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Benfotiamine, a Lipid-Soluble Analog of Vitamin B1, Improves the Mitochondrial Biogenesis and Function in Blunt Snout Bream (Megalobrama amblycephala) Fed High-Carbohydrate Diets by Promoting the AMPK/PGC-1β/NRF-1 Axis

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This study evaluated the effects of benfotiamine on the growth performance and mitochondrial biogenesis and function of Megalobrama amblycephala fed high-carbohydrate diets. Fish (45.25 ± 0.34 g) were randomly fed six diets: the control diet (30% carbohydrate, C), the high-carbohydrate diet (43% carbohydrate, HC) and the HC diet supplemented with different benfotiamine levels (0.7125 (HCB1), 1.425 (HCB2), 2.85 (HCB3) and 5.7 (HCB4) mg/kg) for 12 weeks. High-carbohydrate levels remarkably decreased weight gain rate, specific growth rate, relative feed intake, feed conversion ratio, p-AMPKα/t-AMPKα ratio, peroxisome proliferator activated receptor-γ coactivator-1β (PGC-1β) and nuclear respiratory factor-1 (NRF-1) protein expression, complexes I, III and IV activities and hepatic transcriptions of cytochrome B (CYT-B) and cytochrome c oxidase-2 (COX-2), whereas the opposite was true for plasma glucose, glycated serum protein, advanced glycation end products and insulin levels, tissue glycogen and lipid contents, hepatic adenosine triphosphate (ATP) and adenosine monophosphate (AMP) contents and ATP/AMP ratio, complexes V activities and the expressions of AMP-activated protein kinase α-2 (AMPKα-2), PGC-1β, NRF-1, mitochondrial transcription factor A (TFAM), mitofusin-1 (Mfn-1), optic atrophy-1 (Opa-1), dynamin-related protein-1 (Drp-1), fission-1 (Fis-1), mitochondrial fission factor (Mff) and ATP synthase-6 (ATP-6). As for benfotiamine supplementation, HCB2 diet remarkably increased weight gain rate, specific growth rate, tissue glycogen and lipid contents, AMP contents, p-AMPKα/t-AMPKα ratio, PGC-1β and NRF-1 levels, complexes I, III, IV and V activities and hepatic transcriptions of AMPKα-2, PGC-1β, NRF-1, TFAM, Mfn-1, Opa-1, CYT-B, COX-2 and ATP-6, while the opposite was true for the remaining indicators. Overall, 1.425 mg/kg benfotiamine improved the growth performance and mitochondrial biogenesis and function of fish fed high-carbohydrate diets through the activation of AMPK/PGC-1β/NRF-1 axis, the up-regulation of mitochondrial complexes activities and transcriptions as well as the enhancement of mitochondrial fusion coupled with the depression of mitochondrial fission.
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fphys-09-01079 August 30, 2018 Time: 17:3 # 1
ORIGINAL RESEARCH
published: 03 September 2018
doi: 10.3389/fphys.2018.01079
Edited by:
Nour Eissa,
University of Manitoba, Canada
Reviewed by:
Josefina Blasco,
University of Barcelona, Spain
Tiziano Verri,
University of Salento, Italy
*Correspondence:
Xiang-Fei Li
xfli@njau.edu.cn
Specialty section:
This article was submitted to
Aquatic Physiology,
a section of the journal
Frontiers in Physiology
Received: 25 May 2018
Accepted: 19 July 2018
Published: 03 September 2018
Citation:
Xu C, Liu W-B, Zhang D-D, Shi H-J,
Zhang L and Li X-F (2018)
Benfotiamine, a Lipid-Soluble Analog
of Vitamin B1, Improves
the Mitochondrial Biogenesis
and Function in Blunt Snout Bream
(Megalobrama amblycephala) Fed
High-Carbohydrate Diets by
Promoting the AMPK/PGC-1β/NRF-1
Axis. Front. Physiol. 9:1079.
doi: 10.3389/fphys.2018.01079
Benfotiamine, a Lipid-Soluble Analog
of Vitamin B1, Improves the
Mitochondrial Biogenesis and
Function in Blunt Snout Bream
(Megalobrama amblycephala) Fed
High-Carbohydrate Diets by
Promoting the AMPK/PGC-1β/NRF-1
Axis
Chao Xu, Wen-Bin Liu, Ding-Dong Zhang, Hua-Juan Shi, Li Zhang and Xiang-Fei Li*
Key Laboratory of Aquatic Nutrition and Feed Science of Jiangsu Province, College of Animal Science and Technology,
Nanjing Agricultural University, Nanjing, China
This study evaluated the effects of benfotiamine on the growth performance and
mitochondrial biogenesis and function in Megalobrama amblycephala fed high-
carbohydrate (HC) diets. The fish (45.25 ±0.34 g) were randomly fed six diets: the
control diet (30% carbohydrate, C), the HC diet (43% carbohydrate), and the HC diet
supplemented with different benfotiamine levels (0.7125 (HCB1), 1.425 (HCB2), 2.85
(HCB3), and 5.7 (HCB4) mg/kg) for 12 weeks. High-carbohydrate levels remarkably
decreased the weight gain rate (WGR), specific growth rate (SGR), relative feed
intake (RFI), feed conversion ratio (FCR), p-adenosine monophosphate (AMP)-activated
protein kinase (AMPK)α/t-AMPKαratio, peroxisome proliferator-activated receptor-γ
coactivator-1β(PGC-1β) and nuclear respiratory factor-1 (NRF-1) protein expression,
complexes I, III, and IV activities, and hepatic transcriptions of cytochrome b (CYT-
b) and cytochrome c oxidase-2 (COX-2), whereas the opposite was true for plasma
glucose, glycated serum protein, advanced glycation end product and insulin levels,
tissue glycogen and lipid contents, hepatic adenosine triphosphate (ATP) and AMP
contents and ATP/AMP ratio, complexes V activities, and the expressions of AMPKα-2,
PGC-1β, NRF-1, mitochondrial transcription factor A (TFAM), mitofusin-1 (Mfn-1), optic
atrophy-1 (Opa-1), dynamin-related protein-1 (Drp-1), fission-1 (Fis-1), mitochondrial
fission factor (Mff), and ATP synthase-6 (ATP-6). As with benfotiamine supplementation,
the HCB2 diet remarkably increased WGR, SGR, tissue glycogen and lipid contents,
AMP content, p-AMPKα/t-AMPKαratio, PGC-1βand NRF-1 levels, complexes I, III, IV,
and V activities, and hepatic transcriptions of AMPKα-2, PGC-1β, NRF-1, TFAM, Mfn-
1, Opa-1, CYT-b, COX-2, and ATP-6, while the opposite was true for the remaining
indicators. Overall, 1.425 mg/kg benfotiamine improved the growth performance and
Frontiers in Physiology | www.frontiersin.org 1September 2018 | Volume 9 | Article 1079
fphys-09-01079 August 30, 2018 Time: 17:3 # 2
Xu et al. Effect of Benfotiamine on Liver Mitochondria of Fish
mitochondrial biogenesis and function in fish fed HC diets by the activation of the
AMPK/PGC-1β/NRF-1 axis and the upregulation of the activities and transcriptions of
mitochondrial complexes as well as the enhancement of mitochondrial fusion coupled
with the depression of mitochondrial fission.
Keywords: benfotiamine, glucose metabolism, mitochondrial biogenesis, mitochondrial function, Megalobrama
amblycephala
INTRODUCTION
As the most economical energy source, carbohydrates are now
being commonly incorporated into aquafeeds to improve the
physical quality of the feed and reduce the catabolism of proteins
and lipids by aquatic animals (Yang et al., 2018). However,
it is generally acknowledged that fish show poor capability
in utilizing carbohydrates for energy purposes than terrestrial
animals (Hemre et al., 2002;Enes et al., 2009). Furthermore, most
species (especially carnivorous ones) often exhibit prolonged
hyperglycemia after an intake of carbohydrate-enriched diets
or a glucose load (Moon, 2001;Kamalam et al., 2017) as is
similar to the symptoms of type 2 diabetes mellitus observed
in mammals. Previous studies have suggested that the poor
carbohydrate utilization or the persistent hyperglycemia in fish
may be attributed to the relatively low number of insulin
receptors, the low affinity of glucose transporter proteins for
glucose, a poor inhibition of postprandial gluconeogenesis, poor
hepatic lipogenesis from glucose, etc (Panserat et al., 2000;Enes
et al., 2009). Recently, several approaches such as metabolomics
and transcriptomics are being employed to assess diet-induced
metabolic syndromes in fish (Miao et al., 2017;Prathomya
et al., 2017;Prisingkorn et al., 2017). Findings from such studies
suggest that the disruption of the energy homeostasis in fish
is closely implicated in the development of disturbances in
the glucose metabolism. Therefore, further biochemical and
molecular investigations of the energy metabolism are necessary
and will undoubtedly facilitate better understanding of the
utilization of carbohydrates by fish.
In eukaryotes, the mitochondria are regarded as the most
important organelles responsible for cellular ATP synthesis and
metabolism (Scheffler, 2008). Mitochondrial function mainly
depends on the mitochondrial content and shape (Tang et al.,
2014). Generally, increased mitochondrial content has been
observed in response to challenges involving high-energy
demands, such as in electrical stimulation, exercise, cold,
heat, stress, etc. (Booth and Thomason, 1991;Cannon and
Nedergaard, 2004;Bremer and Moyes, 2011;Gao and Moyes,
2016). Mitochondrial biogenesis is a complex and precise process
involving the replication of mitochondrial DNA (mtDNA)
and the expression of nuclear and mitochondrial genes (Tang
et al., 2014). In this regard, adenosine monophosphate (AMP)-
activated protein kinase (AMPK), known as the cellular “fuel
gauge,” may play a central role, since the energy homeostasis
mediated by it is closely related to the biosynthesis and
function of mitochondria (Zhao et al., 2013). Also, the
AMPK can be activated by a variety of metabolic stresses
that typically increase the cellular AMP/ATP ratio, such as
hypoxia, exercise, a glucose load, energy restriction, and so on
(Hardie et al., 2003). Once activated, AMPK phosphorylates
the mitochondrial master regulator: peroxisome proliferator-
activated receptor γcoactivator-1 (especially PGC-1αand PGC-
1βisoforms) (Reznick and Shulman, 2006;Bremer et al.,
2016). Subsequently, the phosphorylated PGC-1 activates the
nuclear respiratory factor-1 (NRF-1), which, in turn, regulates
the expressions of both mitochondrial and nuclear genes
encoding respiratory chain subunits and other proteins that
are responsible for mitochondrial biogenesis and function (Wu
et al., 1999;Tang et al., 2014). However, these activities
have been mainly observed in mammals. Relevant information
in fish has been quite limited until now. Recently, some
differences have been identified between fish and mammals
related to the mitochondrial biogenesis pathway. Accordingly,
PGC-1βhas been demonstrated to be more effective than PGC-
1αwhen the mitochondrial gene expression was observed,
although they have similar capabilities to induce mitochondrial
biogenesis in mammals (Bremer et al., 2016). In addition,
the mitochondrial function in fish also has been reported to
be affected by a large number of factors, including genetics,
growth and/or developmental stages, water temperature, diet
composition, etc. (LeMoine et al., 2008;Eya et al., 2011, 2012,
2017;Liao et al., 2016). However, information concerning
carbohydrate metabolism is still unknown. Considering this, it is
of great significance to investigate the potential effects of high-
carbohydrate (HC) feeding on the mitochondrial function of
fish and characterize the underlying mechanisms. This might
facilitate the discovery of effective approaches to improve the
mitochondrial function in fish as well as open a new approach
to promote its carbohydrate utilization.
