jussara euterpe edulis mart supplementation during pregnancy and lactation modulates the uncoupling protein 1 ucp 1 and inflammation biomarkers induced by trans fatty acids in the brown adipose tissue of offspring

Accepted Manuscript Jussara (Euterpe edulis Mart.) Supplementation During Pregnancy and Lactation Modulates the Uncoupling Protein (UCP-1) and Inflammation Biomarkers Induced by trans-Fatty Acids in the Brown Adipose Tissue of Offspring Perla Pizzi Argentato, Carina Almeida Morais, Aline Boveto Santamarina, Helena de Cássia 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: 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, Estadella D, de Rosso VV, Pisani LP, Jussara (Euterpe edulis Mart.) Supplementation During Pregnancy and Lactation Modulates the Uncoupling Protein (UCP-1) and Inflammation Biomarkers Induced by trans-Fatty Acids in the Brown Adipose Tissue of Offspring, Clinical Nutrition Experimental (2017), doi: 10.1016/j.yclnex.2016.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT 10 11 12 13 14 15 16 Jussara (Euterpe edulis Mart.) Supplementation During Pregnancy and Lactation Modulates the Uncoupling Protein (UCP-1) and Inflammation Biomarkers Induced by trans-Fatty Acids in the Brown Adipose Tissue of Offspring 17 E-mail: lucianapisani@gmail.com (L.P Pisani) a RI PT Programa de Pús Graduaỗóo em Alimentos Nutriỗóo e Saỳde, Universidade Federal de São Paulo (UNIFESP), Santos, SP, Brazil b Departamento de Biociências Universidade Federal de São Paulo (UNIFESP), SantosSP, Brazil c Programa Interdisciplinar em Ciências da Saúde Universidade Federal de São Paulo (UNIFESP), Santos-SP, Brazil EP TE D M AN U SC *Correspondence author: Silva Jardim, 136 Laboratório 311, 3° andar, Vila Mathias, Santos/SP, 11015020, Brazil Tel./fax: +55 13 38783700 AC C 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Perla Pizzi Argentatoa, Carina Almeida Moraisc, Aline Boveto Santamarinac, Helena de Cássia Césarc, Débora Estadellab, Veridiana Vera de Rossob, Luciana Pellegrini Pisanib* ACCEPTED MANUSCRIPT 49 50 51 Abstract 52 thermogenesis by uncoupling protein (UPC-1) We investigated the effect of the 53 maternal diet enriched with trans fatty acids (TFAs) and/or supplemented with jussara 54 fruit on the 21d-old offspring Specifically, we looked at the proinflammatory state and 55 the expression of UCP-1 in the offsprings’ BAT Methods: We divided dams into four 56 groups during pregnancy and lactation: control diet (C), C diet with 0.5% of jussara 57 fruit (CJ), a diet enriched with TFAs (T), or T diet with 0.5% of jussara fruit (TJ) 58 Results: We found that TFAs reduced growth and increased weight, total cholesterol, 59 TNF-α, TNFRI and UCP-1 in BAT of pups Conversely, maternal supplementation with 60 jussara preserved lean mass, decreased weight gain, carcass lipid, blood glucose and 61 triacylglycerol in the offsprings It also increased IL-10 and UCP-1 levels in BAT 62 Conclusions: Either TFAs and jussara fruit increased the expression of UCP-1 in BAT 63 However, the TFAs are detrimental for the offsprings' health We believe that the 64 bioactive compounds of jussara fruit helped to improve such parameters Our results 65 showed that keeping the same maternal dietetic caloric amount but modifying the fatty 66 acids composition can program the BAT in offspring Jussara fruit supplementation 67 could be used as an alternative treatment for obesity prevention 68 69 Keywords: Uncoupling Protein Brown adipose tissue Programming Jussara Anthocyanins Trans-fatty acids 72 73 74 75 76 77 78 79 80 81 82 83 RI PT SC M AN U TE D EP 71 AC C 70 Background & aims:The brown adipose tissue (BAT) regulates energy expenditure via Abbreviations: Brown adipose tissue (BAT), uncoupling protein (UPC-1), trans-fatty acids (TFAs), tumor necrosis factor-α (TNF-α), tumor necrosis factor receptor (TNFRI), nuclear transcription factor kappab phosphorylated 50 (NF-ߢBp50), toll-like receptor (TLR-4), interleukin (IL-6), interleukin 10 (IL-10), body mass index (BMI), triacylglycerol (TAG), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol ACCEPTED MANUSCRIPT 84 Introduction Inadequate maternal nutrition during pregnancy and lactation is supposed to 86 cause epigenetic changes during the fetal development and neonatal period [1–3] This 87 process, known as metabolic programming or metabolic imprinting, can alter gene 88 expression and affect the structure and function of tissues or organs permanently by 89 increasing the susceptibility of the individual to the development of chronic diseases 90 such as obesity [4,5] RI PT 85 Maternal dietary fat composition and amount are the most important 92 determinants of the degree and type of fatty acids transferred to the fetus and the infant 93 through the placenta