Effects of maternal taurine supplementation on maternal dietary intake, plasma metabolites and fetal growth and development in cafeteria diet fed rats

Background Maternal obesity may disrupt the developmental process of the fetus during gestation in rats. Recent evidence suggests that taurine can exert protective role against detrimental influence of obesogenic diets. This study aimed to examine the effect of maternal cafeteria diet and/or taurine supplementation on maternal dietary intake, plasma metabolites, fetal growth and development. Methods Female Wistar rats were fed a control diet (CON), CON supplemented with 1.5% taurine in drinking water (CONT), cafeteria diet (CAF) or CAF supplemented with taurine (CAFT) from weaning. After 8 weeks all animals were mated and maintained on the same diets during pregnancy and lactation. Results Dietary intakes were significantly different between the groups. Both CAF and CAFT fed dams consumed less water in comparison to CON and CONT dams. Taurine supplementation only increased plasma taurine concentrations in CONT group. Maternal plasma adiponectin concentrations increased in CAF and CAFT fed dams compared to CON and CONT fed dams and there was no effect of taurine. Hyperleptinemia was observed in CAF fed dams but not in CAFT fed dams. Malondialdehyde was significantly increased only in CAF fed dams. Litter size, sex ratio and birth weight were similar between the groups. There was an increase in neonatal mortality in CONT group. Discussion This study showed that maternal taurine supplementation exerted modest protective effects on cafeteria diet induced maternal obesity. The increased neonatal mortality in CONT neonates indicates possible detrimental effects of taurine supplementation in the setting of normal pregnancy. Therefore, future studies should investigate the optimal dose of taurine supplementation and long term potential effects on the offspring.

