Recently, the price escalation of fishmeal has made aquaculture nutritionists to consider using carbohydrate in aquafeed to spare the use of dietary protein [1
]. Teleosts are known to be glucose intolerant with slow serum glucose clearance and hyperglycemia after a high intake of carbohydrate (HC) [3
]. Nevertheless, the use of an appropriate level of carbohydrate as an alternative source of energy can improve oxidative protection in common dentex (Dentex dentex
) juveniles [4
]. The HC diet could induce lipid metabolism disorder, impair antioxidant capacity, reduce nonspecific immunity and decrease resistance to a pathogen in farmed fish [1
]. Therefore, the diet with HC had been consistently linked to the high risk of hypertriglyceridemia, obesity, type 2 diabetes mellitus and fatty liver disease in fish [5
]. Although the HC diet has been widely used in aquaculture, little attention has been paid to the negative effect of dietary HC in fish. With the increasing interest of using more carbohydrate in aquaculture diets, it is necessary to investigate the method to mitigate the negative effects of dietary carbohydrate on fish.
The structure of myo
-inositol (MI) is similar to glucose, and it is a biologically active isomer of inositol in cell membranes [7
]. The MI is the structural base for some secondary messengers, and it is also involved in lipid signaling, osmolarity, glucose and insulin metabolism in land animals [9
]. In mammals, dietary supplementation with MI can effectively ameliorate certain endocrine diseases such as diabetes and insulin resistance as MI is closely related to carbohydrate metabolism [11
]. Due to the de novo synthesis pathway, free MI could be de novo synthesized with glucose-6-phosphate (G6P), which is catalyzed by myo
-inositol-1-phosphate synthase (MIPS
) and myo
-inositol monophosphatase (IMPA1
]. At the same time, G6P is involved in the pathways of carbohydrate metabolism [8
]. Therefore, the myo
-inositol biosynthesis (MIB) pathway is associated with carbohydrate metabolism and dietary supplementation with MI can also regulate lipid metabolism. The dietary MI can reduce the accumulation of triglycerides (TG) and decrease the expression of lipogenic genes and the activity of lipogenic proteinsin in rats with a nonalcoholic fatty liver [15
]. In turbot Scophthalmus maximus
, MI plays a vital role in transmembrane signal transfer, protection of the liver, and lipid metabolism [16
]. In other aquatic animals, dietary supplementation with MI can reduce lipid accumulation in tissues and decrease the chance of becoming a fatty liver [17
]. Although MI can reduce lipid accumulation in aquatic animals, the underlying molecular mechanism is still not clear.
The Oreochromis niloticus
is an excellent farmed fish, which is promoted by Food and Agriculture Organization of the United Nations (FAO) due to its fast growth, high yield potential, low oxygen tolerance, euryhaline habitat, disease resistance and high fecundity [18
]. Therefore, O. niloticus
is an excellent model species for studying carbohydrate metabolism and lipid metabolism. The objective of this study was to investigate the effect of dietary myo
-inositol on alleviating the adverse effects of high carbohydrate diets in O. niloticus
2. Materials and Methods
2.1. Diet Preparation and Experimental Fish
Six semi-purified diets were prepared with a 2 × 2 factorial design are shown in Table 1
. The basal diet contained 38% crude protein and 7% crude lipid. Corn starch was used as the source of carbohydrate. The feed used was the base purified feed. In this experiment, six different diets were prepared with two carbohydrate levels: low carbohydrate (LC 30%) and high carbohydrate (HC 45%) and three levels of MI supplementation (0, 400 and 1200 mg/kg diet) at each level of the carbohydrate diet. The diets were extruded into 2 mm pellets, air-dried and then stored at −20 °C until use.
Nile tilapia (O. niloticus) used in this experiment were obtained from a commercial fish hatchery in Guangdong Province (Guangdong Tianfa Fish Fry Development Co. LTD, Guangzhou, China). Fish were transported to the Biological Experimental Station of East China Normal University. During the acclimation period, the fish were fed with apparent satiety hand-fed twice daily by using a commercial diet. The water temperature was maintained at 27 ± 1 °C. After the two-week acclimation, the fish were fasted for 24 h prior to the experiment. A total of 540 juvenile Nile tilapias (1.45 ± 0.5 g) were selected and randomly distributed into eighteen 200-L tanks with 30 fish per tank. During the eight-week trial, all fish were hand-fed twice daily at 08:30 and 17:30 at a daily ration of 4% body weight. During the feeding trial, the environmental condition was maintained at 27 ± 1 °C, 5.0-6.0 mg L−1 dissolved oxygen, 7.3–7.6 pH, and a period of 12 h light and 12 h dark.
