Aquaculture Reports 21 (2021) 100820

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Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

Effects of taurine supplementation in a high-carbohydrate diet on growth

performance, plasma biochemical, digestive and glucose metabolism
enzymes in hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatus)
Jiahao Qian a, b, c, 1, Bin Yin a, b, c, 1, Hongyu Liu a, b, c, *, Beiping Tan a, b, c, Xiaohui Dong a, b, c,
Shuyan Chi a, b, c, Qihui Yang a, b, c, Shuang Zhang a, b, c
a
Laboratory of Aquatic Animal Nutrition and Feed, Fisheries College, Guangdong Ocean University, Zhanjiang, 524088, PR China
b
Aquatic Animals Precision Nutrition and High Efficiency Feed Engineering Research Centre of Guangdong Province, Zhanjiang, Guangdong, PR China
c
Key Laboratory of Aquatic, Livestock and Poultry Feed Science and Technology in South China, Ministry of Agriculture, Zhanjiang, 524088, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: This study conducted an 8-week feeding trial to evaluate the effects of taurine supplementation in a high-
Digestion carbohydrate diet on the growth performance, plasma biochemical indexes and glucose metabolism of hybrid
Glycolysis and gluconeogenesis grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatus). Six iso-nitrogenous (45.94 %) and iso-lipidic (8.71 %)
Growth performance
diets were formulated containing 20 % carbohydrates (positive control group, PC) and 30 % carbohydrates
Hybrid grouper
(negative control group, NC), and were supplemented with 0.4 % (T1), 0.8 % (T2), 1.2 % (T3) of 1.6 % (T4)
Dietary taurine
taurine on the basis of the PC diet. Five hundred forty fish (initial body weight 12.10 ± 0.30 g) were randomly
distributed among the six treatments with triplicate groups of 30 fish in each treatment. The results showed that,
compared with the PC, fish fed the NC diet significantly decreased the weight gain rate (WGR) and specific
growth rate (SGR), the level of insulin-like growth factor-1 receptor (IGF-1R) in the liver, the activities of trypsin
and α-amylase (AMS) in the intestine, and the activity of glucokinase (GK) in the liver. Under taurine supple­
mentation, the highest significant values of WGR and SGR were found at the T3. The optimal taurine re­
quirements in the 30 % carbohydrate diet were found to be 1.31 %, 1.31 % and 1.05 % based on WGR, SGR and
feed coefficient rate (FCR), respectively. Compared with the NC, significantly higher activities of intestinal
trypsin and AMS were found in the T1-T4. Hybrid grouper fed the T3 diet showed the significantly highest levels
of plasma insulin and triglycerides. Significant increase in GK activity in the liver occurred under taurine sup­
plementation above 0.8 %. Hepatocyte oil-red O and hematoxylin-eosin staining showed that 0.4 %~1.2 %
taurine supplementation could release hepatocyte fat deposition and maintain hepatocyte structure. Therefore,
taurine enhances intestinal digestive function and regulates glycolipid metabolic to improve the utilization of
dietary carbohydrates, thus promoting the growth of hybrid grouper.

1. Introduction rate, which is especially obvious after the intake of a carbohydrate-rich

Carbohydrates are an important and inexpensive energy source
among the three major nutrients required by animals. An appropriate
dietary carbohydrate level can promote fish growth performance, pro­
tein and lipid-sparing, and the supply of metabolic intermediates (Xia
et al., 2015). However, ferocious carnivorous fish are known to exhibit
poor glucose utilization and glucose intolerance because of persistent
postprandial hyperglycemia due to a relatively low glucose metabolic
diet or glucose loading (Moom, 2001). It is generally thought that the
carbohydrate requirement of carnivorous fish is no more than 20 % and
that long-term excessive intake of carbohydrates will have a series of
negative consequences in fish, such as slow growth, fatty liver and lower
apparent digestibility coefficients for proteins and lipid (Chen et al.,
2012; Li et al., 2013; Mozanzadeh et al., 2017; Wilson, 1994). Although
many recent reports have explored the physiological basis of such con­
sequences, the underlying mechanisms are still unclear. According to

* Corresponding author at: Laboratory of Aquatic Animal Nutrition and Feed, Fisheries College, Guangdong Ocean University, Zhanjiang, 524088, PR China.
E-mail address: liuhyu@gdou.edu.cn (H. Liu).
1
Co-first author.

