jussara (euterpe edulis mart.) supplementation during
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Jussara (Euterpe edulis Mart.) Supplementation During Pregnancy and LactationModulates the Uncoupling Protein 1 (UCP-1) and Inflammation Biomarkers Inducedby trans-Fatty Acids in the Brown Adipose Tissue of Offspring
Perla Pizzi Argentato, Carina Almeida Morais, Aline Boveto Santamarina, Helena deCássia César, Débora Estadella, Veridiana Vera de Rosso, Luciana Pellegrini Pisani
PII: S2352-9393(16)30028-8
DOI: 10.1016/j.yclnex.2016.12.002
Reference: YCLNEX 26
To appear in: Clinical Nutrition Experimental
Received Date: 2 October 2016
Revised Date: 19 December 2016
Accepted Date: 25 December 2016
Please cite this article as: Argentato PP, Morais CA, Santamarina AB, de Cássia César H, EstadellaD, de Rosso VV, Pisani LP, Jussara (Euterpe edulis Mart.) Supplementation During Pregnancy andLactation Modulates the Uncoupling Protein 1 (UCP-1) and Inflammation Biomarkers Induced bytrans-Fatty Acids in the Brown Adipose Tissue of Offspring, Clinical Nutrition Experimental (2017), doi:10.1016/j.yclnex.2016.12.002.
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Jussara (Euterpe edulis Mart.) Supplementation During Pregnancy and Lactation 1
Modulates the Uncoupling Protein 1 (UCP-1) and Inflammation Biomarkers 2
Induced by trans-Fatty Acids in the Brown Adipose Tissue of Offspring. 3
4
Perla Pizzi Argentatoa, Carina Almeida Moraisc, Aline Boveto Santamarinac, Helena de 5
Cássia Césarc, Débora Estadellab, Veridiana Vera de Rossob, Luciana Pellegrini Pisanib* 6
7 a Programa de Pós Graduação em Alimentos Nutrição e Saúde, Universidade Federal de 8
São Paulo (UNIFESP), Santos, SP, Brazil. 9 b Departamento de Biociências. Universidade Federal de São Paulo (UNIFESP), Santos-10
SP, Brazil. 11 c Programa Interdisciplinar em Ciências da Saúde. Universidade Federal de São Paulo 12
(UNIFESP), Santos-SP, Brazil. 13
14
*Correspondence author: Silva Jardim, 136. Laboratório 311, 3° andar, Vila Mathias, 15
Santos/SP, 11015020, Brazil. Tel./fax: +55 13 38783700. 16
E-mail: [email protected] (L.P. Pisani). 17
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Abstract 49
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Background & aims:The brown adipose tissue (BAT) regulates energy expenditure via 51
thermogenesis by uncoupling protein 1 (UPC-1). We investigated the effect of the 52
maternal diet enriched with trans fatty acids (TFAs) and/or supplemented with jussara 53
fruit on the 21d-old offspring. Specifically, we looked at the proinflammatory state and 54
the expression of UCP-1 in the offsprings’ BAT. Methods: We divided dams into four 55
groups during pregnancy and lactation: control diet (C), C diet with 0.5% of jussara 56
fruit (CJ), a diet enriched with TFAs (T), or T diet with 0.5% of jussara fruit (TJ). 57
Results: We found that TFAs reduced growth and increased weight, total cholesterol, 58
TNF-α, TNFRI and UCP-1 in BAT of pups. Conversely, maternal supplementation with 59
jussara preserved lean mass, decreased weight gain, carcass lipid, blood glucose and 60
triacylglycerol in the offsprings. It also increased IL-10 and UCP-1 levels in BAT. 61
Conclusions: Either TFAs and jussara fruit increased the expression of UCP-1 in BAT. 62
However, the TFAs are detrimental for the offsprings' health. We believe that the 63
bioactive compounds of jussara fruit helped to improve such parameters. Our results 64
showed that keeping the same maternal dietetic caloric amount but modifying the fatty 65
acids composition can program the BAT in offspring. Jussara fruit supplementation 66
could be used as an alternative treatment for obesity prevention. 67
Keywords: 1. Uncoupling Protein. 2. Brown adipose tissue. 3. Programming. 4. 68
Jussara. 5. Anthocyanins. 6. Trans-fatty acids. 69
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Abbreviations: Brown adipose tissue (BAT), uncoupling protein 1 (UPC-1), trans-fatty 78
acids (TFAs), tumor necrosis factor-α (TNF-α), tumor necrosis factor receptor 1 79
(TNFRI), nuclear transcription factor kappab phosphorylated 50 (NF-�Bp50), toll-like 80
receptor 4 (TLR-4), interleukin 6 (IL-6), interleukin 10 (IL-10), body mass index 81
(BMI), triacylglycerol (TAG), total cholesterol (TC), high-density lipoprotein (HDL) 82
cholesterol. 83
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1. Introduction 84
Inadequate maternal nutrition during pregnancy and lactation is supposed to 85
cause epigenetic changes during the fetal development and neonatal period [1–3]. This 86
process, known as metabolic programming or metabolic imprinting, can alter gene 87
expression and affect the structure and function of tissues or organs permanently by 88
increasing the susceptibility of the individual to the development of chronic diseases 89
such as obesity [4,5]. 90
Maternal dietary fat composition and amount are the most important 91
