Maternal obesity alters fatty acid oxidation, AMPK activity, and associated DNA methylation in mesenchymal stem cells from human infants

Abstract

Objective: Infants born to mothers with obesity have greater adiposity, ectopic fat storage, and are at increased risk for childhood obesity and metabolic disease compared with infants of normal weight mothers, though the cellular mechanisms mediating these effects are unclear.

Methods: We tested the hypothesis that human, umbilical cord-derived mesenchymal stem cells (MSCs) from infants born to obese (Ob-MSC) versus normal weight (NW-MSC) mothers demonstrate altered fatty acid metabolism consistent with adult obesity. In infant MSCs undergoing myogenesis in vitro, we measured cellular lipid metabolism and AMPK activity, AMPK activation in response to cellular nutrient stress, and MSC DNA methylation and mRNA content of genes related to oxidative metabolism.

Results: We found that Ob-MSCs exhibit greater lipid accumulation, lower fatty acid oxidation (FAO), and dysregulation of AMPK activity when undergoing myogenesis in vitro. Further experiments revealed a clear phenotype distinction within the Ob-MSC group where more severe MSC metabolic perturbation corresponded to greater neonatal adiposity and umbilical cord blood insulin levels. Targeted analysis of DNA methylation array revealed Ob-MSC hypermethylation in genes regulating FAO (PRKAG2, ACC2, CPT1A, SDHC) and corresponding lower mRNA content of these genes. Moreover, MSC methylation was positively correlated with infant adiposity.

Conclusions: These data suggest that greater infant adiposity is associated with suppressed AMPK activity and reduced lipid oxidation in MSCs from infants born to mothers with obesity and may be an important, early marker of underlying obesity risk.

Keywords: AMPK; Lipid metabolism; Maternal/fetal; Mesenchymal stem cells; Obesity.

Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Figures

Figure 1

Greater lipid content and lower FAO in Ob-MSCs, see also Supplemental Figure 1. Cells were grown to 90% confluence in growth medium (GM) and underwent myogenic induction (MIM) for 21 days. (A) ORO staining was quantified spectrophotometrically at day 21 of myogenesis in NW- and Ob-MSCs, with representative light microscope images taken at 20× magnification. Data are presented in arbitrary units as the fold-difference in Ob-MSCs relative to NW-MSCs with mean ± SEM (5 technical replicates per subject). (B) Simple Western measures of DGAT1 protein content was measured cells following 21 days of MIM. β-actin serves as a loading control. Data are presented in arbitrary units as the fold-difference in Ob-MSCs relative to NW-MSCs with mean ± SEM (2 culture replicates per subject, pooled). (C) At d21 of myogenesis, fatty acid tracer measurements were made using 14C[oleate:palmitate] in conditions depicted. Total cellular 14C lipid uptake, total 14C esterified lipids, and total 14C FAO were measured. All 14C data are presented as mean ± SEM for NW and Ob-MSC (3 technical replicates per subject). (D) Rates of glycolysis were determined for basal (untreated), minimal (2DG, 100 mM 2-deoxyglucose), and maximal (Olig, 1 μg/mL oligomycin) conditions by measuring accumulation of l-lactate in the culture media following 2 h of incubation. Data are presented as mean ± SEM for NW and Ob-MSC (2 technical replicates per subject). *P < 0.05 for difference in NW- vs. Ob-MSC by 2-tailed Student's t-test or Mann–Whitney U Test, where appropriate. For all measures n ≥ 12 subjects/group. ˆP < 0.05 for difference from untreated condition for both NW- and Ob-MSC groups, combined using 2-way ANOVA. GM, growth Medium; MIM, myogenic induction medium; d, days; ORO, Oil Red O.

Figure 2

Distinct metabolic phenotypes identified among NW- and Ob-MSCs, see also Supplemental Figure 2. At d21 of myogenesis, FAO measurements were made using 14C-[oleate:palmitate] in conditions depicted. (A) The ratio of incomplete FAO (ASM) and complete FAO to CO2 (CO2), reveals two distinct groups of Ob- and NW-MSCs which were further examined as separate groups to gain insight into the metabolic functioning of these cells. Frequency distributions of these data are shown in Supplemental Figure 2. (B) Incomplete FAO as determined by acid soluble metabolites (ASM). (C) Complete FAO to CO2. (D–F) Maternal fasting insulin, HOMA-IR, and FFA levels were measured after overnight fast at median 17 weeks gestation. (G) Insulin was measured in cord blood collected immediately after birth. (H) Infant fat mass was measured using air displacement plethysmography within 24–48 h after birth. All data are presented as mean ± SEM. For each panel, bars with different letters indicate differences for those groups at P < 0.05 as calculated by ANOVA analysis with a priori planned comparisons for three independent groups, and comparison of NW-MSC versus all Ob-MSCs. MIM: myogenic induction media; FAO, fatty acid oxidation; ASM, acid soluble metabolite; HOMA-IR, homeostatic model assessment for insulin resistance; FFA, free fatty acids.