2.2. Sample Collection and Chemical Analysis
At the end of the trial, before fish were weighed and sampled, we stopped feeding for 24 h. The fish were weighed by tank, and the number of fish was counted to determine weight gain (WG) and survival (SR). Twelve fish of each treatment (four per tank) were euthanized (MS-222 at 20 mg/L) (tricaine methanesulfonate, Western Chemicals, Inc., Ferndale, WA, USA) and blood was rapidly collected from the caudal vein with a 1 mL syringe (Klmediacal, Haimen, China) and centrifuged for serum preparation (4,500 rpm, 10 min and 4 °C). The serum was immediately frozen at −80 °C for further analysis. Then the body length, viscera and liver weight of each fish were measured to calculate viscerosomatic index (VIS), hepatosomatic index (HSI) and condition factor (CF) respectively. The liver and muscle were collected for biochemical and molecular assays. The liver tissue was fixed in 4% paraformaldehyde for histological analysis. After that, 12 fish were collected in each treatment group and temporarily stored at −20 °C for the analysis of fish body composition.
2.3. Methods of Measurement
2.3.1. Growth Performance and Body Composition
Weight gain (WG %) = 100 × (final body weight—initial body weight)/initial body weight;
Survival rate (SR %) = 100 × (final fish number/initial fish number);
Feed conversion ratio (FCR) = total feed intake weight/(final body weight—initial body weight);
Condition factor (CF %) = 100 × wet body weight/body length;
Hepatosomatic index (HSI %) = 100 × wet hepatopancreas weight/wet body weight;
Visceral index (VIS %) = 100 × wet visceral weight/wet body weight [13
2.3.2. Proximate Composition
Proximate composition of the whole body was determined by the standard methods (AOAC, 135 1995). Moisture was determined by gravimetric analysis following oven-drying at 105 °C. Crude protein and total lipid were determined by the Kjeldahl method (KjeltecTM 8200, Foss, Sweden) and the chloroform/methanol method, respectively.
2.3.3. Histological Analysis
Three fish livers per tank were fixed in 4% paraformaldehyde solution for 48 h, washed in 70% ethanol solution, and then transferred to a 70% ethanol solution for storage until histological analysis. The paraffin production process, image collection and sample measurement of were determined according to the methods in previous studies, and digital images were taken using Image-Pro plus 6.0 [20
2.3.4. Biochemical Indicators
The contents of glucose (F006-1-1), triglyceride (TG, A110-1-1), high-density lipoprotein (HDL-C, A112-1-1), low-density lipoprotein (LDL-C, A113-1-1) and total cholesterol (T-CHO, A111-1-1) in the serum were all determined using the corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The relevant steps were carried out according to the instructions. The total protein (A045-4-2) in the liver was also determined using the commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s guidelines. Serum insulin levels were analyzed by the ELISA kit following the manufacturer’s protocol (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). The liver tissue was weighed and mixed with saline water at the ratio of 1:9 by weight (pH = 7.4). Ice bath homogenization was performed, and the homogenate was centrifuged at 4500 rotations/min at 4 °C for 10 min. The supernatant was pipetted and put on ice for the test. The liver TG (A110-1-1) content and the activities of superoxide dismutase (SOD, A001-3-2), glutathione peroxidase (GSH-Px, A005-1-2), alkaline phosphatase (AKP, A059-2-2), acid phosphatase (ACP, A060-2-1), aspartate aminotransferase (AST/GOT, C010-2-1), glutamic-pyruvic transaminase (ALT/GPT, C009-2-1) and muscle glycogen (A043-1-1) were determined using the corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
2.3.5. Gene Expression Analysis
Primers designed based on the O. niloticus
transcriptome genome sequences are presented in Table 2
. The primer amplification efficiency of all genes was between 90% and 110%. Total RNA was extracted by TRIzol®
reagent (RN0101, Invitrogen, Shanghai, China). The quantity and concentration of total RNA were measured by the Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, NC, USA). The first-strand cDNA synthesis was performed using the PrimeScriptTM RT Reagent kit (KR116, Tiangen Biotech, Beijing, China).