Mitochondrial dysfunction is a crucial triggering factor of
metabolic diseases such as insulin resistance and diabetes
(Kelley et al., 2002;Petersen et al., 2003). At present,
accumulating evidence has indicated that the supplementation
of mitochondrial nutrients (mt-nutrients) could bring about
a series of physiological benefits on mitochondrial structure
and function, such as (1) the improvement of mitochondrial
membrane structure; (2) the enhancement of mitochondrial
enzymes activities by elevating cofactors levels; (3) increase
in antioxidant defenses by scavenging excess of free radicals,
and so on (Liu and Ames, 2005). Among these mt-nutrients,
vitamin B1has attracted considerable attention as it can
serve as the essential cofactor to regulate the activity of
various mitochondrial enzymes, thereby, improving intracellular
mitochondrial function (Depeint et al., 2006). However, vitamin
B1is excreted quickly from the body due to its water-
soluble characteristic, which can lead to a reduction in its
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Xu et al. Effect of Benfotiamine on Liver Mitochondria of Fish
biological functions (Beltramo et al., 2008). Benfotiamine is a
lipid-soluble analog of vitamin B1with higher absorption and
bioavailability than vitamin B1, and is commonly used as a
food supplement for the treatment of diabetic complications
(Beltramo et al., 2008). It can improve glucose homeostasis by
blocking three major pathways associated with hyperglycemic
damage: the hexosamine, the advanced glycation end products
(AGEs) formation, and the diacylglycerol (DAG)–protein
kinase C pathways (Hammes et al., 2003). In addition,
benfotiamine administration can remarkably enhance the activity
of dehydrogenase enzyme complexes by increasing intracellular
thiamine diphosphate (TPP) levels, thereby increasing glucose
oxidation in mitochondria (Fraser et al., 2012). Moreover,
benfotiamine also has been demonstrated to be able to alleviate
the stress caused by the overproduction of superoxide anion in
the mitochondrial electron transport chain (Chung et al., 2014).
Despite the fact that significant improvements of mitochondrial
function have been confirmed in mammals, such information
in aquatic animals is extremely scarce. Whether benfotiamine
can improve the glucose homeostasis in fish through the
enhancement of mitochondrial function still needs to be
elucidated.
Blunt snout bream (Megalobrama amblycephala) is a
commercially important freshwater fish in China (Xu et al.,
2016). Due to its herbivorous feeding habits, diets formulated
for this fish usually contain large proportions of carbohydrates
to reduce the feed cost and maximize the profit. However,
severe metabolic burden coupled with the compromised glucose
homeostasis is usually observed in this species after the feeding of
HC diets (Li et al., 2014). Previously, our study had demonstrated
that long-term administration of benfotiamine at 2.85 mg/kg
could significantly improve the glucose homeostasis of this
species being fed HC diets. However, the underlying mechanisms
are still poorly understood. In addition, the growth performance
was slightly compromised, suggesting that this dosage might be
too high for this species (Xu et al., 2017a). Hence, we speculated
that (1) a dose-dependent effect of benfotiamine might exist on
the growth performance and intermediary metabolism of fish
and (2) benfotiamine might benefit the glucose homeostasis of
fish through the promotion of mitochondrial function. Bearing
these facts in mind, the present study was conducted to (1)
evaluate the effects of different dietary levels of benfotiamine on
the growth performance, tissue glycogen and lipid deposition,
and levels of plasma metabolites in juvenile blunt snout bream
fed an HC diet and (2) investigate its beneficial effects on
mitochondrial biogenesis and function in the liver of this species
of fish. The findings obtained here might provide us with some
new insights into carbohydrate metabolism in fish as well as
facilitate the development of nutritional strategies to improve the
carbohydrate utilization by aquatic animals.
MATERIALS AND METHODS
Ethics Statement
The care and use of animals in the present study followed
the ethical guidelines of the Nanjing Agriculture University in
China [permit number: SYXK (Su) 2011-0036]. All experimental
procedures involving animals were conducted following the
Guidelines for the Care and Use of Laboratory Animals in
China.
Benfotiamine and Diets
Benfotiamine was obtained from Xian Reain Biomedical
Company (Xian, China) with a purity of at least 98%. Six
isonitrogenous and isolipidic diets were formulated, including
a control diet (30% carbohydrate, C), an HC diet (43%
carbohydrate), and the HC diet supplemented with different
benfotiamine levels [0.7125 (HCB1), 1.425 (HCB2), 2.85 (HCB3),
and 5.7 (HCB4) mg/kg, respectively]. Dietary carbohydrate levels
were adopted according to our previous studies (Li et al.,
2014). Feed formulation and proximate composition of the
experimental diets are presented in Table 1. Proteins were derived
from fish meal, soybean meal, rapeseed meal, and cottonseed
meal. Dietary lipids were derived from fish oil and soybean oil.
Corn starch was adopted to meet the dietary carbohydrate levels
required. Microcrystalline cellulose was included as the filler.
Animals, Experimental Conditions, and
Sampling
Juvenile blunt snout bream were purchased from the National
Fish Hatchery Station in Yangzhou (Jiangsu, China). Before
the experiment, fish were acclimatized to the experimental
conditions for 2 weeks, during which they were fed a commercial
diet (32% protein, 6% lipids, and 33% carbohydrates) to apparent
satiation, manually, three times daily. After acclimatization, 360
fish of similar size (average weight: 45.25 ±0.34 g) were randomly
distributed among 24 indoor tanks (300 L volume) at a number
of 15 fish per tank. Fish in each tank were randomly fed with one
of the six experimental diets. Each diet was tested in four tanks.
Fish were fed to visual satiation thrice daily (07:00, 12:00, and
17:00 h) for 12 weeks. Throughout the experimental period, water
temperature averaged 27.4 ±0.6 C, pH 7.4–7.5, photoperiod 12:
12 h (dark: light), and dissolved oxygen was maintained above
5.0 mg/L.
After the last meal, all the fish in each tank were fasted for
24 h to empty gut content, and then counted and weighed.
Subsequently, 4 fish from each tank were randomly selected and
anesthetized with MS-222 (tricaine methanesulfonate; Sigma,
United States) at 100 mg/L. Blood was drawn into heparinized
tubes as described by Xu et al. (2017b). Liver, muscle, and adipose
tissue were removed and immediately frozen in liquid N2, and
then stored at 80C until assayed.
Analysis of Proximate Composition,
Plasma and Liver Metabolites, and
Tissue Glycogen and Lipid Contents
The proximate composition of diets was determined as follows:
dry matter by drying in an oven at 105C to a constant weight;
protein content (nitrogen ×6.25) using the Kjeldahl method
after acid digestion (FOSS KT260, Höganäs, Sweden); crude lipid
content by ether extraction in a Soxtec System HT (Soxtec System
HT6, Tecator, Höganäs, Sweden); ash by incineration in a muffle
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Xu et al. Effect of Benfotiamine on Liver Mitochondria of Fish
TABLE 1 | Formulation and proximate composition of the experimental diets.
C HC HCB1 HCB2 HCB3 HCB4
Formulation (%)
Fish meal 8.00 8.00 8.00 8.00 8.00 8.00
Soybean meal 26.00 26.00 26.00 26.00 26.00 26.00
Rapeseed meal 17.00 17.00 17.00 17.00 17.00 17.00
Cottonseed meal 17.00 17.00 17.00 17.00 17.00 17.00
Fish oil 2.00 2.00 2.00 2.00 2.00 2.00
Soybean oil 2.00 2.00 2.00 2.00 2.00 2.00
Corn starch 12.00 25.00 25.00 25.00 25.00 25.00
Benfotiamine (mg/kg) 0 0 0.7125 1.425 2.85 5.7
Microcrystalline cellulose 13.00 0.00 0.00 0.00 0.00 0.00
Calcium biphosphate 1.80 1.80 1.80 1.80 1.80 1.80
Premix1.20 1.20 1.20 1.20 1.20 1.20
Proximate composition (% air-dry basis)
Moisture 6.96 6.85 6.92 6.95 6.90 6.87
Crude lipid 5.93 5.71 5.78 5.66 5.77 5.87
Ash 8.46 8.28 8.12 8.23 8.34 8.20
Crude protein 29.82 30.12 30.31 30.03 30.02 30.11
Crude fiber 16.97 6.18 6.29 6.30 6.23 6.28
Nitrogen-free extract31.86 42.75 42.58 42.83 42.74 42.67
Energy (MJ/kg) 19.09 19.24 19.38 19.31 19.23 19.30
C, the control diet; HC, the high-carbohydrate diet; HCB1, the HC diet supplemented with 0.7125 mg/kg benfotiamine; HCB2, the HC diet supplemented with 1.425 mg/kg
benfotiamine; HCB3, the HC diet supplemented with 2.85 mg/kg benfotiamine; HCB4, the HC diet supplemented with 5.7 mg/kg benfotiamine (the same below). Premix
supplied the following minerals and/or vitamins (per kg): CuSO4·5H2O, 2.0 g; FeSO4·7H2O, 25 g; ZnSO4·7H2O, 22 g; MnSO4·4H2O, 7 g; Na2SeO3, 0.04g; KI, 0.026 g;
CoCl2·6H2O, 0.1 g; Vitamin A, 900,000 IU; Vitamin D, 200,000 IU; Vitamin E, 4500 mg; Vitamin K3, 220 mg; Vitamin B1, 320 mg; Vitamin B2, 1090 mg; Vitamin B5,
2000 mg; Vitamin B6, 500 mg; Vitamin B12, 1.6 mg; Vitamin C, 5000 mg; Pantothenate, 1000 mg; Folic acid, 165 mg; Choline, 60,000 mg. Calculated by difference
(100 - moisture - crude protein - crude lipid - ash - crude fiber).
furnace at 550C for 4 h; gross energy content by an adiabatic
bomb calorimeter (PARR 1281, United States), and crude fiber
was determined by the fritted glass crucible method using an
automatic analyzer (ANKOM A2000i, United States).