Figure 3

Ob-MSCs demonstrate lower AMPK protein content. (A) Citrate synthase (CS) activity was determined spectrophotometrically in cell lysates (2 technical replicates per subject). (B) Western blot analysis of ETS complex proteins (complexes I–V) was measured using the MitoProfile Antibody cocktail. Data are presented in arbitrary units as the fold-difference in Ob-MSCs relative to NW-MSCs with mean ± SEM (2 culture replicates per subject, pooled; n ≥ 5 subjects/group). (C) Total CPT activity was measured spectrophotometrically in isolated mitochondria (4 technical replicates per subject; n ≥ 3 subjects/group). (D) Simple Western measures of phosphorylated (Thr172) and total protein content of AMPKα1/α2 were measured. β-actin serves as a loading control. Data are presented in arbitrary units as the fold-difference relative to NW-MSCs (2 culture replicates per subject, pooled; n ≥ 5 subjects/group). All data are presented as mean ± SEM. For each panel, bars with different letters indicate differences for those groups at P < 0.05 as calculated by ANOVA analysis with a priori planned comparisons for three independent groups and comparison of NW-MSC versus all Ob-MSCs. MIM: myogenic induction media; CS, citrate synthase; CPT, carnitine palmitoyltransferase; AMPK, AMP-activated protein kinase.

Figure 4

Lower AMPK activation in ObHi-MSCs, see also Supplemental Figure 3. At d20 of myogenesis, cells were incubated with or without excess fatty acids for 24 h in conditions depicted (24hFA), then Simple Western measures were made for (A) phosphorylated (Thr172) relative to total protein content of AMPKα1/α2 and (B) phosphorylated (Ser79) relative to total protein content of ACC. Simple Western data are presented in arbitrary units as the fold-difference in ObLo and ObHi groups relative to NW-MSCs (2 culture replicates per subject, pooled). Individual phosphorylated and total protein measures are shown in Supplemental Figure 3a–d. At d20 of myogenesis, cells were incubated with or without AICAR for 24 h in conditions depicted, followed by western blot analysis for (C) phosphorylated (Thr172) relative to total protein content of AMPKα1/α2, (D) phosphorylated (Ser79) relative to total protein content of ACC, and (E) phosphorylated (Ser792) relative to total protein content of Raptor. Western blot data are presented in arbitrary units as the fold-difference in ObLo and ObHi groups relative to NW-MSCs (2 culture replicates per subject, pooled). β-actin serves as a loading control. Individual phosphorylated and total protein measures are shown in Supplemental Figure 3e–j. All data are presented as mean ± SEM. For all measures n ≥ 4 subjects/group. For each panel, bars with different letters indicate differences for those groups at P < 0.05 as calculated by ANOVA analysis with a priori planned comparisons for three independent groups and comparison of NW-MSC versus all Ob-MSCs. ˆP < 0.05 for difference from untreated condition for both NW- and Ob-MSC groups, combined using 2-tailed paired t-test. MIM: myogenic induction media; AMPK, AMP-activated protein kinase; ACC, acetyl CoA carboxylase.

Figure 5

Ob-MSCs exhibit DNA hypermethylation of genes specific to FAO, see also Supplemental Tables 3–5 and Supplemental Figure 4. (A) Graphic depicting epigenetic analysis workflow. Based on metabolic phenotype identified for FAO, we selected curated genesets specific to oxidative metabolism and employed candidate gene analysis of Illumina Infinium 450 K measures specific to these genes (see Supplemental Tables 2–3 for geneset details), four genes contained significantly differentially methylated regions (DMRs): PRKAG2, ACACB, CPT1A and SDHC (see Supplemental Table 5 and Supplemental Figure 4 for DMR/full gene details). Select CpG sites confirmed with bisulfite pyrosequencing, followed by qPCR to evaluate expression of identified genes (data shown in Figure 6). (B–E) Graphics depicting DMR loci with respect to the transcription start site (TSS); For each gene graphic, the gene is shown in blue (introns in light blue and exons in dark blue). All CpG sites measured by the 450 K are depicted with bar (450 K Data) showing. CpGs comprising significant DMRs are indicated by red circles. Location of known common SNPs, active histone methylation for similar cell types as indicated in Supplemental Figure 4, and CpG islands are shown. Full genes are shown in Supplemental Figure 4. (F) Graphic depicting metabolic pathways measured with location of proteins encoded by differentially methylated genes marked with stars. For all 450 K measures, n = 12 NW-MSCs and n = 12 Ob-MSC.

Figure 6

Ob-MSC DNA hypermethylation corresponds to lower mRNA content and is correlated with MSC and infant outcomes, see also Supplemental Figure 5, Supplemental Table 7. (A–B) Select CpG sites are shown in bar graph for bisulfite pyrosequencing measures for CPT1A and ACACB. (C) qPCR was used to measure mRNA content of targeted genes in the undifferentiated cells (GM; growth media). See Supplemental Figure 5 for mRNA content of all AMPK subunits. (D) Pearson correlation for percent methylation of PRKAG2 cg20534694 (GM condition) and change in phospho/total AMPK protein in response to 24hFA exposure (MIM condition) was performed in the Ob-MSCs. Pearson correlation for percent methylation of PRKAG2:cg20534694, ACACB:cg02022322, CPT1A:17058475, and SDHC:08716396 vs. maternal fasting insulin and FFA levels, infant cord blood insulin levels, and infant body composition were performed for all subjects (Supplemental Table 7). (D) Significant relationship is depicted for PRKAG2:cg20534694 and infant adiposity. Individual data points are depicted with group coloring. For all measures, n ≥ 12 NW-MSCs and n ≥ 12 Ob-MSC. For pyrosequencing and qPCR measures there were 3 technical replicates per subject. All data are presented as mean ± SEM. For all measures n ≥ 4 subjects/group. For each panel, bars with different letters indicate differences for those groups at P < 0.05 as calculated by ANOVA analysis with a priori planned comparisons for three independent groups and comparison of NW-MSC versus all Ob-MSCs.