Life Sciences 199 (2018) 16–22

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Life Sciences
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Hypolipidemic, antioxidant and antiatherogenic property of sardine T

by-products proteins in high-fat diet induced obese rats
Fouad Affane, Sabrine Louala, Nour el Imane Harrat, Fatima Bensalah, Hadjera Chekkal,
⁎
Amine Allaoui, Myriem Lamri-Senhadji
Laboratory of Clinical and Metabolic Nutrition (LNCM), Faculty of Nature and Life Sciences, University of Oran 1 Ahmed Ben Bella, BP 1524 El m'nouer, 31100 Oran,
Algeria

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

Keywords: Aims: Fish by-products valorization on account of their richness in bioactive compounds may represent a better
Fish by-products alternative to marine products with a view to economic profitability and sustainable development. In this study,
High-fat diet we compared the effect of sardine by-product proteins (SBy-P), with those of the fillets (SF-P) or casein (Cas), on
Hyperlipidemia growth parameters, serum leptin level, lipids disorders, lipid peroxidation and reverse cholesterol transport, in
Lipid peroxidation
diet-induced obese rats.
Obese rats
Main methods: Obesity was induced by feeding rats a high-fat diet (20% sheep fat), during 12 weeks. At body
Sardine proteins
weight (BW) of 400 ± 20 g, eighteen obese rats were divided into three homogenous groups and continue to
consume the high-fat diet for 4 weeks containing either, 20% SBy-P, SF-P or Cas.
Key findings: The results showed that SBy-P, compared to SF-P and Cas, efficiently reduced food intake (FI), BW
gain and serum leptin level, and improved blood lipids levels and reverse cholesterol transport by reducing total
cholesterol (TC), triacylglycerols (TG) and low-density lipoprotein cholesterol (LDL-HDL1-C) serum levels, in-
creasing the level of high-density lipoprotein cholesterol (HDL2-C and HDL3-C), and enhancing lecithin: cho-
lesterol acyltransferase (LCAT) activity. Furthermore, they attenuated lipid peroxidation by increasing ather-
oprotective activity of the paraoxonase-1 (PON-1).
Significance: Sardine by-product proteins due to their richness in certain essential amino acids, highlight weight-
loss, lipid-lowering, antioxidant and anti-atherogenic potentials, contributing to the improvement of the com-
plications associated with obesity.

1. Introduction
Obesity is one of the most chronic metabolic diseases in the world
with an increasing prevalence [1]. According to World Health Organi-
zation [2], there are approximately 600 million obese people in the
world, including 3.4 million adults who die each year from its com-
plications. Long-term exposure to an unbalanced high-fat, high-energy
and micronutrient-poor diet associated with a sedentary lifestyle, in-
volves abnormal or excessive body fat accumulation with a defective
energy balance regulation system [3], and causes development of
obesity and emergence of many cardiovascular risk (CVR) factors which
are associated [4]. Hyperlipidemia, which is characterized by high TC
and TG and decreased HDL-C levels, is an independent CVR factor
which is also considered to be responsible for atherogenicity of obesity
[5]. In addition, oxidative stress resulting from an imbalance between
oxygenated reactive species and antioxidant defense is induced also by
obesity [6]. This may be the consequence of stimulating white fat de-
posits, altering feed intake and increasing pre-adipositary proliferation,
adipocyte differentiation and mature adipocyte size [7]. In order to
study the different mechanisms involved in cardiometabolic risk and
obesity, many experimental studies carried out on rats have used un-
balanced diets including high-fat, high-caloric or high-carbohydrate
diets [8–11].
In humans, several anti-obesity drugs have been approved to im-
prove weight loss; however their potential toxicity may limit their long
term use [12]. Besides that, prevention and treatment of obesity is es-
sentially based on improving lifestyle behaviors including a regular
physical activity combined with good dietary habits, which are char-
acterized by consumption of marine products, source of high nutritional
value protein, minerals, trace elements, vitamins and lipids, in parti-
cular omega-3 polyunsaturated fatty acids (PUFA n-3) thus giving fish
their cardioprotective effects [13]. Therefore, developing nutraceutical

⁎
Corresponding author.
E-mail address: mylamri@hotmail.fr (M. Lamri-Senhadji).

