Mitochondrial respiratory chain adjustment to cellular energy demand

Because adaptation to physiological changes in cellular energy demand is a crucial imperative for life, mitochondrial oxidative phosphorylation is tightly controlled by ATP consumption. Nevertheless, the mechanisms permitting such large variations in ATP synthesis capacity, as well as the consequence on the overall efficiency of oxidative phosphorylation, are not known. By investigating several physiological models in vivo in rats (hyper- and hypothyroidism, polyunsaturated fatty acid deficiency, and chronic ethanol intoxication) we found that the increase in hepatocyte respiration (from 9.8 to 22.7 nmol of O(2)/min/mg dry cells) was tightly correlated with total mitochondrial cytochrome content, expressed both per mg dry cells or per mg mitochondrial protein. Moreover, this increase in total cytochrome content was accompanied by an increase in the respective proportion of cytochrome oxidase; while total cytochrome content increased 2-fold (from 0.341 +/- 0.021 to 0.821 +/- 0.024 nmol/mg protein), cytochrome oxidase increased 10-fold (from 0.020 +/- 0.002 to 0.224 +/- 0.006 nmol/mg protein). This modification was associated with a decrease in the overall efficiency of the respiratory chain. Since cytochrome oxidase is well recognized for slippage between redox reactions and proton pumping, we suggest that this dramatic increase in cytochrome oxidase is responsible for the decrease in the overall efficiency of respiratory chain and, in turn, of ATP synthesis yield, linked to the adaptive increase in oxidative phosphorylation capacity.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

Adjustment of juvenile tuatara to a cooler,southern climate: operative temperatures,emergence behaviour and growth rate

Translocations for conservation often involve species limited to relict distributions. However, uncertainty can exist regarding the ability of source individuals to acclimatise following a shift to a distant location. We investigated the ability of captive-reared juvenile tuatara (Sphenodon punctatus) of Cook Strait stock (41°S) to adjust to outdoor, predator-protected pens within Orokonui Ecosanctuary (45 °S). We examined potential basking and within burrow temperatures, the influence of temperature on emergence, and growth rates in comparison with other locations. Tuatara at Orokonui reached their preferred temperature when basking over summer, and burrows provided protection from freezing over winter. Emergence was temperature-dependent and essentially ceased during winter. Growth rates of Orokonui-held juveniles were within the range for four other captive-rearing facilities and faster than for wild juveniles from a Cook Strait population. As all Orokonui-held juveniles have survived and grown we conclude that the climate at this southern location is suitable to consider a free-release.     

Histochemical changes in neonatal liver caused by vaginal instillation of magnetic nanoparticles in pregnant mice

Drug delivery through the vagina is a novel and effective approach for treating embryonic diseases. Magnetic nanoparticles (MNPs) currently are used as drug delivery systems. The safety of MNPs for use with embryonic tissues remains unclear. We used pregnant mice to investigate the possible toxicity of MNPs toward neonatal liver at three embryonic ages using histochemical and immunohistochemical techniques. MNPs were instilled through the vaginas of pregnant mice at days 12 (E12), 15 (E15) and 17 (E17) after fertilization. We found MNPs in the neonatal liver parenchyma after delivery of the pups on day 20. We observed that MNPs caused mild apoptosis of hepatocytes, cytoplasmic vacuolation and lymphocytic infiltration in the neonatal liver after treatment at E15 compared to instillation at E12 and E17. We observed also that MNPs increased the production of caspase proteins and tumor necrosis factor receptor 2 proteins, which are indicators of apoptosis, in the neonatal liver after instillation of MNPs at E15 compared to instillation at E12 and E17. MNPs also increased the number of collagen fibers and the amounts of connective tissue growth factors in the neonatal liver parenchyma after instillation at E15 compared to instillation at E12 and E17. The general carbohydrates in the neonatal liver were decreased in a time-dependent manner after instillation at E17, E15 and E12 owing to the presence of MNPs in the parenchyma. Overall, we determined that MNPs were mildly toxic to neonatal liver.     

In vivo fluxes in the ammonium-assimilatory pathways in corynebacterium glutamicum studied by 15N nuclear magnetic resonance

Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)-glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.     

Oxidative phosphorylation in intact hepatocytes: Quantitative characterization of the mechanisms of change in efficiency and cellular consequences

Two mechanisms may affect the yield of the oxidative phosphorylation pathway in isolated mitochondria: (i) a decrease in the intrinsic coupling of the proton pumps (H⁺/2e^- or H⁺/ATP), and (ii) an increase in the inner membrane conductance (proton or cation leak). Hence three kinds of modifications can occur and each of them have been characterized in isolated rat liver mitochondria (see preceding chapter by Rigoulet et al.). In intact isolated hepatocytes, these modifications are linked to specific patterns of bioenergetic parameters, i.e. respiratory flux, mitochondrial redox potential, DY, and phosphate potential.(1) The increase in H⁺/ATP stoichiometry of the mitochondrial ATP synthase, as induced by almitrine [20], leads to a decrease in mitochondrial and cytosolic ATP/ADP ratios without any change in the protonmotive force nor in the respiratory rate or redox potential. (2) In comparison to carbohydrate, octanoate metabolism by -oxidation increases the proportion of electrons supplied at the second coupling site of the respiratory chain. This mimics a redox slipping. Octanoate addition results in an increased respiratory rate and mitochondrial NADH/NAD ratio while protonmotive force and phosphate potential are almost unaffected. The respiratory rate increase is associated with a decrease in the overall apparent thermodynamic driving force (2'o - np) which confirms the redox-slipping-like effect. (3) An increase in proton conductance as induced by the protonophoric uncoupler 2,4-dinitrophenol (DNP) leads to a decrease, as expected, in the mitochondrial NADH/NAD and ATP/ADP ratios and in while respiratory rate is increased.Thus, each kind of modification (proton leak, respiratory chain redox slipping or increase in H⁺/ATP stoichiometry of ATPase) is related to a specific set of bioenergetic parameters in intact cells. Moreover, these patterns are in good agreement with the data found in isolated mitochondria.From this work, we conclude that quantitative analysis of four bioenergetic parameters (respiration rate, mitochondrial NADH/NAD ratio, protonmotive force and mitochondrial phosphate potential) gives adequate tools to investigate the mechanism by which some alterations may affect the yield of the oxidative phosphorylation pathway in intact cells.