1. Introduction

2. Materials and Methods

3. Results

3.2. Effect of Overnutrition during Pregnancy and Lactation on Metabolic Function in the Offspring

3.2.1. Insulin Action and Glucose Homeostasis

Female and male offspring born from overnourished mothers during pregnancy and lactation periods were evaluated for body weight, basal glycemia, insulin sensitivity and glucose tolerance (Figure 2).

HF feeding to mothers promoted a significant increase in body weight of both female and male descendants during all the postnatal periods analyzed (Figure 2A). Female offspring from HF mothers increased 65.1%, 33.3% and 9.6% at PD 21, 28 and 60, respectively (Figure 2A, left panel). Male offspring from overnourished mothers showed a significant increase in 63.9%, 45.6% and 20.5% at PD 21, 28 and 60, respectively (Figure 2A, right panel).

Maternal overnutrition during pregnancy and lactation significantly increased basal glycemia of female offspring at PD21 and 28 in comparison to female offspring from NC mothers (basal glycemia: NC PD21= 74.5±6.5, HF PD21=95.6±7.8, p<0.01; NC PD28= 61.0±2.4, HF PD28= 78.8±2.6, p<0.05) without changing basal glycemia at PD60 (Figure 2B, left panel). Interestingly, we found that during litter development, NC female offspring showed an increase in basal glycemia that is indeed significant at PD60 (basal glycemia: NC PD21= 81.3±2.6, p<0.01) when compared to PD28 values and that, in contrast, HF female offspring decreased basal glycemia from PD21 to PD28 (p<0.05), a tendency maintained at PD60 (basal glycemia: HF PD60= 85.6±2.0, p<0.01). Basal glycemia was not altered by HF maternal feeding in male offspring in the period of development analyzed (Figure 2B, right panel).

Figure 2. Effect of overnutrition during pregnancy and lactation on metabolic phenotype of the offspring, evaluated by weight gain (A), insulin sensitivity (B) and glucose tolerance (C), at postnatal day 21 (PD21), 28 (PD28) and 60 (PD60). A) from the left to the right represents body weight in g during offspring development for females and males. B) from the left to the right represents basal glycemia in mg/dL during early development for females and males. C) Insulin sensitivity is represented by glycemia profiles during ipITT (C1) and the area under the curve of those glycemia curves during early descendants’ development for females and males, from the left to the right, respectively (C2). D) glucose tolerance is represented by glycemia profiles during OGTT (D1) and the area under the curve of those glycemia curves during early descendants’ development for females and males, from the left to the right, respectively (D2). PD21 – postnatal day 21; PD28 – post-natal day 28; PD60 – post-natal day 60. Data represents mean ± SEM. Two-Way ANOVA with with Bonferroni and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

Figure 2. Effect of overnutrition during pregnancy and lactation on metabolic phenotype of the offspring, evaluated by weight gain (A), insulin sensitivity (B) and glucose tolerance (C), at postnatal day 21 (PD21), 28 (PD28) and 60 (PD60). A) from the left to the right represents body weight in g during offspring development for females and males. B) from the left to the right represents basal glycemia in mg/dL during early development for females and males. C) Insulin sensitivity is represented by glycemia profiles during ipITT (C1) and the area under the curve of those glycemia curves during early descendants’ development for females and males, from the left to the right, respectively (C2). D) glucose tolerance is represented by glycemia profiles during OGTT (D1) and the area under the curve of those glycemia curves during early descendants’ development for females and males, from the left to the right, respectively (D2). PD21 – postnatal day 21; PD28 – post-natal day 28; PD60 – post-natal day 60. Data represents mean ± SEM. Two-Way ANOVA with with Bonferroni and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