https://doi.org/10.1016/j.lfs.2018.03.001
Received 11 November 2017; Received in revised form 26 February 2018; Accepted 1 March 2018
Available online 02 March 2018
0024-3205/ © 2018 Elsevier Inc. All rights reserved.
F. Affane et al. Life Sciences 199 (2018) 16–22

foods from natural sources, such as marine products, may be more at- Table 1
tractive to counter the adverse effects of this disease. Composition of the experimental dietsa (g·kg−1).
Currently the mass recovery methods of marine products are or-
Ingredients SBy-P SF-P Cas
iented primarily to production of oils, proteins and their hydrolysates
from fish muscle with hypoglycemic, hypolipidemic, antioxidant and Caseinb – – 200
cardioprotective properties [11,14]. However, fish by-products may Sardine fillets proteinc – 200 –
Sardine by-products proteinc 200 – –
represent a better alternative to deal with global demand for marine
Corn starchd 450 450 450
products that cease to grow and meet the population needs [15]. They Sucrosee 40 40 40
are considered as non-consumable parts (head, skin, viscera, tail, fins, Mutton fatf 200 200 200
crests and falls) but recoverable and usable after treatment and con- Celluloseg 50 50 50
stitute > 60% of the whole product, while only 40% of marine products Mineral mixh 40 40 40
Vitamin mixi 20 20 20
are intended for human consumption [16]. If these materials are al-
ready valued in part for animal feed or aquaculture, they can find after a
Diets are hyperlipidic (19.08 MJ/Kg of diet) and given in powdered form.
transformation more rewarding opportunities [15]. Indeed, these sub- b
Prolabo, Fontenay sous bois, France.
stances also contain numerous valuable molecules, including vitamins c
By-products and fillets proteins purified from sardine in our laboratory.
d
and minerals (hydroxyapatite) which are incorporated into fertilisers ONAB (Sidi Bel Abbes, Algeria).
e
[17], flours with anti-atherogenic properties [18], oils rich in PUFA n-3 f
Cevital (SPA, Bejaia, Algeria).
Sheep fat was obtained from a local market.
which can be used for the prevention of cardiovascular disease (CVD) g
Prolabo Paris, France.
[8], as well as proteins of high nutritional value, source of essential h
UAR 205 B (Villemoisson, 91360, Epinay/S/Orge, France), mineral mix (mg/kg of
amino acids (AA) and bioactive peptides [19–22], intended for devel- diet) CaHPO4, 17200; KCl, 4000; NaCl, 4000; MgO2, 420; MgSO4, 2000; Fe2O3, 120;
opment of new substances that can be used in dietetics, nutraceuticals, FeSO4, 7H2O, 200; MnSO4, H2SO4, H2O, 98; CuSO4, 5H2O, 20; ZnSO4, 7H2O 80; CuSO4
pharmaceuticals and therapeutics such anti-diabetic [23], antioxidants 7H2O; KI, 0,32.
i
UAR 200 (Villemoisson 91360, Epinay/S/Orge, France), vitaminic mixture (mg/kg
and anti-hypertensive products [10,20,22]. In contrast, very few studies
diet): vit A, 39600 UI; vit D3, 5000UI, vit B1, 40; vit B2, 30; vit B3, 140; vit B6, 20; vit B7,
are related in the literature on hypolepidemic, anti-obesity, anti- 300; vit B12, 0,1; vit C, 1600; vit E, 340; vit K, 3,80; vit PP, 200; choline, 2720; folic acid,
atherogenic and cardioprotective effects of proteins and peptides de- 10; para-aminobenzoїc acid, 180; biotine, 0,6; cellulose, qst, 20 g.
rived from different tissues of fish by-products [24]. In this context, the
aim of this study was to determine the effect of protein isolated from and sardine proteins was determined by the Nessler method after mi-
sardine by-product compared to those obtained from the edible portion neralization [27], and their purity was estimated by multiplying total
(fillet), and with casein, on growth parameters, serum leptin level, li- nitrogen content by the factor 6.25. The crude fat content was mea-
pids disorders, reverse cholesterol transport and lipid peroxidation by sured by Folch et al. [28] method. The moisture content was estimated
measuring the activity of certain enzymes involved in their mechanisms as the loss in weight drying at 105 °C for 24 h [29]. The ash amount was
namely, LCAT and PON-1 in high-fat diet induced obese rats. analyzed by incineration at 550 °C for 6 h [29]. The AA composition
was determined by high pressure liquid chromatography (HPLC) [30].
2. Materials and methods