Plasma glucose level was determined using the glucose
oxidase method (Asadi et al., 2009). Plasma glycated serum
protein (GSP) and AGEs levels were assayed by the method
detailed by David and John (1984) and Monnier et al.
(1986), respectively. Plasma insulin level was measured using a
heterologous radioimmunoassay method using bonito (Thunnus
thynnus) insulin as the standard and rabbit antibonito insulin
as antiserum (Gutierrez et al., 1984). This method has
been confirmed in Cyprinus carpio (Hertz et al., 1989),
which shares the same classification (Cyprinidae family) with
M. amblycephala. Hepatic contents of adenosine triphosphate
(ATP) and AMP were assessed as described by Jaworek et al.
(1974) and Lund et al. (1975), respectively. Tissue glycogen
and lipid contents were analyzed following the methods
detailed by Folch et al. (1957) and Keppler et al. (1974),
respectively.
Analysis of Mitochondrial Respiratory
Chain Complex Enzyme Activities
Liver mitochondria isolation was performed using a commercial
kit (G006, Nanjing Jiancheng Bioengineering Institute, Nanjing,
China). Briefly, after the last meal, fresh liver samples
were obtained from another tank with 4 fish, and were
then placed immediately in the ice-cold extraction medium
consisting of 10 mM KH2PO4, 250 mM sucrose, and 5 mM
ethylenediaminetetraacetic acid. Subsequently, 1 g of liver tissue
was homogenized in 10 mL of cold medium. The homogenates
were spun down for 10 min at 1,500 gin a refrigerated centrifuge.
The supernatants were retained in a new centrifuge tube. For
the preparation of the mitochondrial fraction, the remaining
supernatants were centrifuged by a second spin. The sediment
was washed three times with the previously mentioned medium,
and was then resuspended in a small volume of medium
plus fatty acid-free bovine serum albumin (1 mg/mL). The
mitochondrial suspensions were immediately stored at 80C
for subsequent analysis. Mitochondrial protein concentration
was determined using the method of Bradford and Dodd (1977).
The activities of complex I–III were measured following the
methods detailed by Jeejeebhoy (2002). Furthermore, complex IV
and V activities were analyzed following the methods of Kirby
et al. (2007).
Western Blot and Quantitative
Polymerase Chain Reaction (qPCR)
Analysis
Hepatic protein extraction was performed according to our
previous study (Xu et al., 2018). Subsequently, protein lysates
(20 µg of protein) were separated on a sodium dodecyl
sulfate–polyacrylamide gel electrophoresis gel using a Mini-
PROTEAN system (BioRad, Spain) for 1–2 h at 100 V. Then,
the electroblotted proteins were transferred to a polyvinylidene
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Xu et al. Effect of Benfotiamine on Liver Mitochondria of Fish
difluoride (PVDF) membrane (Millipore Corp., Bedford, MA,
United States). The specific primary antibodies used were
anti-β-actin (BM3873, Boster, China, 1:5000 dilution), anti-
AMPKα(#2532, Cell Signaling Technology, United States,
1:2000 dilution), anti-phospho-AMPKα(#2535, Cell Signaling
Technology, United States, 1:2000 dilution), anti-PGC-1β
(22378-1-AP, Proteintech, United States, 1:1000 dilution), and
anti-NRF1 (ab34682, Abcam, Cambridge, MA, United States,
1:1000 dilution). After washing, PVDF membranes were
incubated with anti-rabbit (#7074, Cell Signaling Technology,
United States, 1:2000 dilution) and anti-rabbit (BA1054, Boster,
China, 1:50,000 dilution) secondary antibodies, respectively.
Immune complexes were detected by a chemiluminescent
substrate (Gel Imagine CHEMI-SMART-3126, France) based
on the manufacturer’s instructions, and visualized with a
luminescent image analyzer (Fujifilm LAS-3000, Japan). The
protein levels were normalized by β-actin, and the intensities
of each lane were quantified using the densitometry band
analysis tool in Image J 1.44p (U.S. National Institutes of Health,
Bethesda, MD, United States).
Total RNA extraction and cDNA synthesis were performed
using the liver samples according to our previous studies
(Xu et al., 2017a, 2018). Briefly, the quantity and purity of
isolated RNA were determined by absorbance measures at 260
and 280 nm, and its integrity was tested by electrophoresis in
1.0% formaldehyde denaturing agarose gels. Specific primers for
PGC-1β, NRF-1, Mfn-1, Mfn-2, Opa-1, Drp-1, Fis-1, Mff, and
uncoupling protein (UCP)-2 were designed using primer premier
5.0 based on the partial cDNA sequences of the target genes
using the transcriptome analysis of blunt snout bream (Gao et al.,
2012). The AMPKα-1, AMPKα-2, TFAM, ND-1, CYT B, COX-1,
COX-2, and ATP-6 were designed using the published sequences
of blunt snout bream (Table 2). All primers were synthesized
by Invitrogen BioScience & Technology Company (Shanghai,
China). Subsequently, the real-time qPCR was run on an ABI
7500 real-time PCR system (Applied Biosystems, Carlsbad, CA,
United States) using the SYBR Green II Fluorescence Kit (Takara
Bio. Inc., Japan). The fluorescent quantitative PCR reaction
solution consisted of 2.00 µL template (equivalent to 500 ng
cDNA), 10.00 µL SYBRR
premix Ex TaqTM (2×), 0.40 µL
Rox Reference Dye II, 0.40 µL PCR Forward Primer (10 µM),
0.40 µL PCR Reverse Primer (10 µM), and 6.80 µL dH2O. The
qPCR consisted of 40 cycles with the first step of denaturation
at 95C for 30 s and a final extension of 95C for 5 s and
annealing at 60C for 34 s. All amplicons were initially separated
by agarose gel electrophoresis to ensure that they were of the
correct size. Finally, the relative transcripts of target mRNA were
analyzed by elongation factor 1 α(EF1α) gene using the 211CT
method.
Statistical Analysis
All results are presented as mean ±standard error of the mean
(SEM). Before statistical analysis, all data were tested for the
normality of distribution and homogeneity of variances among
different treatments. Then, data were subjected to one-way
ANOVA and Tukey’s multiple tests. Differences were considered
significant if a P-value of less than 0.05 was obtained. All the
statistical analyses were done with SPSS 22.0 for Windows (SPSS
Inc, Chicago, IL, United States).
RESULTS
Growth Performance
Growth performances of blunt snout bream are shown in Table 3.
During a 12-week feeding trial, no mortality was observed
among all the groups. The final weights (FWs), weight gain rate
(WGR), specific growth rate (SGR), relative feed intake (RFI),
feed conversion ratio (FCR), and hepatosomatic index (HIS) of
fish fed the HC diet were all lower than that of the C group,
but significant differences were observed in FW, RFI, and FCR
(P<0.05). Additionally, FW, WGR, and SGR all increased
significantly (P<0.05) as benfotiamine levels increased from
0 to 1.425 mg/kg, but decreased with further increasing levels.
However, both RFI and FCR showed an opposite trend, but
no significant difference (P>0.05), with the minimum value
observed in fish fed the HCB2 diet. Moreover, benfotiamine
supplementation further increased HIS.
Plasma Metabolites, Tissue Glycogen
and Lipid Contents, and Liver
Biochemistry Parameters
As can be seen from Table 4 and Figure 1, plasma glucose, GSP,
AGES and insulin levels, ATP and AMP contents and ATP/AMP
ratio as well as liver and muscle tissue glycogen and lipid contents
of fish fed the HC diet were all significantly (P<0.05) higher than
that of the C group. As for the HC groups, the supplementation
of benfotiamine led to a significant (P<0.05) increase of
plasma insulin levels, tissue glycogen and lipid contents and
AMP contents, whereas the opposite was true for plasma glucose,
GSP and AGES levels, and ATP contents and the ATP/AMP
ratio.
Hepatic Protein Expressions of AMPKα,
PGC-1β, and NRF-1
As can be seen from Figure 2, fish fed the HC diet had
significantly (P<0.05) lower p-AMPKα/t-AMPKαratio and
PGC-1βprotein expressions than that of the C group. As for the
HC groups, the supplementation of benfotiamine significantly
increased (P<0.05) the p-AMPKα/t-AMPKαratio and PGC-1β
protein expression, while little difference (P>0.05) was observed
in the NRF-1 content.
Hepatic Transcriptions of the Genes
Involved in Mitochondrial Biogenesis
As can be seen from Figure 3, no statistical difference (P>0.05)
was observed in the transcriptions of AMPKα-1 and Mfn-2
among all the treatments. Fish fed the HC diet had significantly
(P<0.05) higher transcriptional levels of AMPKα-2, Drp-
1, Fis-1, and Mff than that of the C group, whereas the
opposite was true for PGC-1βexpression. As for the HC groups,
the supplementation of benfotiamine significantly increased
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TABLE 2 | Nucleotide sequences of the primers used to assay gene expressions by real-time PCR.
