Maternal High Fat Diet and Diabetes Disrupts Transcriptomic Pathways That Regulate Cardiac Metabolism and Cell Fate in Newborn Rat Hearts

Abstract

Background: Children born to diabetic or obese mothers have a higher risk of heart disease at birth and later in life. Using chromatin immunoprecipitation sequencing, we previously demonstrated that late-gestation diabetes, maternal high fat (HF) diet, and the combination causes distinct fuel-mediated epigenetic reprogramming of rat cardiac tissue during fetal cardiogenesis. The objective of the present study was to investigate the overall transcriptional signature of newborn offspring exposed to maternal diabetes and maternal H diet. Methods: Microarray gene expression profiling of hearts from diabetes exposed, HF diet exposed, and combination exposed newborn rats was compared to controls. Functional annotation, pathway and network analysis of differentially expressed genes were performed in combination exposed and control newborn rat hearts. Further downstream metabolic assessments included measurement of total and phosphorylated AKT2 and GSK3β, as well as quantification of glycolytic capacity by extracellular flux analysis and glycogen staining. Results: Transcriptional analysis identified significant fuel-mediated changes in offspring cardiac gene expression. Specifically, functional pathways analysis identified two key signaling cascades that were functionally prioritized in combination exposed offspring hearts: (1) downregulation of fibroblast growth factor (FGF) activated PI3K/AKT pathway and (2) upregulation of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC1α) mitochondrial biogenesis signaling. Functional metabolic and histochemical assays supported these transcriptome changes, corroborating diabetes- and diet-induced cardiac transcriptome remodeling and cardiac metabolism in offspring. Conclusion: This study provides the first data accounting for the compounding effects of maternal hyperglycemia and hyperlipidemia on the developmental cardiac transcriptome, and elucidates nuanced and novel features of maternal diabetes and diet on regulation of heart health.

Keywords: PI3K/Akt pathway; cardiovascular disease; functional genomics; high fat diet; maternal diabetes; mitochondrial biogenesis.

Figure 1

Maternal diabetes and maternal high fat diet impart distinct cardiac transcriptome signatures in newborn rat offspring. (A) Schematic showing experimental model of exposed groups. Female rats had at least 28 days of either control or high-fat diet prior to breeding. Female diet continued throughout pregnancy. At gestational day (GD) 14 a single injection of citrate buffer (CB) or streptozotocin (STZ) was delivered to a subset of females with high-fat or control diet. At GD22 newborns were delivered and hearts were extracted for study from four exposed groups: controls (green), diabetes exposed (blue), high fat diet exposed (yellow), and combination exposed (orange). (B) Principal components analysis (PCA) plots depicting distinct transcriptome signature among all the exposed groups. Data is visualized here in two plots with different components plotted in each: components 1–3 are plotted on the left (C1 = 25.1%, C2 = 8.9%, and C3 = 4.4%); and components 3–4 are used in PCA plot on right (C2 = 8.9%, C3 = 4.4% and C4 = 5.1%). (C) Class prediction model used individual sample expression signatures to demonstrate with 100% accuracy the identification of each sample to their respective exposure group.

Figure 2

Differential gene expression of diabetes and high-fat (HF) diet exposed groups. Depicted are the differentially expressed genes (DEG) from all exposed groups visualized through volcano plots. In general, the different exposed groups compared to controls depicted different levels of DEGs. (A) Diabetes exposed group revealed the smallest changes with 6 downregulated and 31 upregulated DEGs when compared to controls. (B) HF diet group depicted 38 and 94 down and upregulated, respectively. (C) Combination of both diabetes and HF diet exposed the largest number of DEGs (323), with 122 downregulated and 201 upregulated. Significance threshold was set at > 1.25 and < −1.25 fold change (FC, green vertical lines) and p < 0.05 (green horizontal line).

Figure 3

Functional pathways prioritized in differentially expressed genes from combination exposed group. Functional annotation and pathways analysis was done using Reactome pathway database in down and upregulated differentially expressed genes (DEGs) of combination exposed group when compared to control. (A) Analysis of the upregulated DEGs elicited four mitochondrial translation-related pathways, associated with Dap3, Mrpl38, Mrps10, Mrpl19, Mrps27, Mrpl40. (B) Downregulated list revealed nine functional pathways directly associated with fibroblast growth factor receptor 2 (Fgfr2).

Figure 4

Akt is a critical hub within the network of transcriptome remodeling that characterizes hearts of combination exposed offspring. Interactions of merged functional networks, constructed in IPA from statistically significant genes in combination exposed group, were uploaded into Cytoscape for network analysis. (A) Circular network layout depicting node interactions and colored by degree. Magnification of region with highest edge density (dark gray) showing Akt as the network node with the highest degree, followed by Erk1/2, Erk, NfkB (complex), Edn1, Ras, Ampk, Cd3, Alp, Fgfr2, Stat5a/b, Hdac, Pdgf-AA, Sos, Pgk1, Nxf1, Nppb and Mrps27 (highest to lowest degree, respectively). (B) IPA Molecule Predictor Analysis of the highest priority network (Network 1, Score 39, top functions: cell cycle, protein synthesis, hair and skin development/function) predicted inhibition (blue) of Akt in the combination exposed newborn heart by net expression of differentially expressed genes. Right: Magnification of Fgfr2 (downregulated in green) and Mrps27 (upregulated in red) hubs and immediate subnetwork neighborhood. (C,D) Graph theory analysis of Network 1 (Combination vs. control) revealed Akt as a network hub with the highest betweenness and closeness centrality metrics, indicative of its importance to overall network structure and communication. Among the top 10 nodes prioritized include: Cdk1, Caspase, Dap3, Mrps27, Cyclin A, Pgk1, Fgf7, Fgfr2, Cdc2, E2f and Rb. IPA, Ingenuity Pathways Analysis; Degree (k) = neighborhood connectivity distribution.

