Expression of cholesterol packaging and transport genes in human and rat placenta: impact of obesity and a high-fat diet.

Evidence suggests that sub-optimal maternal nutrition has implications for the developing offspring. We have previously shown that exposure to a low-protein diet during gestation was associated with upregulation of genes associated with cholesterol transport and packaging within the placenta. This study aimed to elucidate the effect of altering maternal dietary linoleic acid (LA; omega-6) to alpha-linolenic acid (ALA; omega-6) ratios as well as total fat content on placental expression of genes associated with cholesterol transport. The potential for maternal body mass index (BMI) to be associated with expression of these genes in human placental samples was also evaluated. Placentas were collected from 24 Wistar rats at 20-day gestation (term = 21-22-day gestation) that had been fed one of four diets containing varying fatty acid compositions during pregnancy, and from 62 women at the time of delivery. Expression of 14 placental genes associated with cholesterol packaging and transfer was assessed in rodent and human samples by quantitative real time polymerase chain reaction. In rats, placental mRNA expression of ApoA2, ApoC2, Cubn, Fgg, Mttp and Ttr was significantly elevated (3-30 fold) in animals fed a high LA (36% fat) diet, suggesting increased cholesterol transport across the placenta in this group. In women, maternal BMI was associated with fewer inconsistent alterations in gene expression. In summary, sub-optimal maternal nutrition is associated with alterations in the expression of genes associated with cholesterol transport in a rat model. This may contribute to altered fetal development and potentially programme disease risk in later life. Further investigation of human placenta in response to specific dietary interventions is required.


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Maternal nutrition can have a profound impact on fetal development and future physiological 39 function and metabolic health (1) . A number of dietary perturbations, including maternal 40 undernutrition and low protein diets, have been associated with increased risk of obesity and 41 cardiovascular disease in the adult offspring (2,3) . In the context of the growing epidemic of 42 obesity, focus has shifted towards understanding the effects of nutritional excess and obesity 43 on offspring programming of disease. Studies have consistently demonstrated that these 44 exposures are associated with a substantial increase in the risk of poor metabolic health in the 45 offspring in both humans (4) and animal models (5) . There is emerging evidence from animal 46 studies, however, that maternal high-fat diets also have the potential to program metabolic 47 outcomes in the offspring independent of the effects of maternal obesity. In addition, these 48 effects appear to depend not only on the amount of fat in the diet (6) , but also on the fatty acid 49 composition (7,8) . The majority of studies to date that have investigated the effects of a 50 maternal high fat diet have utilised diets high in saturated fat. However, due to changes in 51 population level patterns in dietary consumption (9,10) , attention has now shifted toward the 52 roles of polyunsaturated fats within the diet. 53 The mechanisms underlying this early life programming of obesity and metabolic disease are 54 not completely understood. However, as the sole interface between the mother and the fetus, 55 structural and functional changes within the placenta have been implicated as playing a key 56 role (11) . Cholesterol is present in every cell of the human body and an adequate supply is 57 therefore critical for supporting normal fetal development. As the precursor for all steroid 58 hormone synthesis, cholesterol also plays an important role in placental function. During 59 pregnancy, the fetus obtains cholesterol via endogenous synthesis as well as transfer across 60 the placenta from the maternal circulation, disturbances to either of these processes have 61 negative impacts on fetal growth, cell proliferation, metabolism and the organisation of 62 tissues (12,13) . The endogenous synthesis of cholesterol appears to be most critical for the 63 developing fetus, as defects in this pathway are known to be lethal (14) . Sub-optimal maternal 64 contribution of cholesterol across the placenta, however, has been associated with lower 65 birthweight (15,16) and microcephaly (16) in humans, highlighting the importance of this 66 exogenous cholesterol supply.

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Transport of cholesterol across the placenta is a complex process in both humans and rodents 68 (17,18) . Briefly, the majority of cholesterol circulates the body in the form of HDL, LDL and 69 VLDL cholesterol, which are associated with specific structural apolipoproteins (ApoA2,

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ApoB and ApoC2 respectively). The layer of trophoblast cells, located closest to maternal 71 circulation, take in LDL and VLDL through their respective receptors. HDL cholesterol can 72 be taken up via a specific receptor (scavenger receptor class B type 1; SR-B1) or by binding 73 to proteins such as megalin and cubilin (Cubn). Once within the cell, cholesterol is 74 hydrolysed into free cholesterol, bound to sterol carrier proteins and then transferred to the 75 basolateral membrane where it passes through the fetoplacental endothelium. The processes 76 governing cholesterol efflux from the endothelial layer are poorly understood, although it has 77 been shown that exogenous cholesterol is secreted into fetal circulation, through association 78 with various transporters (19) , where it is repackaged into fetal lipoproteins. This process is 79 facilitated by microsomal triglyceride transfer protein (Mttp). The finding that the placenta 80 expresses and secretes its own apolipoproteins such as ApoB (20) also raises the possibility that 81 cholesterol is repackaged into HDL, LDL and VLDL cholesterol within the placenta itself.

