We tested the hypothesis that gestational diabetes mellitus (GDM) alters the DNA methylation pattern of the fetal serotonin transporter gene (SLC6A4), and examined the functional relevance of DNA methylation for regulation of the SLC6A4 expression in the human placenta. The study included 50 mother-infant pairs. Eighteen mothers were diagnosed with GDM and 32 had normal glucose tolerance (NGT). All neonates were of normal birth weight and born at term by planned Cesarean section. DNA and RNA were isolated from samples of tissue collected from the fetal side of the placenta immediately after delivery. DNA methylation was quantified at 7 CpG sites within the SLC6A4 distal promoter region using PCR amplification of bisulfite treated DNA and subsequent DNA sequencing. SLC6A4 mRNA levels were measured by reverse transcription—quantitative PCR (RT-qPCR). Functional SLC6A4 polymorphisms (5HTTLPR, STin2, rs25531) were genotyped using standard PCR-based procedures. Average DNA methylation across the 7 analyzed loci was decreased in the GDM as compared to the NGT group (by 27.1%, p = 0.037) and negatively correlated, before and after adjustment for potential confounder/s, with maternal plasma glucose levels at the 24th to 28th week of gestation (p<0.05). Placental SLC6A4 mRNA levels were inversely correlated with average DNA methylation (p = 0.010) while no statistically significant association was found with the SLC6A4 genotypes (p>0.05). The results suggest that DNA methylation of the fetal SLC6A4 gene is sensitive to the maternal metabolic state in pregnancy. They also indicate a predominant role of epigenetic over genetic mechanisms in the regulation of SLC6A4 expression in the human placenta. Longitudinal studies in larger cohorts are needed to verify these results and determine to which degree placental SLC6A4 changes may contribute to long-term outcomes of infants exposed to GDM.

Combining of LC n-3 PUFAs and pregnancy yields immunological tolerance and stimulates the production of endogenous insulin in women with T1DM.

Central and peripheral pools of biogenic monoamine serotonin (5-hydroxytryptamine [5HT]) exert opposite effects on the body weight regulation: increase in brain 5HT activity is expected to decrease body weight, whereas increase in peripheral 5HT activity will increase body weight and adiposity. In a genetic model of rats with constitutionally highor low-5HT homeostasis (hyperserotonergic/hyposerotonergic rats), we have studied how individual differences in endogenous 5HT tone modulate net energy balance of the organism. The high-5HT and low-5HT sublines of the model were developed by selective breeding toward extreme platelet activities of 5HT transporter, a key molecule determining 5HT bioavailability/activity. In animals from high-5HT and low-5HT sublines, we assessed physiological characteristics associated with body weight homeostasis and expression profile of a large scale of body weight-regulating genes in hypothalamus, a major brain region controlling energy balance. Results showed that under standard chow diet animals from the high-5HT subline, as compared to low-5HT animals, have lifelong increased body weight (by 12%), higher absolute daily food intake (by 9%), and different pattern of fat distribution (larger amount of white adipose tissue and lower amount of brown adipose tissue). A large number of body weight-regulating hypothalamic genes were analyzed for their mRNA expression: 24 genes by reverse transcription-quantitative polymerase chain reaction (n = 9-10 rats/subline) including neuropeptides and their receptors, growth factors, transcriptional factors, and receptors for peripheral signals, and a total of 84 genes of various classes by polymerase chain reaction array (pools of six rats/subline). Only few genes showed significant differences in mRNA expression levels between 5HT sublines (e.g. neuropeptide Y receptor, fibroblast growth factor 10), but high-5HT animals displayed a clear trend to upregulation of mRNAs for a number of orexigenic signaling peptides, their receptors, and other molecules with orexigenic activity. Receptors for peripheral signals (leptin, insulin) and molecules in their downstream signaling were not altered, indicating no changes in central insulin/leptin resistance. At the protein level, there were no differences in the Frontiers in Neuroscience | www.frontiersin.org 1 March 2020 | Volume 14 | Article 219 Kesić et al. Serotonin and Body Weight Regulationcontent of hypothalamic leptin receptor between 5HT sublines, but significant sex and age effects were observed. Results show that higher constitutive/individual 5HT tone favors higher body weight and adiposity probably due to concurrent upregulation of several hypothalamic orexigenic pathways.