In Figure 2C is represented the curves of glycemia during the ipITT performed to the offspring (Figure 2C1) and the correspondent AUC of those curves (Figure 2C2). From the curves it can be seen that at PD21 male offspring from HF mothers present a significant increase in glycemia levels throughout all the ipITT (Figure 2C1, left panel) demonstrating a decrease in insulin sensitivity. At PD28 neither HF maternal feeding, nor sex, impacted glycemia profile during ipITT (Figure 2C1, central panel). At PD60, glycemia profiles during ipITT showed that male offspring have a significant increase in glycemia throughout the test, independently to the mothers’ diet during pregnancy and lactation (Figure 2C1, right panel). By the analysis of glycemia AUC from the ipITT during offspring development in the initial 60 days, insulin sensitivity significantly increased in the female descendants of NC mothers (AUC glycemia during ipITT: NC PD21=900.4±36.2; NC PD28=677.2±41.6, p<0.01; NC PD60=724.8±22.5, p<0.05) (Figure 2C2, left panel). In male offspring AUC glycemia during ipITT decreased from PD21 to PD28 and increased from PD28 to PD60 in both NC and HF descendants (AUC glycemia during ipITT: NC PD21=807.8±66.4; NC PD28=677.6±23.9, p<0.01; NC PD60=862.6±19.5, p<0.05; HF PD21=1022.2±81.6; HF PD28=663.9±41.2, p<0.01; HF PD60=878.6±34.9, p<0.05) (Figure 2C2, right panel), showing a significant increase in insulin sensitivity from PD21 to PD28 and a significant decrease in insulin sensitivity from PD28 to PD60 independent of maternal diet.

Finally, the impact of maternal diet and offspring sex on descendants’ glucose tolerance was evaluated by the OGTT (Figure 2D). The glycemia profiles showed that neither mother diet nor offspring sex impacted glucose tolerance at each PD evaluated (Figure 2D1). However, female offspring from HF mothers showed a significant decrease in glucose tolerance in comparison to female offspring of NC mothers at PD21 (AUC glycemia during OGTT: NC PD21=19955.2±1033.4; HF PD21=23999.4±857.5; p<0.05), an effect that is attenuated during offspring development (AUC glycemia during OGTT: HF PD21=23999.4±857.5; HF PD28=20866.1±345.6; HF PD60=20076.8±598.9; p<0.05) (Figure 2D2, left panel). Male offspring glucose tolerance was not altered during development by the diet fed to the mothers (Figure 2D2, right panel).

3.2.2. Liver and Adipose Tissue Depots Weight

In Table 2 are depicted the liver and adipose tissue depots weight of descendants of mothers submitted to NC or HF diet during gestation and lactation periods. Maternal overnutrition during pregnancy and lactation impacted both male and female offspring AT depots weight mainly at PD60.

In female offspring, overnutrition in mothers increased significantly visceral and perinephric AT at PD60 (visceral AT weight: NC Females PD60= 0.406 ± 0.038; HF Females PD60= 0.527 ± 0.055, p<0.001; perinephric AT weight: NC Females PD60= 0.196± 0.017; HF Females PD60= 0.374 ± 0.068, p<0.01, mg).

Regarding male offspring, maternal overnutrition promoted an increase in liver weight at PD21 (NC Males PD21= 2.286 ± 0.086; HF Males PD21= 2.717 ± 0.126, p<0.01) and at PD60 (NC Males PD60= 6.377 ± 0.326; HF Males PD60= 7.218 ± 0.465, p<0.05). Also, HF diet in mothers during pregnancy and lactation promoted an increase in the weight of visceral AT (NC Males PD60= 1.216 ± 0.182; HF Males PD60= 1.864 ± 0.152, p<0.01) and genital AT (NC Males PD60= 2.324 ± 0.375; HF Males PD60= 3.642 ± 0.503, p<0.01) in male descendants at PD60.

Brown AT weight did not alter significantly in both females and males descendants of HF mothers, at least till PD60.

Table 2. Impact of overnutrition in mothers during pregnancy and lactation on the weight (mg) of the liver and of the different adipose tissue (AT) depots in male and female descendants at postnatal day 21, 28 and 60.

Table 2. Impact of overnutrition in mothers during pregnancy and lactation on the weight (mg) of the liver and of the different adipose tissue (AT) depots in male and female descendants at postnatal day 21, 28 and 60.

