Two-month-old female B6129SF2/J mice (Envigo, Indianapolis, IN) were fed diets containing either 16% (normal-fat, NF) or 45% fat (high-fat, HF) for 12 weeks ( Table S1). Following 12 weeks of diet intervention, dams were bred with normal chow fed males and pregnancy identified by the presence of a vaginal plug. Midday identification of the vaginal plug was considered embryonic day 0.5 (E0.5). Dams were maintained on their respective diets throughout mating and pregnancy. Dam euthanasia was by CO₂ inhalation according to the animal use and care protocol approved by the USDA Agricultural Research Service, Grand Forks Human Nutrition Research Center Animal Care and Use Committee. Fetuses and placentae were harvested in mid-gestation (E12.5-E13.5) or late gestation (E18.5-E19.5), weighed, measured, and then immediately frozen in liquid nitrogen.

The uterine horn was dissected from the dam and cut between each implantation site separating each amniotic sac containing individual fetus. Removed placenta and fetus were blotted dry before removal of umbilical cords. Weights were recorded and the placenta length and width were measured with the maternal convex side up using a digital caliper (Marathon Watch Company LTD., Richmond Hill, ON Canada). Samples were then flash frozen in liquid nitrogen and stored at -80°C.

HPLC-grade isopropanol, chloroform, butylated hydroxytoluene, and hexane were purchased from Sigma Aldrich (St. Louis, MO, USA) and used as received. HPLC-grade methanol was purchased from Honeywell (Muskegon, MI, USA). Silicic acid (200-325 mesh) was ordered from Clarkson Chromatography Products Inc. (South Williamsport, PA, USA). Internal standards for TAG and CE analysis were purchased from NuChek Prep Inc. (Elysian, MN, USA), phospholipid and sphingolipid standards and LIPIDMAPS standards were purchased from Avanti Polar Lipids (Alabaster, AL, USA) ( Table S2 ).

Briefly, frozen placental samples were homogenized in aqueous buffer. Neutral lipid internal standards were added, and lipids were extracted in 3:2 hexane: isopropanol (50 µmol/L BHT). Extracts were isolated and dried under, then redissolved in 1 mL chloroform. The solution was divided in half, with 500 µL retained for phospholipid analysis (below). Phospholipids were removed from the neutral lipid fraction by dispersive SPE with silicic acid, and100 µL of the supernatant was combined with 100 µL of methanol (20 mM ammonium acetate). This sample (Solution 1) was analyzed for TAG and CE content as described below. A 500 µL aliquot of the SPE supernatant was used for the assay of FC following the method of Liebisch et al. (17).

Analysis of polar lipids was performed using the method adapted from Sundaram et al. with modifications made to allow for automation (18).

Data were collected on an AB Sciex 5500 QTRAP hybrid mass spectrometer equipped with a Turbo V electrospray ion source and SelexION ion mobility device (AB Sciex, Framingham, MA, USA). Samples were infused using a Shimadzu Prominence UPLC system (Shimadzu Scientific Instruments, Columbia, MD, USA) equipped with an LC20XR autosampler (50 µL stainless steel sample loop) and two solvent delivery units following a configuration modified from Bukowski and Picklo as detailed in the Supplemental Information (19).

Multiple instrument modes were used based upon lipid class and preparation ( Table S2 ). TAG were characterized and quantitated by brutto structure as ammoniated cations, [TAG + NH₄]⁺ in enhanced mass spectrum (EMS) mode as previously published (19, 20). Cholesterol esters (CE) were assayed by neutral loss scan for 20 fatty acid neutral losses with confirmatory detection of the cholesterol head group by product ion scan for mass-to-charge ratio (m/z) = 369, as previously published ( Table S3 ) (20, 21). Acylated samples for FC determination were analyzed using multiple reaction monitoring for the acylated d7-cholesterol species (m/z = 453.4376.3) and the endogenous acylated cholesterol (m/z = 446.4369.3) (17). Ceramide species (Cer) were detected using the product ion m/z 264, selective to the sphingosine backbone, and quantified following the method of Picklo et al. (22). Phosphatidylethanolamine, and phosphatidylcholine species were quantified as brutto species using our previously published methods (20, 23). Monitoring the neutral loss of 141 Da allowed for the selective measurement of PE species, while monitoring the product ion m/z = 184 was selective for PC and SM species.

SM species were isolated for characterization and quantitation using the Selexion ion mobility device (24).

