Maternal High-Protein Diet during Pregnancy Modifies Rat Offspring Body Weight and Insulin Signalling but Not Macronutrient Preference in Adulthood

Diet of mothers during gestation may impact offspring phenotype. This study evaluated the consequences of a maternal High-Protein (HP) diet during gestation on food preferences and phenotypic characteristics in adult rat offspring. Dams were fed a HP or a Normal-Protein (NP) isocaloric diet during gestation only. Weaned female pups were divided into 3 diet groups: NP control or one of two dietary self-selection (DSS) conditions. In DSS1, offspring had a free choice between proteins (100%) or a mix of carbohydrates (88%) and lipids (12%). In DSS2, the choice was between proteins (100%), carbohydrate (100%) or lipids (100%). DSS2 groups consumed more of their energy from protein and lipids, with a decreased carbohydrate intake (p < 0.0001) compared to NP groups, regardless of the maternal diet. Offspring from HP gestation dams fed the DSS2 diet (HPDSS2) had a 41.2% increase of total adiposity compared to NPDSS2 (p < 0.03). Liver Insulin receptor and Insulin substrate receptor 1 expression was decreased in offspring from HP compared to NP gestation dams. These results showed the specific effects of DSS and maternal diet and data suggested that adult, female offspring exposed to a maternal HP diet during foetal life were more prone to adiposity development, in response to postweaning food conditions.


Introduction
During the perinatal period, foetus exposure to challenging environmental conditions and particularly inappropriate nutrition, is critical and generates programming effects often revealed birth to PND105 (see details on animal procedure in Supplementary Materials Methods S1). From PND28, food intake was monitored at 24 h intervals, except weekends, throughout the entire study. The total energy intake (in kcal) of each rat was determined from food weighing data and the known energy content of each diet. Since the exact macronutrient composition of each diet was known, the percentage of the total energy intake attributed to each macronutrient was calculated as well. animal procedure in Supplementary Materials Methods S1). From PND28, food intake was monitored at 24 h intervals, except weekends, throughout the entire study. The total energy intake (in kcal) of each rat was determined from food weighing data and the known energy content of each diet. Since the exact macronutrient composition of each diet was known, the percentage of the total energy intake attributed to each macronutrient was calculated as well.

Body Composition Measurement by Magnetic Resonance Imaging (MRI)
Fat pads (total, visceral and subcutaneous) were measured by in vivo MRI (Platform Imagerie du Vivant, Paris-Descartes, France) on PND28, 70 and 105 (eve of euthanasia). Images were defined with a Nutrients 2019, 11, 96 4 of 17 4.7 T Bruker Biospec system (running Paravision 5, Bruker BioSpin GmbH, Ettlingen, Germany) using a Bruker 70 mm i.d. tunable quadrature RF resonator. The anaesthesia was performed with isoflurane in oxygen-supplementary air, breathing rate being monitored and body temperature being maintained at 36-38 • C. Images registered were segmented (by fuzzy c-means) with MIPAV 7.3.0 (national institutes of health, Bethesda, MD, USA). Volume of adipose tissues was determined by quantification of the voxel's mask directly converted to mm 3 (see the example in Supplementary Materials Methods F1) and then to grams using a density value of 0.92 kg/dm 3 .

Sampling
On PND105 after overnight fasting, a first blood sample (~500 µL) was taken by tail vein puncture. Then animals were fed a calibrated meal (3 g of dry matter) during light phase, composed according to the previous week of each one's macronutrient intake when included in a DSS group. The calibrated meal was removed after 30 min and over the following 60 min rats were anesthetized with an intraperitoneal injection of pentobarbital sodium before decapitation, (time + 90 min). A second blood sample was collected during dissection (fed state). The peri-ovarian adipose tissue, liver, pancreas, hypothalamus and isolated Nucleus Accumbens (NAcc) [29] were sampled, snap-frozen in liquid nitrogen and stored at −80 • C. Body composition was assessed by weighing the carcass, adipose tissues (subcutaneous, peri-ovarian, retroperitoneal and mesenteric) and other organs. Blood was centrifuged (3000 g, 4 • C, 15 min) and plasma was directly stored at −80 • C.