	Liver	Visceral AT	Perinephric AT	Genital AT	Brown AT
NC Females PD21	0.963 ± 0.066	0.262 ± 0.026	0.105 ± 0.017	0.128 ± 0.015	0.087 ± 0.010
HF Females PD21	1.646 ± 0.139	0.278 ± 0.039	0.222 ± 0.026	0.318 ± 0.098	0.130 ± 0.021
NC Females PD28	0.978 ± 0.056	0.257 ± 0.021	0.139 ± 0.012	0.114 ± 0.012	0.103 ± 0.009
HF Females PD28	1.965 ± 0.115	0.344 ± 0.042	0.303 ± 0.053	0.286 ± 0.050	0.143 ± 0.015
NC Females PD60	1.892 ± 0.118	0.406 ± 0.038	0.196± 0.017	0.214 ± 0.033	0.135 ± 0.009
HF Females PD60	2.670 ± 0.158	0.527 ± 0.055 ***	0.374 ± 0.068 **	0.472 ± 0.075	0.139 ± 0.010
NC Males PD21	2.286 ± 0.086	0.376 ± 0.042	0.376 ± 0.059	0.305 ± 0.033	0.146 ± 0.016
HF Males PD21	2.717 ± 0.126 **	0.561 ± 0.043	0.472 ± 0.042	0.528 ± 0.054	0.144 ± 0.012
NC Males PD28	4.872 ± 0.405	1.029 ± 0.135	1.109 ± 0.212	1.993 ± 0.405	0.249 ± 0.025
HF Males PD28	4.566 ± 0.178	1.472 ± 0.065	1.879 ± 0.281	2.183 ± 0.386	0.226 ± 0.026
NC Males PD60	6.377 ± 0.326	1.216 ± 0.182	2.599 ± 0.381	2.324 ± 0.375	0.269 ± 0.025
HF Males PD60	7.218 ± 0.465 *	1.864 ± 0.152 **	3.162 ± 0.422	3.642 ± 0.503 **	0.284 ± 0.026

*p<0.05, **p<0.01; ***p<0.001 Two-way ANOVA with Sidak’s multicomparison test comparing animals submitted to normal chow (NC) and animals submitted to high-fat diet (HF) of the same age. PD21 – post-natal day 21; PD28 – post-natal day 28; PD60 – post-natal day 60.

3.3. Effect of Overnutrition during Pregnancy and Lactation on Behaviour Phenotype in the Offspring

In order to assess the impact of overnutrition during pregnancy and lactation on the offspring, a series of behavioral tests were conducted on offspring at PD60. These tests were categorized into three main groups: 3.2.1. Anxiety and Stress; 3.2.2. Memory and Learning; and 3.2.3. Food/Drink behavior.

3.3.1. Anxiety and Stress

In this battery of tests (Figure 3), we incorporated the OF, EPM, and the LDB test, all designed to measure anxiety and stress levels in the offspring [19].

Regarding the OF results, significant differences (p<0.05) were evident among the female offspring groups concerning the distance covered within the inner zone. This suggests that female offspring from mothers on a HF diet spend notably less time in the inner zone compared to those from control mothers. However, no significant differences were observed among the other groups for the remaining three parameters: total distance, total immobility/total test time, and immobility within the inner zone/total test time (Figure 3A).

In other hand, concerning the results of the Elevated Plus Maze (EPM) test, there were no significant differences detected between the groups (NC vs. HF) for the five parameters assessed: ratio (open arm/open+closed arm), ratio (closed arm/open+closed arm), number of poops, number of pees, and the frequency of alternations between arms (Figure 3B).

Finally, when it comes to the outcomes of the light-dark box test, there were no significant differences observed between the groups (NC vs. HF) in the three parameters analyzed: the proportion of time spent in the light zone relative to the total test time, the proportion of time spent in the dark zone relative to the total test time, and the number of alternations (Figure 3C).

