1,3].Past research has shown that the antioxidant abilities were diminished by elevated starch levels in black carp [131], black sea bream (Acanthopagrus schlegelii) [12], GIFT tilapia [5], and largemouth bass [132].Similarly, our results also found a notable reduction in the amounts of SOD, CAT and T-AOC along with a significant increase in the amounts of GST, MDA, and H 2 O 2 in the D5 and D7 groups that were given a highstarch diet.Moreover, a specialized thioredoxin system within mitochondria, consisting of Trx2, TrxR2, and Prx3, has been identified as crucial for protecting against oxidative stress induced by H 2 O 2 through disulfide reductase functions [133,134].Our results show that a high-starch diet could inhibit the genetic expressions of Trx2, TrxR2, and Prx3 in the liver in largemouth bass.Considering these findings and our data, it indicated that high starch could aggravate ROS damage and disturb mitochondrial redox homeostasis by decreasing antioxidant capabilities in largemouth bass [128].
Numerous research studies have shown that an overabundance of NOX-derived ROS can lead to inflammation by boosting the generation of pro-inflammatory cytokines in the liver, such as IL-1β, TNF-α, IFN-γ, IL-6, IL-8, IL-12, IL-17, and others [24,29].Prior studies have discovered elevated amounts of inflammatory markers such as IL-1β, TNF-α, and IFNγ, which were presented in human with diabetes [135][136][137], pig with malnutrition [138], and mice fed high-carbohydrate diets [139].Likewise, our results showed elevated concentrations of IL-1β, IL-6, IL-8, IL-12, IL-17, TNF-α, and IFN-γ in the hepatic cells of largemouth bass with increasing time of feeding high-starch diet in this study.Combined with previous results and our findings, it further proved that inflammatory responses could be caused by ROS excessively generated during the metabolism of higher dietary starch in largemouth bass.In addition, the secretion and maturation of these pro-inflammatory cytokines were mediated by the typical NLRP3 inflammasome in human and animal cells [24,33].The NLRP3 inflammasome consists of the NLRP3 sensor molecule, the ASC adaptor protein, and pro-caspase-1 [35].Various molecular and cellular events, including ROS overload, can lead to the stimulation of the NLRP3 inflammasome [27,140].In the formation of NLRP3 inflammasomes, pro-caspase-1 can transform into cleaved-caspase-1, triggering the processing of pro-IL-1β precursors into their active states [135,141].Recent research has shown that elevated glucose levels can stimulate the generation of ROS, boost the triggering of the NLRP3 inflammasome, and increase IL-1β release in macrophages [34,142] as well as in RSC96 (rat Schwann cell line) cells [27].Moreover, additional research has shown that elevated glucose levels can lead to the depletion of retinal pericytes through a process of inflammatory cell death involving NLRP3 and caspase-1 [141].The research also revealed increased ASC, NOD1, NOD2, GSDME, and caspase-1 expression in fish groups exposed to high starch, mirroring findings in human and rat cells [27,34].By combining these discoveries with our own results, we can conclude that inflammatory reactions may be initiated by the activation of the NLRP3 inflammasome induced by ROS overload in largemouth bass fed with high starch (Figure 11).caspase-1 [141].The research also revealed increased ASC, NOD1, NOD2, GSDME, a caspase-1 expression in fish groups exposed to high starch, mirroring findings in hum and rat cells [27,34].By combining these discoveries with our own results, we can c clude that inflammatory reactions may be initiated by the activation of the NLRP3 infla masome induced by ROS overload in largemouth bass fed with high starch (

Conclusions
In summary, our results found high starch could increase body physical indicat (HSI, VSI, and ISI) and counts of leukocytes (WBC, NEU, MON, EOS, and BAS).And h starch could cause insufficient secretion of INS and ADPN and then induce glucose m abolic disorders and abnormal glycogen accumulation.Meanwhile, high starch co cause abnormal TAG and cholesterol accumulation via SREBP1/2 signal pathways.In dition, high starch could induce oxidative stress by heightening ROS contents and red ing antioxidant capabilities.Oxidative stress or ROS overload induced by high starch largemouth bass can activate the NLRP3 inflammasome, leading to inflammatory sponses (Figure 11).

Conclusions
In summary, our results found high starch could increase body physical indicators (HSI, VSI, and ISI) and counts of leukocytes (WBC, NEU, MON, EOS, and BAS).And high starch could cause insufficient secretion of INS and ADPN and then induce glucose metabolic disorders and abnormal glycogen accumulation.Meanwhile, high starch could cause abnormal TAG and cholesterol accumulation via SREBP1/2 signal pathways.In addition, high starch could induce oxidative stress by heightening ROS contents and reducing antioxidant capabilities.Oxidative stress or ROS overload induced by high starch in largemouth bass can activate the NLRP3 inflammasome, leading to inflammatory responses (Figure 11).