Gene Expression by Quantitative PCR
Total RNA from hypothalamus, NAcc, peri-ovarian adipose tissue (100 mg) and liver (100 mg) were extracted using 1 mL Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and synthesis of cDNA was performed, as previously described [30], with 400 µg total RNA. Real-time PCR was performed on a StepOnePlus TM real-time PCR (Applied Biosystems, Foster City, CA, USA) using SYBR green fast reagent PCR master mix (Applied Biosystems) under the following conditions: 10 min at 95 • C, 40 cycles of 95 • C for 15 s and 1 min at 60 • C. Primers sequences used are detailed in Supplementary Materials Table S1. Gene expression was determined using the formula below. Results of this gene expression are presented as an arbitrary unit using the NPNP group as reference sample (NPNP gene expression = 1).

Statistical Analysis
Data on birth weight. One-way analysis of variance (ANOVA) was performed to determine the effect of the maternal diet during gestation (gestation factor).
Data after weaning. Two-way ANOVA (model W) was performed to determine the effect of the maternal diet during gestation (gestation factor), the postweaning diet (postweaning factor) and the interaction between both (gestation × postweaning). For repeated measurements(model Wt), a 'time factor' was added as a repeated factor and a 'pup factor' was added as a random factor to take into account that data were collected at differing time points on the same pup. In the two postweaning models (W and Wt), a random factor was added to correct for possible correlations between pups from the same litter.
Pairwise comparisons were adjusted with multiple comparisons using a Tukey post-hoc test. The statistical analyses were performed using R studio 1.1.456 (Boston, MA, USA) and the differences between groups were considered significant at p < 0.05. All data are expressed as means ± SEMs (Standard Error of the Mean).

Offspring Energy Intake and Macronutrient Self-Selection
The cumulative energy intake (energy intake summed day by day) of the offspring fed either the NP or DSS diets (PND28 to PND105) is reported in Figure 2a. Macronutrient self-selection during the DSS post-weaning period of pups is reported in Figure 2b.
The statistical analyses were performed using R studio 1.1.456 (Boston, MA, USA) and the differences between groups were considered significant at p < 0.05. All data are expressed as means ± SEMs (Standard Error of the Mean).

Offspring Energy Intake and Macronutrient Self-Selection
The cumulative energy intake (energy intake summed day by day) of the offspring fed either the NP or DSS diets (PND28 to PND105) is reported in Figure 2a. Macronutrient self-selection during the DSS post-weaning period of pups is reported in Figure 2b.
The maternal HP diet, compared to NP diet, did not affect cumulative energy intake (Figure 2a) or the contribution of macronutrients (Figure 2b) to the overall intake in offspring.

Offspring Body Weight Gain and Adiposity Development
Body weight between PND28 and PND105 and final weight gain are reported in Figure 3a. Weights (in g) of visceral (VAT) and subcutaneous (SAT) adipose tissues determined by MRI, on  The maternal HP diet, compared to NP diet, did not affect cumulative energy intake (Figure 2a) or the contribution of macronutrients (Figure 2b) to the overall intake in offspring.

