Myo-inositol alters the effects of glucose, leptin and insulin on placental palmitic acid and oleic acid metabolism.

Well-regulated placental palmitic acid (PA) and oleic acid (OA) metabolism is vital for optimal placental function and fetal development, but dysregulation occurs with gestational diabetes (GDM). We hypothesized that such dysregulation might arise from increased maternofetal glucose, leptin or insulin concentrations present in GDM, and that dysregulated PA and OA lipid metabolism could be moderated by myo-inositol, a natural polyol and potential GDM intervention. Placental explants from 21 women were incubated with stable isotope-labelled 13 C-PA or 13 C-OA for 48 h. Explants were treated with glucose (5, 10 mm) or leptin (13 nm) or insulin (150 nm) in combination with myo-inositol (0.3, 30, 60 µm). Forty-seven 13 C-PA lipids and 37 13 C-OA lipids were measured by liquid chromatography-mass spectrometry (LCMS). Compared with controls (5 mm glucose), glucose (10 mm) increased 19 13 C-OA lipids and nine 13 C-PA lipids, but decreased 13 C-OA phosphatidylethanolamine 38:5 and 13 C-PA phosphatidylethanolamine 36:4. The effects of leptin and insulin were less prominent than glucose, with leptin increasing 13 C-OA acylcarnitine 18:1, and insulin increasing four 13 C-PA triacylglycerides. Most glucose, leptin and insulin-induced alterations in lipids were attenuated by co-incubation with myo-inositol (30 or 60 µm), with attenuation also occurring in all subgroups stratified by GDM status and fetal sex. However, glucose-induced increases in acylcarnitine were not attenuated by myo-inositol and were even exaggerated in some instances. Myo-inositol therefore appears to generally act as a moderator, suppressing the perturbation of lipid metabolic processes by glucose, leptin and insulin in placenta in vitro. Whether myo-inositol protects the fetus and pregnancy from unfavourable outcomes requires further research. KEY POINTS: Incubation of placental explants with additional glucose, or to a lesser extent insulin or leptin, alters the placental production of 13 C-lipids from 13 C-palmitic acid (PA) and 13 C-oleic acid (OA) in vitro compared with untreated controls from the same placenta. Co-incubation with myo-inositol attenuated most alterations induced by glucose, insulin or leptin in 13 C-lipids, but did not affect alterations in 13 C-acylcarnitines. Alterations induced by glucose and leptin in 13 C-PA triacylglycerides and 13 C-PA phospholipids were influenced by fetal sex and gestational diabetes status, but were all still attenuated by myo-inositol co-incubation. Insulin differently affected 13 C-PA triacylglycerides and 13 C-PA phospholipids depending on fetal sex, with alterations also attenuated by myo-inositol co-incubation.

Sex-Dependent Regulation of Placental Oleic Acid and Palmitic Acid Metabolism by Maternal Glycemia and Associations with Birthweight.

Pregnancy complications such as maternal hyperglycemia increase perinatal mortality and morbidity, but risks are higher in males than in females. We hypothesized that fetal sex-dependent differences in placental palmitic-acid (PA) and oleic-acid (OA) metabolism influence such risks. Placental explants (n = 22) were incubated with isotope-labeled fatty acids (13C-PA or 13C-OA) for 24 or 48 h and the production of forty-seven 13C-PA lipids and thirty-seven 13C-OA lipids quantified by LCMS. Linear regression was used to investigate associations between maternal glycemia, BMI and fetal sex with 13C lipids, and between 13C lipids and birthweight centile. Placental explants from females showed greater incorporation of 13C-OA and 13C-PA into almost all lipids compared to males. Fetal sex also influenced relationships with maternal glycemia, with many 13C-OA and 13C-PA acylcarnitines, 13C-PA-diacylglycerols and 13C-PA phospholipids positively associated with glycemia in females but not in males. In contrast, several 13C-OA triacylglycerols and 13C-OA phospholipids were negatively associated with glycemia in males but not in females. Birthweight centile in females was positively associated with six 13C-PA and three 13C-OA lipids (mainly acylcarnitines) and was negatively associated with eight 13C-OA lipids, while males showed few associations. Fetal sex thus influences placental lipid metabolism and could be a key modulator of the impact of maternal metabolic health on perinatal outcomes, potentially contributing toward sex-specific adaptions in which females prioritize survival.