or maternal milk [6] In this regard, the maternal intake of hydrogenated vegetable fat during M AN U 94 SC 91 pregnancy and lactation increases the tumor necrosis factor-α (TNF-α) mRNA, 96 plasminogen activator inhibitor- (PAI-1) mRNA, and TNF receptor-associated factor- 97 (TRAF-6) protein in the adipose tissue of 21-day-old offspring [7,8] Moreover, it has 98 shown to increase PAI-1 mRNA [8], serum endotoxin levels, subunit p65 of nuclear 99 transcription factor Kappa-B (NF-ߢBp65), toll-like receptor (TLR-4), and myeloid 100 differentiation primary response 88 (MyD88) protein expression in the adipose tissue 101 And also, it increases hypothalamic interleukin (IL-6), TNF-ߙ, and IL-1ߚ in the adult 102 offspring [9] 103 accrued weight and adiposity TE D 95 EP Furthermore, these pro-inflammatory changes were accompanied by Although weight gain and obesity have multifactorial etiology, a positive energy 105 balance is one of the determinants of weight and adiposity gain [10] Though, the 106 regulation of energy expenditure occurs, in part, in the brown adipose tissue (BAT) 107 through thermogenesis and it is mediated by uncoupling protein (UPC-1)[11] 108 Recently, BAT was recognized in adults and it inversely correlated with body mass 109 index (BMI) [12] It also showed beneficial effects on the glucose and lipid metabolism 110 [13] Additionally, the BAT responds to external stimulus, as well as, age, sex, genetic 111 traits and diet [14,15] AC C 104 112 Foods rich in bioactive compounds such as polyphenols, especially flavonoids, 113 have been identified as a promising buffer against inflammation and oxidative stress 114 [16–19] It is known that bioactive food compounds can cross the placenta and reach ACCEPTED MANUSCRIPT 115 fetal tissues [20] Many studies have shown that polyphenols can exert metabolic 116 programming effects on the offspring through maternal intake [21–29] Dams treated with a high-fat diet and extracts rich in anthocyanins, a potent 118 polyphenol, during lactation protected their descendants, either male and female, against 119 oxidative stress, body fat gain, hypertriglyceridemia and insulin resistance in adulthood 120 [24,25] The same beneficial effects were also observed after polyphenol 121 supplementation during lactation in male offspring of dams fed a protein restricted diet 122 during pregnancy These animals had decreased body weight, hepatic triacylglycerol 123 levels and increased adiponectin levels [26] SC RI PT 117 Our research group has been studying the fruit of jussara palm (Euterpe edulis 125 Mart.), which is rich in anthocyanins and native species of the Atlantic 126 Rainforest/Brazil [30] Our research has shown promising effects of jussara fruit 127 supplementation on the metabolic programming of the colon, hypothalamus and white 128 adipose tissues in the offspring [31,32] However, studies on the impact of bioactive 129 food compound supplementation on the programming models of BAT are less common 130 in the literature TE D M AN U 124 Thus, the aim of this study was to evaluate the effect of dietary supplementation 132 with 0,5% of jussara fruit pulp during pregnancy and lactation in the presence or 133 absence of hydrogenated vegetable fat and the UCP-1 expression and proinflammatory 134 state in BAT of 21-day-old male offspring Materials and Methods AC C 135 136 137 138 139 140 EP 131 2.1 Animals and Treatments All experimental procedures were approved by the Experimental Research 141 Committee at Federal University of São Paulo (CEUA protocol n°5252010715) Rats 142 were kept under controlled conditions of light (12:12h light-dark cycle with lights on at 143 07:00) and temperature (24 ± 1oC), with ad libitum water and food 144 Twelve-week-old female Wistar rats of first-order parity were allocated 145 overnight to breeding Copulation was verified the following morning by the presence 146 of sperm in vaginal smears On the first day of gestation, rats were isolated in individual ACCEPTED MANUSCRIPT 147 cages and randomly assigned to one of the four groups receiving a control diet (C 148 group), a control diet supplemented with jussara 0.5% freeze-dried powder (CJ group), 149 a diet enriched with hydrogenated vegetable fat (T group), or a T diet supplemented 150 with 0.5% jussara freeze-dried powder (TJ group) The diets were prepared according to the recommendations of the American 152 Institute of Nutrition (AIN-93G) [33,34] and had similar caloric and lipid contents The 153 fat source for the C and CJ groups was soybean oil; the main fat source for the T and TJ 154 groups was hydrogenated vegetable fat, rich in trans-fatty acids (TFAs) The CJ and TJ 155 groups were prepared by adding 0,5% of jussara freeze-dried powder to each diet 156 Jussara pulp (Euterpe edulis Mart.) was obtained from the agroecological Project 157 Jussara/IPEMA - Institute of Permaculture and Ecovillages of the Atlantic (Ubatuba, 158 SP, Brazil) and then freeze-dried to powder using a lyophilizer Diets were then stored 159 at -20°C The phenolic compounds and anthocyanin contents of the jussara pulp were 160 previously analyzed in our laboratory [30] The total levels of anthocyanin, phenolic 161 compounds and the concentrations of their major constituents are shown in Table The 162 centesimal composition of the diets is presented in Table The fatty acid profile of C 163 and T diets was previously described by Pisani et al., 2008 [8] M AN U SC RI PT 151 Dams’ diets were maintained during pregnancy and lactation After birth, litter 165 sizes were adjusted to eight pups for each mother The pups were weighed and 166 measured (nasoanal length) at birth and on postnatal days 7, 14, and 21 In the 21st day 167 of life, offsprings were decapitated; trunk blood was collected and centrifuged Serum 168 was separated and stored at -80°C for later determination of the triacylglycerol (TAG), 169 total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, glucose, and 170 adiponectin The BAT was removed from the subscapularis region, isolated and stored 171 at -80°C 174 EP AC C 172 173 TE D 164 Table 1: Phenolic compounds detected in jussara pulp 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 ACCEPTED MANUSCRIPT Table 2: The composition of the control diet (C), control diet supplemented with 0.5% freeze-dried jussara powder (CJ), diet enriched with hydrogenated vegetable fat, TFAs (T), and diet enriched with TFAs supplemented with 0.5% freeze-dried jussara powder (TJ) according to AIN-93 CJ 20.0 0.3 62.0 8.0 – 0.0014 3.5 1.0 5.0 0.25 0.5 4.02 Diet (g/100 g) T 20.0 0.3 62.0 1.0 7.0 0.0014 3.5 1.0 5.0 0.25 – 4.00 TJ 20.0 0.3 62.0 1.0 7.0 0.0014 3.5 1.0 5.0 RI PT C 20.0 0.3 62.0 8.0 – 0.0014 3.5 1.0 5.0 0.25 – 4.00 SC Ingredient Caseina* L-cystine Cornstarch Soybean oil Hydrogenated vegetable fat$ Butylhydroquinone Mineral mixture§ Vitamin mixture# Cellulose Choline bitartrate Freeze-dried jussara powder Energy (kcal/g) M AN U 175 176 177 178 179 180 0.5 4.02 181 182 *Casein was obtained from Labsynth, São Paulo, Brazil 183 184 L-cystine, cornstarch, butylhydroquinone, cellulose and choline bitartrate were obtained from Viafarma, São Paulo, Brazil 185 186 $ §Mineral TE D Soybean Oil was supplied from (Lisa/Ind Brazil) Hydrogenated vegetable fat was supplied from Unilever, São Paulo, Brazil mix (9mg/kg diet): calcium, 5000; phosphorus, 1561; potassium, 3600; sodium, 1019; chloride, 1571; sulfur, 300; magnesium, 507; iron, 35; copper, 6.0; manganese, 10.0; zinc, 30.0; chromium, 1.0; iodine 0.2; selenium, 0.15; fluoride, 1.00; boron, 0.50; molybdenum, 0.15; silicon, 5.0; nickel, 0.5; lithium, 0.1; vanadium, 0.1 (AIN-93G, mineral mix, Rhoster, Brazil) # Vitamin mix (mg/kg diet): thiamin HCL, 6.0, riboflavin, 6.0; pyridoxine HCL, 7.0; niacin, 30.0; calcium pantothenate, 16.0; folic acid, 2.0; biotin, 0.2; vitamin B12, 25.0; vitamin A palmitate 4000 IU; vitamin E acetate, 75; vitamin D3, 1000 IU; vitamin KI, 0.75 (AIN-93G, vitamin mix, Rhoster, Brazil) Freeze-dried jussara powder: jussara pulp (Euterpe edulis Mart.) was obtained from agroecological Project Jussara/IPEMA - Institute of Permaculture and Ecovillages of the Atlantic (Ubatuba, SP, Brazil) - and by freeze-drying to powder using a lyophilizer 203 with an enzymatic colorimetric method using commercial kits (Labtest Brazil) 204 Serumadiponectin concentration was analyzed by ELISA using the DuoSet kit Mouse 205 Adiponectin / Acrp30 (R & D Systems, Minneapolis, MN, USA) AC C EP 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 2.2 Biochemical Serum Analysis Glucose, TAG, TC and HDL-cholesterol serum concentrations were measured ACCEPTED MANUSCRIPT 206 207 208 209 2.3 Carcass Lipid and Protein Contents The carcasses were eviscerated, and the remnants were weighed and stored at 20 C The lipid content was measured as described by Stansbie et al 1976 [35] and 211 standardized using the method described by Oller Do Nascimento and Williamson [36] 212 The carcass was autoclaved at 120 C for 90 and homogenized with water at a 213 volume twice the carcass mass Triplicate aliquots of approximately g were digested 214 in 3mL of 30% KOH and 3mL of ethanol for 215 cooling, mL of 12NH2SO4 was added, and the samples were washed three times with 216 petroleum ether to extract the lipids RI PT 210 SC 2h at 70°C in capped tubes After The results are expressed in grams of lipid per 100 g of the carcass To measure 218 the protein content, aliquots of the same homogenate, approximately g, were heated at 219 37°C for h in 0.6NKOH with constant