Target gene Forward (50-30) Reverse (50-30) Accession numbers or reference
AMPKα-1 AGTTGGACGAGAAGGAG AGGGCATACAAAATCAC ARF07712.1
AMPKα-2 ACAGCCCTAAGGCACGATG TGGGTCGGGTAGTGTTGAG KX061841
PGC-1βGTGAGGAACGGGGAGATTG AGGGGGGTGAACAGGAAAC Gao et al., 2012
NRF-1 CACAAGCCCTGAGGACTA ACCTGTATGAGCGAGACG Gao et al., 2012
TFAM TCCGAAAGTTAGCAGAGA ATGAAGATGTTGAAGGCG KT380498.1
Mfn-1 CTCCAGATGCTCATTCCCT TTCCTTGGCTTTGGTTGTC Gao et al., 2012
Mfn-2 ACGCCTCTCCGCTCAAACAC CTTCCCCAATCCCTGCCACT Gao et al., 2012
Opa-1 CTTGTTGACTTGCCTGG TTCATTACGGATGTGCT Gao et al., 2012
Drp-1 CAGAGGGACTGCGAGGTT GGCTTGAGCAAAAGGGAA Gao et al., 2012
Fis-1 ATACAAGCAAAAAAGACGAT ATACAAAATAAAAAAAGGGG Gao et al., 2012
Mff CCCGAGAGAATCGTAGTGG GGCGTCTTGAGGGACAGTG Gao et al., 2012
ND-1 CTGACCACTAGCCGCAATA GGAAGAAGAGGGCGAAGG NC010341
CYT-b CATACACTATACCTCCGACAT TCTACTGAGAAGCCACCT NC010341
COX-1 CATACTTTACATCCGCAACA TCCTGTCAATCCACCCAC NC010341
COX-2 AACCCAGGACCTTACACCC CCCGCAGATTTCAGAACA NC010341
ATP-6 TGCTGTGCCACTATGACT ATTATTGCCACTGCGACT NC010341
UCP-2 CCAAAGGTCCTGCGAACA AACCCTACTGCCAATCCC Gao et al., 2012
EF1αCTTCTCAGGCTGACTGTGC CCGCTAGCATTACCCTCC X77689.1
AMPKα-1, AMP-activated protein kinase α-1; AMPKα-2, AMP-activated protein kinase α-2; PGC-1β, Peroxisome proliferator activated receptor-γcoactivator-1β; NRF-
1, Nuclear respiratory factor-1; TFAM, Mitochondrial transcription factor A; Mfn-1 and 2, Mitofusin-1 and 2; Opa-1, Optic atrophy-1; Drp-1, Dynamin-related protein-1;
Fis-1, Fission-1; Mff, Mitochondrial fission factor; ND-1, NADH dehydrogenase-1; CYT-b, Cytochrome-b; COX-1 and 2, Cytochrome c oxidase-1 and 2; ATP-6, ATP
synthase-6; UCP-2, uncoupling protein 2; EF1α, Elongation factor 1α.
TABLE 3 | Growth performance of blunt snout bream fed different experimental diets.
Parameters Diets
C HC HCB1 HCB2 HCB3 HCB4
IW 46.38 ±0.63 45.17 ±0.72 44.81 ±0.78 47.15 ±1.11 43.11 ±1.32 44.79 ±0.64
FW 167.76 ±9.54b140.53 ±9.36d152.62 ±7.61c185.08 ±7.75a129.71 ±8.45e120.30 ±8.81e
WGR 241.71 ±6.49ab 218.19 ±9.72bc 232.84 ±7.77ab 262.03 ±11.11a218.10 ±12.41bc 195.20 ±10.86c
SGR § 1.46 ±0.02ab 1.38 ±0.03bc 1.43 ±0.03ab 1.53 ±0.02a1.38 ±0.02bc 1.29 ±0.05c
RFI | | 2.46 ±0.03a2.00 ±0.09b1.96 ±0.07b1.90 ±0.07b1.94 ±0.09b1.99 ±0.05b
FCR ¶ 1.89 ±0.03a1.61 ±0.08bc 1.53 ±0.05bc 1.41 ±0.04c1.57 ±0.07bc 1.70 ±0.06ab
HSI †† 1.06 ±0.07 1.15 ±0.11 1.21 ±0.15 1.23 ±0.16 1.21 ±0.19 1.23 ±0.12
IW, Initial weights; FW, Final weights. Values are means ±SEM of four replications. Values in the same line with superscripts are significantly different (P <0.05). Weight
gain rate (WGR, %) = (Wt- W0)×100 / W0.§Specific growth rate (SGR, % / day) = (LnWt– LnW0)×100 / T, where W0and Wtare the initial and final body weights,
and T is the culture period in days. ||Relative feed intake (RFI, % body weight d1) = Feed intake (g) ×100/[(initial fish weight (g) +final fish weight (g) +dead fish weight
(g)) ×days reared/2]. Feed conversion ratio (FCR) = Feed consumption (g)/fish weight gain (g). ††Hepatosomatic index (HSI, %) = liver weight (g) ×100/body weight (g).
(P<0.05) the transcriptional levels of AMPKα-2, PGC-1β, NRF-
1, TFAM, Mfn-1, and Opa-1 (with the maximum values all
observed in fish fed the HCB2 diet), whereas the opposite was
true for Drp-1, Fis-1, and Mff.
Hepatic Activities of the Enzymes
Involved in Mitochondrial Function
As can be seen from Figure 4, no significant difference (P>0.05)
was observed in the activities of complex II among all the
treatments. Fish fed the HC diet had significantly (P<0.05)
lower activities of complex I, III, and IV than that of the C
group, whereas the opposite was true for complex V activities.
As for the HC groups, the supplementation of benfotiamine
significantly increased (P<0.05) the activities of complexes
I–V, with the maximum values all observed in fish fed the
HCB2 diet.
Hepatic Transcriptions of the Enzymes
Involved in Mitochondrial Function
As can be seen from Figure 5, no significant difference (P>0.05)
was observed in the transcriptions of ND-1 and COX-1 among all
the treatments. Fish fed the HC diet had significantly (P<0.05)
lower transcriptional level of CYT-b than that of the C group,
whereas the opposite was true for ATP-6 and UCP-2 expression.
As for the HC groups, the supplementation of benfotiamine
significantly increased (P<0.05) the mRNA levels of CYT-b,
COX-2, and ATP-6, with the maximum values all observed in fish
fed the HCB2 diet.
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TABLE 4 | Plasma metabolites and tissue glycogen and lipid contents of blunt snout bream fed different experimental diets.
Parameters Diets P-value
C HC HCB2 HCB3
Glucose (mmol/L) 3.13 ±0.02b5.32 ±0.11a3.31 ±0.02b3.11 ±0.13b<0.05
GSP (mmol/L) 14.32 ±0.12b21.02 ±0.13a13.27 ±0.03bc 12.76 ±0.06bc <0.05
AGEs (ng/mL) 3.46 ±0.03b4.89 ±0.04a3.31 ±0.01b3.34 ±0.02b<0.05
Insulin (ng/mL) 42.31 ±0.54c73.20 ±0.82b96.57 ±0.61a98.19 ±0.91a<0.05
Tissue glycogen contents (µmol glycosyl units/g wet tissue)
Liver 14.31 ±0.38c23.32 ±0.30b45.81 ±0.34a40.44 ±0.23a<0.05
Muscle 2.46 ±0.03c3.23 ±0.02b3.67 ±0.03a3.81 ±0.02a<0.05
Adipose tissue 1.42 ±0.04b1.79 ±0.15ab 2.12 ±0.07a2.05 ±0.01a<0.05
Tissue lipid contents (percentage of wet weight)
Liver 5.92 ±0.02c7.54 ±0.03b8.86 ±0.08a8.72 ±0.05a<0.05
Muscle 5.42 ±0.32c7.22 ±0.11b7.55 ±0.21b9.13 ±0.45a<0.05
Adipose tissue 76.03 ±0.41ab 81.21 ±0.15ab 85.23 ±0.20a84.58 ±0.19a<0.05
GSP, glycated serum protein; AGEs, advanced glycation end products. Values are means ±SEM of four replications. Values in the same line with superscripts are
significantly different (P <0.05).
FIGURE 1 | Hepatic ATP (A) and AMP (B) contents and the ATP/AMP ratio (C) of blunt snout bream fed different experimental diets. Each data represent the
means ±SEM of four replicates. Bars assigned with different superscripts are significantly different (P<0.05). ATP, adenosine triphosphate; AMP, adenosine
monophosphate.
DISCUSSION
In the present study, HC intake led to a decreased value of
FW, WGR, SGR, RFI, and FCR in blunt snout bream, but
HSI is on the opposite trend. This result is consistent with the
fact that high-energy diets easily reduce feed palatability, and
usually accelerate animal satiety, thereby, leading to low feed
consumption (Ali and Jauncey, 2004). Moreover, high dietary
carbohydrates usually results in persistent hyperglycemia, which
is regarded as a physiological stress response, retarding the
growth of fish (Hemre et al., 2002). As for the HIS, it showed
similar trends as the results of liver glycogen and lipid contents
although no statistical difference was observed, which is similar
to the results of our previous study (Xu et al., 2017a). This may
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FIGURE 2 | The protein expressions of AMPKα(A), PGC-1β(B), and NRF-1
(C) in the liver of blunt snout bream fed different experimental diets. Two blots
were presented for each treatment. Gels were loaded with 20 µg total protein
per lane. Each data represent the means ±SEM of four biological replicates.
Bars assigned with different superscripts are significantly different (P<0.05).
be ascribed to the fact that as a herbivorous species, blunt snout
bream has higher glucose tolerance, which can quickly remove
excessive glycogen and lipid from the liver. In addition, FW,
WGR, and SGR were significantly improved by benfotiamine
supplementation with increasing level up to 1.425 mg/kg, while
the opposite was observed in RFI and FCR. These findings
suggested a beneficial effect of benfotiamine on the growth
performance of fish fed the HC diet at a suitable dosage (namely
1.425 mg/kg). According to a previous study, adequate thiamine
(the analog of benfotiamine) levels could promote the activities
of intestinal digestive and brush border enzymes of fish, thus
improving nutrients absorption and feed efficiency, and this
might promote the growth performance (Huang et al., 2011).
In addition, benfotiamine administration could also diminish
the hyperglycemic damage in mammals through the inhibition
of AGEs formation and other metabolic pathways (Hammes
et al., 2003). It is possible that a similar mechanism also exists
in fish. However, further studies are warranted to elucidate
these facts. Together, these effects might be beneficial to the
glucose homeostasis of fish fed carbohydrate-enriched diets,
thereby, improving the carbohydrate utilization by fish. In
addition, benfotiamine supplementation further increased the
HIS, but with no significant difference, indicating benfotiamine
can accelerate the removal of excessive nutrients deposited in the
liver. This result is also beneficial for enhancing liver metabolic
function, since the excessive accumulation of nutrients will result
in histological and pathological damage of the liver (Hilton and
Hodson, 1983;Caballero et al., 2004). Meanwhile, the HCB3
diet led to a decrease of FW, WGR, RFI, and FCR compared
with the HC group. This showed similar trends to the results
of our previous work (Xu et al., 2017a), although no statistical
difference was observed here. Generally, this difference may be
ascribed to the following differences between the previous and the
current study, which might include the initial body size of fish, the
stocking density, the feed consumption, the coefficient variation
of the data collected within each treatment, the water temperature
during the feeding trial, and so on. Furthermore, both the
WGR and SGR decreased significantly with further increasing
benfotiamine levels, indicating that the overdose of this substance
could impair the growth performance of fish. The physiological
basis for such growth retardation is still absent in fish until now.