2.3. Animals and diets

2.1. Sardine proteins purification process

Male Wistar rats (Pasteur Institute, Algiers, Algeria) (n = 18),

Fresh sardine (Sardina pilchardus) was purchased from a local public
weighing at the beginning of the experiment, 120 ± 10 g were sub-
fish market (Oran, Algeria), and proteins were isolated from by-pro-
jected for three months to a high-fat diet (20% of sheep fat). At
ducts and fillets according to Guillaume et al. [25] method. By-products
400 ± 20 g, obese rats were divided into three homogeneous groups
(viscera, heads, skins and edges) were separated from the fillet and then
and continue to consume the high-fat diet for 28 days containing either,
washed, kneaded and placed in an oven (Tan Steril, Italy) at 80–85 °C
20% SBy-P, SF-P or Cas. The SF-P and Cas are taken as controls. The
during 20 min. Has this step, a first separation takes place between a
diets composition is shown in Table 1. Food and water were given ad
solid phase (coagulated proteins) and a liquid phase (water and oil) and
libitum. The animals were placed in metabolic cages under standard
which is completed by a pressing. The press water was decanted and
environmental conditions (temperature of 24 °C, a humidity of 60% and
centrifuged (Eppendorf, Centrifuge 5702, Germany), the proteins in the
a 12–hours light/dark cycle). Advice for the protection and use of la-
pellet were re-incorporated into the solid phase then dried in an oven
boratory animals were followed [31]. The experimental protocol was
(40–45 °C) for 24 h, then ground into flour. To remove the lipids and
approved by the Animal Care and Use Committee of Oran 1 University
other organic solvent-soluble elements, sardine by-products and fillets
and the Algerian Ministry of Higher Education and Scientific Research.
flours were purified with n-hexane (Biochem chemopharma, UK) in oil
Rats were weighed weekly, while the food intake was recorded daily.
extractor (Soxhlet Labo-Tech LT.6, Germany) during 5 h at 50 °C. The
The food efficiency ratio (FER), used to express the animal's ability to
delipidated flour was purified by isoelectric precipitation according to
transform calories ingested into body mass [32], was calculated ac-
Undeland et al. [26] method as follow. Delipidated sardine flour was
cording to formula: FER = [body weight gain (g) / food intake
mixed with cold distilled water (1/10 w/v) containing Na2SO3 to pre-
(g)] × 100. Organs relative weight (RW) was determined referring to
serve sulfur proteins, pH was adjusted to 10.8 with NaOH (2 N). After
the equation given below: Organs RW = [organ weight (g) / BW
an overnight incubation at 4 °C, the mixture was centrifuged at
(g)] × 100.
3000 rpm for 20 min at 4 °C. Supernatant containing proteins was pre-
cipitated with H2SO4 at 98% at pH 4.5 then centrifuged at 10,000 rpm
for 30 min at 4 °C. The proteins were washed, freeze dried (Christ, 2.4. Sample collection
Alpha 1-2; LD) and reduced to powder.
At the last week of the experience, feces of each rats group were
2.2. Chemical and AA composition of casein and sardine proteins collected, dried, weighed then stored at 4 °C. After 28 days of the ex-
periment and overnight fasting, six rats of each group were anesthe-
All measurements were performed in triplicate at the Quality tized by intraperitoneal injection of a 10% chloral solution (3 mL/kg
Control and Compliance Laboratory in Oran (El Feth Etablissement). BW). Blood was collected from the abdominal aorta into tubes and
The crude protein level in casein (Prolabo, Fontenay sous bois, France) serum was obtained by low-speed centrifugation (3000 rpm for 20 min,

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F. Affane et al. Life Sciences 199 (2018) 16–22