shaking After clarification by centrifugation, 220 protein content was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA) 221 with bovine serum albumin as a reference 2.4 Brown adipose tissue TNF-α, IL-6, and IL-10 Levels by ELISA TE D 222 223 224 225 M AN U 217 The BAT was homogenized and centrifuged at 12,000 rpm for 40min at 4°C; the supernatant was saved and the protein concentration determined using the BCA assay 227 (Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as a reference 228 Quantitative assessment of TNF-α, IL-6, and IL-10 proteins was carried out by ELISA 229 (DuoSet 230 recommendations of the manufacturer ELISA, R&D Systems, Minneapolis, MN, USA) following the AC C 231 232 233 234 EP 226 2.5 Brown adipose tissue UCP-1, TNFRI and p-NFߢBp50 by Western Blotting The brown adipose tissue was removed and placed in the extraction buffer (100 235 mM Trizma base pH 7.5, 20 mM EDTA, 100 mM sodium fluoride, 100 mM sodium 236 pyrophosphate, 10 mM sodium orthovanadate, mM PMSFphenylmethylsulfonyl 237 fluoride and 0.1 mg of aprotinin per mL) 238 The total protein content was determined by the Bradford method using the Bio- 239 Rad reagent (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as the reference 240 The samples were treated with the LDS Sample Buffer and Reducing Agent (Life ACCEPTED MANUSCRIPT Technologies) The proteins (100 µg) were heated for 10 before loading onto the 242 Bolt 4-12% Bis-Tris Plus in a Bolt mini gel tank (Novex, Life Technologies, CA, USA) 243 Electrotransfer of proteins from the gel to the nitrocellulose membrane was 244 performed for min/2 gels at 20 V for min, 23 V for and 25 V for the 245 remainder in an Iblot gel transfer device (Life Technologies, CA, USA) Nonspecific 246 protein binding to the nitrocellulose membrane was reduced by preincubation at 22 °C 247 in blocking buffer containing 1% BSA The nitrocellulose membranes were incubated 248 overnight at 15°C with antibodies against UCP-1, TNFRI (ABCAM, MA, USA) and the 249 phosphorylated form of p-NFߢBp50 (Santa Cruz Biotechnology, CA, USA) The 250 antibodies were diluted 1:1000 with blocking buffer The blots were subsequently 251 incubated with a peroxidase-conjugated secondary antibody (ABCAM, MA, USA) for 252 h at 15°C SC RI PT 241 Specific bands were detected by chemiluminescence using Alliance 4.7 254 equipment (Uvitec, Cambridge, UK) The band intensity was quantified by optical 255 densitometry (Scion Image-Release Beta 3b, NIH, USA) The signals were normalized 256 to β-actin (ABCAM, MA, USA) 2.6 Statistical Analysis TE D 257 258 259 260 M AN U 253 Statistical analyses were performed using the software SPSS version 22 Data were submitted to the quality tests Shapiro-Wilk (normality), Levenne (homogeneity) 262 and/or Mauchly (sphericity) If necessary, data were standardized to Z score To verify 263 the interactions between groups, we used two-way ANOVA analysis followed by a 264 Bonferroni posthoc test All results are presented as the means ± SEM (standard error 265 mean) and p < 0.05 was considered statistically significant AC C 266 267 268 269 270 271 EP 261 Results 3.1 Body Weight, Body Weight Gain, Length of the Animal 21-day-old offsprings from TJ group had higher body weight at birth (p = 0.013) 272 compared to the CJ group All animals showed similar length at birth In the first week 273 of treatment, TJ was heavier than CJ group (p = 0.0001) Similarly, length size followed 274 the same pattern, and TJ had higher length than T group (p = 0.01) In the following 275 week, the TJ group maintained higher body weight (TJ > CJ, p = 0.0001), and length 276 (TJ > T, p = 0.01) In the last week, CJ had lower weight gain compared to C group (p = ACCEPTED MANUSCRIPT 277 0.01) and T group had a smaller increase in length than the C group (p = 0.0001) and TJ 278 group (p = 0.01) (Figure 1A and B) Regarding weekly weight gain, in the first week of treatment, T group gained 280 more weight than C group (p = 0.023), the same happened to TJ group compared to CJ 281 group (p = 0.001) In the last week of treatment, TJ group showed lower weight gain 282 compared to CJ group (p = 0.032) (Figure 1C) About total weight gain, the offspring of 283 the CJ group had smaller weight gain than C group (p = 0.001) (Figure 1D) RI PT 279 285 EP TE D M AN U SC 284 Figure 1: (A) Body weight, (B) Length of the animal, (C) Body weight gain, (D) Total weight gain in 21-day-old offspring C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 0.5% freeze-dried Jussara powder The number in parentheses refers to the sample size *p < 0.05 versus C $p < 0.05 versus CJ #p < 0.05 versus T &p < 0.05 versus TJ 298 compared with C group (p = 0.0001 and p = 0.008, respectively) (Figure 2A) The AC C 286 287 288 289 290 291 292 