1
Conjugated linoleic acid affects lipid composition, metabolism and gene
2
expression in gilthead sea bream (Sparus aurata L)
3 4
A. Diez1, D. Menoyo2, S. Pérez-Benavente1, J.A. Calduch-Giner3, S. Vega-Rubin de Celis3, A.
5
Obach4, L. Favre-Krey5, E. Boukouvala5, M. J. Leaver6, D. R. Tocher6, J. Pérez-Sanchez3, G.
6
Krey5 and J. M. Bautista1*
7 8
1
9
Facultad de Veterinaria, Madrid, Spain.
Departamento de Bioquímica y Biología Molecular IV, Universidad Complutense de Madrid,
10
2
11
Agrónomos, Ciudad Universitaria, Madrid, Spain
12
3
Instituto de Acuicultura de Torre de la Sal, (CSIC), Ribera de Cabanes, Castellón, Spain.
13
4
Nutreco Aquaculture Research Centre AS (ARC), Stavanger, Norway.
14
5
National Agricultural Research Foundation, Fisheries Research Institute, Nea Paramos, Kavala,
15
Greece.
16
6
Institute of Aquaculture, University of Stirling, Stirling, United Kingdom.
17
*
To whom correspondence should be addressed: Jose M. Bautista, Dpto. de Bioquímica y
18
Biología Molecular IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Ciudad
19
Universitaria, 28040 Madrid, Spain. Tel + 34 91 394 3823; Fax + 34 91 394 3824, e-mail:
20
[email protected]
21
Total word count: 6486 / Figures: 3 / Tables: 2
22
Running title: Effect of dietary CLA in a fish
23
Key Words: Conjugated linoleic acid; Sparus aurata; Peroxisome proliferator activated
24
receptors; Endocrine signalling
Departamento de Producción Animal, Universidad Politécnica de Madrid, ETS de Ingenieros
25
1
26
Research financed by the European Commission (Q5RS-2000–30360) and Spanish MEC
27
(AGL2004-06319-C02-01). Part of the analyses performed were supported by an award to AD
28
and JMB within the Access to Research Infrastructure Action of the Improving Human
29
Potencial Programme from the European Commission (contract HPRI-CT-2001-00180).
30 31
Abbreviations:
32
ACO: AcylCoA oxidase; BSA: bovine serum albumin; CHO: Cholesterol; CLA: Conjugated
33
linoleic acid; DES: ∆6-fatty acyl desaturase; ELO: Fatty acyl elongase; FAME: Fatty acid
34
methyl ester; FAS: Fatty acid synthase; FFA: Free fatty acids; G6PD: Glucose 6-phosphate
35
dehydrogenase; GH: Growth hormone; HPTLC: High-performance thin-layer chromatography;
36
HIS: Hepatosomatic index; HUFA: n-3 highly unsaturated fatty acids; IGF-I: Insuline-like
37
growth factor; L3HOAD: L-3-hydroxyacyl-CoA dehydrogenase; LA: Linoleic acid; ME: Malic
38
enzyme;
39
phosphatidylethanolamine; PPARs: Peroxisome proliferator acitvated receptors; SCD-1:
40
stearoyl-CoA-desaturase-1; SL: Somatolactin; SM: sphingomyelin; TAG: Triglyceride; VSI:
41
Viscerosomatic index
MUFA:
monounsaturated
fatty
acids;
42
2
PC:
Phosphatidyl
choline;
PE:
42
ABSTRACT
43 44
In order to maximise growth, farmed fish are fed diets with high levels of lipid. This can lead to
45
high levels of lipid in tissues and impacts on carcass quality. Because feeding of conjugated
46
linoleic acid (CLA) reduces body fat in mammals, this study aimed to determine the effects of
47
dietary CLA on growth, composition and postprandial metabolic parameters in sea bream, a
48
major farmed fish species. Three diets were formulated containing 48% protein and 24% fat,
49
using fish oil and a combination of fish oil with 2 and 4% CLA (c9, t11-CLA; t10, c12-CLA;
50
1:1 mixture). Fish were fed the experimental diets for 12 wk and sampled 6 h and 24 h after
51
final feeding. Dietary CLA decreased feed intake and growth but had no effects on VSI or HSI.
52
Feed efficiency was increased by 2% CLA and total body fat was decreased. There were no
53
changes in circulating growth hormone (GH) but somatolactin (SL) was lower than control in
54
CLA diets. Changes in tissue fatty acid composition were associated with decreases in hepatic
55
fatty acyl desaturase (DES) and elongase (ELO) mRNA. Triglyceride level was greater in liver
56
with 4% CLA diets and less than control in muscle with both CLA diets. Major metabolic
57
effects were seen 6 h, but not at 24 h after feeding. These included a decrease in circulating
58
triglyceride, increases in hepatic acyl CoA-oxidase (ACO), and decreases in L-3-hydroxyacyl-
59
CoA dehydrogenase (L3HOAD), markers of peroxisomal and mitochondrial β-oxidation
60
respectively. On lipogenesis markers, CLA had no effect on hepatic fatty acid synthase (FAS) or
61
malic enzyme (ME) but decreased glucose 6-phosphate dehydrogenase (G6PD) activity. Thus,
62
the major physiological effect of CLA in sea bream appears to be the channeling of dietary lipid
63
away from adipose tissue to liver, and the switch from hepatic mitochondrial to peroxisomal β-
64
oxidation, possibly as a detoxification response.
65
3
65
INTRODUCTION
66 67
Lipids and especially fatty acids are, along with proteins, the major macronutrients for fish.
68
Carbohydrates are quantitatively less important as nutrients in most fish, particularly
69
carnivorous and marine species, as they do not constitute a major part of their natural diet.