determinants of the degree and type of fatty acids transferred to the fetus and the infant 92
through the placenta or maternal milk [6]. 93
In this regard, the maternal intake of hydrogenated vegetable fat during 94
pregnancy and lactation increases the tumor necrosis factor-α (TNF-α) mRNA, 95
plasminogen activator inhibitor- 1 (PAI-1) mRNA, and TNF receptor-associated factor-96
6 (TRAF-6) protein in the adipose tissue of 21-day-old offspring [7,8]. Moreover, it has 97
shown to increase PAI-1 mRNA [8], serum endotoxin levels, subunit p65 of nuclear 98
transcription factor Kappa-B (NF-�Bp65), toll-like receptor 4 (TLR-4), and myeloid 99
differentiation primary response 88 (MyD88) protein expression in the adipose tissue. 100
And also, it increases hypothalamic interleukin 6 (IL-6), TNF-�, and IL-1� in the adult 101
offspring [9]. Furthermore, these pro-inflammatory changes were accompanied by 102
accrued weight and adiposity. 103
Although weight gain and obesity have multifactorial etiology, a positive energy 104
balance is one of the determinants of weight and adiposity gain [10]. Though, the 105
regulation of energy expenditure occurs, in part, in the brown adipose tissue (BAT) 106
through thermogenesis and it is mediated by uncoupling protein 1 (UPC-1)[11]. 107
Recently, BAT was recognized in adults and it inversely correlated with body mass 108
index (BMI) [12]. It also showed beneficial effects on the glucose and lipid metabolism 109
[13]. Additionally, the BAT responds to external stimulus, as well as, age, sex, genetic 110
traits and diet [14,15]. 111
Foods rich in bioactive compounds such as polyphenols, especially flavonoids, 112
have been identified as a promising buffer against inflammation and oxidative stress 113
[16–19]. It is known that bioactive food compounds can cross the placenta and reach 114
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fetal tissues [20]. Many studies have shown that polyphenols can exert metabolic 115
programming effects on the offspring through maternal intake [21–29]. 116
Dams treated with a high-fat diet and extracts rich in anthocyanins, a potent 117
polyphenol, during lactation protected their descendants, either male and female, against 118
oxidative stress, body fat gain, hypertriglyceridemia and insulin resistance in adulthood 119
[24,25]. The same beneficial effects were also observed after polyphenol 120
supplementation during lactation in male offspring of dams fed a protein restricted diet 121
during pregnancy. These animals had decreased body weight, hepatic triacylglycerol 122
levels and increased adiponectin levels [26]. 123
Our research group has been studying the fruit of jussara palm (Euterpe edulis 124
Mart.), which is rich in anthocyanins and native species of the Atlantic 125
Rainforest/Brazil [30]. Our research has shown promising effects of jussara fruit 126
supplementation on the metabolic programming of the colon, hypothalamus and white 127
adipose tissues in the offspring [31,32]. However, studies on the impact of bioactive 128
food compound supplementation on the programming models of BAT are less common 129
in the literature. 130
Thus, the aim of this study was to evaluate the effect of dietary supplementation 131
with 0,5% of jussara fruit pulp during pregnancy and lactation in the presence or 132
absence of hydrogenated vegetable fat and the UCP-1 expression and proinflammatory 133
state in BAT of 21-day-old male offspring. 134
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2. Materials and Methods 136
137
2.1. Animals and Treatments 138
139
All experimental procedures were approved by the Experimental Research 140
Committee at Federal University of São Paulo (CEUA protocol n°5252010715). Rats 141
were kept under controlled conditions of light (12:12h light-dark cycle with lights on at 142
07:00) and temperature (24 ± 1oC), with ad libitum water and food. 143
Twelve-week-old female Wistar rats of first-order parity were allocated 144
overnight to breeding. Copulation was verified the following morning by the presence 145
of sperm in vaginal smears. On the first day of gestation, rats were isolated in individual 146
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cages and randomly assigned to one of the four groups receiving a control diet (C 147
group), a control diet supplemented with jussara 0.5% freeze-dried powder (CJ group), 148
a diet enriched with hydrogenated vegetable fat (T group), or a T diet supplemented 149
with 0.5% jussara freeze-dried powder (TJ group). 150
The diets were prepared according to the recommendations of the American 151
Institute of Nutrition (AIN-93G) [33,34] and had similar caloric and lipid contents. The 152
fat source for the C and CJ groups was soybean oil; the main fat source for the T and TJ 153
groups was hydrogenated vegetable fat, rich in trans-fatty acids (TFAs). The CJ and TJ 154