https://doi.org/10.1016/j.aqrep.2021.100820
Received 18 March 2021; Received in revised form 28 July 2021; Accepted 3 August 2021
Available online 7 August 2021
2352-5134/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Qian et al.
previous speculation, the cause of poor carbohydrate utilization of fish
may be similar to type 2 diabetes mellitus (T2DM) in mammals (Yan
et al., 2020). In mammals, the insulin signaling pathway is effectively
activated by glucose, the main form of carbohydrate transport in the
body, maintaining blood glucose homeostasis by regulating glucose
transport, glycolysis and gluconeogenesis (Saadeldeen et al., 2020).
Unfortunately, the insulin secretions of mammals with T2DM and fish
are less sensitive to carbohydrates than amino acids, and the insulin
receptors of mammals with T2DM and fish are characterized by rela­
tively insufficient quantities or functional defects (Enes et al., 2009).
Clinically, a possible solution is the used of oral medication to correct
this unconventional phenotype, which provides a reference for relaxing
restrictions on the application of carbohydrates in carnivorous fish
(Wang, 2012). Based on a large number of screening trails, certain
specific amino acids, such as taurine maybe a good choice for this pur­
pose (Kamalam et al., 2017).
Taurine (2-amino ethane sulfonic acid) is a conditional essential
amino acid that was first discovered and isolated from bovine bile by the
German scientist Tiedemann in 1827 (Tiedemann and Gmelin, 1827).
Although taurine is not involved in protein synthesis, its functions have
been gradually revealed since it was identified and discovered, including
growth promotion, immune regulation, antioxidant activity and even
roles in metabolism and absorption of proteins, lipids and carbohydrates
(Surai et al., 2019). The absence of taurine can cause a series of diseases,
such as diabetes and cardiovascular and retinal neuronal damage
(Chung et al., 2012). In nature, the distribution of taurine in different
species is not uniform. It has been reported that most of higher plants
and bacteria contain hardly any taurine, whereas taurine is relatively
abundant in marine organisms and mammals (Huxtable, 1992). For
example, the taurine concentrations of clams, shrimp and tilapia are
41.4, 12.4 and 9.1 μmol/g wet weight, respectively (Pansantes-Morales
et al., 1989). Compared to calves and cows, yellowtail (Seriola quin­
queradiata) exhibits higher taurine levels (Sakai and Nagasawa, 1992).
Although the above animals have the ability to synthesize taurine, they
all present the characteristics of low taurine synthase activity that
cannot meet their needs through synthesis and need to be supplemented
by diet (Larkin and Place, 2017). The above species have also become
important raw materials for taurine extraction, and taurine products
have been successfully applied for different uses in mammals (Kim et al.,
2007; X. Liu et al., 2020; T. Liu et al., 2020). However, in earlier research
on aquatic animals, scholars paid less attention to how taurine regulates
the metabolism of nutrients, especially carbohydrates (Coutinho et al.,
2017; Garcia-organista et al., 2019). It has been reported that the insulin
structure of taurine shows a synergistic effect with insulin, stimulates
insulin secretion, promotes the absorption of glucose by cells and reg­
ulates glycolysis and gluconeogenesis activities to balance glucose pro­
duction and consumption (Coutinho et al., 2017).
Hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatus) is a
typical farmed carnivorous fish with the advantages of rapid growth,
high disease resistance and high market value. It has become the main
farmed grouper species in Southeast China in recent years (Dennis et al.,
2020; Yin et al., 2018). Recently, research on the taurine and carbohy­
drate requirements of Epinephelus coioides and Epinephelus akaara has
been discussed (Mao et al., 2014; Wang et al., 2016; Zhou et al., 2015a).
In summary, the experiment reported here was conducted to investigate
the effect of taurine on the growth, antioxidant properties, and digestive
and glucose metabolism enzyme activities of hybrid grouper fed a
high-carbohydrate diet, to determine the appropriate requirements, and
to provide a theoretical basis for the increase in the utilization of car­
bohydrates induced by taurine in fish.
2. Material and methods
2.1. Experimental diets and animals
Six iso-nitrogenous (45.94 %) and iso-lipidic (8.71 %) diets were
2
Aquaculture Reports 21 (2021) 100820
designed in this experiment. Taurine-free diets with 20 % and 30 %
carbohydrate levels were used as the positive control and negative
control, respectively. Four graded taurine supplementation levels added
to the negative control diet were used as four other experimental dietary
treatments. The formulation and ingredients of the experimental diets
are shown in Table S1. After dry ingredients were finely ground, fish oil
and water were slowly added to the mixing well. Then, the dough was
pelleted through an F-26 twin-screw extruder (South China University of
Technology, Guangzhou), 3− 4 mm in diameter. The dietary pellets were
dried at room temperature for 48 h and stored at − 20 ◦ C until use.
The hybrid grouper used in the experiment were obtained from a
grouper fry farm on the East Island, Zhanjiang, China, and cultured at
the Donghai Island marine biology base of Guangdong Ocean University.
Before initiation of the feeding trials, all fish were fed a commercial diet
twice daily at 08:30 and 16:30 for two weeks to acclimate the experi­
mental conditions. Then, healthy fish without significant weight dif­
ferences were selected and randomly divided into three replicate groups
per treatment with 30 fish in each 500 L tank. They were fed to apparent
satiation with experimental diets for 8 weeks. The initial weight of
hybrid grouper was 12.1 ± 0.3 g. The water temperature was between
28 and 31 ◦ C, the salinity was 26–28 g/L, the pH value was 7.8–8.2 and
the dissolved oxygen was more than 6.5 mg/L. The procedures of this
study involving animals and their care were conducted in accord with
NIH guidelines (NIH Pub. No. 85-23, revised 1996) and were approved
by the Animal Care and Use Committee of Guangdong Ocean University.
2.2. Sample collection
At the end of the 8-week feeding trial, hybrid groupers were starved
for 24 h. After being deeply anesthetized in diluted eugenol (1:10000;
Shanghai Reagent Corp., Shanghai, China), the total weight and quan­
tity of fish in each tank were counted. Then three fish in each tank were
collected for whole-body composition assessment. Another three fish per
tank were measured to determine morphological indexes, then the liver
of fish was dissected and stored in 4% paraformaldehyde solution for
histological analyses. Six fish per tank were randomly selected and blood
was taken from the tail vein with a 1 mL hypodermic syringe and treated
with saline solution of 7% heparin (Shanghai Yuanye Bio-Technology
Co., Ltd., S12004, 150 U/mg) after weighing. The collected blood was
transferred into a sterilized 1.5 mL centrifuge tube and centrifuged at
4000 rpm at 0–2 ◦ C for 10 min to obtain the plasma. Then the fish were
dissected rapidly for liver, intestine and muscle collection for enzyme
activity analysis. After pretreatment, all samples were immediately store
at − 80 ◦ C until analysis.
2.3. Chemical composition and enzyme activity analysis
The compositions of the diets and fish were determined according to
standard methods. Moisture was detected by oven drying at 105 ◦ C until
constant weight. Crude protein and crude lipid contents were measured
with an Automatic Kjeldahl system (Kjeletc 8400, FOSS Technology and
Trade Co., Ltd., Beijing, China) and an Automatic Fat Extractor (ANKOM
XT15I Extractor, ANKOM Technology Co., Ltd., USA) respectively. The
plasma glucose (F006-1-1), triglyceride (TG, A110-1-1), total choles­
terol (T-CHO, A111-1-1), high-density lipoprotein cholesterol (HDL,
A112-1-1), low-density lipoprotein cholesterol (LDL, A113-1-1), alanine
aminotransferase (GPT, C009-2-1), and aspartate aminotransferase
(GOT, C010-2-1) levels, total antioxidant capacity (T-AOC, A015-2-1),
and malondialdehyde (MDA, A003-1-1), superoxide dismutase (SOD,
A001-3-1), alkaline phosphatase (AKP, A059-2-2) and glycogen content
of liver and muscle (A043-1-1) were tested by using assay kits obtained
from the Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
The levels of intestinal trypsin (ml036384), lipase (ml036371),
α-amylase (AMS, ml036449), hepatic insulin-like growth factor-1 re­
ceptor (IGF-1R,ml028391), hexokinase (HK, ml076603), glucokinase
(GK, ml024808), pyruvate kinase (PK, ml037329),
J. Qian et al. Aquaculture Reports 21 (2021) 100820

phosphoenolpyruvate carboxykinase (PEPCK, ml036430), glucose-6-

phosphatase (G6Pase, ml076993), fatty acid synthetase (FAS,
ml036370), plasma insulin (ml022831) and insulin-like growth factor-1
(IGF-1,ml022803) were determined using commercial enzyme-linked
immunosorbent assay (ELISA) kits obtained from the Shanghai
Enzyme-link Biotech Co., Ltd., Shanghai, China. All operations were
performed strictly according to the product introduction.

2.4. Histological analyses

Histological analyses were performed according to the method

described in previous studies (Yin et al., 2020). Tissue was trimmed with
a scalpel and placed in a dehydration box. The dehydration box was put
into a hanging basket and a gradient of alcohol used for dehydration in a
dehydrator (Wuhan Junjie Electronics Co., Ltd., JJ-12 J). The samples
were embedded in paraffin wax in an embedding machine (Wuhan
Junjie Electronics Co., Ltd., JB-P5) after dehydration. The
wax-impregnated tissue was embedded in an embedding machine
(Wuhan Junjie Electronics Co., Ltd., JB-P5). The trimmed wax blocks
were sectioned on a paraffin microtome to a thickness of 4 μm (Shanghai
Leica Instrument Co., Ltd., RM-2016), which were stained with
hematoxylin-eosin (HE) and then observed under a Nikon ECLIPSE 80i
microscope (Nikon Corporation, Kanagawa, Japan).
The trimmed tissue was dehydrated in graded concentrations of su­
crose solutions at 4 ◦ C. Embedding agent (OCT) was dripped around the
dehydrated tissue, which was placed on the quick-freezing embedding
table of a freezing microtome. After embedding and freezing, the OCT
become white and hard and could be sliced, sections were made of
8–10 μm thickness. The frozen sections were warmed and dried, fixed in
fixing solution for 15 min, washed with tap water, and dried. Subse­
quently, the fixed sections were stained with oil red O and sealed with
glycerinated gelatin for observation and photography under the
microscope.

2.5. Statistical analysis

All data were analyzed by one-way analysis of variance (ANOVA)

using SPSS 20.0 for Windows and are represented as the means ± SE
(standard error of the mean). Tukey’s test was used to compare the mean
values between individual treatments. The linear and quadratic effects
of the treatments were also tested. P-values <0.05 were considered
significant.

3. Results
Fig. 1. The conic curves of WGR, SGR and FCR of hybrid grouper to the dietary
3.1. Growth performance and morphological indexes taurine level.