To further characterize the underlying mechanisms, molecular
investigations were conducted in certain groups (namely the C,
HC, HCB2, and HCB3).
In this study, fish fed the HC diet exhibited relatively high
values of plasma glucose, GSP, AGES, and insulin as well as
tissue glycogen and lipid compared with the control group.
This suggested that high dietary carbohydrate intake induced
a hyperglycemic state of fish. It is generally acknowledged that
high glucose levels induced by HC intake usually stimulate
the synthesis and release of insulin, thus accelerating glucose
disposal in peripheral insulin target tissues by enhancing
glycolysis and glycogenesis (Polakof et al., 2012;Kamalam
et al., 2017). Meanwhile, the elevated intracellular glucose
levels also enhanced the Maillard reaction, thereby, increasing
AGES levels (Beltramo et al., 2008). In addition, the results of
GSP, an accurate and easily detectable intermediate marker of
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FIGURE 3 | Continued
glycemia, in this study, further supported the afore-mentioned
facts (Selvaraj et al., 2008). The enhanced tissue glycogen and
lipid deposition is also justifiable, since HC intake generally
promotes the glycogenesis and lipogenesis of fish (Enes et al.,
2009). In addition, dietary supplementation of benfotiamine
further increased plasma insulin levels and tissue glycogen
and lipid content, whereas the opposite was true for plasma
glucose, GSP, and AGES levels. These results suggested that
benfotiamine could improve the glucose homeostasis of blunt
snout bream fed a HC diet. This may be due to the following
facts: (1) benfotiamine could stimulate insulin synthesis by
improving the function of pancreatic β-cells, thus resulting
in low plasma glucose and GSP levels (Rathanaswami and
Pourany, 1991); (2) benfotiamine could accelerate the removal
of intracellular glycerhaldeyde-3-phosphate (G3P), thereby
decreasing the formation of AGES (Beltramo et al., 2008); and (3)
benfotiamine could enhance the pentose phosphate pathway and
fatty acid synthesis, thus increasing lipid accumulation (Berrone
et al., 2006;Beltramo et al., 2008). However, these are the
cases for mammals. Whether fish show a similar mechanism is
still uncertain, and this warrants further study. Meanwhile, the
elevated insulin levels usually enhance the activities of glycogen
synthase (GSase) and the dephosphorylation of glycogen
phosphorylase (GPase), which might promote glycogen storage
in tissues in fish (Moon, 2001).
Generally, ATP is the major energy “currency” in cells, and
the ATP/AMP ratio is a sensitive indicator of the alterations of
the energy status (Yoshida et al., 2012). In the present study, the
hepatic ATP and AMP contents and ATP/AMP ratio of fish fed
the HC diet were all significantly higher than that of the control
group. The most plausible explanation would be that high dietary
carbohydrate intake usually elevates the energy state of cells, thus
increasing ATP contents (Sathanoori et al., 2015). The excessive
ATP was hydrolyzed consequently, thus leading to the increased
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FIGURE 3 | The relative transcriptions of mitochondrial biogenesis-related genes in the liver of blunt snout bream fed different diets. The transcriptional levels of
AMP-activated protein kinase α-1 and 2 (AMPKα-1 and 2) (A,B), Peroxisome proliferator activated receptor-γcoactivator-1β(PGC-1β)(C), Nuclear respiratory
factor-1 (NRF-1) (D), Mitochondrial transcription factor A (TFAM) (E), Mitofusin-1 and 2 (Mfn-1 and 2) (F,G), Optic atrophy-1 (Opa-1) (H), Dynamin-related protein-1
(Drp-1) (I), Fission-1 (Fis-1) (J) and Mitochondrial fission factor (Mff) (K) were all evaluated using real-time RT-PCR. Expression levels were normalized to
EF1α-expressed transcripts and are presented as fold-change against the control (C) group set to 1. Each data represent the means ±SEM of four replicates. Bars
assigned with different superscripts are significantly different (P<0.05).
AMP content. Moreover, dietary benfotiamine supplementation
led to the increased AMP contents, while the opposite was true
for ATP contents and the ATP/AMP ratio. This indicated that
benfotiamine could modify the intracellular energy state of fish.
According to a previous study, as a thiamin analog, benfotiamine
could accelerate ATP hydrolysis via the following reaction:
thiamine +ATP thiamine diphosphate (ThDP) +AMP,
thereby decreasing the ATP/AMP ratio (Makarchikov, 2009).
In addition, the decreased ATP/AMP ratio by benfotiamine
administration is regarded as a positive signal for glucose
homeostasis, since it could activate some specific energy sensors
(such as AMPK), thereby coordinating the glycolipid metabolism
of fish (Rutter and Leclerc, 2009).
Current studies have demonstrated that mitochondrial fusion
and fission processes play a prominent role in the modulation
of mitochondrial biogenesis and function (Rossignol and
Karbowski, 2009;Su et al., 2009). Additionally, mitochondrial
fusion and fission enzymes have also attracted considerable
attention due to their critical roles in controlling the dynamic
events mentioned earlier (Su et al., 2009). However, such
information in fish is still barely understood. In this study,
hepatic transcriptions of AMPKα-1, Drp-1, Fis-1, and Mff
were all upregulated by the intake of carbohydrate-rich diets
compared with the control group, whereas the opposite was
true for PGC-1βand NRF-1 transcriptions as well as PGC-1β
and NRF-1 protein expressions and the p-AMPKα/t-AMPKα
ratio. According to previous studies, the increased intracellular
ATP contents by HC intake usually inhibit the activity of
AMPK in fish (Polakof et al., 2011a,b). Subsequently, the inactive
AMPK could weaken mitochondrial biogenesis signals by
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FIGURE 4 | The activities of mitochondrial respiratory chain complexes (Complex I (A),II(B), III (C),IV(D) and V (E)) in the liver of blunt snout bream fed different
experimental diets. Each data represent the means ±SEM of four replicates. Bars assigned with different superscripts are significantly different (P<0.05). Complex
I: NADH–ubiquinone oxidoreductase; Complex II: Succinate–ubiquinone oxidoreductase; Complex III: Ubiquinone–ferricytochrome-c oxidoreductase; Complex IV:
Cytochrome c oxidase; Complex V: F1F0–ATP synthase. They were expressed as nanomolars per minute per milligram protein.
reducing the activity of the PGC-1/NRF-1 pathway, resulting
in a decrease of mitochondrial content in cells (Yu and
Yang, 2010). Meanwhile, excessive carbohydrate intake also
amplifies mitochondrial oxidative stress by increasing the
generation of intracellular reactive oxygen species (ROS),
thereby accelerating mitochondrial fission characterized by
the upregulation of related genes (namely Drp-1, Fis-1, and
Mff) (Zhuang et al., 2017). In addition, dietary supplementation
of benfotiamine at 1.425 mg/kg significantly upregulated the
transcriptions of AMPKα-2, PGC-1β, NRF-1, TFAM, Mfn-
1, and Opa-1, the protein contents of PGC-1βas well as
the p-AMPKα/t-AMPKαratio, whereas the opposite was true
for Drp-1, Fis-1, and Mff. These results indicated that a
long-term administration of benfotiamine at the appropriate
dosages effectively enhanced the mitochondrial biosynthesis
of fish fed the HC diet. According to a previous study,
benfotiamine could decrease the ATP/AMP ratio by accelerating
ATP hydrolysis, thereby increasing the activity of AMPK
(Makarchikov, 2009). Once activated, AMPK conveys its signals
to induce mitochondrial biogenesis via targeting the PGC-
1β/NRF-1 pathway that regulates mtDNA replication and
expression (Wu et al., 1999). Furthermore, these results were
further supported by the fact that AMPK activation can prevent
high glucose-induced mitochondrial fission by inhibiting the
activity of the proteins involved in mitochondrial fission (such
as Drp-1 and Mff), thus coordinating this organelle shape and
function (Wikstrom et al., 2013;Ducommun et al., 2015;Craig
et al., 2017). It should be stated here that this information was
mainly derived from mammals. The underlying mechanisms
in fish still need further in-depth studies. Nevertheless, the
enhanced mitochondrial biogenesis by benfotiamine may be
helpful for the maintenance of glucose homeostasis in this fish,
thus improving its carbohydrate utilization.
Mitochondrion is the main site of energy production in
cells, and responsible for the production of ATP for the basic
activities of life (Wang et al., 2017). Generally, ATP synthesis
in mitochondria depends on the oxidative phosphorylation
(OXPHOS) by means of an enzyme pathway consisting
of five multisubunit enzyme complexes located within the
mitochondrial inner membrane (Vedel et al., 1999). Therefore,
investigations of these enzyme complexes will facilitate our
understanding of the mitochondrial function. In the present
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Xu et al. Effect of Benfotiamine on Liver Mitochondria of Fish
FIGURE 5 | The relative transcriptions of mitochondrial function-related genes in the liver of blunt snout bream fed different diets. The transcriptional levels of NADH
dehydrogenase-1 (ND-1) (A), Cytochrome-b (CYT-b) (B), Cytochrome c oxidase-1 and 2 (COX-1 and 2) (C,D), ATP synthase-6 (ATP-6) (E), and Uncoupling protein
2 (UCP-2) (F) were all evaluated using real-time RT-PCR. Expression levels were normalized to EF1α-expressed transcripts and are presented as fold-change against
the control (C) group set to 1. Each data represent the means ±SEM of four replicates. Bars assigned with different superscripts are significantly different (P<0.05).
study, the hepatic complexes I–IV activities of fish fed the HC
diet were all significantly lower than that of the control group,
whereas the opposite was true for the activities of complex V.
These results might be attributed to the fact that the oxidative
stress induced by HC intake would cause mitochondrial damage,
then leading to the decreases in mitochondrial respiratory
enzyme activities (Li et al., 2001;Lin et al., 2009). As for
the increased complex V activities, it might be a result of
the increased energy intake by fish due to HC feeding, which
is in line with the trend of ATP content. This is due to
the fact that complex V (ATP synthase) is the last rate-
limiting enzyme in ATP synthesis, driving the phosphorylation
of ADP to ATP (Vedel et al., 1999;Tang et al., 2014).