groups were prepared by adding 0,5% of jussara freeze-dried powder to each diet. 155
Jussara pulp (Euterpe edulis Mart.) was obtained from the agroecological Project 156
Jussara/IPEMA - Institute of Permaculture and Ecovillages of the Atlantic (Ubatuba, 157
SP, Brazil) and then freeze-dried to powder using a lyophilizer. Diets were then stored 158
at -20°C. The phenolic compounds and anthocyanin contents of the jussara pulp were 159
previously analyzed in our laboratory [30]. The total levels of anthocyanin, phenolic 160
compounds and the concentrations of their major constituents are shown in Table 1. The 161
centesimal composition of the diets is presented in Table 2. The fatty acid profile of C 162
and T diets was previously described by Pisani et al., 2008 [8]. 163
Dams’ diets were maintained during pregnancy and lactation. After birth, litter 164
sizes were adjusted to eight pups for each mother. The pups were weighed and 165
measured (nasoanal length) at birth and on postnatal days 7, 14, and 21. In the 21st day 166
of life, offsprings were decapitated; trunk blood was collected and centrifuged. Serum 167
was separated and stored at -80°C for later determination of the triacylglycerol (TAG), 168
total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, glucose, and 169
adiponectin. The BAT was removed from the subscapularis region, isolated and stored 170
at -80°C. 171
172
Table 1: Phenolic compounds detected in jussara pulp. 173
Phenolic compound Concentration (mg/100 g fresh matter) Cyanidin 3-rutinoside 191.0 ± 6.5 Cyanidin 3-glucoside 71.4 ± 2.1 Total anthocyanins 262.4 ± 8.6
Apigenin deoxyhexosyl-hexoside 25.4 ± 1.5 Luteolin deoxyhexosyl-hexoside 37.6 ± 1.9
Dihydrokaempferol-hexoside 66.4 ± 2.6 Total phenolics compounds 415.1 ± 22.3
174
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175
Table 2: The composition of the control diet (C), control diet supplemented with 0.5% 176
freeze-dried jussara powder (CJ), diet enriched with hydrogenated vegetable fat, TFAs 177
(T), and diet enriched with TFAs supplemented with 0.5% freeze-dried jussara powder 178
(TJ) according to AIN-93. 179
180
Diet (g/100 g) Ingredient C CJ T TJ Caseina* 20.0 20.0 20.0 20.0 L-cystine 0.3 0.3 0.3 0.3
Cornstarch 62.0 62.0 62.0 62.0
Soybean oil 8.0 8.0 1.0 1.0
Hydrogenated vegetable fat$ – – 7.0 7.0 Butylhydroquinone 0.0014 0.0014 0.0014 0.0014
Mineral mixture§ 3.5 3.5 3.5 3.5
Vitamin mixture# 1.0 1.0 1.0 1.0 Cellulose 5.0 5.0 5.0 5.0
Choline bitartrate 0.25 0.25 0.25
Freeze-dried jussara powder – 0.5 – 0.5
Energy (kcal/g) 4.00 4.02 4.00 4.02 181
*Casein was obtained from Labsynth, São Paulo, Brazil. 182
L-cystine, cornstarch, butylhydroquinone, cellulose and choline bitartrate were obtained from 183
Viafarma, São Paulo, Brazil. 184
Soybean Oil was supplied from (Lisa/Ind. Brazil). 185 $Hydrogenated vegetable fat was supplied from Unilever, São Paulo, Brazil. 186
§§§§Mineral mix (9mg/kg diet): calcium, 5000; phosphorus, 1561; potassium, 3600; sodium, 1019; 187
chloride, 1571; sulfur, 300; magnesium, 507; iron, 35; copper, 6.0; manganese, 10.0; zinc, 30.0; 188
chromium, 1.0; iodine 0.2; selenium, 0.15; fluoride, 1.00; boron, 0.50; molybdenum, 0.15; 189
silicon, 5.0; nickel, 0.5; lithium, 0.1; vanadium, 0.1 (AIN-93G, mineral mix, Rhoster, Brazil). 190 #Vitamin mix (mg/kg diet): thiamin HCL, 6.0, riboflavin, 6.0; pyridoxine HCL, 7.0; niacin, 191
30.0; calcium pantothenate, 16.0; folic acid, 2.0; biotin, 0.2; vitamin B12, 25.0; vitamin A 192
palmitate 4000 IU; vitamin E acetate, 75; vitamin D3, 1000 IU; vitamin KI, 0.75 (AIN-93G, 193
vitamin mix, Rhoster, Brazil). 194
Freeze-dried jussara powder: jussara pulp (Euterpe edulis Mart.) was obtained from 195
agroecological Project Jussara/IPEMA - Institute of Permaculture and Ecovillages of the 196
Atlantic (Ubatuba, SP, Brazil) - and by freeze-drying to powder using a lyophilizer. 197
198
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2.2. Biochemical Serum Analysis 200
201
Glucose, TAG, TC and HDL-cholesterol serum concentrations were measured 202
with an enzymatic colorimetric method using commercial kits (Labtest Brazil). 203
Serumadiponectin concentration was analyzed by ELISA using the DuoSet kit Mouse 204
Adiponectin / Acrp30 (R & D Systems, Minneapolis, MN, USA). 205
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206
2.3. Carcass Lipid and Protein Contents 207
208
The carcasses were eviscerated, and the remnants were weighed and stored at 209
20 C. The lipid content was measured as described by Stansbie et al. 1976 [35] and 210
standardized using the method described by Oller Do Nascimento and Williamson [36]. 211
The carcass was autoclaved at 120C for 90 min and homogenized with water at a 212
volume twice the carcass mass. Triplicate aliquots of approximately 3 g were digested 213
in 3mL of 30% KOH and 3mL of ethanol for 2h at 70°C in capped tubes. After 214