293 294 295 296 297 3.2 Carcass Lipid and Protein Contents The carcass lipid content was lower in the offspring from the CJ and T group ACCEPTED MANUSCRIPT 299 protein content was lower in T group compared to C group (p = 0.016) and TJ group (p 300 = 0.004) (Figure 2B) The carcass lipid and protein ratio was higher in the offspring 301 from T group than C group (p = 0.0001) and TJ group (p = 0.0001) (Figure 2C) 303 TE D M AN U SC RI PT 302 Figure 2: (A) Carcass lipid, (B) Protein content, and (C) Lipid/Protein ratio in 21-day-old offspring C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 0.5% freeze-dried Jussara powder The number in parentheses refers to the sample size *p < 0.05 versus C $p < 0.05 versus CJ #p < 0.05 versus T &p < 0.05 versus TJ 315 Regards to relative weight of the BAT, there was no significant difference 316 317 318 AC C EP 304 305 306 307 308 309 310 311 312 313 314 3.3 Weight of Brown adipose tissue between the groups (Table 3) Table Weight of brown adipose tissue of 21-day-old pups Brown adipose tissue C (9) CJ (11) T (11) Relative weight (g/100 g 0,40 ± 0,01 0,38 ± 0,02 0,33 ± 0,01 body weight) TJ (13) 0,35 ± 0,01 ACCEPTED MANUSCRIPT 319 320 321 322 323 324 325 326 C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 0.5% freeze-dried jussara powder; T: offspring of dams fed diet enriched with hydrogenated vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 0.5% freeze-dried jussara powder The number in parentheses refers to the sample size 327 Serum glucose concentration of the 21-day-old offspring in TJ group was lower 328 compared to the T group (p = 0.011) The total cholesterol was higher in the T group 329 compared to C group (p = 0.003) and TJ group compared CJ group (p = 0.0001) Serum 330 triacylglycerol concentration was higher in T group compared to C group (p = 0.04) and 331 TJ group (p = 0.0001) However, no differences were seen in the serum HDL- 332 cholesterol and adiponectin concentrations among groups RI PT SC M AN U Table 4: Serum glucose, total cholesterol, HDL-cholesterol, triacylglycerols and adiponectin in 21-day-old offspring Parameters C (10) CJ (10) T (10) TJ (10) Glucose(mg/dL) 112,52±3,56 108,44±5,98 115,70±4,13 95,59±6,90# Total cholesterol (mg/dL) 93,67±4,93 84,85±3,42 112,55±2,98* 107,9±4,80$ 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 2,33±3,38 2,42±2,95 2,10±1,61 2,21±1,92 (µg/mL) TE D 333 334 335 3.4 Serum Biochemical Analyses C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 0.5% freeze-dried jussara powder The number in parentheses refers to the sample size *p < 0.05 versus C $p < 0.05 versus CJ #p < 0.05 versus T &p < 0.05 versus TJ 346 group compared with C group (p = 0.007) and, also in TJ group compared with T group 347 (p = 0.004) and CJ group (p = 0.0001) (Figure 3A) For the anti-inflammatory cytokine 348 IL-10, there was a higher level in the CJ group than C group (p = 0.0001) and TJ group 349 (p = 0.001), the same happened in the offspring from T group compared to C group (p = 350 0.021) (Figure 3B) However, the cytokine IL-6 remained unchanged between groups 351 (Figure 3C) Regarding IL-10/TNF-α ratio, the CJ group had a higher ratio compared AC C EP 336 337 338 339 340 341 342 343 344 345 3.5 Brown adipose tissue TNF-α, IL-6 and IL-10 level The results showed an increased level of TNF-α protein in the BAT of the T ACCEPTED MANUSCRIPT 352 to the TJ group (p = 0.004) the reverse was found in T group compared C group (p = 353 0.049) (Figure 3D) 355 TE D M AN U SC RI PT 354 356 357 358 359 360 361 362 363 364 365 366 Figure 3: (A) IL-6 protein expression, (B) IL-10, (C) TNF-ߙ, and (D) IL-10/TNF-ߙ ratio in 367 the TJ group compared to the T group (p = 0.047) and CJ (p = 0.009) (Figure 4A) The 368 levels of p-NFߢBp50 remained unchanged between the groups (Figure 4B) 369 Furthermore, the TNFRI protein levels were significantly lower in the CJ group 370 compared to TJ group (p = 0.041) (Figure 4C) 371 372 373 AC C EP 21-day-old offspring at the brown adipose tissue C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 0.5% freeze-dried Jussara powder The number in parentheses refers to the sample size *p < 0.05 versus C $p < 0.05 versus CJ #p < 0.05 versus T &p < 0.05 versus TJ 3.6 Phosphorylated NFߢBp50 subunit, TNFRI and UCP-1 in brown adipose tissue UCP-1 levels in brown adipose tissue of the 21-day-old offspring were higher in M AN U SC RI PT ACCEPTED MANUSCRIPT 374 Figure (A) UCP-1 protein expression in brown adipose tissue, (B) Protein expression of the phosphorylated form p-NFߢBp50 in brown adipose tissue and (C) TNFRI protein expression in brown adipose tissue C: offspring of dams fed control diet; CJ: offspring of dams fed control diet supplemented with 0.5% freeze-dried Jussara powder; T: offspring