There were no significant differences (P > 0.05) in survival rate (SR),

Table 1
Growth performance of hybrid grouper treated with different diets.
Diet WGR (%) SGR (%) SR (%) FCR PER

PC 456.75 ± 10.46ab 3.07 ± 0.03ab 91.11 ± 5.09 0.70 ± 0.02 3.20 ± 0.08
NC 411.63 ± 18.77a 2.91 ± 0.07a 80.00 ± 16.67 0.73 ± 0.05 3.19 ± 0.30
T1 447.35 ± 21.99ab 3.03 ± 0.07ab 86.67 ± 0.00 0.70 ± 0.03 3.14 ± 0.12
T2 468.35 ± 16.07ab 3.10 ± 0.05ab 90.00 ± 6.67 0.69 ± 0.01 3.11 ± 0.03
T3 514.35 ± 42.95b 3.24 ± 0.13b 91.11 ± 8.39 0.67 ± 0.03 3.16 ± 0.14
T4 484.59 ± 30.11b 3.15 ± 0.09b 83.33 ± 3.33 0.71 ± 0.05 3.03 ± 0.19

P-values
Treatment 0.007 0.006 0.522 0.439 0.833
Linear 0.003 0.003 0.957 0.410 0.254
Quadratic 0.398 0.365 0.903 0.876 0.835

All data were expressed as mean ± SE. Different superscript letters means the significant difference (P < 0.05). Weight gain rate (WGR, %) = 100 × (final body weight
− initial body weight)/initial body weight; Specific growth rate (SGR, %) = 100 × (ln final body weight − ln initial body weight/experimental period; Survival rate
(SR, %) = 100 × (final fish number)/(initial fish number); Feed coefficient rate (FCR) = feed intake/(final body weight − initial body weight); Protein efficiency rate
(PER) = (final body weight − initial body weight)/protein intake.

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J. Qian et al. Aquaculture Reports 21 (2021) 100820

Table 2 Table 3
Morphological index of hybrid grouper treated with different diets. Body component of hybrid grouper treated with different diets.
Diet VSI (%) HSI (%) K (g/cm3) Diet Moisture (%) Crude protein (%) Crude lipid (%)
bc ab ab
PC 11.07 ± 0.30 3.94 ± 0.08 5.81 ± 0.45 PC 71.51 ± 1.30 16.56 ± 0.55 5.57 ± 0.03
NC 11.42 ± 0.52c 4.61 ± 0.72b 5.62 ± 0.35ab NC 72.42 ± 1.08 16.32 ± 0.39 5.56 ± 0.05
T1 10.42 ± 0.83abc 3.66 ± 0.62a 5.29 ± 0.91a T1 72.21 ± 0.38 16.45 ± 0.11 5.42 ± 0.08
T2 10.16 ± 0.80ab 3.58 ± 0.67a 5.41 ± 0.17ab T2 71.11 ± 1.40 17.01 ± 0.41 5.71 ± 0.44
T3 10.27 ± 0.67ab 3.27 ± 0.48a 6.18 ± 0.32b T3 71.26 ± 0.46 16.91 ± 0.27 5.90 ± 0.44
T4 10.04 ± 0.50a 3.51 ± 0.54a 5.67 ± 0.42ab T4 71.22 ± 1.76 16.69 ± 0.12 5.79 ± 0.19

P-value P-value
Treatment 0.001 0.002 0.030 Treatment 0.651 0.186 0.335
Linear <0.001 0.001 0.483 Linear 0.307 0.100 0.085
Quadratic 0.490 0.834 0.118 Quadratic 0.607 0.655 0.571

All data were expressed as mean ± SE. Different superscript letters means the
significant difference (P < 0.05). Viscera somatic index (VSI, %) = 100 ×
(viscera weight)/(body weight); Hepatic somatic index (HSI, %) = 100 × (liver
weight)/(body weight); Fulton’s condition factor (K, g/cm3) = 100 × (body
weight)/(body length)3.
feed conversion rate (FCR) or protein efficiency rate (PER) among all the
experimental groups (Table 1). Compared with the 20 % dietary car­
bohydrate group, hybrid groupers in the 30 % carbohydrate group
showed lower WGR and SGR (P > 0.05). The WGR and SGR of the hybrid
groupers in the groups supplemented with 1.2 % and 1.6 % dietary
taurine were significantly higher than those in the 30 % carbohydrate
group. By fitting the conic curves of WGR, SGR and FCR to the dietary
taurine levels, the optimal taurine levels for this experiment were found
to be1.31 %, 1.31 % and 1.05 %, respectively (Fig. 1).
Additionally, the visceral somatic index (VSI), hepatic somatic index
(HSI) and Fulton’s condition factor (K) values of the hybrid groupers
showed an upward trend (P > 0.05) with the increase in dietary car­
bohydrates (Table 2). With taurine supplementation in a 30 % carbo­
hydrate diet, the VSI and HSI values of the hybrid groupers gradually
decreased and began to show significant differences under taurine
supplementation levels above 0.8 % and 0.4 %, respectively (P < 0.05).
In contrast, the K value of the hybrid groupers decreased first and then
increased with taurine supplementation, and the K value in the 1.2 %
taurine supplementation group was significantly higher than that in the
0.4 % taurine supplementation group (P < 0.05).
3.2. Body component, hepatic amino acid profile and tissue glycogen
As shown in Table 3, no obvious changes were seen in the moisture,
crude protein or crude lipid contents of the hybrid groupers fed different
experimental diets (P > 0.05). The data in Table 4 show that the
experimental diets significantly affected the hepatic taurine content,
whereas no significant changes were observed in the other amino acids
(P > 0.05). First, the hepatic taurine content of hybrid groupers in the 30
% carbohydrate group was lower than that of the hybrid groupers in the
20 % carbohydrate group (P > 0.05). However, the hepatic taurine
content of hybrid groupers increased with the gradient of taurine in the
experimental diets and showed obvious increases in the 0.4 % and 0.8 %
supplementation groups (P < 0.05). The changes in hepatic and muscle
glycogen content are presented in Fig. 2. It was shown that a 30 %
carbohydrate diet effectively promoted the accumulation of muscle
glycogen (P < 0.05) instead of hepatic glycogen. With the supplemen­
tation of taurine in a 30 % carbohydrate diet, the hepatic glycogen levels
of the hybrid groupers in the 0.4 %–1.2 % taurine supplementation
groups showed an upward trend (P > 0.05), and the highest muscle
glycogen levels of hybrid groupers were seen in the 30 % carbohydrate
group and the 0.8 % taurine supplementation group, which differed
significantly from those in the other groups.
3.3. Intestinal digestive enzymes
The intestinal digestive enzyme activities of hybrid groupers fed
All data were expressed as mean ± SE. Different superscript letters means the
significant difference (P < 0.05).
diets with different carbohydrate and taurine levels are shown in Fig. 3.
The hybrid groupers fed a 30 % carbohydrate diet exhibited lower in­
testinal trypsin and AMS activities than those fed a 20 % carbohydrate
diet (P < 0.05). After 0.4 %–1.6 % taurine supplementation in a 30 %
carbohydrate diet, the hybrid groupers had significantly increased ac­
tivities of intestinal trypsin and AMS. In addition, although the lipase
activity of the hybrid groupers also showed an increasing trend with
increasing taurine supplementation, lipase activities only showed a
significant increase when the taurine supplementation gradient reached
1.6 %.
3.4. Hepatic IGF-1R level, plasma biochemistry and antioxidant
parameters
As shown in Table 5, hybrid groupers fed a 30 % carbohydrate diet
exhibited higher plasma HDL content and lower plasma T-CHO content
and hepatic IGF-1R level than those fed a 20 % carbohydrate diet (P <
0.05). As taurine supplementation increased, there was a linear increase
in IGF-1R levels, including a significant difference in the 1.6 % taurine
supplementation group. In contrast, plasma insulin and IGF-1 levels
tended to decrease and then increase with the taurine gradient, with the
minimal values occurring in the 0.4 % taurine supplementation group.
Supplementation with 1.2 %–1.6 % taurine in a 30 % carbohydrate diet
significantly increased plasma insulin levels, and 0.8 %–1.6 % taurine
supplementation significantly increased plasma IGF-1 levels. The
plasma glucose levels decreased and then increased as the addition of
taurine increased, and a significant maximum was observed in the high-
dose taurine group (1.6 %). The TG and T-CHO levels of hybrid groupers
increased and then decreased as the amount of taurine increased, and
significant peaks were found in the 1.2 % and 0.8 % taurine supple­
mentation groups. Additionally, the levels of plasma LDL decreased
linearly with increased supplementation of taurine, and significant dif­
ferences were seen under taurine supplementation above 0.8 %. The
plasma HDL levels of hybrid groupers showed a trend of increasing and
then decreasing after taurine supplementation, but the plasma HDL
levels in the taurine-supplemented groups were significantly higher than
those in the nonsupplemented groups (PC and NC).
As shown in Table 6, compared with the 20 % carbohydrate group,
plasma AKP activity in the 30 % carbohydrate group was significantly
increased, whereas SOD activity was significantly decreased. After
taurine supplementation in a 30 % carbohydrate diet, plasma GPT ac­
tivity and the GOT/GPT ratio exhibited a trend of rising and then falling,
and peaked in the 0.4 % (T1) taurine group (P < 0.05). Furthermore,
dietary taurine levels did not significantly affect plasma GPT activity,
but high levels of taurine addition obviously increased the plasma GPT/
GOT ratio. The plasma SOD activity and T-AOC level increased and then
decreased with an increasing taurine content, reaching a maximum in
the 0.4 % taurine supplementation group (P < 0.05). The highest levels
of plasma AKP activities were found in the NC (30 % carbohydrate) and