cooling, 2 mL of 12NH2SO4 was added, and the samples were washed three times with 215
petroleum ether to extract the lipids. 216
The results are expressed in grams of lipid per 100 g of the carcass. To measure 217
the protein content, aliquots of the same homogenate, approximately 1 g, were heated at 218
37°C for 1 h in 0.6NKOH with constant shaking. After clarification by centrifugation, 219
protein content was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA) 220
with bovine serum albumin as a reference. 221
222
2.4. Brown adipose tissue TNF-α, IL-6, and IL-10 Levels by ELISA. 223
224
The BAT was homogenized and centrifuged at 12,000 rpm for 40min at 4°C; the 225
supernatant was saved and the protein concentration determined using the BCA assay 226
(Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as a reference. 227
Quantitative assessment of TNF-α, IL-6, and IL-10 proteins was carried out by ELISA 228
(DuoSet ELISA, R&D Systems, Minneapolis, MN, USA) following the 229
recommendations of the manufacturer. 230
231
2.5. Brown adipose tissue UCP-1, TNFRI and p-NF�Bp50 by Western Blotting 232
233
The brown adipose tissue was removed and placed in the extraction buffer (100 234
mM Trizma base pH 7.5, 20 mM EDTA, 100 mM sodium fluoride, 100 mM sodium 235
pyrophosphate, 10 mM sodium orthovanadate, 2 mM PMSFphenylmethylsulfonyl 236
fluoride and 0.1 mg of aprotinin per mL). 237
The total protein content was determined by the Bradford method using the Bio-238
Rad reagent (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as the reference. 239
The samples were treated with the LDS Sample Buffer and Reducing Agent (Life 240
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Technologies). The proteins (100 µg) were heated for 10 min before loading onto the 241
Bolt 4-12% Bis-Tris Plus in a Bolt mini gel tank (Novex, Life Technologies, CA, USA). 242
Electrotransfer of proteins from the gel to the nitrocellulose membrane was 243
performed for 7 min/2 gels at 20 V for 1 min, 23 V for 4 min and 25 V for the 244
remainder in an Iblot 2 gel transfer device (Life Technologies, CA, USA). Nonspecific 245
protein binding to the nitrocellulose membrane was reduced by preincubation at 22 °C 246
in blocking buffer containing 1% BSA. The nitrocellulose membranes were incubated 247
overnight at 15°C with antibodies against UCP-1, TNFRI (ABCAM, MA, USA) and the 248
phosphorylated form of p-NF�Bp50 (Santa Cruz Biotechnology, CA, USA). The 249
antibodies were diluted 1:1000 with blocking buffer. The blots were subsequently 250
incubated with a peroxidase-conjugated secondary antibody (ABCAM, MA, USA) for 1 251
h at 15°C. 252
Specific bands were detected by chemiluminescence using Alliance 4.7 253
equipment (Uvitec, Cambridge, UK). The band intensity was quantified by optical 254
densitometry (Scion Image-Release Beta 3b, NIH, USA). The signals were normalized 255
to β-actin (ABCAM, MA, USA). 256
257
2.6. Statistical Analysis 258
259
Statistical analyses were performed using the software SPSS version 22. Data 260
were submitted to the quality tests Shapiro-Wilk (normality), Levenne (homogeneity) 261
and/or Mauchly (sphericity). If necessary, data were standardized to Z score. To verify 262
the interactions between groups, we used two-way ANOVA analysis followed by a 263
Bonferroni posthoc test. All results are presented as the means ± SEM (standard error 264
mean) and p < 0.05 was considered statistically significant. 265
266
3. Results 267
268
3.1. Body Weight, Body Weight Gain, Length of the Animal 269
270
21-day-old offsprings from TJ group had higher body weight at birth (p = 0.013) 271
compared to the CJ group. All animals showed similar length at birth. In the first week 272
of treatment, TJ was heavier than CJ group (p = 0.0001). Similarly, length size followed 273
the same pattern, and TJ had higher length than T group (p = 0.01). In the following 274
week, the TJ group maintained higher body weight (TJ > CJ, p = 0.0001), and length 275
(TJ > T, p = 0.01). In the last week, CJ had lower weight gain compared to C group (p = 276
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0.01) and T group had a smaller increase in length than the C group (p = 0.0001) and TJ 277
group (p = 0.01) (Figure 1A and B). 278
Regarding weekly weight gain, in the first week of treatment, T group gained 279
more weight than C group (p = 0.023), the same happened to TJ group compared to CJ 280
group (p = 0.001). In the last week of treatment, TJ group showed lower weight gain 281
compared to CJ group (p = 0.032) (Figure 1C). About total weight gain, the offspring of 282
the CJ group had smaller weight gain than C group (p = 0.001) (Figure 1D). 283
284
285
Figure 1: (A) Body weight, (B) Length of the animal, (C) Body weight gain, (D) Total weight 286
gain in 21-day-old offspring. 287
C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 288