of dams fed diet enriched with hydrogenated vegetable fat, TFAs; TJ: offspring of dams fed diet enriched with TFAs supplemented with 0.5% freeze-dried Jussara powder The number in parentheses refers to the sample value (Fig 4A: T and TJ group presented one outlier sample) Data are means ± SEMs Results are expressed in arbitrary units, stipulating 100 as the control value *p < 0.05 versus C $p < 0.05 versus CJ #p < 0.05 versus T &p < 0.05 versus TJ 386 387 388 Discussion 389 [22,28,29] but only a few have determined the effect of their consumption, in 390 physiological doses, on the UCP-1 expression in the offspring Here we evaluated the 391 role of a highly nutritive fruit and its interaction with the weight control mechanisms 392 by studying the effect of food in the BAT and on fetal programming model with 393 hydrogenated vegetable fat AC C EP TE D 375 376 377 378 379 380 381 382 383 384 385 There are many studies about fetal programming and polyphenols intake 394 Thus, we found that hydrogenated vegetable fat supplementation increased birth 395 weight, and this event continued in the first weeks of lactation (Fig 1A and 1C) Souza ACCEPTED MANUSCRIPT et al 2012 using 6% of hydrogenated vegetable fat during pregnancy and lactation 397 found increased body weight of male offspring on the 7th and 14th day of life [37] 398 Already, Bishop et al., 2015 using hydrogenated vegetable oil, soybean oil and palm oil 399 on a programming model found no change in the body weight of offsprings; it might 400 be because they used 90-day old rats [38] On the other hand, the authors showed that a 401 better fatty acid profile such as fish oil as the main dietary fat source during pregnancy 402 and lactation was associated with decreased body weight from birth up to the 12th 403 week of life [39] RI PT 396 We found that maternal jussara supplementation reduced body weight gain (Fig 405 1A and 1C) and had a protective role against stunting According to our findings, we 406 found the stunted growth of offpring from T group during the first and second week of 407 treatment, and jussara fruit supplementation has inhibited this effect in the TJ group 408 (Fig 1B) In addition, jussara supplementation reduced fat carcass deposits (Fig 2A) 409 and prevented muscle wasting (Fig 2B and 2C) Wu et al., 2014 treated rodents with a 410 high-fat diet and 40 and 200 mg / kg of body weight with anthocyanins isolated from 411 cherries for 12 weeks, which showed reduced weight gain by 5.2% and 11.2% 412 respectively [19] Similarly, Mukai et al., 2013 found that rats fed a protein restricted 413 diet during pregnancy and supplemented with Azuki bean anthocyanins during lactation 414 had lasting beneficial effects on reducing the weight of 23 weeks old offsprings [26] 415 Dolinoy et al., 2006, found the same results in mice supplemented with genistein, the 416 main soy phytoestrogen, administered during pregnancy and lactation in doses of 250 417 mg/ kg of diet [21] As with doses of mg/kg of diet in rats, according to Ball et al., 418 2010 [23] An in vitro study elucidated the role of anthocyanins on weight reducing and 419 its anti-obesogenic properties, through its involvement in suppressing 420 accumulation 421 lipogenesis [40] M AN U TE D EP AC C 422 SC 404 in the adipocytes lipid by inhibiting transcription factors that regulate Some authors argue that the beneficial effects of a fruit similar to jussara are due 423 to not just its anthocyanin content itself, but its nutritional characteristics [41,42] It is 424 known that jussara has high levels of fiber, 28.3 ± 0.3g/100g dry basis It is rich in 425 anthocyanins, 262.4 ± 8.6 mg/100g wet basis, especially Cyanidin 3-rutinoside and 426 Cyanidin 3-glucoside [30] and can be an excellent dietary source of essential fatty acids 427 The oil extracted from the jussara pulp has about 36% oleic acid (monounsaturated fatty 428 acid - MUFA) and 19% linoleic (omega-6 polyunsaturated fatty acid - n-6 PUFA) (Silva ACCEPTED MANUSCRIPT et al., 2013) The literature reveals that dietary fibers exert glycemic control, and 430 improve lipid profile by reducing intestinal absorption of carbohydrates and cholesterol, 431 decrease gastric emptying, and insulin secretion and promote the production of short- 432 chain fatty acids in the colon [44] PUFAs have shown a benefit in attenuating TAG by 433 reducing hepatic lipogenesis [45] Besides, the polyphenols are associated with 434 increased tissue glucose uptake [46], lower cholesterol absorption and synthesis, and 435 increased gene expression 436 supplementation was effective in lowering blood glucose and triacylglycerol levels in 437 the offspring (Table 4) There is evidence that phenolic compounds in fruit, especially 438 flavonoids can