Offspring Body Weight Gain and Adiposity Development
Body weight between PND28 and PND105 and final weight gain are reported in Figure 3a. Weights (in g) of visceral (VAT) and subcutaneous (SAT) adipose tissues determined by MRI, on PND28, 70 and 105 are reported in Table 2. Total adipose tissue (TAT) weights (in g) determined by MRI on PND28 and 70 are also reported in Table 2. TAT weights determined by MRI and dissection on PND105 are presented in Figure 3b. Results from both MRI and dissection for final TAT were in accordance, although MRI results indicated significant differences (p < 0.0001) with approximately 15% lower final weights of TAT in each animal group.    Birth weight of pups was comparable between HP and NP gestation groups (NP, 5.38 ± 0.17 g; HP, 5.50 ± 0.17 g). During lactation, growth and pup weight were not affected by the gestation diet (NP, 47.8 ± 1.5 g; HP, 47.8 ± 0.8 g). On PND28, all pups (randomly distributed in groups) had a similar body weight (NPNP, 78.2 ± 3.4 g; HPNP, 80.3 ± 2.3 g; NPDSS1, 81.4 ± 3.8 g; HPDSS1, 80.4 ± 2.9 g; NPDSS2, 83.2 ± 2.9 g; HPDSS2, 79.5 ± 2.8 g). The gestation diet had no effect on the offspring body weight (Figure 3a). Adipose tissue weights were not significantly different between groups on PND28 ( Table 2).
The postweaning diet over time significantly affected body weight (postweaning × time, p <0.0001) evidenced mainly by a lower body weight in the DSS1 groups (Figure 3a). On PND70, SAT was significantly increased in the DSS2 groups (p < 0.003) evidenced mainly by a higher increase in the HPDSS2 group ( Table 2).
The gestation diet, in conjunction with the postweaning diet and time, affected body weight and displayed a significant effect mainly driven by a difference between NPDSS2 and HPDSS2 groups (gestation × postweaning × time, p < 0.0001) (Figure 3a). On PND105, the final VAT was significantly higher in the HPDSS2 group compared to NPDSS2 group (gestation × postweaning diet, p ≤ 0.02) regardless of the measurement method (Table 2).

Maternal HP Diet during Gestation Affects Fasting Leptin Levels
Blood, plasma glucose, insulin and leptin levels, from both fasting and fed states, are reported in Figure 4. Other blood and plasma parameters are reported in Table 3.

Maternal HP Diet during Gestation Affects Fasting Leptin Levels
Blood, plasma glucose, insulin and leptin levels, from both fasting and fed states, are reported in Figure 4. Other blood and plasma parameters are reported in Table 3.
Blood and plasma parameters presented in Figure 4 and in Table 3 were not affected by the maternal diet during gestation.
Regarding postweaning feeding effects, plasma glucose levels in the fed state were higher in the DSS2 groups and lower in the DSS1 groups, compared to control groups (p < 0.05). Fed plasma insulin was lower, by just by enough to be considered significant (p = 0.05), in the DSS1 groups compared to the other postweaning groups (Figure 4). Fasted plasma TG levels were significantly decreased in both DSS groups, especially in the DSS2 groups (p < 0.05). However, no effects in fed TG plasma concentrations were found. The fed plasma cholesterol level was decreased in the DSS1 and DSS2 groups, compared to the control group (p < 0.05). Protein levels were not affected during fasting but decreased in fed plasma in the DSS1 and DSS2 groups (p < 0.01) ( Table 3).
Unlike the fed plasma, fasting plasma concentrations of leptin on PND105 were significantly affected by the interaction between the gestation and postweaning diets (P < 0.05) (Figure 4).    Table S2).
Blood and plasma parameters presented in Figure 4 and in Table 3 were not affected by the maternal diet during gestation.
Regarding postweaning feeding effects, plasma glucose levels in the fed state were higher in the DSS2 groups and lower in the DSS1 groups, compared to control groups (p < 0.05). Fed plasma insulin was lower, by just by enough to be considered significant (p = 0.05), in the DSS1 groups compared to the other postweaning groups (Figure 4). Fasted plasma TG levels were significantly decreased in both DSS groups, especially in the DSS2 groups (p < 0.05). However, no effects in fed TG plasma concentrations were found. The fed plasma cholesterol level was decreased in the DSS1 and DSS2 groups, compared to the control group (p < 0.05). Protein levels were not affected during fasting but decreased in fed plasma in the DSS1 and DSS2 groups (p < 0.01) ( Table 3).
Unlike the fed plasma, fasting plasma concentrations of leptin on PND105 were significantly affected by the interaction between the gestation and postweaning diets (P < 0.05) (Figure 4).

Maternal HP Diet during Gestation Impacts Liver Insulin Signalling But Not Central Markers of Food Intake Regulation
In the liver, insulin receptor (Ir) and insulin receptor substrate (Irs1) expression significantly decreased with the HP maternal diet (p < 0.05) (Figure 5a).