236 slowed in rats fed the CAFT (243.48±4.58 g) diet during lactation in comparison with those fed 237 the CONT (276.14±4.58 g) (P=0.035). Both CAF and CAFT groups exhibited weight loss during 238 lactation. 239 Dietary treatment during pre-gestational, gestational and lactation periods significantly 240 influenced maternal liver, kidney and adipose tissue weights at the end of the lactation period 241 (Table 2). Taurine addition to cafeteria diet or chow diet did not alter liver weight between CON 242 and CONT or CAF and CAFT. However, CAF and CAFT had significantly lower liver weights 243 than CON and CONT (P<0.001). Both of the right and left kidney weights exhibited a similar 244 pattern as CAF and CAFT had significantly lower kidney weights than CON and CONT 245 (P<0.05). Fat depot mass data indicated that taurine addition to cafeteria diet did not exert a 246 protective effect on gonadal and peri-renal fat mass since CAF and CAFT had significantly 247 heavier values than CON and CONT (P<0.001) and there were no significant difference between 248 CAF and CAFT groups. 249 Maternal plasma glucose, insulin, IGF-1, C-peptide, cholesterol and triglyceride were 250 unaffected by dietary treatment at the end of the lactation (Table 3). CONT, CAF and CAFT 251 displayed higher levels of HbA1c than CON. Leptin was significantly higher in CAF compared 252 to CON and CONT whereas it was similar to all other three groups in CAFT. Adiponectin was 253 significantly higher in CAF and CAFT than CON and CONT. Malondialdehyde was 254 significantly higher only in CAF in comparison to CON and CONT. Maternal plasma taurine 255 was increased in CONT compared to CON, CAF and CAFT (Table 4). Also, taurine was 256 significantly lower in CAF than CON, CONT and CAFT. CAF and CAFT exhibited higher 257 levels of serine and lower levels of tyrosine than CON. Phenylalanine was lower and aspartic 258 acid higher in CAFT than CON. 259 Litter size (CON: 11.00±1.19, CONT: 10.43±1.11, CAF: 9.57±1.11, CAFT: 9.57±1.11, 260 P=0.775) and sex ratio (male:female) (CON: 0.76±0.49, CONT: 1.22±0.46, CAF: 1.56±0.46, 261 CAFT: 1.79±0.46, P=0.464) were similar between the study groups. Similarly, birth weights of 262 pups did not differ between groups (p=0.532) but male offspring's birth weights were higher 263 than female offspring (CON male: 5.87±0.13 g, female: 5.40±0.10 g; CONT male: 5.70±0.11 g, 264 female: 5.36±0.11 g; CAF male: 5.64±0.11 g, female: 5.30±0.12 g and CAFT male: 5.64±0.12 g, 265 female: 5.37±0.11 g, P<0.001). Maternal taurine supplementation significantly increased the 266 proportion of neonatal deaths per litter only in the CONT group and sex of the animals had no 267 effect on this outcome (CONT: 12.3% versus CON: 3.0%, CAF: 4.5% and CAFT: 1.5%, 268 P=0.023). Those offspring died during the first week of lactation. 269 Offspring weight gain was significantly influenced by maternal diet (P<0.001) and study 270 weeks (P<0.001) during lactation but there was no effect of sex ( Figure 4). Maternal diet and 271 study weeks exhibited a significant interaction (P<0.001). Although birth weight did not vary 272 between the groups, this situation disappeared over time and CAF and CAFT offspring displayed 273 lower body weights in comparison to CON and CONT offspring in both genders ( .001). In addition, maternal diet significantly influenced liver, brain, kidney and heart 277 weights of offspring in terms of percentage of body weight in both genders at the end of lactation 278 (Table 5). Fetal exposure to cafeteria diet led to lower liver and kidney weights compared with 279 CON and CONT groups (P<0.001). Liver and kidney weights of CAFT offspring exhibited 280 similar patterns like CAF offspring (P<0.001). However, CAFT offspring had significantly 281 heavier brain weight than CON (P=0.004) and CONT offspring (P=0.001). Offspring that were 282 exposed to maternal cafeteria diet exhibited heavier heart weights in comparison to CON and 283 CONT groups (P<0.001). Furthermore, a reduction in heart weight was observed in the offspring 284 of CAFT (P=0.011). The influence of maternal dietary strategies to prevent the development of chronic diseases on 288 the developing offspring is relatively unknown in contrast to the more direct inferences of 289 neonatal health. Few animal studies support the notion that taurine supplementation may trigger 290 differences in metabolic functions and physiology (Rashid, Das & Sil, 2013;Zheng et al., 2017). 291 However, it is not clear whether such effects can continue on throughout pre-pregnancy, 292 pregnancy and lactation. The maternal metabolic health during these periods is an important 293 determinant of health status of offspring. Both animal and human studies revealed that gestation 294 period does not terminate with birth but with end of lactation (Stuebe AM &Rich-Edwards JW, 295 2009) Hence, the primary aim of this study was to compare the effect of a maternal cafeteria diet 296 with taurine supplemented cafeteria diet in terms of pre-pregnancy nutritional status, pregnancy 297 progression, outcomes and fetal growth and development throughout lactation. In this regard, 298 firstly, the present study showed significant nutritional and metabolic changes in dams in 299 response to a maternal cafeteria diet at weaning. More specifically, maternal taurine 300 supplementation reversed some of these changes at a modest level through preventing maternal 301 hyperleptinemia, reducing malondialdehyde and increased plasma taurine levels. Furthermore, 302 increased neonatal mortality was observed in taurine supplemented-control group. 303 Previous studies reported that ingestion of the cafeteria diet before pregnancy led to 304 hyperphagia and increased energy intake (Akyol, Langley-Evans & McMullen, 2009;Crew, 305 Waddell & Mark, 2016;Sanchez-Blanco et al., 2016). Unlike this finding, energy intake of 306 cafeteria fed dams did not differ from other groups prior to gestation in this study. One 307 explanation might become the expression of the data as energy intake was normalized to body 308 weight in the current study. Furthermore, it was suggested that providing more food items (for 309 example 40 highly palatable energy-dense human food) made the cafeteria diet more successful 310 at sustained hyperphagia and greater weight gain (George et al., 2019). Gugusheff et al. (2016) 311 reported similar energy intakes between cafeteria and control dams throughout pre-gestation and 312 gestation periods which may be associated with presenting less food items in cafeteria diet. Also, 313 energy intake of cafeteria fed dams did not differ from other groups during gestation in the 314 current study. Similarly, one study reported that energy intakes of cafeteria fed dams were 315 similar to other groups (Ferro Cavalcante et al., 2014) whereas other studies showed higher 316 energy intakes during gestation in cafeteria group (Akyol, Langley-Evans & McMullen, 2009;317 Sanchez-Blanco et al., 2016;Vithayathil et al., 2018) .The present study demonstrated that 318 energy intake was not different between groups during lactation, which has been reported 319 previously (Ferro Cavalcante et al., 2014), but not in all studies (Bayol, Farrington & Stickland, 320 2007;Speight et al., 2017;Vithayathil et al., 2018). These contradictory results may be due to 321 type of foods used in the cafeteria diet, differences in the duration and timing of intervention. 322 Cafeteria diet is an unbalanced diet with a higher percentage of total energy coming from fat and 323 a lower percentage coming from carbohydrates and proteins compared to control diet (Sampey et 324 al., 2011). Thus, both CAF and CAFT groups consumed lower protein and carbohydrate and 325 remarkably greater fat compared to CON and CONT groups. Overall, taurine supplementation 326 did not affect food consumption, energy intake and food preferences during gestation and 327 lactation. This is consistent with the findings of previous studies (Li et al., 2013;Li et al., 2015). 328 Since the total energy intake of dams fed the cafeteria diet was similar to dams fed the 329 control diet throughout pre-gestation, gestation and lactation, total body weights did not differ 330 between cafeteria and control groups at the end of lactation. Some studies reported that 331 consumption of cafeteria diet during 8 weeks before gestation might be failed to trigger 332 significant weight gain (Jacobs et al., 2014;Rossetti et al., 2020). Rossetti et al. (2020) indicated 333 that they maintained feeding cafeteria diet until 14th week of the experiment before mating in 334 order to detect significantly increased body weights in CAF group. Therefore, differences in the 335 duration may lead to contradictory results regarding pre-gestational body weights. Also, at 336 mating no significant differences were observed regarding body weight between groups but 337 previous data showed that at day 20 of gestation cafeteria diet fat dams had greater body fat 338 accumulation which is an essential component of obesity (Akyol et al., 2009). In a previous 339 study, profound adiposity was showed although no difference was observed in terms of body 340 weight between cafeteria and control groups (Buyukdere, Gulec & Akyol, 2019). Furthermore, 341 the organ weight data at the end of lactation showed that CAF and CAFT animals had 342 significantly increased gonadal and peri-renal fat depots (72% increase in gonadal fat and 77% in 343 peri-renal fat in CAF). Therefore, it can be suggested that cafeteria diet led to increased adiposity 344 at the end of eight weeks of pre-gestational feeding and this model produced an efficient model 345 of maternal obesity.