at 4 °C). A portion of fresh serum was used to measure LCAT and PON1 Table 2
activities, 0.1% EDTA-Na2 (w/v) was added to the serum remainder as Chemical and amino acids (AA) composition (%) of sardine by-product proteins (SBy-P),
sardine fillets proteins (SF-P) and casein (Cas).
a curator, for carrying out the remaining dosages. Liver and adipose
tissue were removed immediately, rinsed with cold saline and weighed. SBy-P SF-P Cas
Harvested samples were aliquoted and stored at −70 °C until use.
Chemical composition
Crude protein 93.7 ± 0.15 94.8 ± 0.15 95.9 ± 0.32
2.5. Serum leptin evaluation and lipids analysis
Crude fat 1.8 ± 0.11 1.3 ± 0.02 0.2 ± 0.02
Ash 2.1 ± 0.12 1.9 ± 0.01 2.3 ± 0.02
Serum leptin level was measured by radioimmunoassay using a rat Moisture 2.3 ± 0.39 2.0 ± 0.16 1.6 ± 0.27
leptin-specific kit from Assay Designs Inc. (MI, USA). Liver and fecal AA composition
lipids were extracted following Folch et al. [28] method. After total Alanine 7.0 ± 0.05 8.3 ± 0.05 2.9 ± 0.17
lipids determination, the lipids extract was redisolved in isopropanol Arginine 9.1 ± 0.05 6.4 ± 0.08 3.4 ± 0.15
for lipids analysis. Lipids digestibility was calculated as the ratio be- Aspartic acid 10.1 ± 0.05 10.9 ± 0.05 6.2 ± 0.10
Aspergillus 8.9 ± 0.01 9.2 ± 0.06 6.6 ± 0.05
tween the absorbed and the ingested lipids using the following equa-
Cystine 1.1 ± 0.02 0.9 ± 0.05 0.2 ± 0.02
tion: Lipids digestibility (%) = [lipids intake − fecal lipids / lipids in- Glutamic acid 9.9 ± 0.04 11.9 ± 0.15 19.8 ± 0.15
take] × 100. Commercial kits were used to assay TC (Biocon, Glycine 5.7 ± 0.05 3.6 ± 0.10 1.7 ± 0.10
Diagnostics, Germany), TG (GPO-POD Spinreact, Spain), unesterified Histidine 2.2 ± 0.05 3.1 ± 0.10 5.2 ± 0.15
cholesterol (UC) (CHOD-PAP Biolabo, France) and phospholipids (PL) Isoleucine 4.6 ± 0.01 5.1 ± 0.05 4.9 ± 0.06
Leucine 8.1 ± 0.01 7.7 ± 0.15 9.2 ± 0.13
(CHO-POD Cypress, Belgium). Lysine 12.1 ± 0.05 10.5 ± 0.05 7.2 ± 0.15
Methionine 2.9 ± 0.05 2.7 ± 0.06 2.4 ± 0.15
2.6. Isolation and characterization of serum lipoproteins Phenylalanine 1.9 ± 0.03 3.2 ± 0.09 4.8 ± 0.05
Proline 2.5 ± 0.04 3.5 ± 0.06 11.5 ± 0.10
Serine 4.1 ± 0.04 4.8 ± 0.10 5.4 ± 0.11
Serum lipoproteins separation was carried out by a selective pre-
Taurine 5.3 ± 0.02 3.0 ± 0.05 1.8 ± 0.05
cipitation method. Serum very low-density lipoprotein (VLDL) and LDL- Threonine 4.4 ± 0.04 4.7 ± 0.04 4.7 ± 0.05
HDL1 were isolated by precipitation using Magnesium chloride and Tyrosine 2.4 ± 0.01 2.4 ± 0.05 5.2 ± 0.15
Phosphotungstate (Sigma Chemical Company, Lyon, France) as de- Valine 5.1 ± 0.01 5.4 ± 0.10 5.9 ± 0.11
scribed by Burstein et al. [33] method. HDL2 and HDL3 were separated Lysine/arginine 1.33 ± 0.01 1.64 ± 0.02 2.1 ± 0.05
Methionine/glycine 0.51 ± 0.01 0.74 ± 0.01 1.4 ± 0.17
by precipitation according to Burstein et al. [34] method using MgCl2
and dextran sulfate (Sigma Chemical Company). TC and TG in fractions, Chemical and AA composition of SBy-P, SF-P and Cas were performed in triplicate at the
UC and PL in HDL subfractions were determined by enzymatic colori- Quality Control and Compliance Laboratory in Oran (El Feth Etablissement).
metric method described previously. The contents of HDL3 in total
apolipoproteins (apo) were estimated by protein analysis according to 2.9. Statistical analysis
the method of Lowry et al. [35]. Esterified cholesterol (EC) levels were
obtained by difference between TC and UC. HDL2-Cholesteryl esters Values were expressed as means ± standard error of mean (S.E.M.)
(CE) amounts were estimated as 1.67 times the HDL2-EC amounts. for six rats per group. Data were analyzed by one-way analysis of
variance (ANOVA) carried out by Statistica (version 5.1, Statsoft Tulsa,
2.7. Determination of lipid peroxidation in serum and lipoproteins OK, USA). After ANOVA analysis, the classification of the means was
performed using Duncan's new multiple range tests [39]. The means
Lipid peroxidation was estimated by measuring thiobarbituric acid with different superscript were considered significantly different
reactive substances (TBARS) concentrations and expressed in terms of (p < 0.05).
malondialdehyde content according to Quintanilha et al. [36] method.
One milliliter of diluted serum or lipoproteins fractions was added to
3. Results
2 mL of thiobarbituric acid solution (final concentration in the mixture,
0.017 mmol/L) containing 0.375% thiobarbituric acid, diluated on a
3.1. Chemical and AA composition of casein and sardine proteins
solvent mixture (15% trichloroacetic acid, 0.25 N chloridric acid and
distilled water to a final volume of 100 mL) plus butylated hydro-
Chemical composition of casein and sardine proteins shown in
xytoluene (concentration, 3.36 mmol/L). The final mixture was in-
Table 2 was approximately similar. Their purity level was: 96% for
cubated for 60 min at 100 °C. After cooling and centrifugation, the
casein, 94% for sardine by-product protein and 95% for the fillet. The
absorbance of the supernatant was measured at 535 nm.
AA composition of sardine proteins, in particular by-product, revealed a
richness in alanine, arginine, aspartic acid, aspergillus, glycine, lysine
2.8. Determination of LCAT and PON-1 enzymatic activities
and taurine, while their glutamic acid, histidine, phenylalanine, proline
and tyrosine levels and their lysine/arginine and methionine/glycine
LCAT activity (enzyme responsible of UC to EC conversion) was
ratios were low compared to casein. However, the levels of branched-
determined on fresh serum by an endogenous method [37]. UC dis-
chain AA, such as valine, leucine, and isoleucine, were nearly identical
appearance was evaluated between two times (4 h) by colorimetric
(Table 2).
enzymatic method (kit CHOD-PAP Biolabo, France). LCAT activity was
expressed in nanomoles of esterified cholesterol/h/mL of serum. It is
calculated according to the following formula: 3.2. Growth parameters, liver and adipose tissue relative weight
LCAT = (UCt0h − UCt4h)/4 h incubation. PON-1 activity was estimated
by Kuo et al. [38] method. This enzyme catalyses the cleavage of phenyl At day 28, a significant reduction of BW gain (−84%, −41%), FI
acetate resulting in phenol formation. To 10 μL of sample was added (−36%, −19%) and FER (−75%, −27%) were observed in SBy-P and
1 mL of 20 mM Tris-HCl buffer solution pH 8, containing 10 mM phenyl SF-P groups, respectively, compared to Cas group (Table 3). Similar,
acetate and 1 mM CaCl2. The arylesterase activity of PON-1 was cal- difference significantly was noted between SBy-P and SF-P for FI
culated relative to the phenol molar absorption coefficient (−21%), BW gain (−73%) and FER (−66%) (Table 3). Furthermore,
(1.310 M−1 cm−1). One unit of activity of this enzyme is equal to final BW, liver and adipose tissue relative RW were reduced by 6%,
1 μmoL of phenol formed/min. 11% and 18% respectively, in SBy-P compared with Cas.