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In addition to its critical role in fetal growth and development, there is emerging evidence 84 that alterations in placental cholesterol transfer capacity may also be a contributing factor to 85 metabolic programming. In a previous study (21) , we showed providing rats with a low protein 86 diet until day 13 of gestation, a dietary treatment previously associated with programming of 87 obesity, hypertension and glucose intolerance in adult offspring (22,23) , resulted in increased 88 placental expression of a number of genes associated with cholesterol and lipoprotein 89 transport and metabolism in the rat.

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Given the similarity in the metabolic phenotype induced by maternal obesity/nutritional 91 excess and low protein diets, we hypothesised that programming of health and disease is 92 driven by perturbations of a small set of common 'gatekeeper' processes (24,25) and changes in 93 placental cholesterol transfer and metabolism may be common mechanisms underlying 94 metabolic programming by different dietary exposures. Therefore, the aim of this current 95 study was to investigate the effect of a high maternal dietary omega-6:omega-3 fatty acid 96 ratio, associated with decreased placental weight (26) , against a lower ratio as well as total fat 97 intake, on the expression of genes associated with cholesterol and lipoprotein transport, 98 known to be affected by maternal diet, in the mature placenta of the rat. We also aimed to 99 investigate whether placental expression of these same genes differed according to maternal 100 body mass index (BMI) in a cohort of pregnant women.

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Animal Experiments and sample collection 103 This paper reports data from the analysis of placentas that were collected as part of a previous 104 study (26) . Virgin female Wistar rats (n = 24; 75-100 g; Charles River, UK) were housed on 105 wood shavings in individually ventilated cages under a 12 h light/12 h dark cycle at a 106 temperature of 20-22 °C and had ad libitum access to food and water throughout the 107 experiment. Female rats were allowed to acclimatise to the unit for 1-2 weeks, during which   Gene targets were chosen based on our previous data (21) where RNASeq analysis of day 13 157 rat placentas revealed differential expression of 91 genes in response to maternal protein

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Human data were analysed using a one-way ANOVA with maternal BMI as a factor. Possible 184 co-variates were identified and corrected for within the analysis. A value of P<0.05 was 185 considered statistically significant.

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Rat placenta 188 The results of the gene expression analyses in the rodent placentas is shown in Table 1.   Table 2. The average age, parity and gestation 207 length of women within this study was similar between women in the three BMI groups, 208 however the birth weights of infants born to women with a BMI >25 kg/m 2 were significantly 209 greater than those of women in the normal BMI range.

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The results of the gene expression analyses in the human placentas is shown in Table 3. All women with a BMI >25 kg/m 2 , although this did was not statistically significant (P=0.053).

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The mRNA of other placental genes was not different between BMI groups.

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This experiment aimed to test the hypothesis that maternal diet, specifically, fatty acid of key genes associated with these pathways, suggesting enhanced cholesterol transport to the 226 fetus in this group. In the human study, however, only two of these genes were differentially 227 expressed in placentas from women in different BMI categories, suggesting that maternal 228 obesity had a limited impact on placental cholesterol transport at the level of gene expression.

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Within this study we have shown that exposure to a high LA, high-fat diet resulted in 230 increased expression of genes involved in the formation of apolipoproteins, cholesterol 231 uptake and cholesterol repackaging. These differences were not observed when either the fat 232 content and/or the fatty acid ratio was altered, suggesting a strong interaction between these 233 variables. Importantly, the effects observed on the placental gene expression profiles show 234 striking resemblances to our previous findings (21) where upregulation of these genes was 235 observed in the placentas of dams exposed to a low protein diet. These data suggest that, not 236 only do these differences persist to a late stage placenta (day 20), but, despite different 237 dietary interventions, the similarities in results suggest a common mechanism of action. The cholesterol during fetal development can be associated with adverse outcomes. In rodent 242 models, maternal hypercholesterolemia has been associated with growth restriction (28) , 243 altered liver development (29) and atherosclerosis (30,31) . In humans, maternal 244 hypercholesterolaemia has been associated with the development of fatty streaks in fetal 245 arteries and cholestasis during pregnancy is associated with programming of an overweight, 246 insulin resistant phenotype in the child (32,33) . It will therefore be important in future studies 247 to determine the longer term consequence of the changes in placental gene expression for the 248 postnatal offspring.