Serotonin (5-HT) plays an extensive role during pregnancy in regulating both the placental physiology and embryonic/fetal development. The uptake of 5-HT into cells is central to the control of local concentrations of 5-HT near its molecular targets. Here, we investigated the mechanisms of 5-HT uptake into human primary placental cells and cord blood platelets, all isolated immediately after birth. Trophoblasts and cord blood platelets showed 5-HT uptake with similar Michaelis constant (Km) values (~0.6 μM), typical of the high-affinity serotonin transporter (SERT). The uptake of 5-HT into trophoblasts was efficiently inhibited by various SERT-targeting drugs. In contrast, the uptake of 5-HT into feto-placental endothelial cells was not inhibited by a SERT blocker and showed a Km value (~782 μM) in the low-affinity range. Consistent with this, SERT mRNAs were abundant in term trophoblasts but sparse in feto-placental endothelial cells, whereas the opposite was found for the low-affinity plasma membrane monoamine transporter (PMAT) mRNAs. Organic cation transporter (OCT) 1, 2, and 3 mRNAs were absent or sparse in both cell types. In summary, the results demonstrate, for the first time, the presence of functional 5-HT uptake systems in feto-placental endothelial cells and fetal platelets, cells that are in direct contact with fetal blood plasma. The data also highlight the sensitivity to various psychotropic drugs of 5-HT transport into trophoblasts facing the maternal blood. The multiple, high-, and low-affinity systems present for the cellular uptake of 5-HT underscore the importance of 5-HT homeostasis at the maternal–fetal interface.

Type 1 diabetes (T1DM) is an autoimmune disease characterized by the gradual loss of β-cell function and insulin secretion. In pregnant women with T1DM, endogenous insulin production is absent or minimal, and exogenous insulin is required to control glycemia and prevent ketoacidosis. During pregnancy, there is a partial decrease in the activity of the immune system, and there is a suppression of autoimmune diseases. These changes in pregnant women with T1DM are reflected by Langerhans islet enlargement and improved function compared to pre-pregnancy conditions. N-3 polyunsaturated fatty acids (n-3 PUFA) have a protective effect, affect β-cell preservation, and increase endogenous insulin production. Increased endogenous insulin production results in reduced daily insulin doses, better metabolic control, and adverse effects of insulin therapy, primarily hypoglycemia. Hypoglycemia affects most pregnant women with T1DM and is several times more common than that outside of pregnancy. Strict glycemic control improves the outcome of pregnancy but increases the risk of hypoglycemia and causes maternal complications, including coma and convulsions. The suppression of the immune system during pregnancy increases the concentration of C-peptide in women with T1DM, and n-3 PUFA supplements serve as the additional support for a rise in C-peptide levels through its anti-inflammatory action.

The serotonin receptor 2A gene (HTR2A) is a strong candidate for the fetal programming of future behavior and metabolism. Maternal obesity and gestational diabetes mellitus (GDM) have been associated with an increased risk of metabolic and psychological problems in offspring. We tested the hypothesis that maternal metabolic status affects methylation of HTR2A in the placenta. The prospective study included 199 pairs of mothers and healthy full-term newborns. Genomic DNA was extracted from feto-placental samples and analyzed for genotypes of two polymorphisms (rs6311, rs6306) and methylation of four cytosine residues (−1665, −1439, −1421, −1224) in the HTR2A promoter region. Placental HTR2A promoter methylation was higher in male than female placentas and depended on both rs6311 and rs6306 genotypes. A higher maternal pre-gestational body mass index (pBMI) and, to a lesser extent, diagnosis of GDM were associated with reduced HTR2A promoter methylation in female but not male placentas. Higher pBMI was associated with reduced methylation both directly and indirectly through increased GDM incidence. Tobacco use during pregnancy was associated with reduced HTR2A promoter methylation in male but not female placentas. The obtained results suggest that HTR2A is a sexually dimorphic epigenetic target of intrauterine exposures. The findings may contribute to a better understanding of the early developmental origins of neurobehavioral and metabolic disorders associated with altered HTR2A function.

No difference was found in AA and DHA percentages in the maternal or in the umbilical vein serum of diabetic pregnant women. Despite of T1DM, a good metabolic control leads to insignificant changes in the AA and DHA levels in diabetic pregnancy.

Objective: To evaluate the concentration and profile of fatty acids (FAs) among macrosomic neonates delivered by healthy pregnant women and pregnant women with type 1 diabetes mellitus (T1DM). Methods:A prospective study of women who delivered macrosomic neonates at a University Hospital Center, Zagreb, Croatia, 2016-2018. Maternal, umbilical vein, and arterial blood samples were collected immediately on delivery. After lipid extraction, total FAs in maternal, umbilical vein, and arterial serum samples were assessed by gas chromatography. Data were compared between women with T1DM and healthy control women. Results:In total, 50 women were enrolled: 22 with T1DM and 28 control women.Neonates in the T1DM group had a higher ponderal index as compared with the control group (P=0.006). Umbilical vein insulin, insulin resistance, and leptin concentration were higher in the T1DM group than in the control group (all P<0.001). Umbilical vein serum concentrations of total saturated, monounsaturated, n-3 polyunsaturated, and n-6 polyunsaturated FAs were higher in the T1DM group (P=0.004, P<0.001, P=0.015, and P=0.014, respectively). Conclusion: Macrosomic neonates delivered by women with T1DM had a higherPonderal index, and higher concentrations of insulin, leptin, and FAs in the umbilical vein and artery as compared with control group newborns. K E Y W O R D S