0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated 289
vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 290
0.5% freeze-dried Jussara powder. The number in parentheses refers to the sample size. *p < 291
0.05 versus C. $p < 0.05 versus CJ. #p < 0.05 versus T. &p < 0.05 versus TJ. 292
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3.2. Carcass Lipid and Protein Contents 295
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The carcass lipid content was lower in the offspring from the CJ and T group 297
compared with C group (p = 0.0001 and p = 0.008, respectively) (Figure 2A). The 298
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protein content was lower in T group compared to C group (p = 0.016) and TJ group (p 299
= 0.004) (Figure 2B). The carcass lipid and protein ratio was higher in the offspring 300
from T group than C group (p = 0.0001) and TJ group (p = 0.0001) (Figure 2C). 301
302
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Figure 2: (A) Carcass lipid, (B) Protein content, and (C) Lipid/Protein ratio in 21-day-old 304
offspring. 305
C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 306
0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated 307
vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 308
0.5% freeze-dried Jussara powder. The number in parentheses refers to the sample size. *p < 309
0.05 versus C. $p < 0.05 versus CJ. #p < 0.05 versus T. &p < 0.05 versus TJ. 310
311
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3.3. Weight of Brown adipose tissue 313
314
Regards to relative weight of the BAT, there was no significant difference 315
between the groups (Table 3). 316
317
Table 3. Weight of brown adipose tissue of 21-day-old pups. 318
Brown adipose tissue C (9) CJ (11) T (11) TJ (13) Relative weight (g/100 g
body weight) 0,40 ± 0,01 0,38 ± 0,02 0,33 ± 0,01 0,35 ± 0,01
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C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 319
0.5% freeze-dried jussara powder; T: offspring of dams fed diet enriched with hydrogenated 320
vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 321
0.5% freeze-dried jussara powder. The number in parentheses refers to the sample size. 322
323
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3.4. Serum Biochemical Analyses 325
326
Serum glucose concentration of the 21-day-old offspring in TJ group was lower 327
compared to the T group (p = 0.011). The total cholesterol was higher in the T group 328
compared to C group (p = 0.003) and TJ group compared CJ group (p = 0.0001). Serum 329
triacylglycerol concentration was higher in T group compared to C group (p = 0.04) and 330
TJ group (p = 0.0001). However, no differences were seen in the serum HDL-331
cholesterol and adiponectin concentrations among groups. 332
333
Table 4: Serum glucose, total cholesterol, HDL-cholesterol, triacylglycerols and 334
adiponectin in 21-day-old offspring. 335
Parameters C (10) CJ (10) T (10) TJ (10) Glucose(mg/dL) 112,52±3,56 108,44±5,98 115,70±4,13 95,59±6,90#
Total cholesterol (mg/dL) 93,67±4,93 84,85±3,42 112,55±2,98* 107,9±4,80$ HDL-cholesterol (mg/dL) 30,91±0,91 29,50±1,17 31,09±0,80 32,06±1,31 Triacylglycerols (mg/dL) 97,3±5,4 92,2±12,1 123,5±8,1* 73,1±7,7#
Adiponectin (µg/mL)
2,33±3,38
2,42±2,95
2,10±1,61
2,21±1,92
C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 336
0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated 337
vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 338
0.5% freeze-dried jussara powder. The number in parentheses refers to the sample size. *p < 339
0.05 versus C. $p < 0.05 versus CJ. #p < 0.05 versus T. &p < 0.05 versus TJ. 340
341
342
3.5. Brown adipose tissue TNF-α, IL-6 and IL-10 level 343
344
The results showed an increased level of TNF-α protein in the BAT of the T 345
group compared with C group (p = 0.007) and, also in TJ group compared with T group 346
(p = 0.004) and CJ group (p = 0.0001) (Figure 3A). For the anti-inflammatory cytokine 347
IL-10, there was a higher level in the CJ group than C group (p = 0.0001) and TJ group 348
(p = 0.001), the same happened in the offspring from T group compared to C group (p = 349
0.021) (Figure 3B). However, the cytokine IL-6 remained unchanged between groups 350
(Figure 3C). Regarding IL-10/TNF-α ratio, the CJ group had a higher ratio compared 351
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to the TJ group (p = 0.004) the reverse was found in T group compared C group (p = 352
0.049) (Figure 3D). 353
354
355
Figure 3: (A) IL-6 protein expression, (B) IL-10, (C) TNF-�, and (D) IL-10/TNF-� ratio in 356
21-day-old offspring at the brown adipose tissue. 357
C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 358
0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated 359
vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 360
0.5% freeze-dried Jussara powder. The number in parentheses refers to the sample size. *p < 361