70
Lipids have important roles both as structural components and in energy provision. In addition,
71
dietary lipids are important as sources of essential fatty acids, as eicosanoid precursors and they
72
assist in the uptake of lipid-soluble vitamins needed for normal growth and development in fish
73
Therefore, the current practice in intensive gilthead sea bream (Sparus aurata, sea bream
74
hereafter) culture is to feed so-called high energy diets, containing 20% of total mass as lipid
75
(1). However, the use of high lipid diets in farmed fish can lead to increased energy storage in
76
adipose tissue, and the resulting excess fat accumulation in the fish is generally not desirable in
77
aquaculture products (1). In addition, as a result of global limits on the supply of fish oil (2),
78
there is a drive to replace fish oils with plant-derived oils in aquaculture diets (3). This has
79
raised concern regarding the potential for reducing levels of human health-promoting n-3 highly
80
unsaturated fatty acids (HUFA) in farmed fish. Therefore, there is increasing interest to
81
understand the physiological mechanisms that control energy metabolism that determine lipid
82
and fatty acid homeostasis in fish.
83
Conjugated linoleic acid (CLA) is a term used to describe positional and geometric isomers of
84
linoleic acid (18:2(n- 6); LA), the two main naturally occurring isomers being cis-9,trans-11 and
85
trans-10,cis-12 (4). These compounds are known to occur particularly in beef and dairy products
86
but are widespread at lower levels in many foodstuffs (4). Dietary inclusion of CLA can cause
87
significant alterations in energy and lipid metabolism in mammals leading to reductions in
88
overall body fat mass. This has been suggested to be a positive effect in a variety of farmed
89
species and animal disease models and by extension, humans (5). CLA has also been shown to
4
90
alter highly unsaturated fatty acid (HUFA) biosynthesis in cellular models (6), and to increase
91
expression of genes involved in the HUFA biosynthetic pathway (7). There are several fish
92
species, in which the effects of dietary CLA have also been studied (8-11). In most of these
93
CLA does not affect performance, and clear effects on lipid metabolism have not been observed.
94
Like in land animals (12), the CLA effects may be isomer-, dose-, time-, and species-dependent.
95
The aim of the present study is to investigate the effects of dietary CLA, consisting of equal
96
amounts of the cis-9, trans-11 and trans-10, cis-12 isomers, on lipid metabolism, HUFA
97
biosynthesis and lipid composition in the liver and flesh of sea bream.
98
5
98
MATERIALS AND METHODS
99 100
Fish and experimental diets.
101
The in vivo feeding trial with sea bream was carried out from July 25th to October 16th 2005 in
102
an indoor marine water system (Instituto de Acuicultura de Torre de la Sal) in running seawater.
103
Oxygen content of outlet water was always higher than 85% saturation, and day length and
104
temperature followed natural changes, with the latter increasing from 17°C to 25°C. Juvenile
105
fish were obtained from a commercial hatchery (Cupimar, Cádiz, Spain), and were graded by
106
size and for the absence of anatomical malformations. After 20 d of acclimatization to the
107
experimental facilities, groups of 90 fish were placed into 12 circular glass fiber tanks (500-L),
108
with four replicates per dietary treatment. Fish were fed once a day (9h am), six days per week.
109
The diets were formulated to contain 24% fat and 48% protein as a proportional of total mass
110
and were produced at the Skretting Aquaculture Research Centre (Stavanger, Norway). The
111
basal composition of the experimental diets consisted of the following ingredients (g/kg):
112
fishmeal, 499; soybean meal, 50; corn gluten, 100; wheat, 175; oil, 164; micronutrients, 2. In all
113
diets
114
supplemented diets, Tonalin® (Natural ASA, Sandvika, Norway), which contains 81% of CLA
115
as FFA (a mixture of the 2 isomers c9, t11 and t10, c12), was added at 2% and 4% of dry
116
weight of diet to produce the 2% CLA and 4% CLA diets, respectively (for dietary fatty acid
117
composition, see Supplementary Table 1).
118
For feeds, dry matter, crude protein and ash content were determined according to AOAC (24).
119
The fatty acid profile was determined after methanolysis and by gas-liquid chromatography
120
(Perkin Elmer Autosystem GC) as described below. Yttrium was measured by ICP-AES after
121
wet ashing the samples (Jordforsk, Ås, Norway).
122
Sampling protocol.
a south American fish oil (Skretting, Stavanger, Norway) was used. In the CLA-
6
123
Whole body composition was determined in a pooled sample of ten fingerlings at the initiation,
124
and in pools of five fish per tank at the end of growth trial. Specimens for whole body analysis
125
were ground, and small aliquots were dried to estimate water content. The remaining samples
126
were freeze-dried and kept frozen until analysis. To assess dietary effects on postprandial and
127
basal metabolism, two sampling times were performed for plasma metabolites and tissue
128
biochemistry and gene expression. Therefore, at the end of the trial and 6 h after the last feed, 12
129
fish per dietary treatment, i.e. three fish per tank, were anaesthetized with MS 222 (1g/10 L),
130
and blood samples were taken. Samples of liver, white muscle, intestine and peri-visceral
131
adipose tissue for biochemical and molecular analyses were rapidly excised, frozen in liquid
132
nitrogen, and stored at -80°C until analysis. Following overnight (24 h after the last feed), 12
133
additional fish per dietary treatment were taken for plasma and tissue samples (24 h samples;
134
basal metabolism). Tissues for gene expression analyses were obtained from individual fish (1-3
135
fish per tank, 3-6 fish per dietary treatment).
136
National and institutional regulations (CSIC-IATS Ethical Committee), in accordance with the
137
European Union’s relevant legislation, have been followed regarding animal experimentation.
138 139
Proximate analyses, lipid extraction and fatty acid analysis.
140
Moisture, crude protein, lipid content and ash of whole body fish were determined according to
141
AOAC (13). Liver and skinned and de-boned flesh samples, each consisting of three fish, were
142
homogenized into pooled “pates”. Total lipid was extracted with chloroform/methanol (2:1, v:v)
143
and prepared according to the method of Folch et al. (14). The weight of lipid was determined
144
gravimetrically after evaporation of solvent and overnight desiccation in vacuo. Separation of
145
lipid classes was performed by high-performance thin-layer chromatography (HPTLC)
146
according to Henderson and Tocher (15).
