Neonatal diet impacts liver mitochondrial bioenergetics in piglets fed formula or human milk

Abstract

Background: Neonatal diet impacts many physiological systems and can modify risk for developing metabolic disease and obesity later in life. Less well studied is the effect of postnatal diet (e.g., comparing human milk (HM) or milk formula (MF) feeding) on mitochondrial bioenergetics. Such effects may be most profound in splanchnic tissues that would have early exposure to diet-associated or gut microbe-derived factors.Methods: To address this question, we measured ileal and liver mitochondrial bioenergetics phenotypes in male piglets fed with HM or MF from day 2 to day 21 age. Ileal and liver tissue were processed for mitochondrial respiration (substrate only [pyruvate, malate, glutamate], substrate + ADP, and proton "leak" post-oligomycin; measured by Oroboros methods), mitochondrial DNA (mtDNA) and metabolically-relevant gene expression analyses.Results: No differences between the diet groups were observed in mitochondrial bioenergetics indices in ileal tissue. In contrast, ADP-dependent liver Complex I-linked OXPHOS capacity and Complex I + II-linked OXPHOS capacity were significantly higher in MF animals relative to HM fed piglets. Interestingly, p53, Trap1, and Pparβ transcript abundances were higher in MF-fed relative to HM-fed piglets in the liver. Mitochondrial DNA copy numbers (normalized to nuclear DNA) were similar within-tissue regardless of postnatal diet, and were~2-3 times higher in liver vs. ileal tissue. Conclusion:While mechanisms remain to be identified, the data indicate that neonatal diet can significantly impact liver mitochondrial bioenergetics phenotypes, even in the absence of a change in mtDNA abundance. Since permeabilized liver mitochondrial respiration was increased in MF piglets only in the presence of ADP, it suggests that formula feeding led to a higher ATP turnover. Specific mechanisms and signals involved with neonatal dietassociated differences in liver bioenergetics remain to be elucidated.

Real‐time OXPHOS capacity analysis in wounded skin from diabetic mice: A pilot study

“…Mass-specific O 2 fluxes and respiratory states in WS and NWS from NDM and STZ-DM mice were evaluated as previously described 18,[26][27][28] (Table S2). NWS comparisons were used as baseline for evaluating skin mitochondrial respiration in NDM or STZ-DM mice.…”

“…After biopsy collection, skin samples (WS and NWS) were immediately immersed in 2 mL of ice‐cold preservation buffer, BIOPS; pH 7.1, with the following composition: Ca 2+ /EGTA buffer, 10 mM (free calcium .1 μM); imidazole, 20 mM; K + /4‐morpholinoethanesulfonic acid, 50 mM; dithiothreitol (DTT), .5 mM; MgCl 2 , 6.56 mM; ATP, 5.77 mM; phosphocreatine, 15 mM 17 . Skin biopsies were cut into smaller pieces and permeabilized with saponin (50 μg/mL), prepared in BIOPS, for 20 min at 4°C in gently shaking 26 . Biopsies were washed for 10 min in mitochondrial respiratory medium, MIR05; pH 7.1, containing: sucrose, 110 mM; potassium lactobionate buffer, 60 mM; EGTA, .5 mM; MgCl 2 .6H 2 O, 3 mM; taurine, 20 mM; KH 2 PO 4 , 10 mM; Hepes, 20 mM; essential fatty acid‐free BSA, 1 g/L 17,27 .…”

“…17 Skin biopsies were cut into smaller pieces and permeabilized with saponin (50 μg/mL), prepared in BIOPS, for 20 min at 4°C in gently shaking. 26 MIR05; pH 7.1, containing: sucrose, 110 mM; potassium lactobionate buffer, 60 mM; EGTA, .5 mM; MgCl 2 .6H 2 O, 3 mM; taurine, 20 mM; KH 2 PO 4 , 10 mM; Hepes, 20 mM; essential fatty acid-free BSA, 1 g/L. 17,27 Permeabilized WS and NWS were then gently dried on filter paper and weighed on a precision scale (Sartorius CP324S Analytical Balance, Canton, MA).…”

“…26 Oxygen (O 2 ) flux measurements were obtained with O 2 concentrations in the range of 200-400 μM in MIR05 buffer to minimize oxygen dependency. 26 O 2 flux was calculated in the picomolar range at 2-4 s intervals. 26 The O 2 flux reflects the first derivate of the oxygen concentration (nmol/mL) per time in the respiration chambers, and it was normalized to the wet weight of permeabilized skin mass in the chamber [pmol/(s*mg)].…”

“…For oxygen consumption measurements, a steady-state rate of respiration was obtained after the addition of each substrate, inhibitor or uncoupler to the chamber (DatLab version 6; Oroboros Instruments, Innsbruck, Austria) and were performed according to the literature. 18,26,28 In both protocols, the outer mitochondrial membrane integrity was evaluated by cytochrome c titration. 27…”