0.05 versus C. $p < 0.05 versus CJ. #p < 0.05 versus T. &p < 0.05 versus TJ. 362
363
3.6. Phosphorylated NF�Bp50 subunit, TNFRI and UCP-1 in brown adipose tissue 364
365
UCP-1 levels in brown adipose tissue of the 21-day-old offspring were higher in 366
the TJ group compared to the T group (p = 0.047) and CJ (p = 0.009) (Figure 4A). The 367
levels of p-NF�Bp50 remained unchanged between the groups (Figure 4B). 368
Furthermore, the TNFRI protein levels were significantly lower in the CJ group 369
compared to TJ group (p = 0.041) (Figure 4C). 370
371
372
373
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374
Figure 4. (A) UCP-1 protein expression in brown adipose tissue, (B) Protein expression of the 375
phosphorylated form p-NF�Bp50 in brown adipose tissue and (C) TNFRI protein expression in 376
brown adipose tissue. 377
C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 378
0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated 379
vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 380
0.5% freeze-dried Jussara powder. The number in parentheses refers to the sample value (Fig. 381
4A: T and TJ group presented one outlier sample). Data are means ± SEMs. Results are 382
expressed in arbitrary units, stipulating 100 as the control value. *p < 0.05 versus C. $p < 0.05 383
versus CJ. #p < 0.05 versus T. &p < 0.05 versus TJ. 384
385
4. Discussion 386
387
There are many studies about fetal programming and polyphenols intake 388
[22,28,29] but only a few have determined the effect of their consumption, in 389
physiological doses, on the UCP-1 expression in the offspring. Here we evaluated the 390
role of a highly nutritive fruit and its interaction with the weight control mechanisms 391
by studying the effect of food in the BAT and on fetal programming model with 392
hydrogenated vegetable fat. 393
Thus, we found that hydrogenated vegetable fat supplementation increased birth 394
weight, and this event continued in the first weeks of lactation (Fig. 1A and 1C). Souza 395
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et al. 2012 using 6% of hydrogenated vegetable fat during pregnancy and lactation 396
found increased body weight of male offspring on the 7th and 14th day of life [37]. 397
Already, Bishop et al., 2015 using hydrogenated vegetable oil, soybean oil and palm oil 398
on a programming model found no change in the body weight of offsprings; it might 399
be because they used 90-day old rats [38]. On the other hand, the authors showed that a 400
better fatty acid profile such as fish oil as the main dietary fat source during pregnancy 401
and lactation was associated with decreased body weight from birth up to the 12th 402
week of life [39]. 403
We found that maternal jussara supplementation reduced body weight gain (Fig. 404
1A and 1C) and had a protective role against stunting. According to our findings, we 405
found the stunted growth of offpring from T group during the first and second week of 406
treatment, and jussara fruit supplementation has inhibited this effect in the TJ group. 407
(Fig. 1B). In addition, jussara supplementation reduced fat carcass deposits (Fig. 2A) 408
and prevented muscle wasting (Fig. 2B and 2C). Wu et al., 2014 treated rodents with a 409
high-fat diet and 40 and 200 mg / kg of body weight with anthocyanins isolated from 410
cherries for 12 weeks, which showed reduced weight gain by 5.2% and 11.2% 411
respectively [19]. Similarly, Mukai et al., 2013 found that rats fed a protein restricted 412
diet during pregnancy and supplemented with Azuki bean anthocyanins during lactation 413
had lasting beneficial effects on reducing the weight of 23 weeks old offsprings [26]. 414
Dolinoy et al., 2006, found the same results in mice supplemented with genistein, the 415
main soy phytoestrogen, administered during pregnancy and lactation in doses of 250 416
mg/ kg of diet [21]. As with doses of 5 mg/kg of diet in rats, according to Ball et al., 417
2010 [23]. An in vitro study elucidated the role of anthocyanins on weight reducing and 418
its anti-obesogenic properties, through its involvement in suppressing lipid 419
accumulation in the adipocytes by inhibiting transcription factors that regulate 420
lipogenesis [40]. 421
Some authors argue that the beneficial effects of a fruit similar to jussara are due 422
to not just its anthocyanin content itself, but its nutritional characteristics [41,42]. It is 423
known that jussara has high levels of fiber, 28.3 ± 0.3g/100g dry basis. It is rich in 424
anthocyanins, 262.4 ± 8.6 mg/100g wet basis, especially Cyanidin 3-rutinoside and 425
Cyanidin 3-glucoside [30] and can be an excellent dietary source of essential fatty acids. 426
The oil extracted from the jussara pulp has about 36% oleic acid (monounsaturated fatty 427
acid - MUFA) and 19% linoleic (omega-6 polyunsaturated fatty acid - n-6 PUFA) (Silva 428