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J. Qian et al. Aquaculture Reports 21 (2021) 100820

Table 4
Hepatic amino acid profile (mg/g of tissue) of hybrid grouper treated with different diets.
Amino acids PC NC T1 T2 T3 T4 P-value

Treatment Linear Quadratic

His 4.36 ± 0.29 3.59 ± 0.25 4.01 ± 0.50 4.04 ± 0.50 4.34 ± 0.42 4.55 ± 0.35 0.117 0.117 0.055
Tau 3.75 ± 1.01a 3.27 ± 0.21a 7.42 ± 0.07b 9.26 ± 0.43c 9.68 ± 0.52c 9.59 ± 0.20c <0.001 <0.001 <0.001
Ser 7.94 ± 0.56 6.44 ± 0.51 7.08 ± 1.13 7.01 ± 0.83 7.34 ± 0.61 7.91 ± 1.01 0.260 0.546 0.055
Arg 8.73 ± 0.58 7.24 ± 0.58 8.31 ± 1.50 7.99 ± 1.13 8.34 ± 0.56 9.29 ± 1.47 0.337 0.280 0.123
Gly 10.56 ± 0.72 9.19 ± 0.68 10.28 ± 1.93 9.71 ± 0.85 9.95 ± 0.71 10.78 ± 1.69 0.632 0.642 0.257
Asp 17.83 ± 1.76 14.50 ± 1.17 16.43 ± 2.31 16.50 ± 2.18 17.00 ± 1.32 18.29 ± 2.21 0.261 0.299 0.106
Glu 29.05 ± 2.91 24.33 ± 1.09 26.97 ± 3.88 25.69 ± 2.52 26.55 ± 1.30 28.30 ± 2.81 0.325 0.900 0.092
Thr 7.93 ± 0.73 6.55 ± 0.52 7.18 ± 1.01 7.19 ± 0.87 7.44 ± 0.56 8.06 ± 0.78 0.239 0.386 0.058
Ala 9.89 ± 0.52 8.36 ± 0.58 9.13 ± 1.36 9.18 ± 0.98 9.68 ± 0.84 10.36 ± 0.91 0.189 0.171 0.059
Pro 7.49 ± 0.55 6.29 ± 0.46 6.94 ± 1.09 6.78 ± 0.74 7.00 ± 0.46 7.60 ± 1.22 0.430 0.536 0.116
Cys 0.32 ± 0.12 0.14 ± 0.12 0.22 ± 0.12 0.16 ± 0.14 0.22 ± 0.04 0.28 ± 0.04 0.325 0.963 0.067
Lys 12.03 ± 0.89 10.07 ± 0.69 11.25 ± 1.33 11.51 ± 1.48 11.92 ± 1.11 13.02 ± 1.39 0.146 0.085 0.075
Tyr 4.85 ± 0.28 4.10 ± 0.41 4.51 ± 0.63 4.21 ± 0.63 4.53 ± 0.31 4.71 ± 0.48 0.415 0.900 0.114
Met 1.69 ± 0.23 2.15 ± 0.17 2.08 ± 0.07 1.85 ± 0.24 2.14 ± 0.10 2.07 ± 0.16 0.282 0.730 0.878
Val 9.62 ± 1.41 8.13 ± 0.81 8.55 ± 1.10 8.91 ± 1.29 9.70 ± 0.98 10.22 ± 1.01 0.259 0.159 0.074
Ile 7.64 ± 1.25 6.72 ± 1.03 6.96 ± 1.30 6.82 ± 0.88 8.14 ± 0.52 8.59 ± 0.88 0.199 0.094 0.059
Leu 13.28 ± 1.55 11.27 ± 1.25 12.16 ± 1.95 12.04 ± 1.45 13.54 ± 1.08 14.43 ± 1.28 0.165 0.102 0.048
Phe 7.79 ± 0.96 6.55 ± 0.73 7.08 ± 1.03 7.11 ± 0.89 7.79 ± 0.64 8.33 ± 0.74 0.199 0.138 0.055

All data were expressed as mean ± SE. Different superscript letters means the significant difference (P < 0.05). His: histidine; Tau: taurine; Ser: serine; Arg: arginine;
Gly: glycine; Asp: aspartic; Glu: glutamic; Thr: Threonine; Ala: alanine; Pro: proline; Cys: cysteine; Lys: lysine; Tyr: tyrosine; Met: methionine; Val: valine; Ile:
isoleucine; Leu: leucine; Phe: phenylalanine.

key liver glucolipid metabolizing enzymes in the 30 % carbohydrate

group were downregulated, especially those of GK and G6Pase
(P < 0.05). However, as the taurine gradient increased, the GK and FAS
activities of the liver showed an upward trend and began to be signifi­
cantly upregulation in the 0.5 % and 1.2 % taurine supplementation
groups, respectively (P < 0.05), The HK activity of the liver first
decreased and then increased, with the minimum value being found in
the 0.4 % taurine supplementation group; and the maximum value in the
1.6 % taurine supplementation group (P < 0.05). Additionally, PEPCK
and G6Pase activities also showed trends of falling and then rising, with
the peaks occurring in the 1.2 % taurine supplementation group.

3.6. Oil red O staining and HE staining patterns

A 30 % carbohydrate diet led to hepatic lipid deposition, which was

gradually reduced after taurine supplementation, with the lowest level
of deposition being found in the 1.2 % taurine supplementation group
(Fig. 7). When taurine supplementation exceeded 1.2 %, hepatic lipid
accumulation was aggravated again. HE staining results (Fig. 8) showed
that the contours of hybrid grouper hepatocytes were obvious, the cell
morphology was similar, and the nuclei were shifted to varying degrees.
The degree of nuclear deviation and swelling in the 30 % carbohydrate
group was greater than that in the 20 % carbohydrate group. However,
the cell swelling and nuclear deviation of the taurine group were
improved compared with those of the NC group. This effect was espe­
cially visible in the 1.2 % taurine supplementation group.