Figure 3. Effect of overnutrition during pregnancy and lactation on anxiety and stress, evaluated by the Open Field test (A), elevated plus maze test (B) and light-dark box test (C), in the offspring at postnatal day 60. A) from the left to the right the panels shows a schematic representation of the open field maze, the distance in the inner zone (cm), total distance covered (cm), total immobility (s), and the immobility in the inner zone (s); B) from left to right the panel shows a schematic representation of the elevated plus maze, the time in the open arms in comparison to the time in all arms (s), the time in the closed arms in comparison to the time in all arms (s), the number of poos in the maze, the number of pees, and the number of alternations between and the and the closed arms. Data represents mean ± SEM. Two-Way ANOVA with with Bonferroni and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals.

Figure 3. Effect of overnutrition during pregnancy and lactation on anxiety and stress, evaluated by the Open Field test (A), elevated plus maze test (B) and light-dark box test (C), in the offspring at postnatal day 60. A) from the left to the right the panels shows a schematic representation of the open field maze, the distance in the inner zone (cm), total distance covered (cm), total immobility (s), and the immobility in the inner zone (s); B) from left to right the panel shows a schematic representation of the elevated plus maze, the time in the open arms in comparison to the time in all arms (s), the time in the closed arms in comparison to the time in all arms (s), the number of poos in the maze, the number of pees, and the number of alternations between and the and the closed arms. Data represents mean ± SEM. Two-Way ANOVA with with Bonferroni and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals.

3.3.2. Memory and Learning

For this set of assessments (Figure 4), we used the Y maze, the novel object recognition (NOR) and the block test, designed to assess memory and learning [19]

The Y-maze test was used to evaluate spatial learning and memory capacity in the offspring of both NC and HF animals. There were no significant differences observed in the four parameters analyzed: the time spent by the animals in the new arm, the ratio of time spent in the novel arm to the sum of time spent in both the novel and home arms, the number of entries into the novel arm, and alternative behaviors. The last parameter calculates the ratio of the number of alternations to the possible number (total number of arm entries minus two) (Figure 4A).

Regarding NOR results, there were no significant differences observed between the NC and HF groups in the three parameters measured: the time required to identify the new object, the ratio of time spent interacting with the novel object over the total time spent interacting with both the novel and familiar objects, and the ratio of time spent interacting with the familiar object over the total time spent interacting with both the novel and familiar objects. Nevertheless, while not statistically significant, there is an observable trend in the time taken to identify the new object (Figure 4B).

Finally, the animal’s olfactory working memory was assessed using the block test that assesses the ability of the animals to discriminate between familiar and novel, innocuous scents [18]. Results are presented in Figure 3C. No significant differences were observed between the experimental groups for the three parameters analyzed: the ratio of time spent sniffing the novel block over the total time spent sniffing both the novel and own blocks, the ratio of time spent sniffing the own block over the total time spent sniffing both the novel and own blocks, and the time required to identify the new block. However, it is worth noting that there is a noticeable trend in the last parameter, where the offspring of HF animals tend to take more time to complete this task.

Figure 4. Effect of overnutrition during pregnancy and lactation on memory and learning, evaluated by the Y-maze test (A), novel object recognition test (B) and block test (C), in the offspring at postnatal day 60. A) from the left to the right panel shows a schematic representation of the y maze, the first arm choice (%), the ratio between the novel arm vs the time in both novel and familiar, the % of novel arm entries, and the alternative behavior (%); B) from the left to the right the panel presents a schematic representation of novel object recognition apparatus, the time to identify the novel object (s), the ration between the time the animals spent interact with the novel object vs the total interaction time (s), and the time the animals spent interact with the familiar object vs the total interaction time (s); (C) from left to right the panel present a schematic representation of the block test, the time to identify the novel block (s), the time the animals spent sniffing the novel block vs the total time sniffing all the blocks, and the time the animals spent sniffing the own blocks vs the total time sniffing all the blocks. Data represents mean ± SEM. Two-Way ANOVA with Bonferroni and Sidak’s multicomparison tests; *p<0.05, **p<0.01 comparing NC animals.