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et al., 2013). The literature reveals that dietary fibers exert glycemic control, and 429
improve lipid profile by reducing intestinal absorption of carbohydrates and cholesterol, 430
decrease gastric emptying, and insulin secretion and promote the production of short-431
chain fatty acids in the colon [44]. PUFAs have shown a benefit in attenuating TAG by 432
reducing hepatic lipogenesis [45]. Besides, the polyphenols are associated with 433
increased tissue glucose uptake [46], lower cholesterol absorption and synthesis, and 434
increased gene expression that favors cholesterol control [42,47]. Jussara 435
supplementation was effective in lowering blood glucose and triacylglycerol levels in 436
the offspring (Table 4). There is evidence that phenolic compounds in fruit, especially 437
flavonoids can induce glucose transporter type 4 (GLUT4) in adipose tissue and skeletal 438
muscle, and to contribute to the glucose homeostasis. Furthermore, anthocyanins may 439
activate the AMP-activated protein kinase (AMPK) engaged in increasing skeletal 440
muscle glucose uptake, reducing lipogenesis, promoting lipolysis and reducing 441
cholesterol syntesis [46]. In agreement with our results, treatment with açai extract in 442
male rabbits fed a diet enriched with 0.5% cholesterol for 12 weeks was effective in 443
reducing triacylglycerol levels [48]. Emiliano et al., 2011 and Resende et al., 2013 gave 444
grape skin extract to rats fed a high fat diet during lactation, and they found that a dose 445
of 200mg/kg/day protected male and female offsprings from hypertriglyceridemia and 446
enhanced glucose metabolism in adulthood [24,25]. Research with human subjects 447
getting 100 mg of açaí pulp, which is a fruit similar to jussara, twice a day for 1 month, 448
showed favorable results in improving blood glucose levels in overweight individuals 449
[41]. Our research took into account the dose of jussara consumed by human subjects. 450
Thus, supplementation with 0.5% lyophilized jussara corresponds to 3.3 mg 451
anthocyanins/kg/day, and it can be obtained by a human consumption of 100g of fresh 452
pulp or 10g of jussara freeze-dried powder per day. 453
Regarding endocrine function, the secretory role of brown adipose tissue is 454
poorly understood in the literature [49]. In general, BAT has lower cytokines levels than 455
white adipose tissue, possibly due to the proinflammatory phenotype of immune cells 456
that infiltrate the white adipose tissue [50]. However, in obesity, proinflammatory 457
cytokines such as TNF-α were found to recruit macrophages in the BAT [51,52]. Our 458
study showed that trans fat supplementation increases TNF-α levels in BAT of 21-day-459
old offspring. It is well described in the literature that saturated fatty acids and trans 460
fatty acids correlate with increased low grade inflammation [53,54]. They activate toll-461
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like receptor 4 (TLR4) pathway, which activates the NFκB dimers of NFκBp50 or 462
NFκBp65. The NFκBp50 translocation to the nucleus results in the induction of gene 463
expression of proinflammatory cytokines such as TNF-α [55]. Already, jussara 464
supplementation in the maternal diet increased the anti-inflammatory cytokine IL-10 in 465
CJ group. Surprisingly, this cytokine appeared increased in the T group; this may be due 466
to an anti-inflammatory reaction of the animal to counterbalance the increased level of 467
TNF-α (Fig. 3B). Indeed, foods rich in anthocyanins are described in the literature as 468
having a potential anti-inflammatory action [18,28,42]. A study revealed that 469
supplementation with 40 and 200 mg/kg of anthocyanins isolated from cherries, for 12 470
weeks, can attenuate gene expression of TNF and other pro-inflammatory genes in 471
rodent treated with a high-fat diet [19]. Graf et al., 2013 showed that anthocyanins have 472
anti-inflammatory action by inhibiting the translocation of NFkB to the nucleus [42]. 473
However, we haven’t found changes in the expression of phosphorylated transcription 474
factor NFκBp50 with jussara supplementation (Fig. 4B). 475
It has been described that TNF-α has an anti-thermogenic effect in obesity [56]. 476
This reduction in the thermogenesis was evidenced by low doses of intraperitoneal 477
TNF-α administration [57]. Romanatto et al., 2009 discovered that diet-induced obesity 478
can be prevented by increasing thermogenesis through rising UCP-1 expression in BAT 479
of 8 week old male TNFR1 knockout rats (TNFR1 knockout) [58]. However, these 480
studies did not take into account the inflammation in the BAT itself. We found 481
increased tumor necrosis factor receptor 1 (TNFRI) expression in BAT of offsprings 482
exposed to maternal hydrogenated vegetable fat supplementation (Fig. 4C) and, unlike 483