Figure 5

Combination exposed offspring hearts enriches discrete classes of mitochondrial genes. Genes that were duplicated or non-annotated were excluded, resulting in 228 differentially expressed genes (DEG) with 94 down and 134 upregulated in combination exposed hearts when compared to controls. (A) The 228 DEGs were plotted in a Venn diagram against 1,158 mitochondrial associated genes (MitoCarta Database, Mouse) and depicted 32 upregulated mitochondrial genes (Table S9) with none downregulated in the combination exposed offspring hearts. (B) Mitochondrial functions associated with the 32 upregulated mitochondrial genes were: cell signaling and cell fate, mitochondrial biogenesis, dynamism, oxidative phosphorylation, metabolism, oxidative repair and other mitochondrial functions. *Genes with multiple functions; shown in more than one category. (C) RNA expression of Ppargc1a and Mrpl19 were significantly upregulated with fold change (FC) of 2.24 and 2.28, respectively (p < 0.05) in combination exposed newborn hearts (red squares) compared to controls (gray circles; n = 6/group). We showed no significant change in RNA expression of Mrps27 (FC = 1.09, p = 0.6) or Dap3 (FC = 1.10, p = 0.75). Consistent with the RNA expression, protein expression of PGC1α was significantly increased in newborn hearts exposed to diabetes and HF diet when compared to controls (n = 4/group, p < 0.05). We observed a non-significant upregulation trend for MRPL19 and DAP3 protein expressions with no change in MRPS27. (D) Relative mtDNA copy number was not different by mtDNA expression of either cytochrome-c oxidase I (COI) or mitochondrial control region (D-loop). However, combination exposed hearts had higher protein expression of Voltage dependent anion channel 1 (VDAC) and a trend toward higher Translocase of outer mitochondrial membrane 20 (TOM20).

Figure 6

Evidence of insulin resistance in diabetes and high fat diet exposed offspring hearts. Chronic exposure to insulin and other growth hormones can cause insulin resistance through downregulation of growth factor receptors and impaired downstream activation of the PI3K/AKT pathway which shifts metabolism from glycolysis to gluconeogenesis/glycogen accumulation. (A) Combination exposed newborn offspring (n = 10/group) had significantly higher circulating insulin and c-peptide levels. Consistent with transcriptome analyses, combination exposed newborn male hearts, had a trend toward lower RNA expression relative to B2m (p = 0.13, n = 7/group) and lower protein expression (p < 0.05, n = 4/group) of FGFR2. (B) Total and phosphorylated AKT (n = 5/group) was not different, but GSK3β was higher (n = 5/group) and there was a trend toward more phosphorylated (active) and ratio of phosphorylated:total GSK3β (p = 0.14 and p = 0.057, respectively) in combination exposed, male hearts. (C) Primary isolated newborn rat cardiomyocytes (NRCM) from combination exposed male offspring had no significant difference in baseline extracellular acidification rate (ECAR), glucose or rotenone/antimycin (Rot/AA) stimulated glycolysis (glycolytic capacity) by XF analyses (top row). The proton production rate (PPR) was calculated to estimate aerobic (PPR from CO₂) and anaerobic (PPR from lactate) glycolysis. At baseline, there was no difference, but aerobic glycolysis was significantly lower in combination exposed NRCM following glucose injection. Combination exposed NRCM had only 34% aerobic glycolysis vs. 79% aerobic glycolysis following glucose in controls. This suggests maternal diabetes and high fat diet exposure impairs aerobic glycolytic capacity. *p < 0.05, n = NRCM pooled from 3 to 4 pups/litter, 4 litters/group. (D) Periodic Acid Schiff (PAS) staining demonstrates more glycogen deposition in combination exposed hearts, which suggests a chronic in utero switch from glucose utilization to storage occurred during development.

Figure 7

Proposed model of maternal combination exposed diet effects on cardiac network signaling in offspring. This illustrative schematic depicts the metabolic pathways in which the triad of maternal hyperglycemia, hyperlipidemia, and fetal hyperinsulinemia leads to an increased mitochondrial biogenesis, autophagy, and a shift from glycolysis to gluconeogenesis. This occurs via a downregulated PI3K/AKT pathway and increased PGC1α expression amongst uninhibited MAPK activity. AKT, protein kinase B; AMPK, 5′ adenosine monophosphate-activated protein kinase; FGF, fibroblast growth factor; GLUT4, glucose transporter type 4; GSK3β, glycogen synthase kinase 3 beta; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; PGC1α, PPARG coactivator 1 alpha; PPARg, peroxisome proliferator activated receptor gamma; Ras, proto-oncogene protein p21.