Increasing maternal age associates with lower placental CPT1B mRNA expression and acylcarnitines, particularly in overweight women.

Older pregnant women have increased risks of complications including gestational diabetes and stillbirth. Carnitine palmitoyl transferase (CPT) expression declines with age in several tissues and is linked with poorer metabolic health. Mitochondrial CPTs catalyze acylcarnitine synthesis, which facilitates fatty acid oxidization as fuel. We hypothesized that the placenta, containing maternally-inherited mitochondria, shows an age-related CPT decline that lowers placental acylcarnitine synthesis, increasing vulnerability to pregnancy complications. We assessed CPT1A, CPT1B, CPT1C and CPT2 mRNA expression by qPCR in 77 placentas and quantified 10 medium and long-chain acylcarnitines by LC-MS/MS in a subset of 50 placentas. Older maternal age associated with lower expression of placental CPT1B, but not CPT1A, CPT1C or CPT2. CPT1B expression positively associated with eight acylcarnitines and CPT1C with three acylcarnitines, CPT1A negatively associated with nine acylcarnitines, while CPT2 did not associate with any acylcarnitine. Older maternal age associated with reductions in five acylcarnitines, only in those with BMI≥ 25 kg/m2, and not after adjusting for CPT1B expression. Our findings suggest that CPT1B is the main transferase for placental long-chain acylcarnitine synthesis, and age-related CPT1B decline may underlie decreased placental metabolic flexibility, potentially contributing to pregnancy complications in older women, particularly if they are overweight.

Myo-inositol moderates maternal BMI and glycemia related variations in in-vitro placental 13C-DHA-metabolism, altering their relationships with birthweight.

Transplacental docosahexaenoic-acid (DHA) supply for fetal development is regulated by placental DHA-lipid metabolism. Both maternal diabetes and obesity are linked to possible decreased fetal circulating DHA and increased placental DHA-lipids. Since myo-inositol is a promising intervention for gestational diabetes (GDM), we aimed to determine whether myo-inositol could rectify perturbations in placental DHA metabolism associated with maternal increasing glycemia and obesity and examine links with birthweight. Term placental villous explants from 17 women representing a range of BMIs and mid-gestational glycemia, were incubated with 13C-labeled-DHA for 48 h, in 0.3 µmol/L (control) or 60 µmol/L myo-inositol. Individual newly synthesized 13C-DHA-labeled lipid species were quantified by liquid-chromatography-mass-spectrometry. Compared with controls, incubation with myo-inositol decreased most 13C-DHA-lipids in placental explants from women with higher BMI or higher glycemia, but increased 13C-DHA-lipids with normal BMI or lower glycemia. Myo-inositol also increased 13C-DHA-labeled lipids in cases of lower birthweight centile, but induced decreases at higher centiles. Myo-inositol therefore lowered DHA-lipids in placenta with high basal placental DHA-lipid production (higher BMI and glycemia) but increased DHA-lipids where basal processing capacity is low. Myo-inositol thus moderates placental DHA metabolism towards a physiological mean which may in turn moderate birthweight.

Myo-Inositol Moderates Glucose-Induced Effects on Human Placental 13C-Arachidonic Acid Metabolism.

Maternal hyperglycemia is associated with disrupted transplacental arachidonic acid (AA) supply and eicosanoid synthesis, which contribute to adverse pregnancy outcomes. Since placental inositol is lowered with increasing glycemia, and since myo-inositol appears a promising intervention for gestational diabetes, we hypothesized that myo-inositol might rectify glucose-induced perturbations in placental AA metabolism. Term placental explants (n = 19) from women who underwent a mid-gestation oral glucose-tolerance-test were cultured with 13C-AA for 48 h in media containing glucose (5, 10 or 17 mM) and myo-inositol (0.3 or 60 µM). Newly synthesized 13C-AA-lipids were quantified by liquid-chromatography-mass-spectrometry. Increasing maternal fasting glycemia was associated with decreased proportions of 13C-AA-phosphatidyl-ethanolamines (PE, PE-P), but increased proportions of 13C-AA-triacylglycerides (TGs) relative to total placental 13C-AA lipids. This suggests altered placental AA compartmentalization towards storage and away from pools utilized for eicosanoid production and fetal AA supply. Compared to controls (5 mM glucose), 10 mM glucose treatment decreased the amount of four 13C-AA-phospholipids and eleven 13C-AA-TGs, whilst 17 mM glucose increased 13C-AA-PC-40:8 and 13C-AA-LPC. Glucose-induced alterations in all 13C-AA lipids (except PE-P-38:4) were attenuated by concurrent 60 µM myo-inositol treatment. Myo-inositol therefore rectifies some glucose-induced effects, but further studies are required to determine if maternal myo-inositol supplementation could reduce AA-associated pregnancy complications.