18
F. Affane et al. Life Sciences 199 (2018) 16–22

Table 3 Table 5
Growth parameters and organs relative weight (RW). Serum leptin level, lipids concentrations and distribution of cholesterol and triacylgly-
cerols in lipoproteins fractions.
SBy-P SF-P Cas
SBy-P SF-P Cas
Growth parameters
Initial BW (g) 400 ± 18 399 ± 19 400 ± 20 Serum leptin (ng·mL−1) 3.35 ± 0.36c 4.08 ± 0.24b 4.73 ± 0.30a
Final BW (g) 405 ± 18b 417 ± 15ab 431 ± 17a Serum lipids (mmol·L−1)
BW gain (g/day/rat) 0.17 ± 0.07c 0.64 ± 0.19b 1.10 ± 0.19a Total cholesterol 1.51 ± 0.23b 1.60 ± 0.21b 2.20 ± 0.33a
Food intake (g/day/rat) 16.13 ± 1.59c 20.49 ± 1.73b 25.27 ± 1.49a VLDL-C 0.39 ± 0,07b 0.46 ± 0.06b 1.03 ± 0.15a
FER (%) 1.07 ± 0.58c 3.16 ± 0.40b 4.35 ± 0.61a LDL-HDL1-C 0.25 ± 0.04c 0.33 ± 0.04b 0.59 ± 0.08a
Organs RW (%) HDL2-C 0.53 ± 0.08a 0.49 ± 0.06a 0.33 ± 0.05b
Liver 2.08 ± 0.16b 2.23 ± 0.11ab 2.35 ± 0.19a HDL3-C 0.33 ± 0.04a 0.30 ± 0.04a 0.24 ± 0.03b
Adipose tissue 2.58 ± 0.18b 2.87 ± 0.40ab 3.14 ± 0.26a Triacylglycerols 0.84 ± 0.06b 0.88 ± 0.05b 1.05 ± 0.09a
VLDL-TG 0.30 ± 0.03b 0.35 ± 0.05b 0.51 ± 0.04a
Data are shown as the mean ± S.E.M. for six values per group. The three groups fed an Liver lipids (μmol·g−1)
obesogenic diet containing 20% sardine by-products proteins (SBy-P), sardine fillets Total cholesterol 19.79 ± 2.02c 25.16 ± 2.53b 37.63 ± 2.16a
proteins (SF-P) or casein (Cas). Statistical analysis was performed using the Duncan's Triacylglycerols 23.52 ± 2.61b 26.47 ± 4.16b 37.13 ± 3.59a
multiple range tests. The means with different superscript were considered significantly
different (p < 0.05). Data are shown as the mean ± S.E.M. for six values per group. The three groups fed an
obesogenic diet containing 20% sardine by-products proteins (SBy-P), sardine fillets
proteins (SF-P) or casein (Cas). Statistical analysis was performed using the Duncan's
3.3. Lipids intake, fecal lipids and lipids digestibility
multiple range tests. The means with different superscript were considered significantly
different (p < 0.05).
At the end of the experiment, lipids intake values were, lower in
SBy-P (−36%) and SF-P (−19%) groups compared to Cas group, with a respectively, by an increase in HDL3-apo (+49%, +19%) and HDL2-CE
significant reductions in SBy-P (−21%) compared to SF-P (Table 4). (+108%, +88%) and a decrease in HDL3-PL (−48%, −43%) and
Furthermore, an increase in fecal lipids and fecal cholesterol were HDL3-UC (−35%, −26%), Furthermore, LCAT activity and HDL3-apo
noted, respectively, between SBy-P (+35%, +47%) and SF-P groups were respectively, +16% and +26% higher in rats fed SBy-P diet
(+21%, +27%) compared with Cas group. Moreover, the values noted compared to SF-P group (Table 6).
in SBy-P were +13% and +15% higher compared to SF-P group.
Whereas there was no significant difference in lipid digestibility be-
3.6. PON-1 activity, serum and lipoprotein lipid peroxidation
tween the three groups (Table 4).