Figure 4. Effect of overnutrition during pregnancy and lactation on memory and learning, evaluated by the Y-maze test (A), novel object recognition test (B) and block test (C), in the offspring at postnatal day 60. A) from the left to the right panel shows a schematic representation of the y maze, the first arm choice (%), the ratio between the novel arm vs the time in both novel and familiar, the % of novel arm entries, and the alternative behavior (%); B) from the left to the right the panel presents a schematic representation of novel object recognition apparatus, the time to identify the novel object (s), the ration between the time the animals spent interact with the novel object vs the total interaction time (s), and the time the animals spent interact with the familiar object vs the total interaction time (s); (C) from left to right the panel present a schematic representation of the block test, the time to identify the novel block (s), the time the animals spent sniffing the novel block vs the total time sniffing all the blocks, and the time the animals spent sniffing the own blocks vs the total time sniffing all the blocks. Data represents mean ± SEM. Two-Way ANOVA with Bonferroni and Sidak’s multicomparison tests; *p<0.05, **p<0.01 comparing NC animals.

3.3.3. Food/Drink Behavior

To assess competition for food and drink, the respective two tests were used: Food Competition and Water/Sucrose Competition (Figure 5).

The food competition test was used to assess both competition for food, the voracity of feeding, as well as the dominance in offspring in both NC and HF animals. No significant differences were observed in any of the five analyzed parameters: time eating, food intake, antagonistic behavior, time of antagonistic behavior, and grooming. However, a trend can be observed for two parameters: time eating and food intake. HF animals tend to feed for a longer period and consume a higher amount of food compared to NC.

In these tests, antagonistic behaviors indicating dominance over the other were taken into account (see methods 2.3.7 and 2.3.8.).

Like the previous test, the Water/Sucrose Competition is used to determine if there is a preference for drinking between sucrose and water. To do so, the amount of liquid ingested, the speed at which animals consume it, as well as the dominance of this resource among NC and HF animals are measured. When comparing significant differences within the same trial (when the liquid is the same) it is clear that when they compete for water, that males born from HF mothers drink water for more time, although without significant differences in the amount of liquid intake. Note also, that no differences were observed in females in the time or amount of water intake. Moreover, animals when exposed to water do not exhibit significant differences on antagonistic behavior, time of antagonistic behavior and grooming. When animals compete for sucrose no significant differences are observed among the five parameters previously mentioned. When comparing the Water trial vs. the Sucrose trial, it seems that the animals spent more time-consuming water than sucrose, exhibit a greater quantity of antagonistic behaviors when exposed to water compared to sucrose, and also engage in a higher amount of grooming during the water trial.

3.4. Effect of Overnutrition during Pregnancy and Lactation on Hypothalamic, Hippocampal and Prefrontal Markers of Synaptic Transmission, Metabolic Signaling and Inflammation

To assess the impact of excessive nutrition during pregnancy and lactation in some areas of the brain related with stress and anxiety - the hypothalamus, hippocampus, and prefrontal cortex - a Western blot technique was employed to examine the levels of some proteins and receptors involved in synaptic dynamics (3.3.1), metabolism (3.3.2), and inflammation (3.3.3).

3.4.1. Protein Markers of Synaptic Transmission on the Hypothalamus, Hippocampus and Prefrontal Cortex

Stress, anxiety and cognition has been associated with altered synaptic transmission and plasticity [20,21]. To investigate synaptic dynamics in the three designated areas, we access three distinct proteins: the glutamate vesicular transporter (vGLUT), the synaptosomal-associated protein 25kDa (SNAP-25), that contributes to the SNARE complex involved in exocytosis and the postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase family that with PSD-93 is recruited to NMDA receptor and potassium channel clusters.