Discussion
The present study evaluated the influence of maternal HP diet during gestation on feeding behaviour using postweaning DSS feeding and on metabolic phenotype characteristics in adult

Discussion
The present study evaluated the influence of maternal HP diet during gestation on feeding behaviour using postweaning DSS feeding and on metabolic phenotype characteristics in adult female offspring. In fact, only few studies highlighted the specific metabolic mechanisms affected by maternal HP diet during gestation on adult offspring. The present work furthers the literature with a novel approach combining maternal protein diet modification and offspring food choices, through DSS, during growth and early adulthood. DSS mimics free choice in feeding by increasing available food options. The present analyses cover a wide range of phenotypic characteristics in an attempt to elucidate the links between maternal diet and food intake regulation and energy metabolism disorders in offspring. First, the results indicated that offspring put on DSS with separate macronutrient options preferentially consumed more lipids and less carbohydrates, irrespective of maternal feeding during gestation. In accordance with these specific food choices made by rats, the DSS1 diet can be considered as a high-protein/low-fat diet and the DSS2 diet as a high-protein/high-fat diet. Second, the HP maternal diet (vs. NP) altered insulin signalling in the liver of offspring. Third, the HP maternal diet (vs. NP) induced higher adiposity in offspring fed either the DSS2 diet or (to a lesser degree) the high-carbohydrate NP diet but not those fed the DSS1 diet.
Birth weight was not affected by the gestation HP diet, as shown in a previous work [20]. The impact of the gestation HP diet on weight gain was most prominent under both the DSS2 condition and, to a lesser extent, the postweaning high-carbohydrate NP diet. Weight gain was higher throughout the study in HPDSS2 compared to NPDSS2 rats, although energy intake was not significantly different between the two. The higher weight gain in HPDSS2 rats was paralleled by increased fat storage and a two fold increase of the fasting plasma leptin level compared to NPDSS2 rats. Accordingly, total body adiposity and circulating leptin levels were affected by the maternal diet during gestation, even though gene expression in adipose tissue was not affected. Adipose tissue is a key endocrine-like organ secreting leptin in proportion to adipose tissue mass. Leptin acts as a signal of long-term energy balance providing feedback to central energy regulation by steering the balance between energy consumption and expenditure [31]. The results showed that, compared to the postweaning NP control diet, the DSS either offered as DSS1 or DSS2 diets led to a higher protein intake of around 30% of the total energy intake, as previously observed after weaning or in adulthood [26,27,32,33]. However, the increase of fat intake at the expense of carbohydrates observed in DSS2 rats was associated with higher energy intake, compared to both NP and DSS1 rats. Although foetal exposure to HP diet was not studied in association to postweaning DSS, it was reported that LP diet during gestation could lead to a preference for fatty foods in 12-week-old female offspring [18]. Hyperphagia, as observed in the DSS2 condition, may be promoted when nutrients are given separately [34,35] or offered as a palatable diet [36]. It could be hypothesized that both NP and DSS2 diets, characterized by high-carbohydrate or high-fat contents, respectively, simulated an obesogenic diet, revealing an increased risk of being overweight as a result of HP foetal programming.
In this study, insulin levels in the fed state did not show any effect of the maternal diet, whereas fasting plasma insulin increased slightly in offspring from the HP maternal group and especially in the HPDSS2 and HPNP groups. Insulin is a key hormone regulating glycemia and nutrient storage. Deregulation of these metabolic processes can signal metabolic disorders [37] and a basal hyperinsulinemic state that can cause insulin resistance [38]. Interestingly, the present study showed that the maternal HP diet induced decreased Insr and Irs1 expression in the offspring liver regardless of postweaning diet. It has been shown that a HP diet during gestation can induce differences in offspring liver transcriptome [39] and modified gene expression of key metabolism genes in the liver [22]. Because the liver is the major site of insulin clearance, dysfunctional insulin capture for degradation is a risk factor for type 2 diabetes and obesity [40]. Inactivation of Ir can induce hyperinsulinemia, reduce insulin clearance and impair carbohydrate homeostasis in liver-specific Ir knockout mice [41]. In addition, hepatic expression of Pepck expression was increased particularly in the HPDSS2 group indicating a higher hepatic glyconeogenesis compared to the NPDSS2 group. Changes in insulin function, for instance through a decrease in receptor expression, could explain overexpression of Pepck in the HPDSS2 group [42]. As in the present work, previous studies showed that a HP diet during gestation was associated with an increase of adiposity in rat offspring [20,21]. The latter is most likely a direct consequence of a foetal nutritional adjustment to the HP and low-carbohydrate environment, fostering increased gluconeogenesis [43]. Moreover, insulin is also a signal of long-term energy balance [44,45]. This finding highlighted that a gestation HP diet had a potential programming effect on offspring liver development that may impact body physiology differently, depending on the postweaning dietary environment.
In the hypothalamic arcuatus nucleus, leptin activates POMC and CARTPT producing neurons and inhibits NPY and AGRP [46]. In the present study, however, no effect was observed on the expression of Pomc, Cartpt, Npy, Agrp on PND105, despite the fact that leptin levels were normally higher in the HP gestation groups, particularly in HPDSS2 rats. Leptin is known to be higher in obese subjects [47] and obesity can promote dysregulation of leptin signalling leading to central leptin resistance [48,49]. The dopamine system plays a central role in reward and in food preference [50]. NAcc dopamine receptor measurements showed that Drd1 and Drd2 expression was significantly decreased by DSS. DRD2 is well known in human and animal models as an actor favouring obesity [51]. The DSS2 groups had a higher intake of lipids, which is in line with previous studies showing that long-term exposure to high-fat food results in downregulation of Drd2 expression in reward centres [52][53][54]. It is tempting to speculate that there is a link between the lipid intake and observed DRD2 changes in our study-but that its significance is unknown. Moreover, these results showed that free-choice models (DSS) played a key role in hedonic food perception and that the central dopamine pathway was not modified by maternal HP diet during gestation. Nevertheless, studies in rodents focused on a maternal high-fat-high-sugar (HFHS) diet during early development, including gestation, showed that a HFHS diet increased the preference for palatable foods through alterations in the central dopamine circuit and a decrease of Drd2 expression in NAcc [55] or in the ventral tegmental area [56].