147
Fatty acid methyl esters (FAME) from tissue total lipid were prepared and purified according to
7
148
Christie (16) and Ghioni et al. (17). FAME were separated and quantified by gas-liquid
149
chromatography (17, 18). Data were collected and processed using the Chromcard for Windows
150
(version 1.19) computer package (Thermoquest Italia S.p.A., Milan, Italy).
151 152
Determination of key lipogenic and β-oxidation enzyme activities.
153
Cytoplasmic extracts from liver homogenates were prepared and the activities of glucose-6-
154
phosphate dehydrogenase (G6PD; EC 1.1.1.49), malic enzyme (ME; EC 1.1.1.40), and fatty
155
acid synthetase (FAS; EC 2.3.1.38) were performed as previously described (19-21). Acyl-CoA
156
Oxidase (EC 1.3.99.3) from the peroxisome-enriched liver fraction was determined according to
157
previously described procedures (22) with the modifications of Ruyter et al., (23). Mitochondrial
158
extracts from livers were prepared following Harper and Saggerson (24). and the activity of L-
159
3-hydroxyacyl-CoA dehydrogenase (L3HOAD; EC1.1.135) was measured according to
160
Bradshaw and Noyes (25) All enzyme assays were performed in duplicate or triplicate. The
161
enzymatic activity units (IU), defined as µmoles of substrate converted to product per minute at
162
the assay temperature were expressed per mg of soluble protein (specific activity). Protein was
163
determined by the Bio-Rad dye method reagent (Bio-Rad, Hercules, CA, USA) using bovine
164
serum albumin as the standard. Total protein content determined in the mitochondrial extracts
165
and peroxisomal enriched fractions showed no differences among experimental groups.
166 167
Assays of plasma metabolites and hormone levels.
168
Plasma glucose, cholesterol (CHO) and triglyceride (TAG) levels were measured
169
spectrophotometrically using commercial kits from Sigma (Cat No. 315-310; 401-25P and 337-
170
B, respectively) levels were measured spectrophotometrically using commercial kits according
171
to manufacturer´s instructions. Plasma growth hormone (GH) and somatolactin (SL) levels
172
were determined by homologous radioimmunoassays (RIAs) as previously described (26,27).
8
173
The midrange of the assay was 1.8 ng/mL for GH and 2.1 ng/mL for SL. Plasma insulin-like
174
growth factor (IGF-I) was extracted by ethanol-cryoprecipitation and measured by fish RIAs.
175
The gilthead sea bream assay was based on the use of bream (Pagrus auratus) IGF-I (GroPep:
176
5PAF-AGU100) as tracer and standard. Anti-barramundi (Lates calcarifer) IGF-I serum
177
(GroPep: 5PAF1-YU100) (1:8000) was used as a first antibody. A goat anti-rabbit IgG (1:20)
178
(Biogenesis: 5196-2104 ) was used as a precipitating antibody. The sensitivity and midrange of
179
the assay were 0.05 and 0.7-0.8 ng/mL, respectively.
180 181
RNA isolation and real-time quantitative RT-PCR (qRT-PCR).
182
Sea bream total RNA was extracted from the fish tissues using a robotic system for nucleic acid
183
isolation (ABI PRISM 6100 Nucleic Acid Prep Station, Applied Biosystems) performed
184
according to the manufacturer’s instructions. Total RNA was quantified with RIBOGreen TM
185
(Molecular Probes, Europe, Leiden, The Netherlands) using a Perkin-Elmer LS-50B fluorimeter
186
and RNA integrity was checked by electrophoresis in 2% agarose gels. First strand cDNA was
187
synthesized using the High-Capacity cDNA Archive Kit (Applied Biosystems) according to the
188
manufacturer’s instructions.
189
Relative abundance of PPAR-mRNA was assessed using the 5’ fluorogenic nuclease assay
190
(TaqMan) in an ABI Prism7000 Sequence Detector System (Applied Biosystems) using primers
191
and protocols that have been previously described (28). All samples were run in triplicate and
192
quantified by normalizing the PPAR signal to that of α-tubulin by the 2 - ∆∆Ct method (28).
193 194
Riboprobes and ribonuclease (RNase) protection assay.
195
For the sea bream ∆-6 fatty acyl desaturase (DES) riboprobe, oligonucleotide primers 5’-GAC
196
CAT GCA GTT ACA AGC CAC C and 5’-TCC CCT GAG TTC TTC AGT GAC C were used
197
for the PCR amplification of a 216 bp fragment (nucleotides 1225 to 1441) of cDNA (Genbank
9
198
AY055749, (29)). For the sea bream fatty acyl elongase (ELO), oligonucleotide primers 5’-TGC
199
CAG GAC ACT CAC AGT GC and 5’-GGA CGA AGC TGT TTA GGG AGG were used for
200
the PCR amplification of a 226 bp fragment (nucleotides 303 to 529) of cDNA (Genbank
201
AY660879, (30)). The RNase protection assay was performed as previously described (28) and
202
relative expression of the genes between individual fish and treatments was normalized to the β-
203
actin expression (28).
204 205
Statistical analysis
206
Results are expressed as means ± SD. Data were analyzed as a completely randomized design,
207
with type of diet as the main source of variation, by using the General Linear Model procedure
208
of the SAS computer package (SAS Institute Inc., Cary, NC, USA). For plasma metabolites
209
(glucose, TAG and CHO), tissue enzymatic analyses, and gene expression, the interactions
210
between diet and sampling time were also analyzed. Arcsine square root transformations of
211
percentage data were conducted for fatty acids not achieving homogeneity of variance.
212
Significant differences between treatments were assessed by the Newman-Keuls multiple
213
comparison procedure. Differences were considered to be significant when P< 0.05.