These trends were supported by the fact that high energy
consumption generally causes the mitochondria to increase
oxidative phosphorylation rate and ATP production (Jonathan
et al., 2010), thereby enhancing the activities of complex V
(namely the ATP synthase) (Hatefi, 1985). Similar results were
also reported in rainbow trout (Oncorhynchus mykiss) (Eya
et al., 2017) and channel catfish (Ictalurus punctatus) (Jonathan
et al., 2010). Moreover, dietary benfotiamine supplementation
led to the increased complexes I–V activities. This indicated
that benfotiamine could enhance mitochondrial function of
fish. According to a previous study, benfotiamine could
prevent mitochondria from oxidative stress by scavenging free
radicals and inhibiting oxidants production, thus enhancing
the mitochondrial respiratory enzyme activities (Liu and Ames,
2005). Furthermore, a previous study investigating the effects
of the B vitamin family on mitochondrial function further
supported this by demonstrating that vitamin B1is an
important cofactor for various mitochondrial enzyme complexes
(Depeint et al., 2006). Meanwhile, we further evaluated the
mitochondrial function by detecting the transcriptional levels
of related genes. The transcription of CYT-b in liver decreased
remarkably with increasing dietary carbohydrate levels, whereas
the opposite was true for ATP-6 expression. In addition, the
transcripts of ND-1, COX-1, and COX-2 were all downregulated
by the HC diet, although no significance was detected.
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Xu et al. Effect of Benfotiamine on Liver Mitochondria of Fish
These results were in accordance with that of the mitochondrial
complexes activities, suggesting that a long-term intake of the
HC diet could result in an impaired mitochondrial function
in this fish. This was supported by the fact that the decreased
expressions of ND-1, CYT-b, COX-1, and COX-2 might affect
the assembly of mitochondrial complexes, then reducing the
catalytic ability of OXPHOS (Eya et al., 2012;Wang et al.,
2017). Furthermore, the excessive production of ROS resulting
due to the HC intake would also accelerate mitochondrial
fission, which might, in turn, depress the transcriptions of these
genes (Qian et al., 2005;Zhuang et al., 2017). The upregulated
ATP-6 expression might be attributed to the increased energy
intake caused by HC diets, which could increase the oxidative
phosphorylation rate and ATP production in mitochondria,
then enhancing its transcriptional level (Jonathan et al., 2010).
Here, an increased UCP-2 expression was also found in the
HC group, suggesting that HC intake induced the uncoupling
reaction in the respiratory chain. This phenomenon seems to
be the opposite of ATP content, but it is not unique to our
system. Infact, previous studies have confirmed that high-sucrose
diet intake could also accelerate the phosphorylation of ADP
to produce ATP by stimulating the coupling reaction in liver
mitochondria despite the overexpression of UCP (Ruiz-Ramírez
et al., 2011). These results may suggest that the expression
of UCPs and mitochondrial respiration are not completely
interconnected (Herlein et al., 2009;Ruiz-Ramírez et al., 2011).
However, the underlying mechanisms involving these aspects
mentioned previously are still unknown. Meanwhile, dietary
supplementation of benfotiamine at 1.425 mg/kg resulted in a
remarkable increase of the transcriptions of CYT-b, COX-2, and
ATP-6. This result indicated an enhanced mitochondrial function
in the liver of blunt snout bream due to the administration of
benfotiamine at suitable dosages. This was further supported by
the fact that benfotiamine could scavenge excess free radicals
as well as inhibit superoxide anion production, thus alleviating
the functional damage of mitochondria caused by high glucose-
induced oxidative stress (Liu and Ames, 2005). The studies
afore-mentioned are mainly focused on mammals. The exact
mechanisms in fish still warrant further in-depth studies.
In summary, the findings of the present study indicated
that dietary supplementation of benfotiamine could improve the
growth performance and mitochondrial biogenesis and function
of M. amblycephala fed HC diets, through the activation of
the AMPK/PGC-1β/NRF-1 pathway, the upregulation of the
fusion-associated genes, and the enhancement of mitochondrial
enzyme complexes activities as well as their transcriptions. The
best value of growth performance and mitochondrial biogenesis
and function were all observed in fish offered 1.425 mg/kg
benfotiamine.
AUTHOR CONTRIBUTIONS
X-FL, CX, and W-BL conceived and designed the experiments.
CX and D-DZ analyzed the data. X-FL, CX, H-JS, and LZ
performed the experiments and contributed reagents, materials,
and analysis tools. CX and X-FL wrote the paper. All authors read
and approved the final version of the manuscript.
FUNDING
This research was funded by the National Technology System of
Conventional Freshwater Fish Industries of China (CARS-45-14)
and the Postgraduate Research and Practice Innovation Program
of Jiangsu Province (KYCX18_0697).
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... Liver mitochondria were isolated using a commercial kit (reference number: G006) produced by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the procedures detailed by Xu et al. (2018). The mitochondrial protein concentration was quantified following the method described by Bradford and Dodd (1977). ...
... These results were supported by the following facts: (1) changes in the activities of the mitochondrial respiratory chain complexes can lead to the alterations of tissue aerobic capacity, thus affecting the aerobic metabolism and the production of ATP (Eya et al., 2017); (2) the electron transfer and proton pump functions of the mitochondrial respiratory chain complex I, II, and IV are crucial for the formation of the proton gradient, which could be used by complex V to generate ATP (Jonathan et al., 2010); and, (3) being the last rate-limiting enzyme in ATP synthesis, complex V (namely the ATP synthase) could stimulate the phosphorylation of ADP to ATP (Neupane et al., 2018). The present results were justifiable, since high energy diets generally lead to the enhanced mitochondrial OXPHOS capacity and ATP generation in fish (Xu et al., 2018); thus, increasing the activity of mitochondrial complexes. Furthermore, in this study, the mitochondrial function of fish was assessed by examining the transcription of several mitochondriarelated genes. ...
... This agreed with the results observed in the mitochondrial complexes activities in this study, which further confirmed that compared with raw starch, gelatinized starch could improve the mitochondrial activity of blunt snout bream. Previous studies reported that the expressions of ND1, CYTB, COX1 and COX2 were closely related to the synthesis of mitochondrial complexes, thus influencing the mitochondrial OXPHOS capability of fish (Lu et al., 2017;Xu et al., 2018). Generally, the increased transcriptions of these mitochondrial genes may be associated with the increased intake of digestible carbohydrates derived from gelatinized starch, which could enhance the mitochondrial OXPHOS capability and ATP generation, as might enhance their transcriptional levels (Jonathan et al., 2010). ...
Article
This study aimed to evaluate the influences of dietary raw and gelatinized wheat starch and their ratios on the glycolipid metabolism, glucose tolerance and mitochondrial function of blunt snout bream Megalobrama amblycephala. Three diets incorporating 100% raw starch (R100), 100% gelatinized starch (G100) and 75% raw starch +25% gelatinized starch (R75/G25) were randomly assigned to 15 cages of fish for 10 weeks, after which fish were sampled. Then the remaining fish within each treatment were combined and later split into 6 tanks for a time series sampling in a glucose tolerance test. Daily gain index, relative feed intake and plasma levels of glucose, triglyceride, pyruvate and lactate all showed little difference among all the treatments. The R75/G25 group obtained the highest feed efficiency and tissue (namely liver and the abdominal fat tissue) glycogen contents. However, the highest tissue (namely muscle, liver and the abdominal fat tissue) lipid contents and plasma total cholesterol levels were both observed in the G100 group. In addition, the G100 group obtained significantly higher transcriptions of glucose transporter 2, glucokinase, peroxisome proliferator activated receptor γ and acetyl-CoA carboxylase α, but relatively low transcripts of the genes involved in gluconeogenesis and fatty acid β-oxidation, compared with the R100 group. The R100 group showed the highest glucose clearance rate, while the area under the glucose curve showed no statistical difference. Furthermore, the R100 group had relatively low activities of mitochondrial complex I, II, III and V as well as the transcriptions of cytochrome c oxidase 2, cytochrome band NADH dehydrogenase 1 and ATP synthase 6 compared with other groups. Overall, our results suggested that high dietary inclusions of gelatinized starch could enhance the glucose transportation, glycolysis, lipogenesis, lipid deposition, glucose tolerance and mitochondrial function of blunt snout bream, while depress the gluconeogenesis and fatty acid oxidation.
... Recently, our study had con rmed that long-term administration of benfotiamine signi cantly improved the glucose homeostasis of Megalobrama amblycephala (an herbivorous freshwater carp) fed with a HC diet (43% carbohydrate levels) [28]. For example, dietary benfotiamine signi cantly decreased plasma glucose levels of this sh fed HC diet [28,29]. However, the underlying mechanisms are barely understood. ...
... Then, 4 sh from each tank were anesthetized by the diluted MS-222 (100 mg/L). Blood was put into heparinized tubes [28,29]. Also, liver samples were collected from these sh, and then snap frozen in liquid nitrogen and stored at −80 °C until analysis. ...
... Total RNA extraction and cDNA synthesis in liver (about 2 g) were performed by our previous studies [28,29,36]. Brie y, total RNA was extracted by Tri Pure Reagent (Aidlab, China). ...
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Background: The impairment of immunity induced by high-carbohydrate diet is closely associated with the development of glucose metabolic disorders. In the study of diabetes, benfotiamine can prevent β-cell dysfunction by inhibiting inflammation, thereby improving insulin resistance. However, information regarding the effects of this substance on aquatic animals is extremely scarce. Methods: A 12-week nutritional research was conducted to evaluate the influences of benfotiamine on the growth performance, oxidative stress, inflammation and apoptosis in Megalobrama amblycephala (45.25 ± 0.34 g) fed high-carbohydrate (HC) diets. Six experimental diets were formulated, containing a control diet (30% carbohydrate, C), a HC diet (43% carbohydrate), and the HC diet supplemented with four graded benfotiamine levels (0.7125 (HCB1), 1.425 (HCB2), 2.85 (HCB3), and 5.7 (HCB4) mg/kg). Results: HC diet intake remarkably decreased daily growth coefficient (DGC), growth rate per metabolic body weight (GRMBW), feed intake (FI), liver antioxidant enzymes activities, sirtuin-1 (SIRT1) protein expression as well as liver mRNA levels of SIRT1, nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (CAT), manganese superoxide dismutase (Mn-SOD), interleukin10 (IL10) than those of the control group, but the opposite was true for plasma activities of alanine transaminase (AST) and aspartate aminotransferase (ALT), and contents of interleukin 1β (IL1β) and interleukin 6 (IL6), liver contents of malondialdehyde (MDA), and mRNA levels of kelch-like ECH associating protein 1 (Keap1), nuclear factor kappa B (NF-κB), tumour necrosis factor α (TNF α), IL1β, IL6, Bax, Caspase 3, Caspase 9 and P53. As with benfotiamine supplementation, HCB2 diet remarkably increased DGC, GRMBW, liver antioxidant enzymes activities, SIRT1 protein expression as well as liver mRNA levels of SIRT1, Nrf2, CAT, Mn-SOD, IL10 and Bcl2, while the opposite was true for plasma activities of AST and ALT, and contents of IL1β and IL6, liver MDA contents as well as mRNA levels of Keap1, NF-κB, TNF α, IL1β, IL6, Bax, Caspase 3, Caspase 9 and P53. Conclusion: Benfotiamine at 1.425 mg/kg can improve the growth performance and alleviate the oxidative stress, inflammation and apoptosis of M. amblycephala fed HC diets through the activation of the SIRT1 pathway.
