Obesity and Metabolic Traits after High-Fat Diet in Iberian Pigs with Low Birth Weight of Placental Origin.

Intrauterine growth restriction (IUGR) and later obesity and metabolic disorders have classically been associated with maternal malnutrition, but most cases of IUGR are related to placental insufficiency. The current study, using a swine model for IUGR and obesity, aimed to determine the interaction of birth weight (categorized as low birth weight [LBW] or normal birth-weight [NBW]) and postnatal diet (categorized as maintenance diet [MD] or fattening diet [FD]) on body weight, adiposity and metabolic traits. FD induced higher body weight and adiposity (both p < 0.0001), with higher fructosamine levels (p < 0.005) and a trend toward higher HOMA-ß index (p = 0.05). NBW pigs remained heavier than LBW pigs during the early juvenile period (p < 0.005), but there were no differences at later stages. There were no differences in metabolic traits during juvenile development, but there were differences in adulthood, when LBW pigs showed higher glucose and lower insulin levels than NBW pigs (both p < 0.05). These results suggest that (a) FD allows LBW offspring to achieve similar obesity in adulthood as NBW offspring, and (b) glucose metabolism is more compromised in obese LBW than obese NBW pigs. The comparison of our data with previous studies highlights significant differences between offspring with LBW induced by maternal malnutrition or placental insufficiency, which should be considered when studying the condition.

A High-Fat Diet Modifies Brain Neurotransmitter Profile and Hippocampal Proteome and Morphology in an IUGR Pig Model.

Intrauterine Growth Restriction (IUGR) hinders the correct growth of the fetus during pregnancy due to the lack of oxygen or nutrients. The developing fetus gives priority to brain development ("brain sparing"), but the risk exists of neurological and cognitive deficits at short or long term. On the other hand, diets rich in fat exert pernicious effects on brain function. Using a pig model of spontaneous IUGR, we have studied the effect on the adult of a long-term high-fat diet (HFD) on the neurotransmitter profile in several brain areas, and the morphology and the proteome of the hippocampus. Our hypothesis was that animals affected by IUGR (born with low birth weight) would present a different susceptibility to an HFD when they become adults, compared with normal birth-weight animals. Our results indicate that HFD affected the serotoninergic pathway, but it did not provoke relevant changes in the morphology of the hippocampus. Finally, the proteomic analysis revealed that, in some instances, NBW and LBW individuals respond to HFD in different ways. In particular, NBW animals presented changes in oxidative phosphorylation and the extracellular matrix, whereas LBW animals presented differences in RNA splicing, anterograde and retrograde transport and the mTOR pathway.

Polyphenols and IUGR Pregnancies: Effects of the Antioxidant Hydroxytyrosol on the Hippocampus Proteome in a Porcine Model.

Supplementation of a mother's diet with antioxidants such as hydroxytyrosol (HTX) has been proposed to ameliorate the adverse phenotypes of foetuses affected by intrauterine growth restriction (IUGR). Our previous studies showed, in a porcine model of IUGR, an effect of maternal HTX supplementation on the neurotransmitter profile of several brain areas and the morphology of the hippocampus in 100 days old foetuses. The present study analyzed the impact of maternal HTX supplementation on the hippocampus proteome at this foetal age by TMT10plex labelling. Eleven differentially abundant proteins were identified by comparing both conditions, and eight of them downregulated and three upregulated in the HTX-treated group. The downregulated proteins were mainly involved in protein synthesis and RNA metabolism and may explain the differences in neuron differentiation in the HTX-treated group. The upregulated proteins were related to cell detoxification and could represent a potential mechanism to explain the neuroprotective effect of HTX.

Polyphenols and IUGR Pregnancies: Intrauterine Growth Restriction and Hydroxytyrosol Affect the Development and Neurotransmitter Profile of the Hippocampus in a Pig Model.

Intrauterine growth restriction (IUGR) refers to poor growth of a fetus during pregnancy due to deficient maternal nutrition or oxygen supply. Supplementation of a mother's diet with antioxidants, such as hydroxytyrosol (HTX), has been proposed to ameliorate the adverse phenotypes of IUGR. In the present study, sows were treated daily with or without 1.5 mg of HTX per kilogram of feed from day 35 of pregnancy (at 30% of the total gestational period), and fetuses were sampled at day 100 of gestation. Fetuses were classified as normal body weight (NBW) or low body weight (LBW) as a consequence of IUGR, constituting four groups: NBW-Control, NBW-HTX, LBW-Control, and LBW-HTX. The brain was removed, and the hippocampus, amygdala, and prefrontal cortex were rapidly dissected. Neuronal markers were studied by immunohistochemistry, and a decrease in the number of mature neurons in the hippocampal Cornu Ammonis subfield 1 (CA1) and the Dentate Gyrus (DG) regions was observed in LBW fetuses together with a higher number of immature neurons and other alterations in neuronal morphology. Furthermore, IUGR conditions altered the neurotransmitter (NT) profile, since an increase in the serotonin (5-HT) pathway was observed in LBW fetuses. Supplementation with HTX was able to reverse the morphological and neurochemical changes, leading both characteristics to values similar to those of NBW fetuses.

Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis.

Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (H2O2), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as H2O2 levels) was determined with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater H2O2 extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular H2O2 levels produced by sperm and NETosis.

Polyphenols and IUGR Pregnancies: Effects of the Antioxidant Hydroxytyrosol on Brain Neurochemistry and Development in a Porcine Model.

Supplementation of a mother's diet with antioxidants, such as hydroxytyrosol (HTX), has been proposed to ameliorate the adverse phenotypes of fetuses at risk of intrauterine growth restriction. In the present study, sows were treated daily with or without 1.5 mg of HTX per kilogram of feed from day 35 of pregnancy (at 30% of total gestational period), and individuals were sampled at three different ages: 100-day-old fetuses and 1-month- and 6-month-old piglets. After euthanasia, the brain was removed and the hippocampus, amygdala, and prefrontal cortex were dissected. The profile of the catecholaminergic and serotoninergic neurotransmitters (NTs) was characterized and an immunohistochemical study of the hippocampus was performed. The results indicated that maternal supplementation with HTX during pregnancy affected the NT profile in a brain-area-dependant mode and it modified the process of neuron differentiation in the hippocampal CA1 and GD areas, indicating that cell differentiation occurred more rapidly in the HTX group. These effects were specific to the fetal period, concomitantly with HTX maternal supplementation, since no major differences remained between the control and treated groups in 1-month- and 6-month-old pigs.

Metabolome and proteome changes in skeletal muscle and blood of pre-weaning calves fed leucine and threonine supplemented diets.

In pre-weaning calves, both leucine and threonine play important roles in growth and muscle metabolism. In this study, metabolomics, proteomics and clinical chemistry were used to assess the effects of leucine and threonine supplementation added to milk replacer on 14 newborn Holstein male calves: 7 were fed a control diet (Ctrl) and 7 were fed the Ctrl diet supplemented with 0.3% leucine and 0.3% threonine (LT) from 5.6 days of age to 53.6 days. At this time, blood and semitendinosus muscle biopsies were collected for analysis. Integrated metabolomics and proteomics showed that branched-chain amino acids (BCAA) degradation and mitochondrial oxidative metabolism (citrate cycle and respiratory chain) were the main activated pathways in muscle because of the supplementation. BCAA derivatives and metabolites related to lipid mobilization showed the major changes. The deleterious effects of activated oxidative phosphorylation were balanced by the upregulation of antioxidant proteins. An increase in protein synthesis was indicated by elevated aminoacyl-tRNA biosynthesis and increased S6 ribosomal protein phosphorylation in skeletal muscle. In conclusion, LT group showed greater BCAA availability and mitochondrial oxidative activity; as the muscle cells undergo greater aerobic metabolism, antioxidant defenses were activated to compensate for possible cell damage. Data are available via ProteomeXchange (PXD016098). SIGNIFICANCE: Leucine and threonine are essential amino acids for the pre-weaning calf, being of high importance for growth. In this study, we found that leucine and threonine supplementation of milk replacer to feed pre-weaning calves led to differences in the proteome, metabolome and clinical chemistry analytes in skeletal muscle and plasma, albeit no differences in productive performance were recorded. This study extends our understanding on the metabolism in dairy calves and helps optimizing their nutritional status.

SWATH-MS quantitative proteomic investigation of intrauterine growth restriction in a porcine model reveals sex differences in hippocampus development.

Intrauterine growth restriction (IUGR) is characterized by reduced growth and weight of the foetus, mainly due to the lack of nutrients and oxygen. Animals affected by IUGR show changes in specific brain areas and several neuronal processes. Female offspring affected by IUGR show increased survival and development compared to males. The objective of this study was to analyse changes in the hippocampus proteome in male and female piglets affected by IUGR. Seven pregnant Iberian sows were fed from Day 35 of pregnancy onwards at 50% of their requirements. At Day 100 of pregnancy, foetuses were obtained and classified by sex and weight, as mild IUGR (Normal Body Weight) versus severe IUGR (Low Body Weight). Hippocampi were dissected and the proteomes analysed by SWATH-MS DIA. In this study, 1497 proteins were identified of which 260 were quantitatively analysed. All differential proteins were more abundant in females versus males and were involved in protein synthesis, neuronal development, metabolism, antiapoptotic signalling and vesicular transport. Our findings support that female foetuses tolerate nutrient limitation better than males, especially under mild IUGR. Under severe IUGR, females still seems to maintain normal lipid metabolism and antiapoptotic signalling, which may be related to the increased female survival. SIGNIFICANCE: In the last years, proteomics have been used to evidence differences related to sex in non-reproductive organs. Intrauterine Growth Restriction (IUGR) can affect female and male offspring differently. Female offspring has stronger protective strategies compared to males, enhancing growth and postnatal survival. Most studies regarding this issue have focused on metabolic organs (i.e. liver). However, the predominance of neurodevelopmental disorders in males suggests that the central nervous system in female offspring adapt better to nutritional stress conditions than that of males. Based on the differential protein expression in hippocampal samples, our work demonstrates that female foetuses indeed adapt better to IUGR than males, especially under mild IUGR conditions. In severe IUGR conditions, differences between males and females were not so evident, but even in this case, the remaining differences suggest increased survival in females than in males.