The effect of maternal dietary fat content and omega-6 to omega-3 ratio on offspring growth and hepatic gene expression in the rat.

Omega-6 fatty acids have been shown to exert pro-adipogenic effects whereas omega-3 fatty acids work in opposition. Increasing intakes of LA (linoleic acid; omega-6) vs ALA (alpha-linolenic acid; omega-3) in Western diets has led to the hypothesis that consumption of this diet during pregnancy may be contributing to adverse offspring health. This study investigated the effects of feeding a maternal dietary LA:ALA ratio similar to that of the Western diet (9:1) compared to a proposed 'ideal' ratio (~1:1.5), at two total fat levels (18% vs 36% fat w/w), on growth and lipogenic gene expression in the offspring. Female Wistar rats were assigned to one of the four experimental groups throughout gestation and lactation. Offspring were culled at 1 and 2 weeks of age for sample collection. Offspring of dams consuming a -36% fat diet were ~20% lighter than those exposed to a 18% fat diet (P<0.001). Male, but not female, liver weight at 1 week was ~13% heavier, and had increased glycogen (P<0.05), in offspring exposed to high LA (P<0.01). Hepatic expression of lipogenic genes suggested an increase in lipogenesis in male offspring exposed to a 36% fat maternal diet and in female offspring exposed to a low LA diet, via increases in the expression of Fasn and Srebf1. Sexually dimorphic responses to altered maternal diet appeared to persist until two weeks-of-age. In conclusion, whilst maternal total fat content predominantly affected offspring growth, fatty acid ratio and total fat content had sexually dimorphic effects on offspring liver weight and composition.

172 Lipogenic pathway and adipokine target genes were chosen based on previous data from our 173 laboratory that indicated that these genes were sensitive to changes in the maternal diet (23) and 174 included; peroxisome proliferator-activated receptor gamma (Pparg), sterol regulatory Page 9 of 26 240 The blood fatty acid profile of the dams at the end of lactation, after a further 6 weeks on their 241 respective experimental diets, were similar to those observed after the first 4 weeks of dietary 242 intervention. A notable difference, however, was that at this time point, relative proportions of 243 DHA, as a percentage of total lipids, were not different between dietary groups (Fig. 2F). LA 244 (1.5-fold), AA (1.8-fold) and total omega-6 (1.5-fold) were all higher in dams consuming a 245 high LA diet irrespective of dietary fat content (P<0.001; Fig. 2E). Conversely, total omega-3 246 levels were 3-fold higher in dams consuming a low LA diet, irrespective of dietary fat content 247 (P<0.001). The proportions of ALA were also higher in the groups consuming the low LA diets 248 and in rats consuming the 36% vs 18% fat diets in the low LA group only (P<0.05; Fig 2F).
249 DPA proportions were higher in the groups consuming the low LA diets, however, unlike ALA, 250 DPA proportions were lower, rather than higher, in dams consuming the 36% fat diets in the 251 low LA group only (P<0.001; Fig. 2F). EPA proportions were higher in groups consuming a 252 low LA diet compared to those consuming a high LA diet (P<0.001; Fig. 2F). EPA proportions 253 were also affected by total dietary fat content, and were lower in dams consuming a high-36% 254 fat (36% fat) diet compared to an lower (18% fat) diet (P<0.001; Fig. 2F). Maternal blood total 255 MUFA levels at the end of lactation were 1.4-fold higher in the dams consuming a low LA diet 256 irrespective of dietary fat content (P<0.001; Fig. 2E).

257
258 Maternal weight, body composition and gene expression 259 There were no significant differences in dam bodyweight between dietary groups prior to the 260 commencement of the dietary intervention or at any time during the experiment (data not 261 shown). Dams consuming the 36% fat diets had heavier lungs relative to bodyweight at the end 262 of lactation compared to those consuming the 18% fat diets, independent of the LA:ALA ratio 263 (P<0.05). There were no differences in the relative weight of the heart, liver, brain, kidney, 264 gonadal or retroperitoneal fat pads between experimental groups (   310 MUFA proportions were 1.2 fold higher in offspring of dams fed the low LA diets, and 1.2 311 fold higher in offspring of dams who consumed a 36% fat vs 18% diet (P<0.001; Fig. 3C).