Figure 2 .
Figure 2. Effect of high-starch diet on related mRNA levels of hepatic glucose metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

Figure 2 . 28 Figure 3 .
Figure 2. Effect of high-starch diet on related mRNA levels of hepatic glucose metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).Metabolites 2024, 14, x FOR PEER REVIEW 11 of 28

Figure 4 .
Figure 4. Effect of high-starch diet on related mRNA levels of signal molecules regulating glucose metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-c ) are significantly different (p < 0.05).

Figure 3 .
Figure 3.Effect of high-starch diet on related mRNA levels of hepatic glycogen metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

28 Figure 3 .
Figure 3.Effect of high-starch diet on related mRNA levels of hepatic glycogen metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

Figure 4 .
Figure 4. Effect of high-starch diet on related mRNA levels of signal molecules regulating glucose metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-c ) are significantly different (p < 0.05).

Figure 4 .
Figure 4. Effect of high-starch diet on related mRNA levels of signal molecules regulating glucose metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-c ) are significantly different (p < 0.05).
tabolites 2024, 14, x FOR PEER REVIEW 12 of the levels of the triglyceride transporter apolipoprotein B-100 (APOB100) were signifi cantly increased to the highest point in the D5 groups, then decreased in the D7 groups < 0.05), with no significant difference between the D1 and D3 groups (p > 0.05) (Figure5

Figure 5 .
Figure 5.Effect of high-starch diet on related mRNA levels of hepatic lipid metabolism in larg mouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different s perscripts ( a-d ) are significantly different (p < 0.05).

Figure 5 .
Figure 5.Effect of high-starch diet on related mRNA levels of hepatic lipid metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-e ) are significantly different (p < 0.05).

Figure 6 .
Figure 6.Effect of high-starch diet on related mRNA levels of hepatic cholesterol metabolism largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with diffe superscripts ( a-d ) are significantly different (p < 0.05).

Figure 6 .
Figure 6.Effect of high-starch diet on related mRNA levels of hepatic cholesterol metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

Figure 7 .
Figure 7. Effect of high-starch diet on related mRNA levels of hepatic bile acid metabolism in l mouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with differen perscripts ( a-d ) are significantly different (p < 0.05).

Figure 7 .
Figure 7. Effect of high-starch diet on related mRNA levels of hepatic bile acid metabolism in largemouth bass.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

Figure 8 .
Figure 8.Effect of high-starch diet on related mRNA levels of hepatic antioxidant index in largemouth bass.(A) Antioxidant and oxidative parameters, (B) ROS generating and oxidative stress indexes.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

Figure 8 .
Figure 8.Effect of high-starch diet on related mRNA levels of hepatic antioxidant index in largemouth

Figure 9 .
Figure 9.Effect of high-starch diet on related mRNA levels of hepatic inflammation in largemouth bass.(A) Inflammatory reaction, (B) The NLRP3 inflammasome activation.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

Figure 9 .
Figure 9.Effect of high-starch diet on related mRNA levels of hepatic inflammation in largemouth bass.(A) Inflammatory reaction, (B) The NLRP3 inflammasome activation.Data are reported as the mean ± SD of three replicates (n = 3).Bars with different superscripts ( a-d ) are significantly different (p < 0.05).

Figure 11 .
Figure 11.The summary of glucose and lipid metabolism, antioxidant and oxidative responses, inflammatory responses in the hepatic cells of largemouth bass.

Figure 11 .
Figure 11.The summary of glucose and lipid metabolism, antioxidant and oxidative responses, and inflammatory responses in the hepatic cells of largemouth bass.

Table 2 .
The list of PCR primers used in this study.

Table 3 .
Effects of dietary days on hematological parameters in largemouth bass fed high (mean ± SD, n = 15).

Table 4 .
Effects of dietary days on biochemical and hormone indicators of serum in largemouth bass fed high starch (mean ± SD, n = 9).

Table 5 .
Effects of dietary days on glucose and lipid metabolism of liver in largemouth bass fed high starch (mean ± SD, n = 9).

Table 6 .
Effects of dietary days on the antioxidative and oxidative indices of liver in largemouth bass fed high starch (mean ± SD, n = 9).

Table 7 .
Effects of dietary days on the anti-inflammatory and pro-inflammatory cytokines of liver in largemouth bass fed high starch (mean ± SD, n = 9).
Institutional Review Board Statement: Animal procedures were strictly performed in accordance with the Regulations of the Experimental Animal Ethics Committee of Huzhou University and approved by the Institutional Animal Care and Use Committee (approval ID: HUZJ-DW-2022-032; approval date: 6 March 2022).