The reaction volume used in qRT-PCR was 20 μL containing 10 μL 2 × Ultra SYBR Mixture (CWbio, Nanjing, China), 1.6 μL of each forward and reverse primers (2.5 μmol/μL), 1 μL of diluted cDNA (200 ng/μL) and 7.4 μL of RNAase free water. All procedures are performed according to the manufacturer’s instruction. β-actin was used as the reference gene. qRT-PCR data were analyzed with the 2−ΔΔCt method.
2.4. Statistical Analysis
All statistical analyses were performed using SPSS Statistics 19.0 software. Normality and homoscedasticity assumptions were checked prior to the statistical analysis. Two-factor analysis of variance (ANOVA) was used to detect the two main factors of carbohydrate level and MI supplementation and their interaction. If significant interactions were detected, post hoc tests were used to assess the dependencies between the six treatments. All data are on average ± standard error (means ± SE) said. An asterisk (*) represents a significant difference of p < 0.05 between different MI levels in the same carbohydrate group. Double asterisks (**) represent a significant difference of p < 0.01 between different MI levels within the same carbohydrate group. A, B, C and a, b, c Values on bars without a common superscript letter are significantly different (p < 0.05), (a/A indicated the lowest value).
2.5. Ethical Statement
This research has been approved by Animal Ethics Committee of East China Normal University in February 2019 (permit number: E20120101).
In the present study, the HC diets significantly affected growth, immunity, carbohydrate metabolism, lipid metabolism, and the health of liver tissue, which are similar to those observed in the blunt snout bream (Megalobrama amblycephala
), Nile tilapia (Oreochromis niloticus
) and European seabass (Dicentrarchus labrax
) fed HC diets [5
]. In the present study, the HC feed increased the weight gain and HSI, but dietary MI supplementation decreased HSI. This result may be due to the reason that HC diets can induce the synthesis of lipid from excess glucose in the liver [25
]. Some studies have suggested that dietary MI deficiency can cause high accumulation of triacylglycerol, cholesterol, and non-esterified lipids in the mammalian liver, indicating that MI plays a crucial role in lipid metabolism [11
]. As expected, the fish crude lipid, vacuolization of the cytoplasm and TG content in the liver were decreased with the increase of dietary MI. The MI is a precursor of inositol phosphates and is a vital second messenger signaling molecule in cellular processes, such as lipid signaling, glucose, and insulin metabolism [11
]. The results in the current study suggest that MI can affect the lipid synthesis and metabolism in the body, thereby relieving lipid accumulation in body [16
]. The qPCR results showed that dietary MI supplementation could down-regulate the expression of genes related with lipid synthesis (FAS
) and up-regulate the expression of genes (CPT, PPAR-α, ATGL
) related with lipid metabolism. The FAS
plays a key role in the opposite process of de novo lipogenesis by converting acetyl-CoA and malonyl-CoA into fat [32
has been identified as a critical regulator for hepatic lipid metabolism to control the transcription of genes involved in fatty acids beta-oxidation, lipoprotein metabolism, glucose metabolism, hepatic inflammation, and hepatocyte peroxisome proliferation [34
is the main regulatory enzyme in mitochondrial fatty acid oxidation because it is the catalyzing enzyme of the reaction from fatty acyl-CoAs into fatty acylcarnitines [35
is a critical lipolysis lipase, and the lack or low expression of ATGL
would result in a defect of lipolysis and the accumulation of triacylglycerols in tissues [37
]. So, the results of the present study showed that MI could alleviate lipid accumulation by promoting lipid decomposition and inhibiting lipid synthesis. A previous study has suggested that MI can regulate lipid metabolism by mediating insulin resistance [42
The decomposition of liver glycogen accelerated with the addition of MI regardless of carbohydrate levels. This may be due to that MI could promote the decomposition of liver glycogen into glucose and increase the glucose content by increasing protein kinase B (PKB)/Akt phosphorylation, increasing the sensitivity of insulin and promoting the utilization of glucose [43
]. However, the specific molecular mechanism remains to be confirmed in future studies. The G6P is an essential intermediate in carbohydrate metabolism. Most G6P comes from the glycolysis process catalyzed by GK
, and continues the glycolysis reaction by the catalysis of PK
. A part of G6P would act as the substrate and enters the MIB pathway by the catalysis of MIPS
The other part of G6P enters the pentose phosphate pathway by the catalysis of G6PDH