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Based on the substantial impact of maternal high-fat high LA feeding, a dietary pattern 250 commonly observed in the modern Western diet, we extended our study to determine if there 251 was any evidence to suggest an effect of maternal obesity on cholesterol transfer in the 252 human. There were, however, relatively few differences in the expression of key genes 253 associated with different BMI categories in human placental samples, although there were 254 some subtle differences in the expression of 3 genes (ApoB, Rbp4 and Ttr) between BMI 255 categories. Ttr is a protein that binds to and transports Rbp4. In the bound state, Rbp4 is 256 protected from glomerular filtration and so levels of these two proteins are often correlated.

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As such, the similar patterns of expression across these two genes observed in this study was 258 anticipated. What was surprising, however, was our finding that women with a BMI above 259 the normal range (>25 kg/m 2 , overweight or obese) exhibited decreased placental expression 260 of these genes since elevated levels of circulating Rbp4 have been associated with many of 261 the co-morbidities linked to obesity including hypertension (34) , insulin resistance and type 2 262 diabetes (35,36) . It is important to note, however, that these observations have all been 263 associated with circulating levels of Rbp4, whereas we measured gene expression in the 264 placenta. There is limited literature evaluating the role of Rbp4 within the placenta during 265 pregnancy, particularly in association with maternal obesity. It may be, however, that in 266 obese mothers, placental expression is reduced to compensate for the increased maternal 267 circulating levels and therefore avoiding fetal exposure to high quantities of Rbp4. Further 268 experimentation, is required to determine expression of Rbp4 and Ttr in both the mother, 269 fetus and the placenta throughout pregnancy and their association with maternal obesity.

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Placental ApoB expression was increased in women whose BMI was above the normal range 271 (>25 kg/m 2 ). A study by Dubé and colleagues (37) , showed increased circulating ApoB  One key limitation of the current study is that direct measurements of cholesterol transport or 279 measurement of the genes of interest at the level of protein were not analysed, and so care 280 must be taken when extrapolating the findings to functional outcomes. Further to this, human 281 participants within this study were stratified based on BMI whereas the animal experiments 282 utilised specific dietary interventions. There are many factors that can affect BMI and,283 although nutrition is a key element, there is still a huge variety of nutritional habits that can 284 lead to individuals resulting in similar BMIs. As such, assumptions cannot be made about 285 specific nutrient intakes of the women based on this data. Finally, it is important to note that, 286 whilst there are many similarities between placental physiology and function in the rat and 287 humans, there are some key differences. Of particular importance to this study is the 288 difference in circulating progesterone levels at the end of pregnancy. In rodents there is a 289 dramatic decrease in circulating progesterone (38) , whereas in humans, progesterone levels are 290 increased or at least maintained at the time of parturition (39) . Progesterone is a key steroid 291 produced from cholesterol within the placenta and has been shown to regulate the expression 292 of some genes including Rbp4 in other tissues (40) . As such, careful consideration of the 293 differences in placental hormone production, particularly steroid hormones, between the two 294 species should be made when drawing comparisons.

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In conclusion, this study aimed to elucidate whether differences in placental expression of 296 genes involved in cholesterol transport and efflux were associated with altered maternal 297 nutrition in a manner similar to our previous observations in the low-protein model. We 298 demonstrated that exposure to high levels of omega-6 as part of a high-fat diet elicited a 299 similar pattern of placental gene expression, suggesting an increase in cholesterol transport 300 across the placenta. This highlights the potential for a common mechanism by which sub-301 optimal maternal nutrition during pregnancy alters placental function, and potentially fetal 302 development, resulting in increased risk of disease in later life. We then carried out a 303 preliminary study which aimed to establish if similar alterations were observed in human 304 placentas. Although BMI was associated with some changes in expression, these observations 305 were not consistent and further experimentation is required on placental samples where the 306 specific nutrient intake of the participants are known.