214
10
214
RESULTS
215 216
Efects of CLA feeding on growth, feeding and lipid content
217
Our results showed that dietary CLA supplementation reduced feed intake, final body and liver
218
weight, growth rates and whole body lipid content and retention (Table 1). These effects were
219
accompanied by an improvement in feed efficiency in fish fed the 2% CLA diet.
220
In the analysis of the lipid class compositions of liver and muscle (Table 2) it was observed that
221
the 4% CLA containing diet resulted in increased TAG levels in the liver, and a reduction of
222
sphingomyelin (SM) and phosphatidylethanolamine (PE). In muscle, the experimental dietary
223
treatments resulted in a lipid class profile opposite to that observed in liver (Table 2). Thus,
224
dietary CLA decreased TAG levels in muscle and increased proportions of SM and PE and
225
phosphatidylserine (PS). Dietary CLA also increased CHO levels in muscle, but not in liver.
226
The fatty acid composition, expressed as percentage of total fatty acids, (Supplementary Table
227
2) showed that both CLA isomers were detected in liver and muscle after dietary CLA inclusion.
228
In fish fed the diet containing 2% CLA, the accumulation of CLA in muscle was almost double
229
that found in liver (5.3% vs. 3% of total lipid). However, similar concentrations, 7.2% in muscle
230
and 6.6% in liver, were found in fish fed the diet containing 4% CLA. Dietary CLA also
231
resulted in some differences in individual fatty acids in both liver and muscle. A decrease of
232
total monounsaturated fatty acids (MUFA) was observed in the liver of fish fed the CLA diets,
233
and accumulation of 18:0 was observed in the liver of fish fed 2% CLA (Suppl. Table 2).
234
Dietary CLA also decreased liver 20:5(n-3) and increased 18:2(n-6) and 18:3(n-3)
235
concentrations in this tissue. In muscle, dietary CLA induced a decrease of 16:0 and an increase
236
of 18:0 concentrations resulting in an overall decrease in total saturated fatty acids (SFA). The
237
concentration of total MUFA was reduced in the muscle of fish fed the CLA diets. Moreover,
238
dietary CLA appeared to increase percentages of 22:6(n-3) in muscle.
11
239 240
Effects of CLA on lipid metabolism and endocrine factors.
241
Plasma glucose levels were not affected by dietary treatment (Supplementary Table 3), as
242
values decreased between 6h and 24 h postprandially, irrespective of diet. Plasma TAG levels
243
also decreased over the course of the postprandial period, and CLA diets reduced circulating
244
TAG relative to controls at 6 h after feeding. Dietary treatment did not affect plasma cholesterol
245
levels although was significantly lower in the 24 h samples. The circulating levels of GH, IGF-I
246
and SL were differentially affected by experimental diets so that dietary CLA reduced plasma
247
SL levels (18.92 ± 1.52, 14.32±1.17 and 15.31± 1.16 for 0, 2 and 4 %CLA respectively) without
248
significant effects on GH and IGF-I (data not shown).
249
Dietary effects on liver lipogenic and β-oxidation enzyme activities (Figure 1) showed that
250
CLA-feeding induced a postprandial decrease in lipogenic liver G6PD activity (Fig. 1A).
251
Although no diet-related differences in the activity of liver ME were observed (data not shown),
252
this lipogenic enzyme exhibited a higher specific activity in the 6 h post-feeding liver samples as
253
compared to the 24h samples (up to four fold). On the other hand, no time or dietary effects
254
were observed in hepatic FAS specific activity (data not shown), another lipogenic indicator. In
255
contrast to lipogenic activities, major differences were observed in lipolytic enzymes following
256
CLA feeding. In fish fed the control diet the activity of hepatic ACO, the rate-limiting enzyme
257
of the peroxisomal fatty acid β-oxidation spiral, did not change over the course of the
258
postprandial period. However, after CLA feeding, ACO activity was increased up to four fold
259
(Fig. 1B). This effect was not observed in the basal ACO activity (P < 0.05 in the interaction).
260
Liver L3HOAD activity, a marker of mitochondrial β-oxidation, exhibited a large increase at 6 h
261
postprandially compared with 24 h in fish fed the control diet (Fig 1C). CLA did not affect the
262
basal enzyme activities at 24 h but it induced a marked suppression of L3HOAD response at the
263
6 h postprandial point (Fig 1C).
12
264 265
Effects of CLA on fatty acyl desaturase (DES) and elongase (ELO) gene expression.
266
The levels of ELO and DES mRNA as assessed by RNase protection assay revealed that these
267
genes were differentially expressed in the tissues of interest (Fig. 2A). Thus, ELO is expressed
268
in both liver and intestine but not in white muscle or adipose tissue, while DES appeares to be
269
liver-specific. Therefore, the analysis of the diet-dependent mRNA expression of these genes
270
was performed only in the tissues in which they appear to be expressed and on samples obtained
271
at 24 h post-final feeding.
272
The mRNA expression of ELO in the liver and intestine (Figures 2B and 2C) showed that
273
dietary CLA resulted in down-regulation of this gene in liver, whilst in the intestine this effect
274
was observed only with the 4% CLA-supplemented diet. Dietary CLA also resulted in decreased
275
liver DES expression (Fig. 2D).
276 277
Effects of CLA on PPAR mRNA expression in fish tissues.
278
The mRNA expression analysis of the PPAR isotypes was asseseed in the muscle, liver and
279
adipose tissue, at 6 and 24 h post-feeding (Figure 3). In muscle, there was no difference in
280
PPARα or PPARβ expression over the postprandial period in the control diet groups. However
281
basal (24h) mRNA expression of both subtypes was increased by CLA feeding and postprandial
282
(6h) expression of PPARβ was increased by dietary CLA inclusion at the 4% level (Fig. 3A). No
283
differences were found between sampling times on muscle PPARγ expression although a
284
significant (P < 0.05) postprandial increase of this isotype was observed in fish fed the 2% CLA
285
diets (Fig. 3A).
