High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo.

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal approximately 4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Hydrogen sulfide mediates the vasoactivity of garlic.

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H(2)S), an endogenous cardioprotective vascular cell signaling molecule. This H(2)S production, measured in real time by a novel polarographic H(2)S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H(2)S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the alpha carbon of the allyl substituent, thereby forming a hydropolysulfide (RS(n)H), a key intermediate during the formation of H(2)S. Organic polysulfides (R-S(n)-R'; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RS(n)H and H(2)S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H(2)S. The vasoactivity of garlic compounds is synchronous with H(2)S production, and their potency to mediate relaxation increases with H(2)S yield, strongly supporting our hypothesis that H(2)S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H(2)S can be used to standardize garlic dietary supplements.

Assessing NO-dependent vasodilatation using vessel bioassays at defined oxygen tensions.

Results from vessel bioassays have provided the foundation for much of our understanding of the mechanisms that control vascular homeostasis and blood flow. The seminal observations that led to the discovery that nitric oxide (NO) is a critical mediator of vascular relaxation were made with the use of such methodology, and many studies have used NO-dependent vessel relaxation as an experimental readout for understanding mechanisms that regulate vascular NO function. Studies have coupled controlling oxygen tensions within vessel bioassay chambers to begin to understand how oxygen-specifically hypoxia-regulate NO function, and this context has identified red cells-specifically hemoglobin within-as critical modulators. Alone, vessel bioassays or measuring oxygen partial pressures (pO2) is relatively straightforward, but the combination necessitates consideration of several factors. We use the example of deoxygenated red cells/hemoglobin-dependent potentiation of nitrite-dependent dilation to illustrate the salient factors that are critical to consider in designing and interpreting experiments aimed at understanding the interplay between oxygen and NO function in the vasculature.

Inhalation exposure model of hydrogen sulfide (H2S)-induced hypometabolism in the male Sprague-Dawley rat.

Hydrogen sulfide (H2S) has been accepted as a physiologically relevant cell-signaling molecule with both toxic and beneficial effects depending on its concentration in mammalian tissues. Notably, exposure to H2S in breathable air has been shown to decrease aerobic metabolism and induce a reversible hypometabolic-like state in laboratory rodent models. Herein, we describe an experimental exposure setup that can be used to define the reversible cardiovascular and metabolic physiology of rodents (rats) during H2S-induced hypometabolism and following recovery.

Metabolic and cardiac signaling effects of inhaled hydrogen sulfide and low oxygen in male rats.

Low concentrations of inhaled hydrogen sulfide (H(2)S) induce hypometabolism in mice. Biological effects of H(2)S in in vitro systems are augmented by lowering O(2) tension. Based on this, we hypothesized that reduced O(2) tension would increase H(2)S-mediated hypometabolism in vivo. To test this, male Sprague-Dawley rats were exposed to 80 ppm H(2)S at 21% O(2) or 10.5% O(2) for 6 h followed by 1 h recovery at room air. Rats exposed to H(2)S in 10.5% O(2) had significantly decreased body temperature and respiration compared with preexposure levels. Heart rate was decreased by H(2)S administered under both O(2) levels and did not return to preexposure levels after 1 h recovery. Inhaled H(2)S caused epithelial exfoliation in the lungs and increased plasma creatine kinase-MB activity. The effect of inhaled H(2)S on prosurvival signaling was also measured in heart and liver. H(2)S in 21% O(2) increased Akt-P(Ser473) and GSK-3ß-P(Ser9) in the heart whereas phosphorylation was decreased by H(2)S in 10.5% O(2), indicating O(2) dependence in regulating cardiac signaling pathways. Inhaled H(2)S and low O(2) had no effect on liver Akt. In summary, we found that lower O(2) was needed for H(2)S-dependent hypometabolism in rats compared with previous findings in mice. This highlights the possibility of species differences in physiological responses to H(2)S. Inhaled H(2)S exposure also caused tissue injury to the lung and heart, which raises concerns about the therapeutic safety of inhaled H(2)S. In conclusion, these findings demonstrate the importance of O(2) in influencing physiological and signaling effects of H(2)S in mammalian systems.

Hydrogen sulfide mediates vasoactivity in an O2-dependent manner.

Hydrogen sulfide (H(2)S) has recently been shown to have a signaling role in vascular cells. Similar to nitric oxide (NO), H(2)S is enzymatically produced by amino acid metabolism and can cause posttranslational modification of proteins, particularly at thiol residues. Molecular targets for H(2)S include ATP-sensitive K(+) channels, and H(2)S may interact with NO and heme proteins such as cyclooxygenase. It is well known that the reactions of NO in the vasculature are O(2) dependent, but this has not been addressed in most studies designed to elucidate the role of H(2)S in vascular function. This is important, since H(2)S reactions can be dramatically altered by the high concentrations of O(2) used in cell culture and organ bath experiments. To test the hypothesis that the effects of H(2)S on the vasculature are O(2) dependent, we have measured real-time levels of H(2)S and O(2) in respirometry and vessel tension experiments, as well as the associated vascular responses. A novel polarographic H(2)S sensor developed in our laboratory was used to measure H(2)S levels. Here we report that, in rat aorta, H(2)S concentrations that mediate rapid contraction at high O(2) levels cause rapid relaxation at lower physiological O(2) levels. At high O(2), the vasoconstrictive effect of H(2)S suggests that it may not be H(2)S per se but, rather, a putative vasoactive oxidation product that mediates constriction. These data are interpreted in terms of the potential for H(2)S to modulate vascular tone in vivo.

Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function.