312
313 Offspring organ weight and liver composition 314 At 1 week of age, heart weight relative to bodyweight was higher in female offspring of dams 315 receiving a high (36%) fat diet compared to the 18% fat diet, independent of the dietary 316 LA:ALA ratio (P<0.05). There were no differences in the relative weight of lung or kidney at 317 1 week of age and no differences in the relative weight of the heart, lung, liver, gonadal or 318 retroperitoneal fat pads in the offspring at 2 weeks of age between treatment groups in either 319 male or female offspring (Table 3). 320 321 Liver weight at 1 week appeared to be influenced by the LA:ALA ratio of the diet to a greater 322 extent than total fat level, at least in males. Thus, male offspring of dams consuming the high 323 LA diets had increased liver weights compared to offspring of dams receiving a low LA diet 324 (P<0.01), irrespective of total dietary fat content. The glycogen content of the livers was also 325 higher in male offspring of dams consuming the high LA diets at 1 week of age (P<0.05). No 326 effect of maternal diet on offspring liver protein or DNA concentration was observed (Table   327 4). These differences were not present in females at 1 week of age and no differences in 328 glycogen content were observed at two weeks of age in male offspring. DNA concentration in 329 females at two weeks of age was marginally increased (1.1-fold) in offspring exposed to a high-330 36% fat diet, irrespective of maternal dietary fatty acid ratio (P<0.05).  343 There were no differences in the expression of Fasn or Lpl in female offspring, or expression 344 of any hepatic genes in male offspring at this time point (Table 3).  (5,9,26,27) , decreasing the dietary LA:ALA ratio resulted in substantial increases in 363 relative maternal ALA and EPA levels but only a very modest increase in DHA proportions 364 after a 4-week exposure, and no difference compared to the higher LA:ALA ratio after 10 365 weeks. Interestingly, and independent of dietary LA:ALA ratio, dams appeared to be more 366 efficient at converting DPA to DHA when total dietary fat load was higher. One possibility is 367 could be that this is simply a result of the higher amount of substrate (i.e. ALA) available for 368 conversion to the longer chain derivatives such as DPA and DHA in diets containing higher 369 total fat levels. This effect did not, however, persist after a further 6 weeks of dietary exposure, 370 at which point EPA and DPA were lower in dams consuming a low LA 36% fat diet compared 371 to a low LA 18% fat diet. This may be a result of saturation of the PUFA metabolic pathway 372 when total fat, and therefore PUFA, levels were higher (10,28) . This apparent decrease in capacity 373 to convert ALA through to EPA and DHA during consumption of a high-36% fat diet coincides 374 with the decreased protein intake observed in these groups. It is possible that the lower 375 consumption of protein in rats fed on the 36% fat diets may have contributed to reduced  428 This finding was, however, consistent with many other studies that reported decreased fetal 429 (40,41) , birth (42) and weaning weight (43) in offspring of dams exposed to a high-36% fat diet 430 during gestation and lactation periods. The differential effects of different high-36% fat diets 431 on offspring growth is likely due to differences in composition of the diet as well as periods of 432 exposure between studies (3) . In those studies that have reported lower offspring weights in 433 offspring fed a high-fat diet, lower protein intakes in dams consuming a high-fat diet have been 434 cited as a likely contributing factor. Further to this, protein restricted diets have been associated 435 with impaired mammary gland development (29,44) leading to impaired milk synthesis (44) , and 436 this may also have contributed to reduced offspring growth observed during the suckling 437 period. It is important to note however, that the reduction in protein intake in high-fat dams 438 consuming a 36% fat diet in the current study were more modest (10-25%) than those typically 439 used in low-protein diet studies (~50% reduction) (45,46,47,48) . 440 441 The lower Fasn expression in the liver and adipose tissue of dams exposed to a high-36% fat 442 diet is consistent with the established role of this enzyme in suppressing lipogenesis in times 443 of energy excess (49) . Surprisingly, this change did not appear to be mediated through changes  (50) . It is important 445 to note that since only mRNA expression was measured, we cannot comment on any 446 differences in protein expression or activity of this transcription factor although mRNA and 447 protein levels have been shown to be closely correlated (23) . Following this up at the protein 448 level is a major priority for future study. In the offspring, however, hepatic Fasn expression 449 was not downregulated by exposure to a maternal high-36% fat diet but was actually higher in 450 male offspring of dams consuming the 36% fat compared to the 18% fat diets at 1 week of age  477 The majority of the hepatic mRNA expression differences, as well as gross differences in liver 478 weight and composition, appeared to be transient and were no longer present at 2 weeks of age. 479 A notable exception was the lower expression of Srebf1 mRNA and higher expression of Pparg 480 in females of dams exposed to a high LA diet compared to the low LA diet, with a similar trend 481 observed in males. Although found in relatively low concentrations in the liver, activation of 482 Pparg has been shown to increase hepatic lipid storage and is elevated in models of hepatic 483 steatosis (52) . As such, decreased Pparg expression can alleviate some of the symptoms of 484 hepatic steatosis leading to a reduced liver weight in conjunction with a reduction in hepatic 485 triglyceride content (53) . Thus, our finding that female offspring of dams exposed to a high LA 486 diet tended towards to have an increased liver weight at one week of age followed by increased 487 hepatic Pparg expression at two weeks of age may suggest that the increase in Pparg 488 expression is a potential response to the increased liver growth observed a week earlier. 498 expression have been suggested as a mechanism for programming changes in metabolic 499 processes within tissues as well as the morphology of the tissues themselves (1) . In this study, 500 offspring are still exposed to the experimental diets via the dams milk, and further studies in 501 offspring at older ages are required to assess whether the changes in growth, hepatic gene 502 expression and liver weights in the current study are associated with phenotypic changes that 503 persist once offspring are no longer exposed directly to the altered diet composition. In 504 addition, analysis of lipogenic pathway and adipokines targets at the protein level, as well as 505 whole transcriptome analysis, may yield useful information about their regulation and the 506 extent to which these experimental diets programme other metabolic and regulatory pathways 507 in the liver. Further to thisFinally, the longevity of these perturbations into later life, especially 508 when presented with secondary metabolic challenges such as aging, prolonged high-fat feeding 509 or in the case of female offspring, pregnancy, remains to be elucidated.

ITEM RECOMMENDATION
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Abstract 2 Provide an accurate summary of the background, research objectives, including details of the species or strain of animal used, key methods, principal findings and conclusions of the study.

INTRODUCTION
Background 3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale. b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.
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Study design 6
For each experiment, give brief details of the study design including: a. The number of experimental and control groups. b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when). c. The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out.

7
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