Mass spectra for all lipid class were examined manually to confirm brutto structure assignment. Isotopic and ionization correction factors were determined as previously described and quantitation of target species was performed using LipidView software (AB Sciex, Framingham, MA, USA) (19, 20, 22, 25). Values were normalized to tissue wet weight. TAG 54:0 and TAG 54:1 were excluded from analysis due to isobaric overlap with a silicone oligomer contaminant which was extracted from the septa and could not be reliably removed by subtraction.

Statistical analyses were performed using MetaboAnalyst 5.0 (26). After range scaling, data were analyzed by one-way ANOVA with an alpha of P<0.05, and a 0.05 false discovery rate. Tukey’s HSD was employed to as a post-hoc test for significant interactions between groups.

Using the combined infusion mass spectrometric methods 190 major lipid species including sphingolipids and phospholipids were identified in the placental samples. In the neutral lipid fraction 34 cholesterol esters, 50 TAG and FC were quantitated (additional details provided in Tables S4, S5 ). In the polar lipid fraction, brutto structures included 49 PC, 36 PE, eight SM, eight Cer, and four HexCer species ( Tables S6–S8 , respectively). A summary of the relationships established by univariate analysis is presented in Table 1 .

Cer and other glycosphingolipids (cerebroside) species were identified based upon the m/z = 264 product ion which was selective for the sphingosine base, d18:1, allowing for identification of the fatty acid moiety based upon the precursor ion scan for the Cer species. As shown in Figure 1 , PC and SM species were both detected in the precursor ion scan. The monoisotopic peaks used for quantitation of SM and PC species were offset one Dalton and presented overlap as in the case of the grouping of peaks from m/z 806-816, in which species PC 38:4, PC 38:3, PC 38:2, and PC 38:1 overlapped with SM 42:3, SM 42:2, and SM 42:1. Removal of PC the contribution using the Selexion ion mobility interface was necessary to accurately quantitate to SM, which represented approximately 4% of the signal. A difference in acyl carbon distribution between PC and PE species was evident ( Figure 1 ).

When compared, PE and PC species with equivalent acyl carbon numbers in placental membrane phospholipids demonstrated an increase in concentration of LC-PUFA moieties in PE ( Figure 1 , dashed lines). The majority of PC species had 34, and 36 acyl carbons with between 0 and 3 double bonds, corresponding to structures containing 16- and 18-carbon fatty acids such as palmitic, palmitoleic, stearic, oleic, and linoleic acids while the dominant PE species had 40 acyl carbons and between 4 to 7 double bonds and were composed largely of PUFA species as detailed in the Supplemental Information ( Table S9 ) (25).

The gestational age of the placenta affected the neutral lipid and sphingolipid content. For neutral lipid species the concentration of FC ( Figure 2A ) increased from mid-gestation to late-gestation irrespective of diet or genotype. While animals on HFD appeared to have greater FC at mid gestation, biological variability rendered this difference non-significant and placental FC concentrations at late gestation were indistinguishable. A gestational age-dependent increase in total CE concentration was observed for WT-NF and TRPC1 -/- -HF animals. For the WT-NF group the increase was due primarily to increases in major species such as CE 16:0, CE 18:0, CE 18:1, CE 18: 2, ( Figure 3A ), though gains in CE 16:1, CE 20:2, CE 22:5, CE 22:6 and CE 24:1 also contributed to the elevated concentration. The major contributor to the increase in CE concentration for TRPC1 -/- -HF animals was CE 20:4, which increased 1.9-fold from mid to late gestation. Gestation-dependent increases in CE 22:5 (4.3-fold, Figure 3B ) and CE 22:6 (2.9-fold, Figure 3B ) and CE 22:4 (2.6-fold, Figure 3A ) were also observed. These trends were not observed for either the TRPC1 -/- -NF or WT-HF animals, though the latter did exhibit a decrease CE 16:1 concentration ( Figure 3B ).

Total placental TAG concentration was not affected by gestational age; however, individual TAG species underwent gestation-dependent changes. Tissue from WT-HF dams demonstrated increased concentrations of TAG 50:1 and TAG 48:0 with a concomitant decrease in TAG 56:7 ( Figures 4A, B ). For WT animals, irrespective of diet, the most concentrated TAG species, (TAG 54:2, TAG 54:3, TAG 56:6, TAG 56:8, TAG 58:6, TAG 58:8) were stable over the observed gestational time scale, however TRPC1-knockout animals experienced decreases in TAG 58:6 and TAG 58:6 ( Figures 4B, C ), though the concentrations of these species were higher at the early gestational time point, as will be discussed below.