346
Ingestion of cafeteria diet resulted in weight loss during lactation and this was not 347 influenced by taurine supplementation. This can be explained by suggesting that dams fed the 348 cafeteria diet during lactation could invest more energy to milk production and hence their milk 349 could be richer than that the produced by the chow diet-fed lactating rats (Bayol, Farrington & 350 Stickland, 2007). Indeed, it was demonstrated that milk from cafeteria diet-fed dams contained 351 higher concentration of fat and lower concentration of protein when compared to controls. This 352 may explain the reduced growth rate of the offspring of cafeteria diet fed dams (Pomar et al., 353 2017). It can be suggested that maternal ingestion of cafeteria diet affected the offspring in a 354 similar setting to the effects of a low protein diet. Lower protein content of the maternal diet 355 could lead to lower body weight at weaning (Bayol, Farrington & Stickland, 2007;Pomar et al., 356 2017). In the present study, both male and female CAF and CAFT offspring were leaner than 357 CON and CONT offspring at weaning. In studies conducted with low protein diet models 358 supplementation with taurine did not prevent growth retardation of the offspring during lactation 359 (Boujendar et al., 2002;Merezak et al., 2014). However, another study reported that weaning 360 weights of offspring exposed to obesogenic diet supplemented with taurine was similar to control 361 offspring (Li et al., 2015). Different dietary exposure models could lead to differential effects on 362 fetal growth and development as protein content of obesogenic diet models exhibited distinct 363 levels of protein.

364
Despite weight loss during lactation, cafeteria diet fed dams exhibited markedly increased 365 adiposity at the end of lactation. Although CAFT group had lower gonadal and peri-renal fat 366 depots than CAF group; this did not reach to statistical significance in the current study. Thus, 367 taurine supplementation did not decrease gonadal and peri-renal fat pad weights. 368 Supplementation of 5% (wt/wt) taurine (estimated taurine intake 3 mg/g body weight/day and 11 369 mg/g body weight/ day respectively) was shown to prevent tissue fat accumulation and obesity 370 with increased energy expenditure (Tsuboyama-Kasaoka et al., 2006;Lin et al., 2013), while 371 high fat diet induced obesity in mice could not be prevented by 1% taurine treatment (estimated 372 taurine intake 1.7 mg/g body weight/day) (Murakami, Kondo & Nagate, 2000). This difference 373 between the studies can be attributable to supplementation dosage and amount of estimated 374 taurine intake. In the current study, estimated taurine intake of CAFT dams was 2.4 mg/g body 375 weight/day. This result may suggest that higher amount of taurine is required to observe anti-376 obesogenic effects.