The results of lipid peroxidation represented in Table 7, showed that

3.4. Serum leptin, lipoproteins and liver lipids concentrations
Serum leptin and LDL-HDL1-C levels were, respectively, lower in
SBy-P (−29%, −56%) and SF-P (−14%, −43%) groups compared
with Cas group (Table 5). Moreover, the values noted in the SBy-P were
−18% and −23% lower than SF-P group. A significant reduction in
serum TC (−31%, −27%), VLDL-C (−62%, −55%), TG (−19%,
−16%), VLDL-TG (−41%, −31%) and liver TG (−37%, −29%)
concentrations, were observed in SBy-P and SF-P groups, respectively,
compared to Cas values (Table 5). Liver TC levels were lower in SBy-P
group (−47%) and SF-P group (−33%) compared with Cas group
(Table 5). Moreover, the value noted in SBy-P was −21% lower than at
SF-P rats. However, an increase in HDL2-C (+62%, +50%) and HDL3-C
contents (+35%, +25%), was noted respectively, in SBy-P and SF-P
groups compared with the Cas group.
3.5. LCAT activity, HDL3-UC, -PL, -apo and HDL2-CE
Reverse cholesterol transport exploration has shown that the LCAT
activity was increased by 57% and 35%, respectively, in SBy-P and SF-P
groups compared with Cas group. This augmentation was accompanied,
serum, LDL-HDL1, HDL2 and HDL3 TBARS levels were, respectively,
reduced by 34%, 54%, 64% and 63% in SBy-P group, and by 20%, 33%,
39% and 32% in SF-P group, compared to Cas group. Moreover, the
values of TBARS noted in SBy-P, were respectively, reduced on serum
(−18%), LDL-HDL1 (−31%), HDL2 (−40%) and HDL3 (−46%) com-
pared with SF-P group (Table 7). Serum PON-1 activity was increased in
rats fed SBy-P (2.2-fold) and SF-P (1.7-fold) diets compared to Cas.
Likewise, this activity was also enhanced in HDL2 (2- and 1.5-fold) and
HDL3 (1.9- and 1.5-fold). Nevertheless, these increases were more
marked in SBy-P group (1.3-fold in HDL2 and 1.3-fold in HDL3) com-
pared to SF-P group (Table 7).
4. Discussion
The purpose of the present work was to investigate whether proteins
isolated from sardine by-products, might reduce growth parameters and
serum leptin level, improve lipids metabolism and attenuate serum and
lipoproteins oxidation in obese rats.
Rats, in response to a high-fat diet develop obesity as characterized
by weight gain, reduced satiety and increased blood lipids disorders
[4,8]. Furthermore, this type of diet stimulates visceral lipogenesis,

Table 4 Table 6
Lipids intake, fecal lipids and lipids digestibility. Serum LCAT activity and HDL3-UC, HDL3-PL, HDL3-apo and HDL2-CE contents.

SBy-P SF-P Cas SBy-P SF-P Cas

c b a −1 −1 a b
Lipids intake (mg/day/rat) 3226 ± 319 4098 ± 347 5055 ± 298 LCAT (nmol·h ·mL ) 19.34 ± 1.26 16.63 ± 1.24 12.35 ± 1.80c
Fecal lipids HDL3-UC (mmol·L−1) 0.05 ± 0.01b 0.06 ± 0.01b 0.08 ± 0.008a
Fecal total lipids (mg/day/rat) 76.8 ± 4.0a 68.5 ± 3.5b 56.9 ± 6.4c HDL3-PL (mmol·L−1) 0.48 ± 0.11b 0.53 ± 0.09b 0.93 ± 0.18a
Fecal CT (mg/day/rat) 6.87 ± 0.33a 5.97 ± 0.45b 4.68 ± 0.61c HDL3-apo (g·L−1) 0.77 ± 0.06a 0.61 ± 0.04b 0.51 ± 0.04c
Lipids digestibility (%) 97.6 ± 0.3 98.3 ± 0.2 98.9 ± 0.1 HDL2-CE (mmol·L−1) 0.75 ± 0.12a 0.68 ± 0.10a 0.36 ± 0.08b

Data are shown as the mean ± S.E.M. for six values per group. The three groups fed an
obesogenic diet containing 20% sardine by-products proteins (SBy-P), sardine fillets
proteins (SF-P) or casein (Cas). Statistical analysis was performed using the Duncan's
multiple range tests. The means with different superscript were considered significantly
different (p < 0.05).
Data are shown as the mean ± S.E.M. for six values per group. The three groups fed an
obesogenic diet containing 20% sardine by-products proteins (SBy-P), sardine fillets
proteins (SF-P) or casein (Cas). Statistical analysis was performed using the Duncan's
multiple range tests. The means with different superscript were considered significantly
different (p < 0.05).