286
In liver a postprandial decrease of PPARα and PPARβ mRNA levels was observed irrespective
287
of dietary treatments (Fig. 3B). In addition, a further postprandial decrease of both PPARβ and
288
PPARγ mRNA levels was observed in fish fed the CLA diets (Fig. 3B). Notably, CLA feeding
13
289
had no consistent effect on PPARα mRNA expression in liver.
290
In adipose tissue, in fish fed the control diet, no significant differences between postprandial and
291
basal states were found for any of the PPARs isotypes (Fig. 3C) , and CLA feeding had no effect
292
on postprandial (6h) expression levels of PPARβ and PPARγ. However, increasing CLA in the
293
diet had opposite effects on PPARβ and PPARγ basal (24h) mRNA levels, with PPARβ
294
decreasing after CLA feeding and PPARγ increasing. The 2% CLA diet significantly increased
295
adipose tissue PPARα expression in the posprandial state (Fig. 3C). The baseline expression of
296
this isotype was increased in fish fed the 4% CLA diet.
297
14
297
DISCUSSION
298 299
In mammals, CLA-feeding affects body weight and fat deposition by reducing body fat,
300
increasing lean body mass and lowering serum lipids (5). Proposed mechanisms involve changes
301
in gene expression and physiology of important lipid homeostatic tissues such as adipose tissue
302
and liver. In this present study, where sea bream juveniles were fed a commercial diet
303
containing 24% fat supplemented with up to 4% CLA (1:1 mixture of c9,t11 and t10,c12) for 12
304
wk, we aimed to assess the practical proposition of including CLA in aquaculture diets as well
305
as investigating effects on lipid deposition and metabolism. Our results demonstrate that CLA
306
feeding induced significant growth as well as metabolic and gene expression changes in these
307
fish, effects which, however, were accompanied by a reduction in feed intake. It could be argued
308
that this reduction in feed intake accounted also for reductions in growth and whole body lipid
309
content and retention. However, it is important to note that there were no apparent CLA-
310
associated changes in condition factors such as VSI or HSI or in total protein retention, i.e. in
311
parameters which would be expected to be reduced in sea bream following reductions in ration
312
size (31). Reduction of ration in sea bream also has well documented effects on the circulating
313
levels of GH and IGF-I (32), neither of which were affected by CLA-feeding. In contrast, the
314
levels of SL, an emerging marker of adiposity regardless of feed intake (33), were reduced by
315
CLA in sea bream. Taken together these results indicate that there were no effects on overall
316
growth physiology and the changes in tissue lipid profiles and metabolic parameters were
317
largely due to CLA accumulation, while reduced feed intake affected these only marginally, if at
318
all. In support of this, the few pair-feed trials involving dietary CLA that have been performed
319
on mammals confirm that the reduction on feed intake cannot solely account for the reductions
320
in fat mass or in a variety of metabolic effects (34, 35). Nevertheless, it is clear that CLA at the
321
dietary levels used here exerts no beneficial effects on growth performance in sea bream.
15
322
Previous studies in fish have demonstrated a similar lack of benefit (11) although, interestingly,
323
as also observed with sea bream, increased feed efficiency has been reported (8).
324
Dietary CLA accumulation in fish tissues varies with species, dietary lipid source, CLA
325
inclusion level and fish size (8-11). In the present study juvenile gilthead sea bream incorporated
326
both CLA isomers (c9,t11 and t10,c12) into liver and muscle tissues. The concentration of a
327
given fatty acid in the fish tissue divided by its concentration in the diet provides a deposition
328
ratio (RD) value which is helpful to evaluate the changes in fatty acid composition of the fish in
329
response to the diet (10). Accordingly, CLA was deposited at a lower rate than expected
330
regarding the amount of dietary supply (RD< 1). Deposition ratio was higher in muscle (RD ∼
331
0.70) than in liver (RD ∼ 0.50) with the level of dietary CLA employed having no effect on this
332
as previously reported in Atlantic salmon (10). Notably the accumulation of CLA in muscle is
333
associated with increases in total phospholipids, particularly PE and PS, indicating that CLA
334
may be incorporated into structural membrane lipid. In both muscle and liver the incorporation
335
of CLA resulted in a decrease in total MUFA and in muscle in an increase in total saturates. This
336
phenomenon is also seen in mammals (12) and in various fish species (8-11) and seems to be
337
one of the most consistent effects of CLA. The main MUFA, 16:1(n-7) and 18:1(n-9), are
338
synthesized through stearoyl-CoA desaturase -1 (SCD-1) and in mammals the above effects
339
have been suggested to be due to suppression or direct inhibition of SCD (6). Thus, it is likely
340
that the effects observed in sea bream involved similar mechanisms.
341
In addition to changes in MUFA and SFA, there was also a reduction in total hepatic 20:5(n-3)
342
levels and an increase in the precursor fatty acid 18:3(n-3). These changes can be directly
343
related to observed reductions in sea bream hepatic DES and ELO expression, genes responsible
344
for HUFA biosynthesis. This reduction in HUFA biosynthesis would not be a desirable effect in
345
aquaculture, particularly where dietary fish oils, rich in HUFA, have been replaced by HUFA-
346
deficient plant oils.
16
347
An important point arising from this study is that some of the most significant CLA-induced
348
changes were observed 6h postprandially and were not evident 24 h postfeeding. Thus careful
349
design of experiments and sampling procedures can provide greater insight into the varied
350
effects of CLA. A clear effect of CLA in sea bream was the increase of TAG in liver (at 4%
351
CLA) and the postprandial decrease in plasma TAG. Moreover TAG levels in muscle were
352
decreased and total body lipid decreased. These effects were not associated with increases in
353
liver lipogenesis and, indeed, there was some evidence for decreased lipogenesis in this tissue.
