Age and Sex Influence Mitochondria and Cardiac Health in Offspring Exposed to Maternal Glucolipotoxicity

Figure 1. Maternal Glucolipotoxicity Increases Offspring Mortality Despite Little Evidence of Metabolic Syndrome (A) Sprague Dawley rat model to study offspring cardiometabolic health following fetal exposure to late-gestation diabetes and maternal high-fat (HF) diet. (B) Perinatal and natural long-term mortality of control and exposed offspring. (C–E) Offspring weight (C), serum triglyceride levels (D), and whole blood glucose levels (E) over time with 12MO offspring delineated by sex. NP1 = 216–250 offspring/group, N3WK = 27–41, N10WK = 21–35, N6MO = 4–9, N12MO = 15–20 (C and E); NP1 = 32–57, N3WK,10WK = 14–24, N6MO = 9–10, N12MO = 15–16 (D). (F) Neonatal insulin, C-peptide, and 12MO hemoglobin A1C levels. NP1 = 75–137, N12MO = 8. Although not marked, body weight (A) and TG levels (D) were significantly higher in males than females. Data represent mean ± SEM. p ≤ 0.05: +diabetes or ∗diet effect by two-way ANOVA, ˆgroup effect by 1-way ANOVA and Dunnett post-hoc test when interaction by two-way ANOVA was significant. See also Tables S1 and S2 and Figure S1.

Figure 2. Exposed Offspring Are Born with Cardiomyopathy That Improves After Birth but Reemerges at 12MO (A) Heart:body weight ratios over time with 12MO offspring separated by sex. NP1 = 216–250 offspring/group; N3WK = 27–41; N10WK = 21–35; N6MO = 4–9; N12MO = 15–20. (B–D) LV mass (B), systolic function per ejection fraction (C), and diastolic function per E:A ratio (D) over time with 12MO offspring separated by sex. NP1 = 119–144 offspring/group; N3WK = 18–30; N10WK = 10–19; N6MO = 17–28; N12MO = 15–18. Although not marked, heart:body weight and ejection fraction are both significantly higher in normal females than males, whereas LV mass is higher in males. Data represent mean ± SEM. p ≤ 0.05: +diabetes or ∗diet effect by two-way ANOVA, ˆgroup effect by one-way ANOVA and Dunnett post-hoc test when interaction by two-way ANOVA. See also Figure S1 and Table S3.

Figure 3. Diabetes- and HF Diet-Exposed Newborns Have Impaired Mitochondrial Respiration that Initially Improves but Reemerges at 6MO as Developmental Reliance on OXPHOS Increases. (A–C) Basal (A), FCCP-stimulated maximal (B), and reserve (C) respiratory capacities over time with 12MO offspring separated by sex. Reserve capacity was calculated by subtracting basal from maximal respiration. (D–F) Mitochondrial stress test was used to calculate respiratory control ratios (D), ATP-linked oxygen consumption (E), and mitochondrial proton leak (F) over time with 12MO offspring separated by sex. Assay medium was supplemented with glucose and pyruvate. NP1 = 4–7 per group; N3WK = 4–5; N10WK = 3–4; N6MO = 3; N12MO = 7–11. Data represent mean ± SEM. p ≤ 0.05: +diabetes or ∗diet effect by two-way ANOVA, ˆgroup effect by one-way ANOVA and Dunnett post-hoc test when interaction by two-way ANOVA, and δsex-specific effect by Student's t test.