Gestational age affected the concentration of sphingolipids in placentae. In all cases the SM concentration increased with gestational age ( Figure 2B ). The major contributors to the net increase in SM concentration were SM 42:1, SM 42:2, and SM 34:1 ( Figure 5B ). Total Cer concentrations were unchanged for WT-NF samples but increased with gestational age for TRPC1 -/- -NF ( Figure 2B ). Cer d18:1/16:0, Cer d18:1/22:0, Cer d18:1/24:0, and Cer d18:1/24:1 were the major contributors to this increase ( Figure 5A ) Contrastingly, the WT-HF and TRPC1 -/- -HF arms exhibited decreased Cer concentration with increasing gestational age, as well as a decrease in total HexCer concentration.

Total concentration PE species were not affected by gestational age in aggregate ( Figure S1 ); however, PE 40:4 decreased in concentration for nearly all groups with increasing gestational age ( Figure S2B ). Only placentae from TRPC1 -/- -HF dams demonstrated a loss in PC concentration ( Figure S1 ) but the concentration major contributors to that change were PC 36:2, PC 36:3, PC 38:3, and PC 34:2 ( Figures S3A, B ).

Cholesterol ester concentrations at the earlier gestational time point were greater (p<0.05) in placentae from HFD fed dams irrespective of phenotype ( Figure 2A ). Species-level univariate analysis revealed the diet-induced increase in cholesterol ester concentration was independent of fatty acid identity, with increases in concentration for nearly all cholesterol ester species ( Figure 3 ). The exceptions to this trend were CE 16:0 and CE 16:1, which demonstrated decreases from WT-NF -L to WT-HF-L. Tissue from TRPC1 -/- -HF-L dams demonstrated increases in CE 18:2, CE 20:4, CE 22:4, CE 20:3 CE 22:5, and CE 22:6 relative to TRPC1 -/- -NF-L though, with the exception of CE 20:4, the concentrations at this time point were not significantly different from WT-HF-L samples.

Differences in genotypic response to HFD were observed between the WT-HF-M and TRPC1 -/- -HF-M arms ( Figures 3A, B ). At the earlier time point the SFA-containing species CE 16:0 and CE 18:0 were only 37% and 53% of the WT-HF-L arm, respectively. This trend held for MUFA-containing CE (CE 18:1 and CE 16:1; 53% and 30% of WT-HF-L, respectively), as well as PUFA-containing CE species such as CE 18:2, CE 20:5, CE 22:5, and CE 22:6 (54%, 54%, 28%, and 39% of WT-HF-L, respectively). These differences were not present at the later time point for the HFD arms. These data, along with the observed increase in CE 20:4 noted earlier suggests a compensatory mechanism for PUFA CE accumulation.

For samples from WT animals consumption of HFD led to a decrease in TAG storage for TAG 54:2, 54:3, 54:7, 54:9, 56:0, 56:1, 56:2, 56:3, 56:9, 56:10, 58:0, 58:1,58:2, 58:4, 58:5, 58:10, 58:11, 60:11, and 60:12 ( Figures 4A–C and Figure S2 ). TAG species which displayed genotypic responses followed the pattern of presenting elevated TAG levels for the TRPC1 -/- placentae relative to the WT at the earlier gestational time point. These species included TAG 56:3, 56:6, 56:7, 56:8, 58:5, 58:6, 58:7, 58:8, 58:10, 60:11, and 60:12 ( Figures 4A–C and Figure S2 ). TAG 48:0 presented as an outlier to this trend presenting greater in WT-HF-L animals that the corresponding TRPC1 -/- arm ( Figure 4B ).

As discussed above, the general trend observed for SM species was an increase in concentration with gestational age irrespective of diet or genotype. For SM 34:1 a genotype-dependent response was observed. At the later gestation time point the concentration of SM 34:1 for TRPC1 -/- -NF-L was 64% of that observed for the corresponding WT-NF-L arm, however TRPC1 -/- -HF-M and TRPC1 -/- -HF-L had SM 34:1 concentrations that were 230% and 160% greater than in the corresponding WT groups ( Figure 5B ).

In addition to the gestational trends indicated above, the concentration of Cer species tended to be higher in placentae from WT-HF animals. For TRPC1 -/- -NF-L subjects the concentrations of Cer d18:1/16:0, Cer d18:1/24:1, and d18:1/24:1 increased with gestational age and were greater than corresponding WT-NF-L subjects ( Figure 5B ).