354
This suggests that TAG derived from dietary sources is being diverted to the liver rather than to
355
muscle for fuel or to the adipose tissue for storage. Furthermore, the increase in liver TAG is
356
associated with major changes in β-oxidation pathways, as shown by the switch from
357
postprandial mitochondrial metabolism to the peroxisomal pathway, as indicated by changes in
358
L3HOAD and ACO activity, respectively. In other species CLA seems to have differing effects
359
on liver TAG levels. For example, CLA increases liver TAG in mouse but decreases it in
360
hamster (36, 37). However, as is the case with sea bream, various studies on rodents have
361
indicated increases in peroxisomal metabolism (38) suggesting that this may be a common effect
362
of CLA feeding. Peroxisomal β-oxidation is a mechanism for metabolizing atypical fatty acids,
363
such as branched chain or very long chain molecules which are structurally incapable of
364
undergoing mitochondrial metabolism (39). In this regard studies on CLA-fed rats have detected
365
significant amounts of probable peroxisomal metabolites of CLA (40).
366
Although the mechanisms for the effects of CLA on lipid metabolism are presently unclear,
367
there is accumulating evidence that these may involve PPAR-dependent gene regulation (41-
368
43). PPARs are ligand-dependent transcription factors that are known to have critical roles in
369
regulating lipid homeostasis in a variety of tissues, principally liver, muscle and adipose. These
370
proteins act by regulating the activity of numerous genes involved in fatty acid storage, uptake
371
and metabolism (44). Studies on PPARα-null mice have shown that most of the effects of CLA
17
372
on genes involved in mitochondrial β-oxidation are independent of PPARα whilst the
373
peroxisomal ACO was increased by CLA via a PPARα-dependent mechanism (42). More
374
recently, it has been suggested that the lipid lowering effects of CLA result from the diminished
375
expression of PPARγ and many of its downstream target genes (43). Sea bream PPAR isotype
376
mRNAs are differentially expressed in a range of tissues (28). Thus, in muscle PPARα and
377
PPARβ are the isotypes predominantly expressed, whilst in adipose tissue PPARγ expression
378
predominates. All three isotypes are expressed in liver. Previous studies have shown that CLA
379
is a highly effective activator of sea bream PPARα and to a lesser extent PPARβ (28). The
380
mRNA levels of PPARs in the present study were affected by CLA feeding, particularly in
381
muscle where the basal levels of both PPARα and PPARβ increased. The levels of PPARα in
382
liver showed a postprandial decrease, which confirms the results of previous studies (28) and
383
were not greatly affected by CLA feeding, indicating that PPARα-dependent gene regulation
384
may not be affected by CLA. Interestingly, the level of PPARβ in liver was postprandially
385
reduced by CLA, which coincides with the observed reduction in mitochondrial β-oxidation
386
capacity. In addition, CLA down-regulated the post prandial PPARγ expression in liver and up-
387
regulated the basal expression of this isotype in adipose tissue. Given the proposed functions of
388
PPARγ in regulating fat accumulation (44), the observed decrease in whole body fat and
389
increase in liver fat in sea bream do not correlate with the CLA-induced expression profile of
390
PPARγ. However, it should be noted that these are gene expression studies and do not
391
necessarily bear any relation to functional protein levels. Therefore, our data do not provide
392
sufficient evidence that the CLA effects on sea bream are mediated through PPARs. In addition,
393
the higher order of complexity of PPAR biology in fish as compared to mammals must also be
394
considered, since the presence of multiple PPAR isoforms in fish species has been inferred
395
previously (28). Thus, it is possible that PPAR isoforms, additional to the ones assayed here,
396
may be functionally expressed in sea bream and more directly involved in the mediation of the
18
397
CLA effects.
398
In summary, the lipid lowering effect of CLA observed in sea bream juveniles may be a
399
combination of decreased feed intake, endocrine status, and diversion of dietary-derived TAG
400
from muscle and adipose tissue to liver and to increased hepatic peroxisomal β-oxidation. This
401
induction of peroxisomal β−oxidation suggests a subtoxic response to CLA which may have
402
unknown consequences for fish health. Thus, despite encouraging decreases in adiposity after
403
CLA feeding, the slightly negative growth effects and reduction in HUFA biosynthesis indicate
404
that inclusion of CLA in aquaculture diets would be of little benefit.
405
Accordingly, the use of CLA in fish diets would not be of general use to intensive sea bream
406
farming. However, its effects on physiology, in different developmental stages of the fish, need
407
to be further evaluated, since the potential of using fish fed on CLA as a functional food for
408
humans, would combine the lipid lowering properties of CLA with the health beneficial effects
409
of HUFA for which fish are a naturally rich source.
410 411
ACKNOWLEDGEMENTS
412 413
We are indebted to Mr Guillermo Borés, DVM, for help, advice and organization of the in vivo
414
growth trial.
415
19
415
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25
Table 1. Growth performance, body composition and nutrient utilization in gilthead sea bream fed the experimental diets for 12 wk1. Diets Initial body weight (g) Final body weight (g) Viscera weight (g) Liver weight (g) VSI (g/100 g body) 2 HSI (g/100g body) 3 Liver fat (g/100 g fish)
0% CLA 34.0 ± 0.10 144.0 ± 1.1 a 10.3 ± 1.4 2.5 ± 0.3 a 7.2 ± 0.69 1.78 ± 0.24 0.19 ± 0.10
2% CLA 34.1 ± 0.06 127.9 ± 1.5 b 9.7 ± 1.4 2.4 ± 0.3 ab 7.1 ± 0.69 1.80 ± 0.35 0.20 ± 0.10
4% CLA 34.0 ± 0.10 124.9 ± 2.3 b 9.2 ± 1.7 2.1 ± 0.3 b 7.2 ± 0.35 1.67 ± 0.17 0.24 ± 0.24
Feed intake (g/fish) SGR (%) 4 FE 5
113.1 ± 1.7 a 1.74 ± 0.01 a 0.96 ± 0.01 b
88.4 ± 0.28 b 1.59 ± 0.01 b 1.04 ± 0.01 a
89.4 ± 0.14 b 1.57 ± 0.03 b 1.00 ± 0.01 ab
Whole body composition6 (% wet matter) Moisture Crude protein Crude fat Ash
63.7 ± 0.42 17.4 ± 0.14 13.1 ± 0.01 a 4.5 ± 0.05
65.1 ± 0.14 16.4 ± 0.14 11.6 ± 0.28 b 4.8 ± 0.07
64.9 ± 0.71 16.8 ± 0.15 11.8 ± 0.14 b 4.4 ± 0.08
Retention (% intake) Nitrogen Lipid
33.9 ± 0.42 56.1 ± 0.71 a
34.3 ± 1.6 51.9 ± 1.1 b
33.7 ± 1.4 51.3 ± 1.6 b
1
Values are means ± SD, n = 4. Experimental unit is the tank (4 tanks). Means in a row without
a common letter differ, P < 0.05. 2
Viscerosomatic index = [100 × (viscera wt/ fish wt)].