induce glucose transporter type (GLUT4) in adipose tissue and skeletal 439 muscle, and to contribute to the glucose homeostasis Furthermore, anthocyanins may 440 activate the AMP-activated protein kinase (AMPK) engaged in increasing skeletal 441 muscle glucose uptake, reducing lipogenesis, promoting lipolysis and reducing 442 cholesterol syntesis [46] In agreement with our results, treatment with aỗai extract in 443 male rabbits fed a diet enriched with 0.5% cholesterol for 12 weeks was effective in 444 reducing triacylglycerol levels [48] Emiliano et al., 2011 and Resende et al., 2013 gave 445 grape skin extract to rats fed a high fat diet during lactation, and they found that a dose 446 of 200mg/kg/day protected male and female offsprings from hypertriglyceridemia and 447 enhanced glucose metabolism in adulthood [24,25] Research with human subjects 448 getting 100 mg of aỗaớ pulp, which is a fruit similar to jussara, twice a day for month, 449 showed favorable results in improving blood glucose levels in overweight individuals 450 [41] Our research took into account the dose of jussara consumed by human subjects 451 Thus, supplementation with 0.5% lyophilized jussara corresponds to 3.3 mg 452 anthocyanins/kg/day, and it can be obtained by a human consumption of 100g of fresh 453 pulp or 10g of jussara freeze-dried powder per day EP TE D M AN U SC that favors cholesterol control [42,47] Jussara AC C 454 RI PT 429 Regarding endocrine function, the secretory role of brown adipose tissue is 455 poorly understood in the literature [49] In general, BAT has lower cytokines levels than 456 white adipose tissue, possibly due to the proinflammatory phenotype of immune cells 457 that infiltrate the white adipose tissue [50] However, in obesity, proinflammatory 458 cytokines such as TNF-α were found to recruit macrophages in the BAT [51,52] Our 459 study showed that trans fat supplementation increases TNF-α levels in BAT of 21-day- 460 old offspring It is well described in the literature that saturated fatty acids and trans 461 fatty acids correlate with increased low grade inflammation [53,54] They activate toll- ACCEPTED MANUSCRIPT 462 like receptor (TLR4) pathway, which activates the NFκB dimers of NFκBp50 or 463 NFκBp65 The NFκBp50 translocation to the nucleus results in the induction of gene 464 expression 465 supplementation in the maternal diet increased the anti-inflammatory cytokine IL-10 in 466 CJ group Surprisingly, this cytokine appeared increased in the T group; this may be due 467 to an anti-inflammatory reaction of the animal to counterbalance the increased level of 468 TNF-α (Fig 3B) Indeed, foods rich in anthocyanins are described in the literature as 469 having a 470 supplementation with 40 and 200 mg/kg of anthocyanins isolated from cherries, for 12 471 weeks, can attenuate gene expression of TNF and other pro-inflammatory genes in 472 rodent treated with a high-fat diet [19] Graf et al., 2013 showed that anthocyanins have 473 anti-inflammatory action by inhibiting the translocation of NFkB to the nucleus [42] 474 However, we haven’t found changes in the expression of phosphorylated transcription 475 factor NFκBp50 with jussara supplementation (Fig 4B) RI PT of proinflammatory cytokines such as TNF-α [55] Already, jussara M AN U SC potential anti-inflammatory action [18,28,42] A study revealed that It has been described that TNF-α has an anti-thermogenic effect in obesity [56] 477 This reduction in the thermogenesis was evidenced by low doses of intraperitoneal 478 TNF-α administration [57] Romanatto et al., 2009 discovered that diet-induced obesity 479 can be prevented by increasing thermogenesis through rising UCP-1 expression in BAT 480 of week old male TNFR1 knockout rats (TNFR1 knockout) [58] However, these 481 studies did not take into account the inflammation in the BAT itself We found 482 increased tumor necrosis factor receptor (TNFRI) expression in BAT of offsprings 483 exposed to maternal hydrogenated vegetable fat supplementation (Fig 4C) and, unlike 484 the above studies, this event was associated with less weight gain in this group by the 485 end of the experiment The same happened in regards to the UCP-1 expression in this 486 group (TJ > CJ, Fig 4B), which it would explain the weight reduction observed, likely 487 by increased thermogenesis via UCP-1 Although jussara activated thermogenesis via 488 increased UCP-1, it did not reduce inflammation in the offsprings which had TFA It 489 could be that it required extra time of treatment exposure to see an overt anti- 490 inflammatory effect in the BAT Considering that the antioxidant action occurs in 491 inflamed individuals and not at absence of this stimulus [59,60] Corroborating our 492 results, administration of black