377
Plasma taurine levels were shown to decrease in obesity since taurine synthesis in white 378 adipose tissue is reduced (Tsuboyama- Kasaoka et al., 2006). Also, Rosa et al. (2014) reported 379 that obese women had lower plasma taurine levels than normal weight control group. However, 380 Li et al. (2013 and2015) indicated that dams fed with maternal obesogenic diets displayed 381 similar plasma taurine concentrations to control dams. These differences might be due to the 382 degree of fat deposition. CAF fed dams exerted decreased plasma taurine levels in this study. 383 Most studies demonstrated that taurine supplementation improved plasma taurine levels in the 384 setting of diet induced obesity (Tsuboyama-Kasaoka et al., 2006;Li et al., 2013;Li et al., 2015). 385 In addition, taurine supplementation resulted in a marked elevation of taurine concentrations in 386 CONT and CAFT dams in the current study.  (Chen et al., 2008) 393 concentrations in cafeteria fed dams. The above-cited studies have some differences in the 394 duration and timing of intervention. Therefore, it becomes very difficult to reach a clear 395 conclusion. In the present study, the concentrations of these plasma metabolites were determined 396 at the end of lactation. Therefore, it is crucial to assess the influence of both cafeteria diet and 397 taurine supplementation on these metabolic parameters during pregnancy in future studies. 398 Previous reports have shown that plasma leptin concentrations of cafeteria diet-fed dams 399 increased in proportion to body fat mass (Chen et al., 2008;Bouanane et al., 2009;Jacobs et al., 400 2014). While CAF dams displayed higher plasma leptin concentrations in comparison to control 401 dams, CAFT dams did not display hyperleptinemia in the current study. Similarly, in one study 402 increased plasma leptin levels were observed in maternal obesogenic diet group but not in taurine 403 supplemented group although there was no significant effect of taurine (Li et al., 2013). Kim et 404 al. (2012) reported that leptin levels were significantly lower in taurine supplemented group 405 despite similar body weight and epididymal fat mass to control groups. They suggested that 406 additional studies are needed to elucidate the possible effect of taurine on leptin signaling in 407 adipose tissue (Kim et al., 2012). This result is consistent with another report which showed that 408 long term taurine supplementation did not reduce fat tissue but decreased mRNA expression 409 levels of leptin in white adipose tissue (Kim et al., 2019). Decreased circulating adiponectin 410 levels have been demonstrated in high fat and cafeteria diet induced murine models of obesity 411 (Chaolu et al., 2011;Suárez-García et al., 2017). Interestingly, plasma adiponectin 412 concentrations of both CAF and CAFT groups were markedly greater than CON and CONT 413 groups in the present study. Some studies have shown that body weight reduction resulted in 414 increased adiponectin levels in obesity (Yang et al., 2001;Esposito et al., 2003). Therefore, 415 weight loss of CAF and CAFT groups during lactation may have lead to higher adiponectin 416 levels in comparison to CON and CONT groups. Similarly, the present study demonstrated that CAF dams had increased plasma 422 malondialdehyde concentrations. Plasma malondialdehyde levels of CAFT dams did not differ 423 from CON and CONT dams, which indicated a partial normalization of the malondialdehyde 424 levels in response to taurine supplementation. It was reported that taurine administration 425 mitigated hepatic oxidative stress through reduction of malondialdehyde levels in the liver of 426 cafeteria fed rats (Abd Elwahab et al. 2017). Also, Ogasawara et al. (1994) reported that taurine 427 inhibited the production of oxidized low density lipoprotein by reacting with malondialdehyde. ). In the current study, both CAF and CAFT dams displayed higher levels of 434 serine and lower levels of tyrosine compared with control dams. Previous studies reported the 435 effects of cafeteria diet on plasma amino acid concentrations with contradictory results (Salvadó,436 Segués & Arola, 1991; Lladó et al., 1995;Pomar et al., 2019). Offspring suckled by cafeteria 437 diet fed dams exhibited higher circulating levels of serine due to increased hepatic 438 gluconeogenesis (Pomar et al., 2019). However, Salvadó, Segués & Arola, (1991) demonstrated 439 that serine concentrations was lower in the pups exposed to maternal cafeteria diet during 440 lactation than in the control pups. The low serine concentrations of the pups exposed to maternal 441 cafeteria diet were related to contribution of this amino acid to glucose synthesis in the suckling 442 offspring. Also, serine was associated with increased growth rate in the pups exposed to maternal 443 cafeteria diet. The amino acid imbalance observed in this study could be related with a possible 444 maternal trade-off to improve growth and development during suckling period.