In the hypothalamus, development induced an increase of 37.5% and of 44.7% (p<0.001) in levels of vGLUT and females showed a non-significant increase of PD28 and PD60. Male HF offspring do not exhibit significant differences with NC offspring, but female offspring at PD21 show a significant increase of 57.5% that attenuates with age (Figure 6A1). Both males and females showed an increase in SNAP-25 levels from PD21 onwards in NC animals, this being statistically significant only for females at PD60 (increase of 99.3%). Moreover, both males and females born from HF mothers exhibit altered levels of SNAP-25 at the hypothalamus, with males exhibiting always higher levels of protein that are significant at PD28 and females exhibiting significant higher levels at PD21 that are maintained at PD28 and PD60 (Figure 6A2). In relation to PSD-95 it is clear that males exhibit a tendency to progressively increase the levels of this protein with age, an effect that is not observed in females. Overnutrition during pregnancy and lactation did not change these results (Figure 6A3).

In the hippocampus, no significant differences were observed regarding age, diet and sex for vGLUT (Figure 6B1) or PSD-95 (Figure 6B3). However, notable differences are evident in SNAP-25 in males and females. Note that there is a clear decrease in the levels of SNAP-25 between PD21 and PD28 and PD60 in NC animals (males: decrease of 32.3% PD28 and 52.6% at PD60; females: decrease of 52.2% at PD28 and 39.9% at PD60). Interestingly, overfeeding of mothers during pregnancy and lactation did not change the levels of SNAP-25 in males at PD21 and PD28, but increased its levels by 37.5% at PD60. Moreover, females born from HF mothers showed significantly increased levels at PD21 and PD60 of 47.7% and 59.8% respectively.

The prefrontal cortex development in males and females produced significant changes in the levels of vGLUT, but not on the other proteins studied where no differences were observed between PD21 and PD28 and PD60 (Figure 6C). Note that vGLUT levels were significantly higher at PD28 (increase of 70.3%) and PD60 (increase of 51.1%) in males and at PD60 (increase of 37.4%%) in females. Interestingly, only male offspring from HF mothers show differences at PD21 that are attenuated with age (Figure 6C1). SNAP-25 levels were increased in males and females born from HF mothers, an effect that was attenuated with age, but there was more marked in males. Note that, at PD21 and PD28 males born from HF mothers show high levels of SNAP-25 that were attenuated at PD60. Interestingly, this increase, although non-significant, was only seen in females at PD21 (increase by 51.1%%) (Figure 6C2). HF diet during pregnancy and lactation did not change the levels of PSD-95 in males or females (Figure 6C3).

Figure 6. Effect of overnutrition during pregnancy and lactation on the levels of proteins involved in synaptic transmission on the hypothalamus (A), hippocampus (B) and prefrontal cortex (C). From the left to the right panel, graphs shows the mean levels of the ratio between vGLUT and β-actin, the ratio between SNAP-25 and β-actin and the ratio between PSD-95 and β-actin in males and females, respectively. On the top of the graphs are shown representative western blot membranes for the proteins of interest and the respective loading control. Data represents mean ± SEM. Two-Way ANOVA with with Tukey’s and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

Figure 6. Effect of overnutrition during pregnancy and lactation on the levels of proteins involved in synaptic transmission on the hypothalamus (A), hippocampus (B) and prefrontal cortex (C). From the left to the right panel, graphs shows the mean levels of the ratio between vGLUT and β-actin, the ratio between SNAP-25 and β-actin and the ratio between PSD-95 and β-actin in males and females, respectively. On the top of the graphs are shown representative western blot membranes for the proteins of interest and the respective loading control. Data represents mean ± SEM. Two-Way ANOVA with with Tukey’s and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

3.4.2. Protein Markers of Metabolism on the Hypothalamus, Hippocampus and Prefrontal Cortex

Figure 7 depicts the impact of hypercaloric diets consumed by mothers in some metabolic markers – insulin receptor and AMPK - in some specific brain areas of the offspring.