4. Discussion

Carnivorous fish utilize carbohydrates poorly due to long-term

Fig. 2. Hepatic and muscle glycogen contents of hybrid grouper.
T3 (1.2 % taurine supplementation) groups, whereas the lowest was
found in T2 (0.8 % taurine supplementation).
3.5. Hepatic metabolic enzymes
The results of the analysis of the key enzymes of glycolysis, gluco­
neogenesis and the fat synthesis pathway in the liver are shown in
Figs. 4–6. Compared with the 20 % carbohydrate group, the activities of
adaptive evolution to a low carbohydrate intake throughout their nat­
ural life (Bangsbo et al., 1997; Lin et al., 2000). A recent report showed
that the carbohydrate requirement of hybrid grouper was approximately
7.22 %. Once the dietary carbohydrate level exceeded 7.22 %, the final
body weight (FBW) and SGR showed a negative correlation with the
increase in dietary carbohydrate levels (Li et al., 2019). In the present
study, fish fed 30 % dietary carbohydrates presented lower WGR and
SGR, which was consistent with the above results and similar to findings
in jaguar guapote (Cichlasoma managuense), redlip mullet (Liza haema­
tocheila) and blunt snout bream (Megalobrama amblycephala). (Jiang
et al., 2016; X. Liu et al., 2020; T. Liu et al., 2020; Sun et al., 2017).
Taurine, an amino acid derivative was classified as a natural food

5
J. Qian et al.
Fig. 3. Intestinal digestive enzyme activities of hybrid grouper treated with
different diets.
attractant, playing a role in simulating the sense of olfactory and gus­
tatory organs, conducive to higher feed intake (FI) and lower FCR for
fish, which was confirmed in yellowfin seabream (Acanthopagrus latus)
and Nile tilapia (Orepchromis niloticus). (Dehghani et al., 2019; Zhou
et al., 2015b).Likewise, Abbasi et al. (2020) also indicated that taurine
provoked yellowfin seabream appetite and can alleviate the negative
influence of substitution of FM with PP in diet. The results of this
experiment showed that 1.01~1.79 % taurine significantly promoted
hybrid grouper WGR and SGR, but the FCR and PER did not show sig­
nificant differences among all treatment groups. Based on a study of the
marine fish goldlined spinefoot (Siganus guttatus) with a similar back­
ground, a high-carbohydrate (36.40 %) diet decreased the FI and
increased the FCR, resulting in lower utilization of carbohydrates by the
fish (Li et al., 2012). Therefore, the lack of improvement in FCR and PER
in this study may be due to the offset of the growth-promoting effect of
6
Aquaculture Reports 21 (2021) 100820
taurine and the inhibition of growth caused by high-carbohydrate stress.
According to the relationship between hybrid grouper growth, feed
utilization and dietary taurine levels, the fitted curve model indicated
that a 1.31 % taurine level had the best effect in promoting growth,
whereas a 1.05 % taurine level was best for the use of a high carbohy­
drate (30 %) diet for hybrid grouper.
Digestive enzymes play the main roles in the decomposition and
metabolism of nutrients, and their activities demonstrate the utilization
of protein, fat and carbohydrates by fish to a certain extent (Moraes
et al., 2020; Thongprajukaew et al., 2011). In the present study, the
activities of trypsin and AMS in hybrid grouper significantly decreased
after 30 % carbohydrate diet intake, and the activities of trypsin, lipase
and AMS gradually recovered and even significantly increased after
supplementation with taurine. A report on the utilization of different
types of carbohydrates by silver perch (Bidyanus bidyanus) confirmed
that a pregelatinized wheat starch diet had higher dry matter, protein
and energy contents than raw wheat starch, which is good for improving
the digestibility of feed (Stone et al., 2003). Nonetheless, the use of
pregelatinized starch in fish feed is still limited. Studies on yellow
croaker (Pseudosciaena crocea) and catla catla (Cyprinidae Catla)
revealed that excessive pregelatinized starch led to declines in fish
growth performance (WGR, SGR) and enzyme activities (trypsin and
α-amylase) in the intestine (Cheng et al., 2013). Interestingly, the in­
fluence of pregelatinized starch on digestive enzymes in hybrid grouper
are found a highly similar in the middle intestine of common carp
(Cyprinus carpio) (Sun et al., 2016). Since taurine was discovered, it has
been determined that taurine combines with bile acid to form taurine
cholic acid, effectively emulsify lipids, increase lipase activity and
enhance the absorption or synthesis of lipids by fish (Salze et al., 2012).
Consequently, the significant enhancement of lipase activity in hybrid
grouper after supplementation was a predictable result. Taurine also
significantly increased the activities of intestinal trypsin and amylase,
which were more sensitive to taurine than lipase from the perspective of
activity changes. A study reported that yellowfin seabream fed low
fishmeal and fish oil diets supplemented with taurine also showed pro­
motion of trypsin and AMS activity, which was thought to be the result
of an increase of protease inhibitors and indigestible carbohydrates in
plant proteins (Abbasi et al., 2020). However, given the consistency of
plant-based ingredients dosage and the decreased proportion of indi­
gestible carbohydrates between NC control and taurine groups in this
study, it was concluded that the promotion of trypsin and AMS activity
was influenced by taurine. However, the specific mechanism of taurine
regulation on trypsin and AMS enzymes remains to be elucidated. In
summary, taurine effectively promoted trypsin, amylase and lipase ac­
tivities in hybrid grouper, which may be the first step whereby taurine
increases the glucose tolerance of hybrid grouper.
In addition, the body composition of fish changes with changes in
nutritional intake and further affects morphological indexes (Wiriduge
et al., 2020; Zhan et al., 2020). It has been reported that moisture, crude
protein and lipid contents show no significant differences after the
treatment of rainbow trout (Oncorhynchus mykiss) with 25.8 %–30.1 %
starch diets (Brauge et al., 1994) or treatment of European sea bass with
diets containing 0–1 % taurine (Kotzamanis et al., 2020). Similar results
were discovered in a study of hybrid lemon fin barb (Barbonymus
gonionotus ♀ × Hypsibarbus wetmorei male), which also proved that sur­
plus carbohydrates would significantly reduce the retention of protein
and carbohydrates in the fish (Sulaiman et al., 2020). In this study, the
examination of the body composition showed that dietary pregelati­
nized starch and taurine levels had no significant effect on the moisture,
crude protein and crude lipid contents of hybrid grouper. Taurine sup­
plementation in a 30 % carbohydrate diet significantly decreased the VSI
and HIS values. The reason for the decrease in HSI and VSI was that
taurine reduced lipid deposition in the viscera and liver by promoting
the lipid metabolic pathway, which could also be indirectly confirmed
by the oil red sections of grouper liver in this study (Han et al., 2020).
Taurine is considered a conditionally essential amino acid due to
J. Qian et al. Aquaculture Reports 21 (2021) 100820

Table 5
Hepatic IGF-1R level and plasma biochemistry parameters of hybrid grouper treated with different diets.
Diet Insulin IGF-1 IGF-1R Glucose TG T-CHO LDL HDL
(mIU/L) (ng/mL) (ng/mgProt) (mmol/L) (mmol/L) (mmol/L) (mmol/L) (mmol/L)

PC 30.54 ± 1.72b 13.06 ± 0.63b 12.08 ± 0.55bc 2.32 ± 1.09a 0.37 ± 0.12a 2.96 ± 1.14bc 0.32 ± 0.03b 1.35 ± 0.12a
NC 29.49 ± 0.82b 11.68 ± 1.29b 9.40 ± 0.64a 3.19 ± 0.11a 0.40 ± 0.06a 1.87 ± 0.20a 0.31 ± 0.02b 1.58 ± 0.05b
T1 23.99 ± 1.81a 8.15 ± 0.36a 9.43 ± 1.62a 2.07 ± 0.10a 0.44 ± 0.14a 2.65 ± 0.57abc 0.30 ± 0.03b 2.40 ± 0.08e
T2 31.18 ± 1.53b 14.51 ± 0.44c 9.86 ± 0.59a 1.95 ± 0.09a 0.44 ± 0.14a 3.32 ± 0.43c 0.22 ± 0.03a 2.14 ± 0.09d
T3 37.18 ± 2.49c 19.49 ± 0.62d 10.63 ± 1.40ab 2.45 ± 0.28a 0.73 ± 0.13b 2.93 ± 0.07abc 0.21 ± 0.02a 1.82 ± 0.06c
T4 38.94 ± 1.14c 15.54 ± 0.97c 13.65 ± 0.76c 7.25 ± 1.65b 0.26 ± 0.17a 1.97 ± 0.59ab 0.19 ± 0.02a 1.79 ± 0.10c

P-value
Treatment <0.001 <0.001 <0.001 <0.001 <0.001 0.001 <0.001 <0.001
Linear <0.001 <0.001 0.001 <0.001 0.333 0.584 <0.001 <0.001
Quadratic <0.001 <0.001 <0.001 <0.001 0.004 0.087 0.396 <0.001

All data were expressed as mean ± SE. Different superscript letters means the significant difference (P < 0.05). IGF-1R: insulin-like growth factor 1 receptor; Plasma
biochemical parameters: insulin, insulin-like growth factor 1 (IGF-1), glucose, triglycerides (TG), total cholesterol (T-CHO), low density lipoprotein (LDL), high density
lipoprotein (HDL).

