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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http://dx.doi.org/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

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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

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*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

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2.1. Animals and Treatments 138

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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

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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

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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

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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

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2.2. Biochemical Serum Analysis 200

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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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2.3. Carcass Lipid and Protein Contents 207

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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

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2.4. Brown adipose tissue TNF-α, IL-6, and IL-10 Levels by ELISA. 223

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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

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2.5. Brown adipose tissue UCP-1, TNFRI and p-NF�Bp50 by Western Blotting 232

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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

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2.6. Statistical Analysis 258

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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

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3. Results 267

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3.1. Body Weight, Body Weight Gain, Length of the Animal 269

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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

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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

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Figure 2: (A) Carcass lipid, (B) Protein content, and (C) Lipid/Protein ratio in 21-day-old 304

offspring. 305






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3.3. Weight of Brown adipose tissue 313

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Regards to relative weight of the BAT, there was no significant difference 315

between the groups (Table 3). 316

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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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0.5% freeze-dried jussara powder; T: offspring of dams fed diet enriched with hydrogenated 320


0.5% freeze-dried jussara powder. The number in parentheses refers to the sample size. 322

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3.4. Serum Biochemical Analyses 325

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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

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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




0.5% freeze-dried jussara powder. The number in parentheses refers to the sample size. *p < 339


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3.5. Brown adipose tissue TNF-α, IL-6 and IL-10 level 343

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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

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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






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3.6. Phosphorylated NF�Bp50 subunit, TNFRI and UCP-1 in brown adipose tissue 364

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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

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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




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

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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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