Figure 7. Effect of overnutrition during pregnancy and lactation on the levels of insulin receptor and AMPK on the hypothalamus (A), hippocampus (B) and prefrontal cortex (C). From the left to the right panel, graphs represent mean levels of the ratio between phosphorylated insulin receptor/total insulin receptor (P-IR/T-IR) and β-actin, and the ratio between phosphorylated AMPK and total AMPK (p-AMPK/T-AMPK) and β-actin in males and females, respectively. On the top of the graphs are shown representative western blot membranes for the proteins of interest and the respective loading control. Data represents mean ± SEM. Two-Way ANOVA with with Tukey’s and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

Figure 7. Effect of overnutrition during pregnancy and lactation on the levels of insulin receptor and AMPK on the hypothalamus (A), hippocampus (B) and prefrontal cortex (C). From the left to the right panel, graphs represent mean levels of the ratio between phosphorylated insulin receptor/total insulin receptor (P-IR/T-IR) and β-actin, and the ratio between phosphorylated AMPK and total AMPK (p-AMPK/T-AMPK) and β-actin in males and females, respectively. On the top of the graphs are shown representative western blot membranes for the proteins of interest and the respective loading control. Data represents mean ± SEM. Two-Way ANOVA with with Tukey’s and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

We can observe that in the hypothalamus development increased significantly and progressively the levels of the ratio IR-P/IR-T in males and females born from NC mothers (PD28 males 31.9% increase; PD60 males 43.0% increase; PD28 females 42.8% increase, PD60 females 44.4% increase), suggesting a higher phosphorylation of the IR with development. Interestingly, while no changes were observed in the levels of this ratio at PD21 and PD28 in males born from HF mothers, a significant reduction was seen at PD60 (49.3% decrease). In contrast, in females HF diet in mothers promoted a higher ratio of IR-P/IR-T at PD21 (40.2% increase) that was attenuated with age (Figure 7A1). Panel A2 shows the ratio between AMPK-P/AMPK-T, a metabolic sensor. Age increases the levels of the ratio AMPK-P/AMPK-T in both females and males, being these only significant in males (Figure 7A2), suggesting higher phosphorylation of AMPK with development. HF diet consumption in mothers did not change this ratio in both sexes (Figure 7A2). In the hippocampus, the levels of the ratio IR-P/IR-T follow more or less the same trend than in hypothalamus, with the phosphorylation of IR increasing by 49.8% at PD60 in males and progressively with development in females although without statistical significance (Figure 7B1). HF diet intake by mothers did not alter significantly the ratio of IR-P/IR-T in the offspring in both sexes (Figure 7B1). Interestingly, we observe sexual dimorphism in the ratio AMPK-P/AMPK-T at the hippocampus, with females NC showing a progressively and statistical significant increase in the phosphorylation of AMPK at PD28 (36.4%) and PD60 (36.3%), but no effect on males (Figure 7B2). HF diet in mothers did significantly change the ratio of AMPK-P/AMPK-T in the offspring in both sexes (Figure 7B2). Figure 7C depicts the levels of the same proteins in the prefrontal cortex. Note that there is a progressive decrease in the ratio IR-P/IR-T with development in the offspring from NC mothers, that is more accentuated and statistically significant in males (PD 28 38.8% decrease and PD60 55.4% decrease compared with PD21) than in females (Figure 7C1). Also note that HF diet intake in mothers promoted a decrease of 48.6% in the phosphorylation of IR at PD21 in males that was not altered with age. In females, no differences were observed (Figure 7C1). In contrast the ratio of AMPK-P/AMPK-T statistically increase in the offspring of NC mothers from PD21 onwards, both in males (PD28 13.6% increase, PD60 15.4% increase), and females (PD28 20.2% increase, PD60 18.3% increase). HF diet intake in mothers induced a higher phosphorylation of AMPK in the offspring at PD21 in both males (14.1% increase) and females (17.5% increase) that was maintained with age (Figure 7C2).

3.4.3. Protein Markers of Inflammation on the Hypothalamus, Hippocampus and Prefrontal Cortex

Inflammation has been implicated in several mood and anxiety disorders as well as in cognitive declive [22,23]. To explore the inflammation as being involved in the effects of overnutrition during pregnancy and lactation in stress and anxiety in the offspring we evaluated the levels of 3 inflammatory markers: GFAP, whose expression was shown to be increased in brain inflammatory states [23]; TNF-α, pro-inflammatory cytokine, expressed in many brain pathologies and associated with neuronal loss [24] and IL6-R, the receptor for IL6, a major cytokine that has anti- and pro-inflammatory in the brain and that has been linked with neurodegenerative diseases and with chronic stress contributing to neurobehavioral complications [25].