Table 6
Plasma liver function and antioxidant indicators of hybrid grouper treated with different diets.
Diet GPT GOT GPT/GOT GOT/GPT AKP SOD T-AOC MDA
(U/L) (U/L) (U/L) (U/mL) (mM) (nmol/mL)

PC 41.87 ± 3.33 22.78 ± 5.40a 1.91 ± 0.38a 0.54 ± 0.11ab 67.88 ± 1.61b 755.36 ± 10.67c 0.54 ± 0.06a 8.17 ± 0.23
NC 40.43 ± 10.13 27.72 ± 9.35a 1.56 ± 0.44a 0.68 ± 0.21ab 130.68 ± 3.10d 692.20 ± 4.62b 0.57 ± 0.02ab 7.29 ± 1.32
T1 44.63 ± 2.29 48.02 ± 14.18b 1.04 ± 0.47a 1.08 ± 0.34c 69.01 ± 1.43b 759.17 ± 4.50c 0.68 ± 0.01c 8.74 ± 0.22
T2 40.12 ± 4.11 27.93 ± 4.90a 1.47 ± 0.25a 0.70 ± 0.12b 63.74 ± 2.05a 606.18 ± 7.64a 0.68 ± 0.02c 8.59 ± 0.91
T3 41.52 ± 2.10 26.79 ± 15.24a 2.50 ± 2.28a 0.64 ± 0.36ab 109.57 ± 1.49d 684.58 ± 6.75b 0.61 ± 0.02b 7.33 ± 1.71
T4 46.51 ± 2.54 15.73 ± 13.43a 5.73 ± 4.66b 0.34 ± 0.28a 72.33 ± 4.43c 691.66 ± 9.10b 0.61 ± 0.01b 7.68 ± 0.93

P-value
Treatment 0.055 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.054
Linear 0.118 0.069 0.001 0.32 <0.001 <0.001 <0.001 0.458
Quadratic 0.172 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 0.201

All data were expressed as mean ± SE. Different superscript letters means the significant difference (P < 0.05). GPT: alanine aminotransferase; GOT: aspartate
aminotransferase; AKP: alkaline phosphatase; SOD: superoxide dismutase; T-AOC: total antioxidant capacity; MDA: Malondialdehyde.

animals’ weak ability to synthesize taurine (Huxtabl and Sebring, 1986).
Although aquatic animals are rich in taurine, juvenile fish must obtain
supplementary taurine through food intake because of the inadequate
amount of synthesized taurine (Yokoyama et al., 2001). Fish show
similar characteristics to typical T2DM patients, and T2DM patients
often suffer from decreased taurine levels (De Luca et al., 2001). The
present study measured the liver taurine content of grouper and found
that a 30 % carbohydrate diet also decreased the liver taurine content of
hybrid grouper. Hybrid grouper hepatic taurine content was positively
correlated with dietary taurine levels, consistent with the outcome of a
Japanese flounder (Paralichthys olivaceus) study (Kim et al., 2007).
Moreover, the taurine content in the liver of hybrid grouper reached the
highest level when consuming a 0.89 %–1.79 % taurine diet, which
showed that the problem of relatively deficient taurine due to high
carbohydrate levels did not exist.
It is well known that GPT and GOT mainly exist in hepatocytes, and
they are released into the blood when liver cell membrane permeability
is impaired (Yan et al., 2016). Clinically, an increase in the plasma
GOT/GPT ratio is generally seen in patients with acute hepatitis and
later chronic hepatitis (Araki and Wada, 1991; Wallerstedt et al., 1974).
A study of the characteristics of T2DM at the time of onset reported that
a high ratio of GPT/GOT in patient plasma indicated a decreased in
FAD-linked glycerophosphate dehydrogenase (m-GDH) activity,
impairing the secretion of pancreatic β-cells after responding to glucose,
which may be the pathogenesis of noninsulin-dependent diabetes mel­
litus (Vidal et al., 1996). In the current study, dietary pregelatinized
starch levels had no significant effects on GPT, GOT, GPT/GOT or
GOT/GPT. After taurine supplementation in a high carbohydrate di­
etary, GOT activity and the GOT/GPT value were significantly increased
in the T1 group, and the GPT/GOT value was significantly increased in
the T4 group, indicating that a low dose (0.4 %) of taurine might in­
crease the chance of chronic hepatitis in grouper, while a high dose
(1.60 %) of taurine reduced carbohydrate utilization in grouper. This
study also examined cell morphology through the HE staining of paraffin
sections of the liver. Vidal et al. (1996) studied the pathology of a liver
section of grass carp (Ctenopharyngodon idellus) fed 28.4–36.9 % car­
bohydrate diet and found that hepatocyte boundaries and nuclei were
clearly observable and that nuclei were mostly located in the center of
the cells; however, the hepatocytes experienced cell volume swelling
and nuclear atrophy as the carbohydrate level in the diet increase. In
another study, a higher carbohydrate level (42.31 %) led to significant
nuclear migration and cavitation denaturation in grass carp, which
might have been caused by the accumulation of fat droplets in the liver
due to high-carbohydrate nutrient stress (Guo et al., 2014). In the pre­
sent study, normal cell morphologies and clear boundaries were
observed among all treatments, but the hepatocytes of the fish in the
high-carbohydrate group (30 %) were large in size, and ORO staining
results showed a significant increase in lipid droplets, which was basi­
cally consistent with the above studies. After taurine supplementation,
the hybrid grouper liver cell volume gradually returned to the standard
of the grouper in the 20 % carbohydrate group, and the number of cells
with nuclear migration and fat droplets in liver cells was visibly reduced.
It has been reported that the protective mechanism of taurine on he­
patocyte morphology and the improvement of fatty deposition are
related to its antioxidant function (Yu et al., 2007).
Insulin is an important hormone in mammals that regulates and
maintains glucose homeostasis (Moom, 2001). In fish, the existence of
an impaired and delayed hormonal response to glucose loading is well
established and is one of the reasons for a low glucose metabolic rate
(Enes et al., 2011; Li et al., 2018). Although long-term adaptation to

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J. Qian et al.
Fig. 4. Hepatic glycolytic enzyme activities of hybrid grouper treated with
different diets.
high-carbohydrates could increase the insulin concentration in fish
plasma, the deviation of blood glucose and insulin concentration was
still observed soon after glucose injection (Enes et al., 2011). IGF-1 is a
type of polypeptide molecule with an insulin-like structure that stimu­
lating tissues (skeletal muscle, fat cell) glucose uptake more effectively
than insulin (Bouraoui et al., 2010). These findings led scholars to
believe that IGF-1 plays a greater role in fish glucose metabolism than
insulin. Insulin and IGF-1 require specific cell surface receptors to exert
their biological activities (Alessi et al., 1996). It has been confirmed that
the number of insulin and IGF-1 receptors varies with fish species and
tissues, and that more IGF-1 receptors are found in some organs, such as
heart and skeletal muscle (Baños et al., 1997; Párrizas et al., 1995;
Pirrizas et al., 1993). Chinook salmon (Oncorhynchus tshawytscha) and
rainbow trout plasma insulin, IGF-1 and corresponding receptor levels
have been shown to be higher under a glucose load or high-carbohydrate
intake (Baños et al., 1998; Furuichi and Yone, 1981). Additionally, in
studies of European sea bass and Chinese perch, dietary carbohydrates
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Aquaculture Reports 21 (2021) 100820
Fig. 5. Hepatic gluconeogenesis enzyme activities of hybrid grouper treated
with different diets.
Fig. 6. Hepatic fatty acid synthesis enzyme activity of hybrid grouper treated
with different diets.
had no effect on plasma insulin and IGF-1 or their receptors, which could
be explained by the complete regulation of carbohydrates after 24 h of
fasting (Borrebaek et al., 2003; Enes et al., 2010). This study showed
that there were no statistically significant differences in the plasma in­
sulin and IGF-1 levels of hybrid grouper fed 30 % pregelatinized starch,
but the abundance of IGF-1 receptors was significantly decreased, and
plasma glucose levels subsequently increased. The lower IGF-1 receptor
abundance under high-carbohydrate stress might be a reason for the
impaired regulation of plasma glucose. The insulinotropic potency of
dietary carbohydrates in carnivorous fish is weaker than that of certain
amino acids, such as taurine, relative to glucose (Kamalam et al., 2017;
Kim and Gupta, 2007). Taurine is a free molecule that is both structur­
ally and functionally related to insulin and IGF-1 and is involved in
glucose and lipid metabolism regulation (Hansen, 2001; Huxtable,
J. Qian et al. Aquaculture Reports 21 (2021) 100820

Fig. 7. Hepatocyte oil red O (ORO) staining section of hybrid grouper treated with different diets. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article).