... Protein extraction and western blot analysis were performed according to the method described in our previous studies [48][49][50]. The specific primary antibodies are anti-Ampk α (#2532, Cell Signaling Technology, United States, 1:2000 dilution), anti-phospho-Ampk α (#2535, Cell Signaling Technology, United States, 1:2000 dilution), anti-SIRT1(13161-1-AP, Proteintech, United States, 1:1000 dilution) and anti-β-actin (BM3873; Boster, China, 1:5000 dilution) antibodies. ...
... The secondary antibody is anti-rabbit antibody (#7074, Cell Signaling Technology, United States, 1:2000 dilution). These antibodies have been shown to successfully cross-react with blunt snout bream proteins [48][49][50][51]. ...
... Total RNA extraction and cDNA synthesis were performed using the liver samples according to our previous studies [35,[48][49][50]. Then, the transcriptions were investigated for several target genes, including AMPKα1, AMPKα2, SIRT1, Nrf2, Keap1 (kelch-like ECH associating protein 1), CAT, Cu/Zn-SOD (copper/zinc superoxide dismutase), Mn-SOD (manganese superoxide dismutase), GPx1 (glutathione peroxidase 1), GST (glutathione S-transferase), IKK α (IkB kinase α), NF-κB, TNF α, IL 1β, IL 6, IL 8, IL 10 and TGF β (transforming growth factor-β). ...
Article
This study investigated the effects of restricted feeding on the growth performance, oxidative stress and inflammation of Megalobrama amblycephala fed high-carbohydrate (HC) diets. Fish (46.94 ± 0.04 g) were randomly assigned to four groups containing the satiation of a control diet (30% carbohydrate) and three satiate levels (100% (HC1), 80% (HC2) and 60% (HC3)) of the HC diets (43% carbohydrate) for 8 weeks. Results showed that HC1 diet remarkably decreased final weight (FW), weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), hepatic activities of total anti-oxidation capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT), the AMP/ATP ratio, the p-AMPKα/t-AMPKα ratio, sirtuin-1 (SIRT1) protein expression and hepatic transcriptions of AMPKα2, SIRT1, nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (CAT), manganese superoxide dismutase (Mn-SOD), glutathione peroxidase 1 (GPx1) and interleukin10 (IL 10) compared to the control group, whereas the opposite was true for protein efficiency ratio (PER), nitrogen retention efficiency (NRE), energy retention efficiency (ERE), plasma glucose levels, alanine transaminase (AST) and aspartate aminotransferase (ALT) activities, hepatic contents of malondialdehyde (MDA), tumour necrosis factor α (TNF α) and interleukin 1β (IL 1β), ATP and AMP contents and hepatic transcriptions of kelch-like ECH associating protein 1 (Keap1), IkB kinase α (IKK α), nuclear factor kappa B (NF-κB), TNF α, IL 1β, interleukin 6 (IL 6) and transforming growth factor β (TGF β). As for the HC groups, fish fed the HC2 diet obtained relatively high values of SGR, PER, NRE, ERE, hepatic activities of T-AOC, SOD and CAT, the AMP/ATP ratio, the p-AMPKα/t-AMPKα ratio, SIRT1 protein expression and hepatic transcriptions of AMPKα2, Nrf2, CAT, copper/zinc superoxide dismutase (Cu/Zn-SOD), Mn-SOD, GPx1, glutathione S-transferase (GST) and interleukin10 (IL 10), while the opposite was true for hepatic content of IL 6 and transcription of IKK α. Overall, an 80% satiation improved the growth performance and alleviated the oxidative stress and inflammation of blunt snout bream fed HC diets via the activation of the AMPK-SIRT1 pathway and the up-regulation of the activities and transcriptions of Nrf2-modulated antioxidant enzymes coupled with the depression of the levels and transcriptions of the NF-κB-mediated pro-inflammatory cytokines.
... Protein extraction and Western blot analysis (20 μg of liver protein) were performed as previously described (26,31,41) using anti-Ampk α (#2532; Cell Signaling Technology), anti-phospho-Ampk α (#2535; Cell Signaling Technology) and anti-β-actin (BM3873; Boster, Wuhan, China) antibodies. These antibodies have been shown to successfully cross-react with Megalobrama amblycephala proteins (26,31,41). ...
... Protein extraction and Western blot analysis (20 μg of liver protein) were performed as previously described (26,31,41) using anti-Ampk α (#2532; Cell Signaling Technology), anti-phospho-Ampk α (#2535; Cell Signaling Technology) and anti-β-actin (BM3873; Boster, Wuhan, China) antibodies. These antibodies have been shown to successfully cross-react with Megalobrama amblycephala proteins (26,31,41). ...
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After a 12-week feeding trial, the glucose tolerance test was performed in Megalobrama amblycephala to evaluate the effects of metformin on the metabolic responses of glycolipids. Plasma insulin peaked at 2 h, then decreased to the basal value at 8-12 h post-injection. Plasma triglycerides levels and liver glycogen contents of the control group a decreased significantly during the first 2 and 1 h, respectively. Then, they returned to basal values at 12 h. During the whole sampling period, the high-carbohydrate groups had significantly higher levels of plasma metabolites and liver glycogen than those of the control group, and metformin supplementation enhanced these changes (except insulin levels). Glucose administration lowered the transcriptions of ampk α1, ampk α2, pepck, g6pase, fbpase, cpt IA and aco, the phosphorylation of Ampk α and the activities of the gluconeogenic enzymes during the first 2-4 h, while the opposite was true of glut 2, gs, gk, pk, accα and fas. High-carbohydrate diets significantly increased the transcriptions of ampk α1, ampk α2, glut 2, gs, gk, pk, accα and fas, the phosphorylation of Ampk α and the activities of the glycolytic enzymes during the whole sampling period, while the opposite was true for the remaining indicators. Furthermore, metformin significantly up-regulated the aforementioned indicators (except accα and fas) and the transcriptions of cpt IA and aco. Overall, metformin benefits the glucose homeostasis of Megalobrama amblycephala fed high-carbohydrate diets through the activation of Ampk and the stimulation of glycolysis, glycogenesis and fatty acid oxidation, while depressing gluconeogenesis and lipogenesis.
... Furthermore, high-fat diets can activate the mitochondrial biogenesis, fusion and oxidation, thereby reducing the lipid accumulation mediated in yellow catfish (Pelteobagrus fulvidraco) . Nonetheless, high dietary carbohydrate levels significantly depressed the mitochondrial fusion, mitochondrial biogenesis and mitochondrial oxidative phosphorylation capability of blunt snout bream (Megalobrama amblycephala) while enhancing mitochondrial fission (Li et al., 2019;Xu et al., 2018). However, to date, the potential mechanisms underlying these physiological processes are still poorly understood. ...
Article
Carbonylcyanide-3-chlorophenylhydrazone (CCCP) is a protonophore, which causes uncoupling of proton gradient in the inner mitochondrial membrane, thus inhibiting the rate of ATP synthesis. However, this information is manly derived from mammals, while its effects on the mitochondrial homeostasis of aquatic animals are largely unknown. In this study, the mitochondrial homeostasis of a carp fish Megalobrama amblycephala was investigated systematically in a time-course manner by using CCCP. Fish was injected intraperitoneally with CCCP (1.8 mg/kg per body weight) and DMSO (control), respectively. The results showed that CCCP treatment induced hepatic mitochondrial oxidative stress, as was evidenced by the significantly increased MDA and PC contents coupled with the decreased SOD and MnSOD activities. Meanwhile, mitochondrial fission was up-regulated remarkably characterized by the increased transcriptions of Drp-1, Fis-1 and Mff. However, the opposite was true for mitochondrial fusion, as was indicative of the decreased transcriptions of Mfn-1, Mfn-2 and Opa-1. This consequently triggered mitophagy, as was supported by the accumulated mitochondrial autophagosomes and the increased protein levels of PINK1, Parkin, LC3-II and P62 accompanied by the increased LC3-II/LC3-I ratio. Mitochondrial biogenesis and function both decreased significantly addressed by the decreased activities of CS, SDH and complex I, IV and V, as well as the protein levels of PGC-1β coupled with the decreased transcriptions of TFAM, COX-1, COX-2 and ATP-6. Unlikely, DMSO treatment exerted little influence. Overall, CCCP treatment resulted in the imbalance of mitochondrial homeostasis in Megalobrama amblycephala by promoting mitochondrial oxidative stress, fission and mitophagy, but depressing mitochondrial fusion, biogenesis and function.
... Total RNA extraction and cDNA synthesis of head kidney and spleen were performed following our previous studies [31][32][33][34]. Then, the transcriptions of tumour necrosis factor α (TNF α), interleukin 1β (IL 1β) and heat shock proteins 7 (HSP 70) were investigated. ...