]. In the present study, the HC diet promoted glycolysis and gluconeogenesis, but these processes decreased with the addition of MI in the feed. Simultaneously, the addition of MI promotes the activities of glucose in the pentose phosphate pathway [52
]. In the current study, MI might change glucose to the pentose phosphate pathway. NADPH is mostly generated by the pentose phosphate pathway being one of the main intracellular reducing agents and an essential co-factor required for the normal function of antioxidant cycles such as the glutathione thioredoxin systems [49
]. In the present study, when a large amount of lipid accumulated in the body and caused lipid peroxidation, the addition of MI can promote more glucose to enter the pentose phosphate pathway to increase the ratio of NADPH/NADP+
, which would increase the activity of the antioxidant system and maintain the cell health [56
]. However, the MIPS
tend to decrease with the addition of MI regardless of the carbohydrates level. It may be due to the feedback regulation in the body, and the addition of MI in the feed meets the normal needs of the body, and there is no need to synthesize excessive MI [8
]. Therefore, the addition of MI to the HC diet can promote the decomposition of liver glycogen into glucose, and then promote glucose metabolism into pentose phosphate pathway, thus providing a large amount of energy for the body and reducing power to alleviate the oxidative damage caused by HC diet.
The contents of lipidemia can reflect whole-body lipid metabolism state was detected [60
]. The TG is the product of one glycerol molecule and three fatty acid molecules esterification. Under a normal condition, the serum TG content maintains the dynamic balance, but a large amount of TG would accumulate when lipid metabolism was disturbed [62
]. The contents of T-CHO, HDL-C and LDL-C in the serum can reflect lipid metabolism and transport capacity of the liver [62
]. Physiologically, HDL-C is good cholesterol due to having an anti-atherogenic effect, because HDL-C is responsible for transporting CHO from extrahepatic tissues to liver for metabolism to prevent free CHO deposition in blood [60
]. The results showed that the addition of MI in the HC diet can improve the transportation of CHO from blood and other peripheral tissues to the liver, which effectively improved the absorption and transportation of CHO on the HC diet in O. niloticus
]. Therefore, the reason why dietary MI decreased the lipid accumulation might be that additional MI accelerated the transportation of CHO back to the liver and promoted the lipolysis reaction.
The results of the present study demonstrate that the HC diet caused lipid deposition, which triggered lipid peroxidation and oxidative stress and then induced liver function damage. Similar findings have been found in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂
), Surubim (Pseudoplatystoma reticulatum × P corruscans
) and Oreochromis niloticus
]. The accumulation of large amounts of lipid will damage the liver structure, physiological function and lipid metabolism disorder, which would lead to excessive free radicals and finally cause oxidative damage to the body [68
]. SOD is a vital antioxidant enzyme and can eliminate excess free radicals, reduce and inhibit lipid peroxidation, and protect cells from oxidative damage [70
]. GSH-Px is a vital peroxidase widely existing in the body, which can protect the structure and function of cell membranes from interference and damage of oxides because it can reduce the toxic peroxides to nontoxic hydroxyl compounds [72
]. In the present study, the SOD and GSH-Px activities were significantly improved with the increase of MI supplementation regardless of dietary carbohydrate levels. At the same time, the MDA content was significantly decreased with the increase of MI supplementation regardless of dietary carbohydrate levels. This may be because that dietary MI decreased the accumulation of lipid in the liver which was easy to cause oxidative stress, so the antioxidant capacity of the body had a corresponding increase [75
]. Moreover, the activities of GST/GOT and ALT/GPT were the most sensitive indicators of liver cell damage, which is increased by dietary MI, this indicates that the addition of MI alleviates the damage of liver cells and avoids the formation of fatty liver [77
]. AKP and ACP are essential enzymes for growth metabolism, homeostasis and health [17
]. Therefore, the activities of AKP and ACP in the liver tissue also indirectly reflect the health of the liver [80
]. The activities of AKP and ACP were consistent with the results of antioxidant-related enzymes in the present study. These results further demonstrate that dietary MI can help avoid oxidative stress caused by lipid peroxidation, increase the antioxidant capacity and thus maintain the normal structure and function of the liver cells [11