The recent discovery that hydrogen sulfide (H(2)S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H(2)S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H(2)S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemia-reperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H(2)S by cardiac-specific overexpression of cystathionine gamma-lyase (alpha-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H(2)S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H(2)S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

Polarographic measurement of hydrogen sulfide production and consumption by mammalian tissues.

The role of nitric oxide (NO) in redox cell signaling is widely accepted. However, the biological role of another candidate small inorganic signaling molecule and the subject of this study, hydrogen sulfide (H2S), is much less known. H2S as a reductant and nucleophile has numerous potential cellular targets; however, its rapid biological oxidation suggests a fleeting cellular existence. The challenge of accurate real-time measurement of H2S at low micromolar or nanomolar concentrations in biological preparations represents a major impediment to H2S investigations. We here demonstrate the use of a novel polarographic H2S sensor (PHSS) to follow rapid changes in H2S concentration in common buffered biological solutions with a detection limit near 10 nM. The PHSS, used in combination with O2 and NO sensors in multisensor respirometry, shows stability, a high signal-to-noise ratio, and signal specificity for H2S. Preparations of rat vascular tissue exhibit H2S production on the addition of sulfhydryl-bearing amino acid substrates and H2S consumption when supplied with exogenous H2S. Taken together, these findings suggest the existence of dynamic steady-state cellular H2S levels. The PHSS should facilitate the investigation of H2S biology by providing a previously unattainable continuous record of H2S under biologically relevant conditions.

Sulfide consumption by mussel gill mitochondria is not strictly tied to oxygen reduction: measurements using a novel polarographic sulfide sensor.

Some organisms that survive in environments rich in hydrogen sulfide possess specific metabolic pathways for sulfide oxidation and subsequent use of reducing equivalents in oxidative phosphorylation, a process called chemolithoheterotrophy. This process is dependent on ambient oxygen partial pressure and environmental sulfide exposure. To define accurately the kinetics of sulfide metabolism and its dependence on cellular conditions, we have developed a polarographic sulfide sensor (PSS) to measure sulfide concentrations directly and continuously under physiological conditions. The ribbed mussel Geukensia demissa, an inhabitant of sulfide-rich coastal sediments, consumes sulfide in a chemolithoheterotrophic metabolic strategy. Gill mitochondria use sulfide as respiratory substrate for ATP production, and sulfide consumption is sufficiently rapid and so kinetically complex that only continuous real-time detection captures these events. Under normoxic conditions, oxygen and sulfide consumption are matched. Under hypoxic to anoxic conditions, however, sulfide consumption continues without commensurate oxygen consumption, and these results can be duplicated at higher oxygen conditions by selective blockade of terminal oxidases. These metabolic capabilities depend on prior environmental sulfide exposure, which suggests substantial mitochondrial metabolic plasticity. The recent finding that endogenous sulfide is a critical cell signaling molecule in all organisms suggests that the metabolic pathways that tightly control cellular sulfide levels are widespread. Sensors that accurately report sulfide concentrations under physiologically relevant conditions are valuable tools with which to explore the expanding role of sulfide in biological systems.

A Physiological Comparison of Bivalve Mollusc Cerebro-visceral Connectives With and Without Neurohemoglobin. II. Neurohemoglobin Characteristics.

Several bivalve mollusc species possess hemoglobin in their nervous systems whereas most species do not. The function of this neurohemoglobin was investigated in situ in cerebro-visceral connectives of Tellina alternata and Spisula solidissima. Both neurohemoglobins, located in glial cells, exhibit high oxygen affinities and relatively high Hill numbers. The rate of oxygen diffusion into the connective begins to fall below the consumption rate near the PO2 at which each neurohemoglobin begins to unload oxygen, assuming the perineural sheath presents an effective barrier to oxygen diffusion. The neurohemoglobin could thus act as an oxygen store during periods of low PO2. Oxygen unloading from the neurohemoglobin proceeds for a considerable length of time at a constant rate. The long duration may be attributed to the geometry of the connective and to the perineural sheath, whose primary function may be to retain oxygen within the connective during anoxic conditions. The constant unloading rate may be attributed to neurohemoglobin cooperativity in situ because the driving force for unloading remains nearly constant at the P50 of each neurohemoglobin. An oxygen supply at a constant rate for an extended period of time would be useful to an animal requiring aerobic nervous function during anoxic conditions.

A Physiological Comparison of Bivalve Mollusc Cerebro-visceral Connectives With and Without Neurohemoglobin. I. Ultrastructural and Electrophysiological Characteristics.

The presence of hemoglobin in tissues of a small number of species is puzzling when homologous tissues of closely related species do not possess hemoglobin. Several species of bivalve molluscs possess nervous systems with nerve hemoglobin (neurohemoglobin). These systems were compared to nervous systems from other bivalve molluscs without neurohemoglobin to determine ultrastructural and electrophysiological characteristics under normoxic conditions in an attempt to locate any differences between these two types of nervous systems. Cerebro-visceral connectives from the bivalves Tellina alternata and Spisula solidissima with neurohemoglobin and Tagelus plebeius and Geukensia demissa without neurohemoglobin possess a perineural sheath, a subjacent peripheral layer of glial cells, and glial cell processes that enwrap bundles of 0.2-0.4 µm diameter axons. Neurohemoglobin-containing cerebro-visceral connectives have smaller axon bundles and more dense perineural sheaths than those without neurohemoglobin. These features may be important in oxygen delivery from the neurohemoglobin to the axons. Action potential traces, conduction velocities, refractory periods, strength-duration relationships, and temperature responses of all four connectives are typical of nerves possessing very small axons. There are no obvious electrophysiological differences between cerebro-visceral connectives with and without neurohemoglobin.

Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state.

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.