Figure 4. Mitochondrial Number, Basal Endogenous FAO, and Aerobic Glycolysis Increase with Age, but HF Diet Exposure Exaggerates Mitochondrial Biogenesis in Females, whereas Diabetes Exposure Incites Mitochondrial Dysfunction-Induced Biogenesis in Males causing Higher Oxygen Consumption and Poor Reserve Capacity in Aged Offspring (A) Mitochondrial DNA copy number at each time point was used to estimate mitochondrial biogenesis from P1 to 12MO. NP1 = 10–15 per group; N3WK = 4; N10WK = 4; N6MO = 3–4; N12MO = 6–9. (B) Basal OCR of CM in media without pyruvate, palmitate-stimulated OCR, and calculated responses to palmitate estimate FAO at each developmental time point. (C) Basal, maximal, and reserve capacity for endogenous FAO at 12MO (Rogers et al., 2014). (D–F) Basal, glucose-stimulated, and maximal extracellular acidification rates (ECAR) with cellular response to glucose (D) and proton production rates (PPR) over time (E) and at 12MO by sex (F). Maximal ECAR and PPR were stimulated with oligomycin in P1–6MO CM and with rotenone/antimycin A + monensin in 12MO CM. NP1 = 4–7 per group; N3WK = 4–5; N10WK = 3–4; N6MO = 3; N12MO = 7–11 (B–F). Data represent mean ± SEM. p ≤ 0.05: +diabetes or…

Figure 5. Diet-Exposed Females and Diabetes-Exposed Males Have Fuel-Specific Complex I Dysfunction at Birth and as OXPHOS Increases with Age Consume More Oxygen (A) Schematic diagram of mitochondrial respiratory complexes and fuels feeding electrons into each. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane. (B) Basal oxygen consumption rates (OCR) of permeabilized P1 and 12MO CM. (C) Oxidative responses to various complex fuels at P1 and 12MO time points by sex. State3 respiration (OCR) is shown as a percent change from basal OCR. (D) Oxidation capacity per respiratory control ratios (RCR) at P1 and 12MO time points by sex. Dashed lines (C and D) separate fuels by complexes (I–IV). (E) Relative expression of mitochondrial complex proteins in 12MO hearts by sex, normalized to beta-actin. For NDUFA2, lower (11kDa) band was used for analyses. Dashed lines separate exposure groups. Data represent mean ± SEM. p ≤ 0.05: +diabetes or ∗diet effect by two-way ANOVA, ˆgroup effect by one-way ANOVA and Dunnett post-hoc test when interaction by two-way ANOVA, and δsex-specific effect by Student's t test. NP1 = 4–5 per group; N12MO = 4–6. See also Figure S4 and Table S5.

Figure 6. Baseline and Stress-Induced Mitophagy in 12MO CM Vary Significantly by Sex and Fetal Exposure (A) Representative images of 12MO CM stained with MitoTracker Green, Hoechst, and LysoTracker Red. Once treated with FCCP, mitochondria lose their membrane potential and recruit lysosomes to undergo mitophagy. This is reflected by increasing number of co-localized mitochondria-containing autolysosomes (mitolysosomes) indicated by orange arrowheads (bottom row, middle-left image). (B) Number of lysosomes at baseline, before FCCP treatment. (C–E) Computer analyses of live video images quantified mitolysosome at baseline and over time (C) providing an estimate of physiologic (D) and stress-induced mitophagy after FCCP (E). Data represent mean ± SEM (B and D–E) and linear regression of Pearson correlation coefficient over time (C). p ≤ 0.05: +diabetes or ∗diet effect by two-way ANOVA, ˆgroup effect by one-way ANOVA and Dunnett post-hoc test when interaction by two-way ANOVA, and δsex-specific effect by Student's t test. N = 7–11 offspring/group. See also Video S1.

Figure 7. Diabetes-Exposed Male CM have Faster MMP Loss and Cell Death Following Metabolic Stress (A) Representative images of 12MO CM stained with MitoTracker Green, Hoechst, and TMRE. Once treated with FCCP, mitochondria lose their MMP and trigger cell death as seen by retraction and pyknosis. (B) MMP loss (75% from baseline) following FCCP-induced stress and the rate of MMP loss, calculated by linear regression analysis. (C and D) Retraction and pyknosis, markers of CM death, were defined as the time from FCCP-induced stress to 50% original cell length (C) or to 10% loss in nuclear area (D), respectively. Time from start of MMP loss to either was calculated from a starting point of 25% MMP loss. Data represent mean ± SEM. p ≤ 0.05: +diabetes effect by two-way ANOVA, δsex-specific effect by Student's t test. N = 8–11 offspring/group. See also Figure S5 and Video S2.