3
Hepatosomatic index = [100 × (liver wt/ fish wt)].
4
Specific growth rate = [100 × (ln final fish wt − ln initial fish wt)] / days.
5
Feed efficiency = wet wt gain / dry feed intake.
6
Initial body composition (% wet matter) was: moisture, 69.9 ± 0.3; protein, 15.5 ± 0.5; lipid,
8.3 ± 0.2.
26
Table 2. Lipid class compositions of liver and white muscle from sea bream fed experimental diets1. Liver
Muscle
Diets
Diets
0% CLA
2% CLA
4% CLA
0% CLA
2% CLA
4% CLA
1.1 ± 0.2a
0.8 ± 0.1ab
0.6 ± 0.0b
0.8 ± 0.1b
1.8 ± 0.2a
1.7 ± 0.1a
PC
6.4 ± 0.4
6.4 ± 0.9
4.9 ± 0.7
11.7 ± 1.0b
16.1 ± 1.0a
14.4 ± 1.2a
PE
5.1 ± 0.1a
4.6 ± 0.6a
3.1 ± 0.29b
7.0 ± 0.5b
9.4 ± 0.8a
8.9 ± 0.3a
CHO
11.1 ± 0.4
10.4 ± 1.3
9.3 ± 0.6
8.8 ± 0.5b
9.9 ± 1.0a
9.7 ± 0.6ab
FFA
13.9 ± 1.7
12.7 ± 2.4
10.7 ± 1.6
2.8 ± 0.7
4.0 ± 0.8
4.4 ± 0.7
TAG
55.0 ± 5.5b
58.3 ± 5.6ab
67.4 ± 5.0a
67.0 ± 2.7a
52.2 ± 3.0b
54.5 ± 1.9b
Lipid class SM
1
Only are shown values for those lipid class that changed or differed between dietary
treatments. Values are means ± SD, n = 4. Experimental unit is the tank (4 tanks). Means in a row without a common letter differ, P < 0.05.
27
LEGENDS TO FIGURES
Figure 1. Effects of dietary CLA on sea bream hepatic enzyme activities. (A) glucose-6phosphate dehydrogenase, (B) acyl-CoA oxidase and (C) L-3-hydroxyacyl-CoA dehydrogenase. Values are means ± SD with individual fish being the experimental unit (n = 6, i.e. three fish from two replicate tanks corresponding to a particular dietary treatment). Means in a panel without a common letter differ, P < 0.05. * Different from 6h post-feeding P < 0.05
Figure 2. Effect of dietary CLA on mRNA expression of potential PPAR target genes in sea bream. (A) Expression of fatty acid elongase (ELO) and desaturase (DES) as assessed by RNase protection assay in liver [L], intestine [I], white muscle [M], and adipose tissue [A]. (B) Expression of ELO mRNA in liver in response to the three dietary treatments, as indicated. (C) Expression of ELO mRNA in intestine in response to the three dietary treatments, as indicated. (D) Expression of DES mRNA in liver in response to the three dietary treatments, as indicated. Relative mRNA expression was normalized to β-actin expression in each tissue. Standarization of mRNA abundance range in fish fed the control diet was set at 100. All values are means, with standard deviation, of three fish per dietary treatment. Means in a panel without a common letter differ, P < 0.05.
Figure 3. Effects of dietary CLA on mRNA levels of sea bream PPARs isotypes in (A) muscle, (B) liver and (C) adipose (mesenteral) tissues. mRNA levels were analyzed by quantitative realtime PCR using specific primers and probes. Values are arbitrary units relative to the reference (α-tubulin). Values are means, with standard deviation, of six fish per dietary treatment (three from each of two replicate tanks, n = 6). Means in a panel without a common letter differ, P < 0.05. Statistical comparison is made between the three diets at each time point. * Different from 6h post-feeding P < 0.05.
28
ONLINE SUPPORTING MATERIAL (OSM)
Supplementary Table 1. Fatty acid composition of experimental diets
0% CLA Fatty acids
2% CLA g/100g fatty acids
4% CLA
14:0 16:0 18:0 Total saturated 16:1(n-7) 18:1(n-9)
7.3 19.1 3.9 31.1 7.0 8.7
6.4 16.9 3.7 27.8 6.3 9.6
5.8 15.6 3.6 25.7 5.7 10.4
20:1 22:1 Total monounsaturated 18:2(n-6) 20:4(n-6) CLA (9c,11t)(*) CLA (10t,12c) (*) Total (n-6) PUFA 18:3(n-3) 20:5(n-3) 22:6(n-3) Total (n-3) PUFA
3.5 3.9 25.9 5.0 0.8 0.0 0.0 6.1 1.3 13.8 15.6 36.5
3.1 3.7 25.3 5.1 0.8 3.6 3.5 13.2 1.3 12.6 14.3 33.5
3.1 3.7 25.5 5.2 0.7 5.9 5.8 17.8 1.3 11.5 13.2 30.9
(*)The commercial CLA supplement, Tonalin®, contains around 4% (in total) of other CLA isomers (c9, c11 CLA, c10,c12 CLA, t9, t11 CLA and t10, t12 CLA) that can only be detected at trace levels.