19
F. Affane et al.
Table 7
Thiobarbituric acid reactive substances (TBARS) and paraoxonase (PON-1) activity in
serum and lipoproteins.
SBy-P SF-P Cas
Serum and lipoproteins
TBARS
Serum-TBARS 18.61 ± 2.10c 22.65 ± 1.29b 28.33 ± 2.24a
(μmol·mL−1)
VLDL-TBARS (μmol·mL−1) 5.39 ± 0.86 4.53 ± 0.85 6.53 ± 0.94
LDL-HDL1-TBARS 3.56 ± 0.80c 5.19 ± 1.07b 7.80 ± 1.05a
(μmol·mL−1)
HDL2-TBARS (μmol·mL−1) 2.77 ± 0.64c 4.65 ± 1.11b 7.64 ± 1.32a
HDL3-TBARS (μmol·mL−1) 3.12 ± 1.00c 5.76 ± 1.09b 8.48 ± 1.23a
Serum and lipoproteins
PON-1
Serum-PON-1 (U·mL−1) 102.3 ± 18.0a 81.3 ± 15.3a 46.8 ± 12.4b
HDL2-PON-1 (U·mL−1) 9.4 ± 1.5a 7.3 ± 1.4b 4.7 ± 1.0c
HDL3-PON-1 (U·mL−1) 16.3 ± 1.4a 12.9 ± 1.7b 8.6 ± 1.0c
Data are shown as the mean ± S.E.M. for six values per group. The three groups fed an
obesogenic diet containing 20% sardine by-products proteins (SBy-P), sardine fillets
proteins (SF-P) or casein (Cas). Statistical analysis was performed using the Duncan's
multiple range tests. The means with different superscript were considered significantly
different (p < 0.05).
thereby causing lipid peroxidation [6,9]. Similar observations were
found in our study after three months of a high-fat diet consumption at
20% of sheep fats [4,8], compared to those observed in normal rats
consuming a normocaloric diet [9].
Interestingly, one of the first results observed in our study discloses
that SBy-P and SF-P exhibited a significant decrease in FI, FER and BW
gain compared to Cas diet. The observed effects are more prominent
with by-products proteins. Similar results were reported in studies
targeting decreased food intake and weight loss with fish by-products
(collagen peptides) and fillet proteins [10,11]; Their low content of
certain AA such as phenylalanine, tyrosine, histidine and proline or
their high content of alanine, arginine, glycine and taurine compared to
casein could be implicated in these effects [19,23]. Bougatef et al. [40]
reported that rats fed smooth hound muscle proteins hydrolysates,
obtained by digestive proteases, showed a significant decrease in BW, as
well as a decrease of FI and explained that by a satietogenic effect of
fish peptides. This results suggested that sardine muscle and especially
by-products proteins release more efficiently peptides in digestive tract
with satietogenic properties, by stimulating Glucagon-like peptide-1
(GLP-1) activity [10,41]. The rise in endogenous GLP-1 signaling was
seen as a strategy leading to weight loss [42]. The regulation of body
weight may also depend on the tissue mass (liver and adipose tissue)
that is reduced in the SF-P group, and particularly in SBy-P group. This
reduction is concomitant with the leptin concentration reduction. Sar-
dine proteins could by their hypolipidemic properties, increase the li-
ver's ability to excrete excess lipids [43] or reduce their accumulation in
adipose tissue [10,41,44]. Furthermore, it's known that leptin is a sa-
tiety-inducing hormone and involved in the adipose tissue regulation,
its blood concentration tends to increase during weight gain and de-
crease after weight loss [45]. As well, SBy-P could mitigate the pro-
gression of obesity by stimulating fatty acid oxidation through the up-
regulation of AMP-activated protein kinase (AMPK) genes, as well as by
inhibiting the synthesis of adipogenic and lipogenic enzymes [46].
In experimental studies, it has been shown that proteins from dif-
ferent fish species have cholesterol-lowering properties compared to
casein [10,11,14]. In this context, our results showed that sardine
proteins (by-products and fillet) improve cholesterolemia by decreasing
serum VLDL-C and LDL-HDL1-C and increasing HDL-C levels, with
higher fecal fat and cholesterol content in rats fed SBy-P. Though, the
exact mechanisms involved in the cholesterol-lowering effect of fish
proteins have not been fully identified, many hypotheses could be
proposed to explain them. An inhibition of endogenous cholesterol
biosynthesis by decreasing 3-hydroxy-3-methyl-glutaryl-coenzyme A
20
Life Sciences 199 (2018) 16–22
reductase activity and/or an increase of its excretion via its hepatic
catabolism by cholesterol 7α-hydroxylase [14,46]. Furthermore, the
high contents in taurine and glycine in SBy-P and SF-P groups, could be
play an important role in bile acids secretion. Indeed, according to
Liaset et al. [43], the fish promotes the excretion of bile acids by their
conjugation with taurine and glycine, which increases their affinity for
membrane hepatic bile acids transporters. Likewise, the inhibition of
lipids and cholesterol absorption in gut is also possible. Indeed, Hosomi
et al. [14], had demonstrated that fish protein have high binding affi-
nity to bile acids which inhibits the cholesterol emulsifying power. As