Fig. 8. Hepatocyte hematoxylin-eosin (HE) staining section of hybrid grouper treated with different diets.

1992). At the hormone level, plasma insulin levels of taurine transporter
knockout (TauTKO) mice were lower than those of wild-type (WT) mice
before and after glucose injection, and the decline in islet β cells in
TauTKO mice was one of the reasons for this difference (Ito et al., 2015).
Another study reported that mice fed 2% taurine water achieved better
Ca2+ uptake than taurine-free mice through the upregulated expression
of the β2 subunit of the L-type voltage-sensitive Ca2+ channel protein,
which improved insulin secretion and sensitivity (Rocco et al., 2009).
Additionally, Amoreaux et al. (2010) discovered a drop in intracellular
insulin levels in pancreatic β cell lines of Syrian hamsters and rats
treated with taurine alone or in combination with glucose (1 mM taurine
or 1 mM glucose plus 1 mM taurine) compared to a control group (1 mM
glucose), whereas a 3 mM glucose plus 1 mM taurine group appeared to
unexpectedly climb, revealing that the addition of taurine to islet β cells
allowed higher glucose levels to accumulate and increased the retention
of insulin. Analogously, diabetes model mice receiving different doses of
oral taurine showed that significantly decreased plasma insulin levels
after low-dose taurine replenishing, and their plasma insulin returned to
normal or even higher levels with a higher dose of taurine (Zeng et al.,
2019). For IGF-1, the surprisingly consistent changes in insulin and
IGF-1 observed in the taurine supplement group were expected because
of the synergistic regulation of glucose metabolism. Similar results have
been reported in many studies (Gaylord et al., 2007; Wardani et al.,
2020). Moreover, taurine can specifically bind to insulin to show