29
Supplementary Table 2. Fatty acid composition of total lipids of liver and white muscle from sea bream fed the experimental diets 1.
Fatty acid
0% CLA
4% CLA
Liver Diets 2% CLA
0% CLA
14:0
5.0 ± 0.2
a
2.8 ± 0.4
b
2.8 ± 0.4
b
4.2 ± 0.1
a
3.7 ± 0.1
16:0
19.9 ± 0.4
a
17.0 ± 0.8
b
17.0 ± 0.9
b
18.3 ± 0.3
a
17.3 ± 0.8
18:0
4.6 ± 0.3
b
5.9 ± 0.4
a
5.9 ± 0.5
a
6.1 ± 0.2
b
9.9 ± 1.2
29.4 ± 0.3
4% CLA b ab a
4.4 ± 0.1 a 16.4 ± 0.6 b 7.1 ± 0.7 b
30.3 ± 0.9
a
26.3 ± 1.0
b
26.3 ± 1.7
b
16:1(n-7)
6.7 ± 0.2
a
4.5 ± 0.3
b
4.5 ± 0.1
b
6.5 ± 0.1
a
5.1 ± 0.1
c
5.7 ± 0.1 b
18:1(n-9)
12.9 ± 0.5
a
10.6 ± 0.3
b
11.0 ± 0.4
b
14.2 ± 1.0
a
11.8 ± 0.8
b
12.2 ± 0.5 b
Total saturated
20:1 22:1 Total monoenes
2.9 ± 0.1
2.7 ± 0.4
2.4 ± 0.1 28.5 ± 1.0
18:2(n-6)
4.4 ± 0.3
20:4(n-6)
0.9 ± 0.1
2.6 ± 0.4
2.1 ± 0.2 a
23.2 ± 0.5
2.5 ± 0.0
2.4 ± 0.3 b
4.4 ± 0.1
23.9 ± 0.6
28.7 ± 1.4
2.1 ± 0.5
2.4 ± 0.4 b
2.4 ± 0.4
1.7 ± 0.1
2.2 ± 0.1
29.7 ± 1.1
a
25.3 ± 1.3
b
26.3 ± 0.5 b
4.2 ± 0.1
b
4.3 ± 0.2
b
4.8 ± 0.2 a
4.4 ± 0.1
1.0 ± 0.1
31.7 ± 1.8
1.0 ± 0.0
1.0 ± 0.1
1.0 ± 0.0
0.8 ± 0.1
0.0 ± 0.0
b
2.6 ± 1.3
a
3.5 ± 0.7
a
0.0 ± 0.0
c
1.4 ± 0.3
b
CLA (10t,12c)
0.0 ± 0.0
b
2.7 ± 1.3
a
3.7 ± 0.9
a
0.0 ± 0.0
c
1.6 ± 0.0
b
3.0 ± 0.6 a
Total (n-6) PUFA
6.5 ± 0.3
b
12.0 ± 2.5
a
13.9 ± 1.6
a
6.5 ± 0.2
c
9.7 ± 0.3
b
13.4 ± 1.4 a
18:3(n-3)
1.1 ± 0.0
1.1 ± 0.0
1.0 ± 0.1
1.0 ± 0.0
b
1.0 ± 0.0
b
1.2 ± 0.1 a
18:4(n-3)
2.2 ± 0.1
2.3 ± 0.0
2.3 ± 0.0
1.7 ± 0.2
1.7 ± 0.2
20:4(n-3)
1.0 ± 0.0
1.0 ± 0.0
1.0 ± 0.1
1.5 ± 0.1
1.6 ± 0.0
CLA (9c,11t)
1
Muscle Diets 2% CLA
20:5(n-3)
10.2 ± 0.5
22:5(n-3)
3.0 ± 0.1
22:6(n-3)
17.0 ± 1.2
Total (n-3) PUFA
34.7 ± 1.9
Total PUFA
41.2 ± 1.9
10.4 ± 0.6
9.6 ± 0.7
3.2 ± 0.2 b
20.3 ± 1.0
2.9 ± 0.3 a
38.4 ± 1.9 b
50.4 ± 1.1
8.6 ± 0.4
18.9 ± 1.3
4.6 ± 0.6 ab
35.9 ± 2.3 a
49.7 ± 2.2
a
a
7.3 ± 0.3
1.6 ± 0.0 1.4 ± 0.1 b
5.1 ± 0.2
16.8 ± 0.4
3.6 ± 0.8 a
7.8 ± 0.4 b 4.2 ± 0.4
16.5 ± 0.2
15.4 ± 0.7
34.4 ± 0.7
a
33.3 ± 0.1
ab
31.6 ± 1.2 b
40.9 ± 0.9
b
43.0 ± 0.5
a
44.9 ± 1.1 a
Values are means ± SD, n = 4. Experimental unit is the tank (4 tanks). Means in a row without a common letter differ, P < 0.05.
30
Supplementary Table 3. Effect of diet on plasma levels of glucose, triglycerides and cholesterol in fish sampled at the end of the trial at 6 and 24 h after feeding1.
Diets
Sampling time (h)
Glucose (mg/dL)
Triglycerides (mmol/L)
Cholesterol (mg/dL)
0% CLA 2% CLA 4% CLA
6h
89.4 ± 10.1 94.4 ± 12.1 99.9 ± 14.1
14.5 ± 5.5a 9.4 ± 2.4b 8.8 ± 4.1b
345.1 ± 65.8 380.7 ± 58.9 338.5 ± 55.4
0% CLA 2% CLA 4% CLA
24 h
63.5 ± 6.2 63.1 ± 7.9 59.9 ± 5.2
4.4 ± 2.7 3.5 ± 0.7 3.7 ± 1.3
296.9 ± 62.2 334.4 ± 76.2 339.5 ± 40.5
0.571