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A 10-week feeding trial was performed to investigate the effects of Streptococcus faecalis on the growth, intestinal microflora composition and expression of immune-related genes of blunt snout bream (Megalobrama amblycephala). Fish (46.32 ± 0.09 g) were fed four experimental diets containing 0 cfu/g (SF0, control), 1 × 10⁵ cfu/g (SF1), 1 × 10⁶ cfu/g (SF2) and 1 × 10⁷ cfu/g (SF3) of S. faecalis, respectively. Results showed that daily growth index (DGI), feed efficiency ratio (FER), plasma glucose level, plasma contents of total protein and albumin as well as intestinal serous layer (SL), muscular layer (ML), submucous layer (SML), villi thickness (VT) and lamina propria (LP) were all no significant difference among all the treatments, whereas their (except plasma albumin content and intestinal ML) relatively high values were found in the SF2 group. Meanwhile, the intake of the SF2 diets significantly increased plasma globulin content and intestinal digestive enzymes activities, the opposite was true for the activities of plasma aspartate aminotransferase (AST) and alanine transaminase (ALT). In addition, the analysis of the intestinal microbiota showed that fish fed the SF2 diet have the highest values of intestinal alpha diversity and intestinal abundances of Actinobacteria, Chlamydiae, Firmicutes, Planctomycetes, Verrucomicrobia, Clostridium and Synechococcus, while the opposite was true for intestinal abundances of Acinetobacter, Anoxybacillus, Flavobacterium, Planctomyces, Plesiomonas, Pseudomonas, Staphylococcus and Clostridium perfringens. At the molecular level, the expression levels of tumour necrosis factor α (TNF α), interleukin 1β (IL 1β) and heat shock proteins 7 (HSP 70) in head kidney and spleen were all decreased significantly with the increasing S. faecalis levels up to 1 × 10⁶ cfu/g, and then they were increased with further increasing S. faecalis levels. Overall, dietary supplementation of S. faecalis at 1 × 10⁶ cfu/g could improve the intestinal health and innate immunity of blunt snout bream.
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Alzheimer's disease (AD) is an age-dependent, incurable mental illness that is associated with the accumulation of aggregates of amyloid-beta (Aβ) and hyperphosphorylated tau fragments (p-tau). Detailed studies on postmortem AD brains, cell lines, and mouse models of AD have shown that numerous cellular alterations, including mitochondrial deficits, synaptic disruption and glial/astrocytic activation, are involved in the disease process. Mitophagy is a cellular process by which damaged/weakened mitochondria are selectively eliminated from the cell. In AD, impairments in mitophagy trigger the gradual accumulation of defective mitochondria. This review will focus on the recent progress in understanding the molecular mechanisms and pathological role of mitophagy and its implications for AD pathogenesis. We will also discuss the novel concept of the regulation of mitophagy as a therapeutic avenue for the prevention and treatment of AD.
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A 12-week nutritional research was conducted to evaluate the influences of benfotiamine on the growth performance, oxidative stress, inflammation and apoptosis in Megalobrama amblycephala (45.25 ± 0.34 g) fed high-carbohydrate (HC) diets. Six diets were prepared, containing the control diet (30% carbohydrate, C), the HC diet (43% carbohydrate) and the HC diet supplemented with four benfotiamine levels (0.7125 (HCB1), 1.425 (HCB2), 2.85 (HCB3) and 5.7 (HCB4) mg/kg). HC diet remarkably decreased DGC, GRMBW, liver T-AOC, SOD and CAT activities, SIRT1 protein expression as well as the mRNA levels of SIRT1, Nrf2, CAT, Mn-SOD and IL10 in liver compared with the C group, but the opposite trend was found in plasma activities of AST and ALT, and contents of IL1β and IL6, liver contents of MDA and mRNA levels of Keap1, NF-κB, TNF α, IL1β, IL6, Bax, caspase 3, caspase 9 and P53. As for benfotiamine supplementation, HCB2 diet remarkably increased DGC, GRMBW, liver T-AOC, SOD and CAT activities, SIRT1 protein expression as well as liver mRNA levels of SIRT1, Nrf2, CAT, Mn-SOD, IL10 and Bcl2, while the opposite was true for plasma AST and ALT activities, and IL1β and IL6 contents, liver MDA contents as well as mRNA levels of Keap1, NF-κB, TNF α, IL1β, IL6, Bax, caspase 3, caspase 9 and P53. In summary, benfotiamine (1.425 mg/kg) promoted the growth, and alleviated the oxidative stress, inflammation and apoptosis of M. amblycephala fed HC diets through the SIRT1-mediated signaling pathway.
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As the cheapest energy source, carbohydrates are used in fish feeds to improve physical quality and reduce catabolism of proteins and lipids. The liver is the primary organ for metabolism and is also an important site of immune regulation. Here, we investigated the effect of different dietary carbohydrate levels on growth and health by evaluating the liver transcriptome of Epinephelus akaara. In this study, E. akaara juveniles were fed diets containing few (0% corn starch), moderate (18% corn starch), and high (30% corn starch) levels of dietary carbohydrate. After an 8-week feeding trial, E. akaara fed 30% dietary carbohydrates exhibited poor growth performance compared with those fed 0% and 18% dietary carbohydrates (P > 0.05). Genes related to the immune system, including IL8, TLR9, CXCR4, CCL4, and NFκB inhibitor alpha, were over-expressed in E. akaara fed the highest level of carbohydrate (30%). This general over-expression could indicate activation of inflammatory processes in the liver. The liver transcriptome data of E. akaara reported here indicate that high carbohydrate level of diet can lead to poor growth and inflammatory immune response in E. akaara.
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Background: Global trend of the introduction of high levels of relatively cheap carbohydrates to reduce the amount of costly protein in the aquatic animal feed production has affected the aquaculture of an economically important cyprinid fish, blunt snout bream (Megalobrama amblycephala). This dietary shift has resulted in increased prevalence of metabolic disorders, often causing economic losses. High dietary intake of carbohydrates, associated with obesity, is one of the major causes of non-alcoholic fatty liver disease (NAFLD) in humans. Results: We have conducted an eight-week feeding trial to better understand how a high-carbohydrate diet (HCBD) affects the liver health in this fish. Hepatosomatic index and lipid content were significantly (P < 0.05) higher in the HCBD group. Histology results also suggested pathological changes in the livers of HCBD group, with excessive lipid accumulation and indication of liver damage. Metabolomics and serum biochemistry analyses showed that a number of metabolites indicative of liver damage were increased in the HCBD group. This group also exhibited low levels of betaine, which is a metabolite crucial for maintaining the healthy liver functions. Transcriptomic and qPCR analyses indicated that HCBD had a strong impact on the expression of a large number of genes associated with the NAFLD and insulin signalling pathways, which may lead to the development of insulin resistance in hepatocytes, pathological liver changes, and eventually the NAFLD. Conclusions: Transcriptomics, metabolomics and histology results all indicate early symptoms of liver damage. However whether these would actually lead to the development of NAFLD after a longer period of time, remains inconclusive. Additionally, a very high number of upregulated genes in the HCBD group associated with several neurodegenerative diseases is a strong indication of neurodegenerative changes caused by the high-carbohydrate diet in blunt snout bream. This suggests that fish might present a good model to study neurodegenerative changes associated with high-carbohydrate diet in humans.
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A 12-week feeding trial was performed to evaluate the effects of metformin on growth performance, energy sensing, insulin signaling pathway, glycolipid metabolism and glucose tolerance of blunt snout bream fed high-carbohydrate diets. Fish were randomly fed four diets containing two dietary carbohydrate levels (30 and 43%) and two metformin levels (0 and 0.25%). High carbohydrate levels remarkably increased tissue glycogen and lipid contents, hepatic adenosine triphosphate (ATP) and adenosine monophosphate (AMP) contents and the ATP/AMP ratio, plasma levels of glucose, insulin, triglyceride, glycated serum protein (GSP), advanced glycation end products (AGES) and lactic acid, hepatic t-AMPK protein levels and the transcriptions of AMPKα1, AMPKα2, target of rapamycin (TOR), insulin receptor (IR), ribosomal protein S6 kinase-polypeptide 1 (S6K1), glucokinase (GK), pyruvate kinase (PK), glucose transporter 2 (GLUT 2), glycogen synthase (GS), acetyl-CoA carboxylase α (ACCα), fatty acid synthase (FAS) and sterol regulatory element-binding protein-1 (SREBP1), whereas the opposite was true for weight gain, relative feed intake (RFI), feed conversion ratio (FCR), pyruvate levels, p-AMPK level, p-AMPK/t-AMPK ratio and the transcriptions of insulin receptor substrate 1 (IRS1), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), fructose-1,6-biphosphatase (FBPase), carnitine palmitoyltransferase IA (CPT IA), peroxisome proliferator activated receptor α (PPARα) and acyl-CoA oxidase (ACO). In addition, metformin supplementation remarkably increased tissue glycogen contents, plasma lactic acid level, hepatic AMP content, t-AMPK and p-AMPK level, p-AMPK/t-AMPK ratio and the transcriptions of AMPKα1, AMPKα2, IR, IRS1, GK, PK, GLUT 2, GS, CPT IA, PPARα and ACO, while the opposite was true for the remaining indicators. Significant interactions between dietary carbohydrate and metformin were also observed in RFI, FCR, tissue glycogen and lipid contents, hepatic ATP/AMP ratio, plasma metabolites, the phosphorylation of AMPK and the transcriptions of TOR, IRS1, S6K1, PK, G6Pase, GLUT 2, GS, ACCα, FAS, SREBP1, CPT IA, PPARα and ACO. Furthermore, metformin improved the glucose tolerance of fish fed high-carbohydrate diets after glucose loading. Overall, our findings indicated that metformin should be benefits the intermediary metabolism of Megalobrama amblycephala fed high-carbohydrate diets through the enhancement of energy sensing, insulin sensitivity, glycolysis, glycogenesis and fatty acid oxidation coupled with the suppression of gluconeogenesis and lipogenesis.
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This study aimed to characterize the full-length cDNA of AMPKα2 in Megalobrama amblycephala, and evaluate its potential role in glucose homeostasis and carbohydrate metabolism. The cDNA obtained covered 1942 bp with an open reading frame of 1635 bp encoding 545 amino acids. Multiple alignments and phylogenetic analysis revealed a high homology (91–100%) among most fish and higher vertebrates. This AMPKα2 mRNA predominantly expressed in muscle, liver and brain, while little in gill and intestine. Then, the AMPKα2 expressions were determined in the muscle, liver and brain of fish subjected to a glucose load (injected intraperitoneally with 0, 1.67 and 3.34 g glucose per kg body weight) and after a 12-week feeding trial (fed two dietary carbohydrate levels: 30% and 43%), respectively. After the glucose load, plasma glycemia peaked at 1 h in fish. Thereafter, it decreased significantly to the basal level at 8 h. However, AMPKα2 expression in muscle, liver and brain all decreased significantly during the first 2 h, then returned to the basal value at 24 h. Unlikely, tissue AMPKα2 expression of fish receiving saline solution increased significantly during the whole sampling period. Additionally, high-carbohydrate diet enhanced its expression in liver and muscle, but not that in brain. These findings indicated that the AMPKα2 gene shared a high degree of conservation with that of the other vertebrates. Muscle, liver and brain AMPKα2 expressions were highly induced by glucose administration. Furthermore, high dietary carbohydrate modified its expressions in these tissues.