soybean seed coat extract, rich in 3-glucoside cyanidin 493 (9.2%), catechins (6.2%) and procyanidins (39.8%) for 14 weeks reduced body weight 494 gain and increased UCP-1 protein expression in BAT of animals challenged with a AC C EP TE D 476 ACCEPTED MANUSCRIPT 495 high-fat diet [61] We also found an increase in the UCP-1 expression after jussara 496 supplementation (TJ > T, Fig 4A) The mechanisms by which polyphenols influence thermogenesis are not 498 understood However, as we found, supplementation of g/kg diet/day with resveratrol, 499 a phenolic antioxidant, for eight weeks increased the UCP-1 levels without changing 500 the relative weight of BAT of weeks old mice [62] It is less frequent in the literature 501 programming studies investigating the relation of bioactive food compounds, isocaloric 502 and normolipidic diets, differing only from their type of dietary fatty acids, and their 503 impact on the thermogenic parameters It is estimated that about 60g of BAT can 504 contribute up to 20% of total daily heat production in humans [63] The UCP-1 505 mechanism of action is through 506 mitochondria and to release energy as heat from the oxidation of fatty acids that have 507 not been coupled to the production of adenosine triphosphate (ATP) [11] It is known 508 that the degree of UCP-1 activation varies with the availability and the flow of fatty 509 acids within the cells [64] One of our hypothesis is that the high fat content of jussara, 510 comprised mainly of oleic acid, palmitic and linoleic with the additional dietary lipids 511 included in the diets, contributed to increase the UCP-1 expression in BAT of TJ group 512 In that sense, Priego et al., 2013 found that better fatty acid profile, such as the oleic 513 acid present in large quantities in olive oil can increase UCP-1 in the BAT of males and 514 females 21 day old offsprings [65] the transport of protons in the TE D M AN U decoupling SC RI PT 497 Therefore, we demonstrated that jussara supplementation during pregnancy and 516 lactation can be a natural way to enhance the expression of UCP-1 in BAT and to 517 improve body composition in the 21- day-old offspring 519 520 AC C 518 EP 515 Conclusion In summary, we show that maternal supplementation with TFAs increased 521 weight, TNF-α, RITNF and UCP-1 levels in BAT of 21-day-old offspring Besides, 522 maternal diet supplementation with 0.5% of jussara during pregnancy and lactation 523 reduced weight gain and fat carcass deposits It further protected against the stunted 524 growth and prevented lean mass loss, decreased glucose and triacylglycerol levels, and 525 increased the anti-inflammatory cytokine IL-10 levels and UCP-1 expression in BAT ACCEPTED MANUSCRIPT 526 Coincidentally, the animals that lost the greatest amount of weight at the end of the 527 treatment had higher UCP-1 levels in BAT Therefore, either TFAs and jussara fruit increased the expression of UCP-1 in 529 BAT However, the TFAs are detrimental for the body composition, the metabolic and 530 the inflammatory parameters We believe that the bioactive compounds of jussara fruit 531 helped to improve such parameters Our results showed that keeping the same caloric 532 amount of the maternal diet but modifying its quality by adding a natural food in 533 physiological doses can program the BAT of the offspring Thus, jussara fruit 534 supplementation can be considered an alternative therapy for the prevention of the 535 development of chronic diseases in adulthood, such as obesity (Fig 5) 536 AC C EP TE D M AN U SC RI PT 528 537 Figure TFAs supplementation and with jussara 0.5% freeze-dried powder on BAT 21-day- 538 old offspring (Fatty acids and cianidinas - from FreeDigitalPhotos.net) 539 540 ACCEPTED MANUSCRIPT 541 542 543 Conflict of Interests The authors declare that they have no conflict of interests regarding the publication of this paper 544 546 547 Acknowledgements RI PT 545 This research was supported by FAPESP (Fundaỗóo de Amparo Pesquisa Estado de São Paulo) number 2015/02602-3 We are grateful to this Institution SC 548 AC C EP TE D M AN U 549 ACCEPTED 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MANUSCRIPT 10 11 12 13 14 15 16 Jussara (Euterpe edulis Mart. ) Supplementation During Pregnancy and Lactation Modulates the Uncoupling Protein (UCP -1) and Inflammation Biomarkers Induced by trans- Fatty. .. al Jussara (Euterpe edulis Mart. ) supplementation during pregnancy and lactation modulates the gene and protein expression of inflammation biomarkers induced by trans- fatty acids in the colon of. .. and UCP -1 in brown adipose tissue UCP -1 levels in brown adipose tissue of the 21- day-old offspring were higher in M AN U SC RI PT ACCEPTED MANUSCRIPT 374 Figure (A) UCP -1 protein expression in