Maintain Adenosine Triphosphate Levels Research Articles

Oxygenated versus non-oxygenated flush out during deceased donor liver procurement: The first proof-of-concept study in humans.

Liver transplantation is used for treating end-stage liver disease, fulminant hepatitis, and oncological malignancies and organ shortage is a major limiting factor worldwide. The use of grafts based on extended donor criteria have become internationally accepted. Oxygenated machine perfusion technologies are the most recent advances in organ transplantation; however, it is only applied after a period of cold ischemia. Due to its high cost, we aimed to use a novel device, OxyFlush®, based on oxygenation of the preservation solution, applied during liver procurement targeting the maintenance of ATP during static cold storage (SCS). Twenty patients were randomly assigned to the OxyFlush or control group based on a 1:1 ratio. In the OxyFlush group, the perfusion solution was oxygenated with OxyFlush® device while the control group received a non-oxygenated solution. Liver and the common bile duct (CBD) biopsies were obtained at three different time points. The first was at the beginning of the procedure, the second during organ preparation, and the third after total liver reperfusion. Biopsies were analyzed, and adenosine triphosphate (ATP) levels and histological scores of the liver parenchyma and CBD were assessed. Postoperative laboratory tests were performed. OxyFlush® was able to maintain ATP levels during SCS and improved the damage caused by the lack of oxygen in the CBD. However, OxyFlush® did not affect laboratory test results and histological findings of the parenchyma. We present a novel low-cost device that is feasible and could represent a valuable tool in organ preservation during SCS.

Distinct roles of the preparatory and payoff phases of glycolysis in hematopoietic stem cells

In physiological conditions, most adult hematopoietic stem cells (HSCs) maintain a quiescent state. Glycolysis is a metabolic process that can be divided into preparatory and payoff phases. Although the payoff phase maintains HSC function and properties, the role of the preparatory phase remains unknown. In this study, we aimed to investigate whether the preparatory or payoff phases of glycolysis were required for maintenance of quiescent and proliferative HSCs. We used glucose-6-phosphate isomerase (Gpi1) as a representative gene for the preparatory phase and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a representative gene for the payoff phase of glycolysis. First, we identified that stem cell function and survival were impaired in Gapdh-edited proliferative HSCs. Contrastingly, cell survival was maintained in quiescent Gapdh- and Gpi1-edited HSCs. Gapdh- and Gpi1-defective quiescent HSCs maintained adenosine-triphosphate (ATP) levels by increasing mitochondrial oxidative phosphorylation (OXPHOS), whereas ATP levels were decreased in Gapdh-edited proliferative HSCs. Interestingly, Gpi1-edited proliferative HSCs maintained ATP levels independent of increased OXPHOS. Oxythiamine, a transketolase inhibitor, impaired proliferation of Gpi1-edited HSCs, suggesting that the nonoxidative pentose phosphate pathway (PPP) is an alternative means to maintain glycolytic flux in Gpi1-defective HSCs. Our findings suggest that OXPHOS compensated for glycolytic deficiencies in quiescent HSCs, and that in proliferative HSCs, nonoxidative PPP compensated for defects in the preparatory phase of glycolysis but not for defects in the payoff phase. These findings provide new insights into regulation of HSC metabolism, which could have implications for development of novel therapies for hematologic disorders.

Increasing neuronal glucose uptake attenuates brain aging and promotes life span under dietary restriction in Drosophila.

SummaryBrain neurons play a central role in organismal aging, but there is conflicting evidence about the role of neuronal glucose availability because glucose uptake and metabolism are associated with both aging and extended life span. Here, we analyzed metabolic changes in the brain neurons of Drosophila during aging. Using a genetically encoded fluorescent adenosine triphosphate (ATP) biosensor, we found decreased ATP concentration in the neuronal somata of aged flies, correlated with decreased glucose content, expression of glucose transporter and glycolytic enzymes and mitochondrial quality. The age-associated reduction in ATP concentration did not occur in brain neurons with suppressed glycolysis or enhanced glucose uptake, suggesting these pathways contribute to ATP reductions. Despite age-associated mitochondrial damage, increasing glucose uptake maintained ATP levels, suppressed locomotor deficits, and extended the life span. Increasing neuronal glucose uptake during dietary restriction resulted in the longest life spans, suggesting an additive effect of enhancing glucose availability during a bioenergetic challenge on aging.

Hibernator-Derived Cells Show Superior Protection and Survival in Hypothermia Compared to Non-Hibernator Cells.