the above studies, this event was associated with less weight gain in this group by the 484
end of the experiment. The same happened in regards to the UCP-1 expression in this 485
group (TJ > CJ, Fig. 4B), which it would explain the weight reduction observed, likely 486
by increased thermogenesis via UCP-1. Although jussara activated thermogenesis via 487
increased UCP-1, it did not reduce inflammation in the offsprings which had TFA. It 488
could be that it required extra time of treatment exposure to see an overt anti-489
inflammatory effect in the BAT. Considering that the antioxidant action occurs in 490
inflamed individuals and not at absence of this stimulus [59,60]. Corroborating our 491
results, administration of black soybean seed coat extract, rich in 3-glucoside cyanidin 492
(9.2%), catechins (6.2%) and procyanidins (39.8%) for 14 weeks reduced body weight 493
gain and increased UCP-1 protein expression in BAT of animals challenged with a 494
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high-fat diet [61]. We also found an increase in the UCP-1 expression after jussara 495
supplementation (TJ > T, Fig. 4A). 496
The mechanisms by which polyphenols influence thermogenesis are not 497
understood. However, as we found, supplementation of 4 g/kg diet/day with resveratrol, 498
a phenolic antioxidant, for eight weeks increased the UCP-1 levels without changing 499
the relative weight of BAT of 4 weeks old mice [62]. It is less frequent in the literature 500
programming studies investigating the relation of bioactive food compounds, isocaloric 501
and normolipidic diets, differing only from their type of dietary fatty acids, and their 502
impact on the thermogenic parameters. It is estimated that about 60g of BAT can 503
contribute up to 20% of total daily heat production in humans [63]. The UCP-1 504
mechanism of action is through decoupling the transport of protons in the 505
mitochondria and to release energy as heat from the oxidation of fatty acids that have 506
not been coupled to the production of adenosine triphosphate (ATP) [11]. It is known 507
that the degree of UCP-1 activation varies with the availability and the flow of fatty 508
acids within the cells [64]. One of our hypothesis is that the high fat content of jussara, 509
comprised mainly of oleic acid, palmitic and linoleic with the additional dietary lipids 510
included in the diets, contributed to increase the UCP-1 expression in BAT of TJ group. 511
In that sense, Priego et al., 2013 found that better fatty acid profile, such as the oleic 512
acid present in large quantities in olive oil can increase UCP-1 in the BAT of males and 513
females 21 day old offsprings [65]. 514
Therefore, we demonstrated that jussara supplementation during pregnancy and 515
lactation can be a natural way to enhance the expression of UCP-1 in BAT and to 516
improve body composition in the 21- day-old offspring. 517
518
5. Conclusion 519
In summary, we show that maternal supplementation with TFAs increased 520
weight, TNF-α, RITNF and UCP-1 levels in BAT of 21-day-old offspring. Besides, 521
maternal diet supplementation with 0.5% of jussara during pregnancy and lactation 522
reduced weight gain and fat carcass deposits. It further protected against the stunted 523
growth and prevented lean mass loss, decreased glucose and triacylglycerol levels, and 524
increased the anti-inflammatory cytokine IL-10 levels and UCP-1 expression in BAT. 525
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Coincidentally, the animals that lost the greatest amount of weight at the end of the 526
treatment had higher UCP-1 levels in BAT. 527
Therefore, either TFAs and jussara fruit increased the expression of UCP-1 in 528
BAT. However, the TFAs are detrimental for the body composition, the metabolic and 529
the inflammatory parameters. We believe that the bioactive compounds of jussara fruit 530
helped to improve such parameters. Our results showed that keeping the same caloric 531
amount of the maternal diet but modifying its quality by adding a natural food in 532
physiological doses can program the BAT of the offspring. Thus, jussara fruit 533
supplementation can be considered an alternative therapy for the prevention of the 534
development of chronic diseases in adulthood, such as obesity (Fig. 5). 535
536
Figure 5. TFAs supplementation and with jussara 0.5% freeze-dried powder on BAT 21-day-537
old offspring. (Fatty acids and cianidinas - from FreeDigitalPhotos.net). 538
539
540
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Conflict of Interests 541
The authors declare that they have no conflict of interests regarding the 542
publication of this paper. 543
544
Acknowledgements 545
This research was supported by FAPESP (Fundação de Amparo à Pesquisa do 546
Estado de São Paulo) number 2015/02602-3. We are grateful to this Institution. 547
548
549
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