In the hypothalamus, no significant changes were observed in GFAP levels in males concerning diet or age. However, although GFAP levels in females did not change with age in NC offspring, they exhibited a 38.5% significant increase from PD21 to PD28 in HF offspring (Figure 8A1). Surprisingly, the levels of TNF-α decrease from PD21 to PD28 and PD60 both in males (PD28 32.7% , PD60 26.2%) and females (PD28 36.4% , PD60 52.4%) in NC offspring. Overnutrition during pregnancy and lactation in mothers did not alter the levels of TNF-α in the hypothalamus in the offspring (Figure 8A2). Considering IL6-R, there was an increase in the levels of this protein with development in both males (PD28 36.4%, PD60 91.8%) and females (PD28 32.5%)in NC offspring. Overnutrition during pregnancy and lactation in mothers did not alter significantly the levels of IL6-R in the hypothalamus in the offspring (Figure 8A3).

Figure 8. Effect of overnutrition during pregnancy and lactation on the levels of proteins involved in inflammation on the hypothalamus (A), hippocampus (B) and prefrontal cortex (C). From the left to the right panel, graphs shows mean levels of the ratio between GFAP and β-actin, the ratio between TNF-α and β-actin and the ratio between IL6-R in males and females, respectively. On the top of the graphs are shown representative western blot membranes for the proteins of interest and the respective loading control Data represents mean ± SEM. Two-way ANOVA with Tukey’s and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

Figure 8. Effect of overnutrition during pregnancy and lactation on the levels of proteins involved in inflammation on the hypothalamus (A), hippocampus (B) and prefrontal cortex (C). From the left to the right panel, graphs shows mean levels of the ratio between GFAP and β-actin, the ratio between TNF-α and β-actin and the ratio between IL6-R in males and females, respectively. On the top of the graphs are shown representative western blot membranes for the proteins of interest and the respective loading control Data represents mean ± SEM. Two-way ANOVA with Tukey’s and Sidak’s multicomparison tests; *p < 0.05, **p < 0.01 comparing NC animals with different ages; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 comparing NC vs HF animals with the same age; and ^$p < 0.05, ^$$p < 0.01, and ^$$$$p < 0.0001 comparing HF animals with different ages.

In the hippocampus, when examining GFAP, no significant differences were concerning age and diet for both sexes in the offspring (Figure 8B1). In the analysis of the TNF-α, it becomes apparent that diet has a notable impact on males. While there was a decrease in TNF with age in male NC offspring, there was an increase in the levels IL6-R in HF offspring at PD28 and PD60 when compared to NC (35.8% and 51.2%, respectively), a trend not observed in females (Figure 8B2). When comparing IL6-R in males, HF diet in mothers increased by 35.8% at PD21, an effect that was attenuated at PD28 and PD60. In females, age did not change IL6-R levels in NC offspring, but HF diet intake in mothers increased significantly by 28.1% IL6-R levels at PD60 days (Figure 8B3).

In the prefrontal cortex GFAP levels increased significantly with age in males from NC offspring (PD28 39.6% and PD60 32.5%), an effect absent in females. Overnutrition during pregnancy and lactation in mothers did not alter significantly the levels of GFAP in the prefrontal cortex in the offspring (Figure 8C1). TNF-α decreases with age in both males and females born from NC mothers, however only in males the decrease is statistically significant ( PD28 41.5%, PD60 47.5% in comparison with PD21). HF diet intake in mothers did not change TNF-α levels in males or females (Figure 8C2). In the analysis of IL6-R, there was a 29.9% increase in IL6-R levels at PD60 in the male offspring from NC mothers, while in females no alterations were observed with age. Overnutrition during pregnancy and lactation in mothers did not alter significantly the levels of IL6-R in the prefrontal cortex in the offspring (Figure 8C3).

4. Discussion

5. Conclusions

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