Conclusions
In conclusion, the present study showed that, in rats, a maternal HP diet during gestation followed by a postweaning DSS2 feeding, composed of three separate macronutrients, increased the susceptibility to energy balance disorders and obesity in female offspring. In the case of the DSS condition, the dopamine circuit was affected through the decrease of Drd1 and Drd2, suggesting that long-term DSS exposure could impact the hedonic food circuit. Offspring macronutrient choices were affected by the way the food was proposed; when the macronutrients were kept separate from each other, pups consumed more proteins and lipids and less carbohydrates, compared to control groups with a complete diet (NP). In addition, the maternal HP diet during gestation induced a decrease of Ir and Irs1 expression in offspring livers, suggesting programming effects on the insulin signalling that may underlie the probable alterations in energy homeostasis. The effects of maternal LP diet [57,58] and maternal HP diet as showed in the present study, suggest the existence of an optimal protein/energy ratio in maternal diet during gestation for offspring health risk. These observations confirm the major role of maternal perinatal nutrition and showed the protein level during gestation as a determinant for phenotype programming with latter consequences on development of metabolic risks.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/1/96/s1, Methods S1: Animal procedure details during each experimental period, Methods F1: Example, for each diet group, of voxel masks of total (a), subcutaneous (b) and visceral adipose (c) tissues on the section 47/95 from pictures obtained by MRI. Volume of adipose tissues was determined by quantification of the voxel's mask, from all sections of each rat, directly converted to mm 3 by the software. MRI, Magnetic resonance imaging, Table S1: P Primers, Table S2: Gut hormones in plasma on PND105, Table S3: Gene expression in adipose tissue and hypothalamus on PND105.