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J. Qian et al.
insulin-like functions, strengthening the transduction of insulin the
signaling pathway, the regulation of glycolysis and the activation of
glucose transporters (Carneiro et al., 2009). IGF-1 receptor is an
RTK-like insulin receptor, and its mode of action is very similar to that of
the insulin receptor. Therefore, whether the hypoglycemic effect of
taurine on grouper occurs mainly via insulin or the IGF-1 pathway re­
mains to be discussed.
Active glucose metabolism in fish beneficially provides abundant
intermediates for synthesizing important compounds (Polakof et al.,
2012). In a report on the herbivorous grass crap, it was obvious that
plasma TG and T-CHO levels increased with increasing carbohydrate
levels (15.90~45.45 %) (Cai et al., 2018). However, studies on marine
fish showed that the level of T-CHO in European sea bass is significantly
increased by carbohydrates (0–18 %), whereas the level in gilthead sea
bream is decreased (0~18.7 %) (Castro et al., 2016, 2015). In this study,
hybrid grouper fed 20~30 % carbohydrate showed plasma TG had no
significant influence and T-CHO had a downward trend. The decrease in
plasma T-CHO might be due to weakened glycolysis reducing the supply
of substances required for T-CHO synthesis. Although the plasma T-CHO
of hybrid grouper decreased with increasing dietary carbohydrate
levels, plasma HDL was significantly upregulated, leading to enhanced
transport of lipids to the liver. After taurine supplementation, plasma TG
and T-CHO levels of hybrid grouper displayed a trend of rising and then
falling, which could be explained by the enhanced absorption of exog­
enous TG and T-CHO in intestinal tissues, as well as endogenous syn­
thesis in the liver (Hoseini et al., 2018; Petrosian and Haroutounian,
2000; Shi et al., 2019). Plasma HDL levels in the taurine supplementa­
tion groups were significantly higher than those in the nontaurine
group, whereas LDL levels significantly decreased in the T2-T4 group.
Therefore, taurine can be considered to increase the transport of lipids to
the liver.
Many studies have reported that high-carbohydrate intake or blood
glucose loading create metabolic disorders as well as a reduction of
antioxidants in fish, which are caused by unequal reactive oxygen spe­
cies (ROS) generation and clearance during the oxidative decomposition
of carbohydrates (Jin et al., 2013; Vielma et al., 2003; Wang et al.,
2014). Once the antioxidant capacity is insufficient, the pressure of lipid
peroxidation and insulin resistance are aggravated, which is also
considered to be one of the mechanisms of fish carbohydrate intolerance
and lipid metabolism disorder. In many fish studies, the evaluation of
the effects of carbohydrates on oxidative stress has produced varying
results. In a previous study, both SOD activity and T-AOC of hybrid
grouper treated with 5.27–25.63 % α-cassava starch significantly
decreased with increasing starch levels, and the MDA content of serum
and liver in the high-carbohydrate group was significantly increased,
suggesting that excessive carbohydrates increase the oxidative stress of
hybrid grouper ((Li et al., 2020). In contrast, the SOD activities and
T-AOC of golden pompano (Trachinotus ovatus) and common dentex
(Dentex dentex) were shown to be significantly increased by carbohy­
drates, and the detected volume of MDA was also significantly reduced,
supporting the antioxidant benefits of carbohydrates to fish
(Pérez-jiménez et al., 2017; Zhao et al., 2020). Interestingly, the SOD
activities of the 30 % pregelatinized starch group in this study showed
significant decreases, which indicated a reduction in antioxidant ca­
pacity. Furthermore, aquatic animal AKP activities have been shown to
increase significantly when fish are exposed to either nutritive or envi­
ronmental stress (Liu et al., 2010; Zhang et al., 2020). In the present
study, hybrid grouper AKP activities were greatly promoted by a 30 %
carbohydrate level diet, which indicated that the hybrid groupers were
stressed by excess carbohydrates. It is known that taurine can contribute
to antioxidant defense through different approaches (Surai et al., 2019).
Weaned pigs, broiler chickens, Chinese mitten crabs (Eriocheir sinensis)
and common carp provide references regarding the taurine-mediated
relief of oxidative pressure and metabolic disorders, such as the
improvement of SOD and T-AOC, and reduced occurrence of MDA
(Abdel-Tawwab and Monier, 2018; Dong et al., 2018; Liu et al., 2014).
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Aquaculture Reports 21 (2021) 100820
After yellow catfish were injected with taurine, there was a significant
rise in plasma AKP activities and MDA concentrations rather than SOD
and T-AOC (Zhang et al., 2018). In this study, the levels of SOD and
T-AOC revealed an upward trend before falling, and there were no
obvious adverse effects on growth performance or liver tissue structure,
which showed an increased antioxidant capacity in hybrid grouper. At
the same time, the decline in AKP activities in the taurine supplemen­
tation groups indicated remission of carbohydrate stress in hybrid
grouper. This is consistent with previous studies in yellow seabream,
which supports the idea that taurine exerts its antioxidant properties by
quenching various radicals or improving or restoring the antioxidant
enzyme activities (Abbasi et al., 2020).
Fish use glucose in a similar way to mammals, in which glycolysis
and gluconeogenesis play alternating roles to maintained glucose ho­
meostasis (Polakof et al., 2012). HK and GK are the first rate-limiting
enzymes in glycolysis, but GK is characterized by a low Km value and
specific distribution in the liver, which make it effective only when the
intracellular glucose concentration is high enough (Polakof and Pan­
serat, 2016). Therefore, the activity of GK could be used as an indicator
to determine glucose utilization in cells. According to previous studies,
Atlantic cod (Gadus morhua) liver HK enzyme activity is not significantly
influenced by different carbohydrate levels (Sundby et al., 1991; Tra­
nulis et al., 1991). Dietary starch levels of 2.67 %–21.39 % can effec­
tively induce the liver GK activity of in hybrid grouper (Li et al., 2019),
but overly high carbohydrate levels have been found to inhibit the
mRNA expression of GK in largemouth bass (Micropterus salmoides)
(Zhang et al., 2019). In terms of PK, gilthead sea bream (Sparus aurata)
and darkbarbel catfish (Pelteobagrus vachelli) fed diets with different
carbohydrate levels showed no significant differences in PK activities
(Enes et al., 2008; Zhang et al., 2012), whereas studies on grass carp and
European sea bass (Dicentrarchus labrax) provided led to the conflicting
observation that PK activities were significantly increased by
high-carbohydrate levels (Enes et al., 2006; Gao et al., 2010). These
differences among glycolytic enzymes can be attributed to the fish
species, carbohydrate type and carbohydrate gradient. This study
showed that a 30 % pregelatinized starch diet only inhibited hybrid
grouper GK activities, whereas PK activities were not significantly
affected, reflecting that the restrictive factors of hybrid grouper using
glucose lessened intracellular glucose in the liver. Elevator blood
glucose levels usually inhibit the gluconeogenesis enzyme activities to
maintain blood glucose stability. However, It has been reported that
some carnivorous marine fish lack the feedback regulation effect of
carbohydrates on gluconeogenesis enzymes (Enes et al., 2008; Moreira
et al., 2008). A previous study reported that hybrid grouper gluconeo­
genesis enzymes (PEPCK, FBPase and G6Pase) showed a positive linear
relationship with dietary carbohydrate levels (5.27~23.65 %) (Li et al.,
2019). In this study, when dietary carbohydrate levels reached 30 %, the
activities of PEPCK and G6Pase showed a downward trend, and a sta­
tistical decline was found in G6Pase. All of these findings seem to
indicate that key glycolysis and gluconeogenic enzymes in hybrid
grouper are very insensitive to glucose stimulation, similar to insulin. In
herbivorous and omnivorous fish, FAS expression is often stimulated by
dietary carbohydrates, whereas FAS expression is inhibited in carnivo­
rous fish (Cai et al., 2018; Wang et al., 2017; Xu et al., 2017). Further­
more, the addition of taurine to the diet promoted GK and HK activities,
while inhibiting PEPCK and G6Pase activities. Although there are few
studies on taurine in fish glucolipid metabolism, some similar results in
mammals can be found. For example, taurine compensation in mice
receiving either oral or injected carbohydrates improved glucose toler­
ance via increase in GLUT and GK mRNA expression (Carneiro et al.,
2009). Similar results were found in alloxan-treated rats, and the effect
was mediated by PI3K and AKT (Rashid et al., 2013). Moreover, it has
been reported that the activities of FBPase, a key gluconeogenesis
enzyme are significantly inhibited in European seabass and Totoaba
(Totoaba macdonaldi) after treatment with taurine (Bañuelos-vargas
et al., 2014; Martins et al., 2021). In this study, hybrid grouper fed a diet
J. Qian et al.
with a suitable taurine level showed increasing activities of HK and GK
and decreasing activities of PEPCK and G6Pase, increasing the glucose
metabolic intensity, which slowed the rise in blood glucose levels. In
mammalian and poultry studies, taurine supplementation has been
shown to significantly reduce FAS mRNA expression and to play a roles
in lowing lipid levels. (Han et al., 2020; Ma et al., 2012). Although some
fish studies have shown that taurine does not necessarily inhibit FAS
activity or expression, the promotion of FAS activity observed in this
experiment was unexpected (Bañuelos-vargas et al., 2014; De Carvalho
et al., 2020). This finding provides evidence that taurine improves
abnormal glucose and lipid metabolism in hybrid grouper.
Fish, like other vertebrates, are able to synthesize glycogen to store
carbohydrates (Zamora-Sillero et al., 2013). Studies of largemouth bass
and yellow croaker illustrated that the accumulation of hepatic and
muscle glycogen was significantly increasing with dietary carbohydrate
levels (0–25 %) (Lin et al., 2018; Zhou et al., 2016). However, another
study reported that the hepatic glycogen content of lebranche mullet
(Mugil liza) stopped increasing when the fish were fed more than 25 %
dextrin (Zamora-Sillero et al., 2013). In the present study, hepatic
glycogen deposition of hybrid grouper was unaffected by 20–30 %
carbohydrate levels, whereas muscle glycogen deposition was increased,
indicating that the hepatic glycogen synthesis capacity of grouper had
reached its upper limit. Taurine could increase the synthesis of glycogen
in the liver rather than muscle. This is mainly attributed to taurine’s
roles in increasing insulin secretion, sensitizing insulin receptors,
interfering with the insulin signaling pathway, and then activating
glycogen synthase kinase (GSK), leading to the deposition of hepatic
glycogen (Gomez et al., 2018; Rashid et al., 2013).
5. Conclusions
In conclusion, the present study showed that the appropriate dietary
taurine levels for hybrid grouper fed a 30 % carbohydrate diet were 1.31
% and 1.05 % for achieving the best growth and feed coefficient rates,
respectively. Taurine also facilitates intermediary metabolism,
including promoting the activities of key enzymes in glycolysis and lipid
synthesis and decreasing the levels of gluconeogenesis rate-limiting
enzymes to maintain glucose homeostasis. Of course, the changes in
LDL and HDL indicated that suitable supplementation with taurine in­
crease transport of lipids to the liver. The examination of hepatocytes
sections showed that taurine prevented damage to the liver through a
decrease in lipid deposition and the maintenance of cell structure.
Furthermore, taurine increased the total antioxidant capacity of hybrid
grouper to deal with lipid peroxidation caused by enhanced nutrient
metabolism. Overall, this experiment demonstrates that taurine has a
positive effect on glucose metabolism and can be used to expand the
upper limit of carbohydrate utilization by hybrid grouper, but the spe­
cific mechanism still needs to be further explored. Interestingly, the
inhibition of IGF-1R by high-carbohydrate intake is of great significance
to hybrid grouper glucose intolerance, and the addition of taurine seems
to increase the secretion of IGF-1 to compensate for the defects in IGF-
1R.
Author Statement
All of the indicated authors have actively contributed to this study.
The contributions of the co-authors are as follows: H. Y. Liu and B. P. Tan
designed the study; J. H. Qian conducted the study and B. Yin analyzed
the data; X. H. Dong participated in the interpretation of results; J. H.
Qian wrote the manuscript; Q. H. Yang, S. Y. Chi, and S. Zhang pur­
chased the reagents; H. Y. Liu revised the manuscript. All authors have
read and approved the final version of the manuscript.
Funding information
This research was funded by NATIONAL KEY R&D PROGRAM OF
11
Aquaculture Reports 21 (2021) 100820
CHINA, grant number 2019YFD0900200; NATIONAL NATURAL SCI­
ENCE FOUNDATION OF CHINA, grant number 31772864; and NATU­
RAL SCIENCE FOUNDATION OF GUANGDONG PROVINCE, grant
number 2018A030313154 & 2020A1515011129.
Data availability statement
The data that support the findings of this study are available from the
corresponding author upon request. The data are not publicly available
due to privacy or ethical restrictions.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
This study was supported by the National Key R&D Program OF
China, grant number 2019YFD0900200; the National Natural Science
Foundation Of China, grant number 31772864; and the Natural Science
Foundation OF Guangdong Province, grant number 2018A030313154 &
2020A1515011129.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.aqrep.2021.100820.
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