Myo-inositol alters 13C-labeled fatty acid metabolism in human placental explants.

We postulate that myo-inositol, a proposed intervention for gestational-diabetes, affects transplacental lipid supply to the fetus. We investigated the effect of myo-inositol on fatty-acid processing in human placental-explants from uncomplicated pregnancies. Explants were incubated with 13C-labeled palmitic-acid, 13C-oleic-acid and 13C-docosahexaenoic-acid across a range of myo-inositol concentrations for 24 h and 48 h. The incorporation of labeled-fatty-acids into individual lipids was quantified by liquid-chromatography-mass-spectrometry. At 24 h, myo-inositol increased the amount of 13C-palmitic-acid and 13C-oleic-acid labeled lipids (median fold-change relative to control=1). Significant effects were seen with 30 µM myo-inositol (physiological) for 13C-palmitic-acid-lysophosphatidylcholines (1.26) and 13C-palmitic-acid-phosphatidylethanolamines (1.17). At 48 h, myo-inositol addition increased 13C-oleic-acid-lipids but decreased 13C-palmitic-acid and 13C-docosahexaenoic-acid lipids. Significant effects were seen with 30 µM myo-inositol for 13C-oleic-acid-phosphatidylcholines (1.25), 13C-oleic-acid-phosphatidylethanolamines (1.37) and 13C-oleic-acid-triacylglycerols (1.32) and with 100 µM myo-inositol for 13C-docosahexaenoic-acid-triacylglycerols (0.78). Lipids labeled with the same 13C-fatty-acid showed similar responses when tested at the same time-point, suggesting myo-inositol alters upstream processes such as fatty-acid uptake or activation. Myo-inositol supplementation may alter placental lipid physiology with unknown clinical consequences.

Metabolism of 13C-Labeled Fatty Acids in Term Human Placental Explants by Liquid Chromatography-Mass Spectrometry.

Placental lipid transport and metabolism are poorly understood despite the importance for fetal development and lifelong health. We aimed to explore fatty acid (FA) processing in human villous placental explants from seven uncomplicated term singleton pregnancies delivered by elective cesarean section. Explants were treated with stable isotope-labeled palmitic acid (13C-PA), oleic acid (13C-OA), or docosahexaenoic acid (13C-DHA) for 3, 24, or 48 hours. Stable isotope-labeled lipids synthesized by placental explants from labeled FA were quantified, alongside endogenous unlabeled placental lipids, by liquid chromatography-mass spectrometry. Labeled phosphatidylcholines (PCs), triacylglycerols (TAGs), and phosphatidylethanolamines were detected in explants, whereas labeled lysophosphatidylcholines were found in both explants and conditioned media. 13C-PA was primarily directed into PC synthesis (74% of 13C-PA-labeled lipids), whereas 13C-OA was directed almost equally into PC and TAG synthesis (45% and 53%, respectively, of 13C-OA-labeled lipids). 13C-DHA was only detectable in TAGs. TAGs demonstrated the highest isotopic enrichment for all 13C-FAs with 13C-OA-TAGs comprising >50% of total OA-TAGs (unlabeled and labeled), consistent with TAGs being a labile and accessible reservoir for FA storage. Variations in lipid incorporation were correlated to maternal glycemia and body mass index, suggesting that this experimental model could be used to investigate the effect of maternal factors on placental lipid metabolism. We conclude that lipid metabolic partitioning of freshly imported FAs into labile and less labile lipid reservoirs in placenta is FA dependent. This process may partly mediate the physiological preferential transplacental transfer of particular FAs to the fetus, but may also be implicated in the fetoplacental pathophysiology of maternal metabolic dysfunction.