445
In the current study no difference was observed in litter size and sex ratio between 446 groups. This is consistent with previous reports ( . Also, a meta regression analysis of 452 animal models investigating the effect of maternal obesogenic diet exposure on birthweight 453 demonstrated that this exposure had no effect on birthweight (Ribaroff et al., 2017). Akyol et al., 454 (2009) reported that exposure to maternal cafeteria diet led to fetal growth restriction, but they 455 showed increased birth weights in their further study (Akyol et al., 2011). These differences 456 might occur due to using different food items in cafeteria diet and duration and time of exposure.

457
Similar to the results of this study, it was reported that maternal taurine supplementation 458 had no effect on birth weights (Li et al., 2013). In addition to these outcomes, rise in neonatal 459 mortality was observed in CONT offspring. Similarly, other reports showed that taurine 460 supplementation in the setting of normal pregnancy resulted in increased neonatal mortality (Li 461 et al., 2013;Li et al., 2015). There are limited data on possible unfavorable effects of taurine in 462 normal pregnancies and underlying mechanisms have not been elucidated, clearly. Boujendar et 463 al. (2002) reported that taurine provided in dams fed a control diet induced fetal hypoglycaemia 464 and decreased pancreatic and postnatal body weights. Although taurine supplementation in vivo 465 exerted protective effects on pancreatic islets of the offspring from low protein diet fed dams 466 against cytokine toxicity, islet sensitivity of control animals has been increased and pancreatic 467 development was impaired (Boujendar et al., 2002;Merezak et al. 2004). These results 468 demonstrated that the effects of taurine supplementation on pregnancy outcomes were closely 469 associated with maternal nutritional background. Future studies should investigate the possible 470 toxicity of taurine supplementation in control pregnancy outcomes. In fact, one limitation of this 471 study could be giving taurine supplementation during pre-gestation since this design complicates 472 translating the current approach and outcomes to human pregnancy. Instead, taurine 473 supplementation could have been administered after mating but this procedure might have 474 masked the potential regressive influence of taurine on obesity development. Therefore, in 475 addition to possible toxicity of taurine supplementation in control pregnancy outcomes, a further 476 investigation can examine addition of taurine supplement after conception in a similar setting.

477
Exposure to maternal high fat diet during gestation may alter the development of various 478 organs and affect several organ systems. These effects differ by the animal model, timing and 479 duration of the high fat diet exposure as well as the offspring's gender (Williams et al., 2014) 480 Previously it has been reported that offspring exposed to maternal obesogenic diet exhibited 481 increased relative heart weight at weaning (Blackmore et al., 2014). This study demonstrated that 482 exposure to maternal cafeteria diet resulted in increased relative heart weight in both male and 483 female CAF offspring at weaning. Taurine supplementation to cafeteria diet normalized relative 484 heart weight. It was shown that taurine supplementation reduced heart weight in hypertensive 485 rats and was associated with decreased cardiac hypertrophy by displaying antioxidant activity 486 (Chahine et al., 2010). Also, both CAFT male and female offspring displayed increased brain 487 weight at weaning. It has been suggested that taurine deficiency might cause decreased 488 proliferation of neural progenitor cell and smaller brain weight in mouse (Shivaraj et al., 2012). 489 In a study of IUGR rats has been reported that prenatal taurine supplementation resulted in 490 increased brain weight above IUGR levels by reducing apoptosis in fetal rat cerebral cells and 491 promotes cerebral cell regeneration (Liu, Liu & Chen, 2011). In conclusion, present data suggest that maternal cafeteria diet led to increased adiposity and 495 malondialdehyde levels, hyperleptinemia and decreased plasma taurine levels. Maternal taurine 496 supplementation did not prevent adiposity but partially normalized cafeteria-induced maternal 497 metabolic dysfunction. Also, taurine supplemented cafeteria diet exerted favorable effects on 498 heart and brain weights of the offspring. The reason why taurine did not have profound 499 protective effects on these metabolic disturbances can be attributable to the amount of taurine 500 consumed by rats. Furthermore, it was showed that taurine supplementation resulted in increased 501 neonatal mortality in control pregnancies, which might be associated with maternal nutritional 502 background. While a few other studies investigated the effects of maternal taurine 503 supplementation on metabolic disturbances induced by high fructose and high fat obesogenic 504 diets (Li et al., 2013;Li et al., 2015), this study reported the influences of taurine 505 supplementation in a model of maternal cafeteria diet for the first time. The long-term effects of 506 maternal taurine supplementation on offspring and adverse maternal effects in normal 507 pregnancies must be further investigated.

Concentrations of biochemical parameters in maternal plasma
Mean values with their standard errors, n=6 (CON), n=7 (CONT, CAF and CAFT).

Concentrations of amino acids in maternal plasma
Mean values with their standard errors, n=6 (CON), n=7 (CONT, CAF and CAFT).