result, the non-reabsorption of bile acids and cholesterol, leading to an
increase in the expression of cholesterol 7α-hydroxylase [46]. Conse-
quently, sardine proteins (by-products and fillet) were characterized by
a low lysine/arginine (1.33 and 1.64, respectively) and methionine/
glycine (0.51 and 0.74, respectively) ratios compared to casein (2,1 and
1.4, respectively), that seem to favor a cholesterol-lowering effect
[14,47], probably by increasing hepatic LDL receptor expression [47].
In hypercholesterolemic rats, Mir et al. [48] found that diet con-
taining sardine proteins increased HDL-C levels, in particular its con-
tents in cholesteryl esters (CE) and apo-HDL3, indicating an important
LCAT activity; an enzyme responsible for reverse cholesterol transport
and which plays an important role in HDL maturation [49]. In this
study, decreased PL-HDL3 (LCAT substrate) and UC-HDL3 (acyl group
acceptor) and increased apo-HDL3 (cofactor-activator) and HDL2-CE
(LCAT product) in response to both SBy-P and SF-P diets could be in-
timately linked to a higher LCAT activity, which could possibly related
to an increased expression of its apo AI cofactor activator and/or its
hepatic genes synthesis in the liver [47]. In an early study, we had
shown that sardine by-products flour (with approximately 60% of
protein contents) effectively reduces the pro-atherogenic risk in rats fed
an obesogenic diet by improving lipid profile and reverse cholesterol
transport [18].
It is well established that visceral fat accumulation is correlated
with abnormalities of lipoprotein metabolism by an increased release of
free fatty acids circulating which are primarily directed to liver [5].
This mechanism could increase TG liver synthesis and VLDL produc-
tion, leading to long-term dyslipidemia [5]. In our study, reduction in
hepatic TG and hypotriglyceridemia associated with decreased VLDL-
TG in obese rats treated with both sources of sardine proteins were
similar to those observed in rats subjected to a high-fat diet treated with
fish proteins and their hydrolysates [11]. This result could be probably
attributed to the increase in lipoprotein lipase activity by decreasing
VLDL hepatic synthesis enriched on TG and/or accelerated VLDL and
apo B-100 catabolism [47].
Recent data suggest that oxidative stress may be the mechanistic
linked between obesity and its complications [6]. Besides that, many
studies reported that LCAT may have a dual role in lipoproteins oxi-
dation, allowing dose-reactive action as a potent pro-oxidant during
VLDL oxidation, but as an antioxidant during LDL oxidation which
would be in favor to reduce an atherogenic acceleration process related
to obesity [50]. In this study, we have found that sardine proteins de-
creased serum and lipoproteins TBARS levels and improved LCAT and
PON-1 activities, probably by their high taurine content and their
richness in AA with an hypocholesterolemic and antioxidant properties
[19,22], by inhibiting the production of oxidized LDL [20]. The in-
creased PON-1 activity could also explain the low TBARS levels in
serum and lipoproteins observed in our study. This antioxidant enzyme
is closely associated with HDL synthesis secondary to the high LCAT
activity and prevents LDL oxidation [51].
Furthermore, high quality fish proteins hydrolysates prepared from
viscera, skin and gelatin by-products, also demonstrated high in vitro
antioxidant properties and inhibitory activity against lipid peroxidation
[20–22], and protect DNA damage caused by hydroxyl radicals [52],
probably due to their richness in certain bioactive peptides. The anti-
oxidant activity of proteins (or their peptides) fish is not limited to a
single mechanism. They contain various AA with different antioxidant
F. Affane et al.
properties. Some are more effective as radical scavengers or lipid per-
oxidation inhibitors, while others are metal chelators or reducers
[19,20].
5. Conclusion
Sardine proteins and in particular those of by-products because of
their richness in certain essential amino acids, favored the weight body
loss by their satiating capacity, and ameliorated lipid profile by de-
creasing the feedback inhibition of cholesterol biosynthesis and/or in-
creasing bile acid excretion. Sardine proteins improved reverse cho-
lesterol transport by increasing LCAT activity, leading to serum
cholesterol regulation. This anti-atherogenic action is particularly fa-
vored by increasing PON-1 activity which prevents lipoproteins from
oxidation, thus reducing the complications related to obesity.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was funded by the Ministry of Higher Education and
Scientific Research- Algeria (CNEPRU number D01N01UN310120150023).
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