Mitochondrial failure is recognized to play an important role in a variety of diseases. We previously showed hibernating species to have cell-autonomous protective mechanisms to resist cellular stress and sustain mitochondrial function. Here, we set out to detail these mitochondrial features of hibernators. We compared two hibernator-derived cell lines (HaK and DDT1MF2) with two non-hibernating cell lines (HEK293 and NRK) during hypothermia (4 °C) and rewarming (37 °C). Although all cell lines showed a strong decrease in oxygen consumption upon cooling, hibernator cells maintained functional mitochondria during hypothermia, without mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential decline or decreased adenosine triphosphate (ATP) levels, which were all observed in both non-hibernator cell lines. In addition, hibernator cells survived hypothermia in the absence of extracellular energy sources, suggesting their use of an endogenous substrate to maintain ATP levels. Moreover, hibernator-derived cells did not accumulate reactive oxygen species (ROS) damage and showed normal cell viability even after 48 h of cold-exposure. In contrast, non-hibernator cells accumulated ROS and showed extensive cell death through ferroptosis. Understanding the mechanisms that hibernators use to sustain mitochondrial activity and counteract damage in hypothermic circumstances may help to define novel preservation techniques with relevance to a variety of fields, such as organ transplantation and cardiac arrest.

Cardioprotective Effects of VCP Modulator KUS121 in Murine and Porcine Models of Myocardial Infarction

No effective treatment is yet available to reduce infarct size and improve clinical outcomes after acute myocardial infarction by enhancing early reperfusion therapy using primary percutaneous coronary intervention. The study showed that Kyoto University Substance 121 (KUS121) reduced endoplasmic reticulum stress, maintained adenosine triphosphate levels, and ameliorated the infarct size in a murine cardiac ischemia and reperfusion injury model. The study confirmed the cardioprotective effect of KUS121 in a porcine ischemia and reperfusion injury model. These findings confirmed that KUS121 is a promising novel therapeutic agent for myocardial infarction in conjunction with primary percutaneous coronary intervention.

Argon preconditioning enhances postischaemic cardiac functional recovery following cardioplegic arrest and global cold ischaemia.

Previous studies demonstrated that preconditioning with argon gas provided a marked reduction in inflammation and apoptosis and increased myocardial contractility in the setting of acute myocardial ischaemia-reperfusion (IR). There is substantial evidence that myocardial IR injury following cardioplegic arrest is associated with the enhancement of apoptosis and inflammation, which is considered to play a role in cardiac functional impairment. Therefore, the present study was designed to clarify whether preconditioning with argon gas enhances recovery of cardiac function following cardioplegic arrest. Sprague-Dawley rats were anaesthetized and ventilated and allocated to (i) the control group (control IR, n = 10) and (ii) the in vivo group (argon IR), which received 3 cycles of argon (50% argon, 21% oxygen and 29% nitrogen, n = 10) administered for 5 min interspersed with 5 min of a gas mixture (79% nitrogen and 21% oxygen). The hearts were excised and then evaluated in an erythrocyte-perfused isolated working heart system. Cold ischaemia (4°C) for 60 min was induced by histidine-tryptophan-ketoglutarate cardioplegia, followed by 40 min of reperfusion. Cardiac functional parameters were assessed. In left ventricular tissue samples, the expressions of extracellular-regulated kinase (ERK1/2), AKT serine/threonine kinase (Akt), jun N-terminal kinase (JNK), endothelial nitric oxide synthase (eNOS) and HMGB1: high-mobility group box 1 (HMGB1) protein were assessed by western blot, and high-energy phosphates were evaluated by high-performance liquid chromatography. At the end of reperfusion, the rats preconditioned with argon showed significantly enhanced recovery of cardiac output (101 ± 6% vs 87 ± 11%; P < 0.01), stroke volume (94 ± 4% vs 80 ± 11%; P = 0.001), external heart work (100 ± 6% vs 81 ± 13%; P < 0.001) and coronary flow (90 ± 13% vs 125 ± 21%; P < 0.01) compared with the control IR group. These results were accompanied by a significant increase in the levels of myocardial phosphocreatine (23.71 ± 2.07 µmol/g protein vs the control IR group, 13.50 ± 4.75; P = 0.001) and maintained adenosine triphosphate levels (13.62 ±1.89 µmol/g protein vs control IR group adenosine triphosphate: 10.08 ± 1.94 µmol/g; P = 0.017). Additionally, preconditioning with argon markedly reduced the activation of JNK (0.11 ± 0.01 vs 0.25 ± 0.03; P = 0.005) and the expression of HMGB1 protein (0.52 ± 0.04 vs 1.5 ± 0.10; P < 0.001) following reperfusion. Preconditioning with argon enhanced cardiac functional recovery in rat hearts arrested with histidine-tryptophan-ketoglutarate cardioplegia, thereby representing a potential novel cardioprotective approach in cardiac surgery.

Influence of perfusate on liver viability during hypothermic machine perfusion.

To optimize the perfusates used for hypothermic machine perfusion (HMP). Sprague-Dawley rats were assigned randomly to three groups (n = 12 per group) that received either saline, University of Wisconsin cold-storage solution (UW) or histidine-tryptophan-ketoglutarate solution (HTK) as the perfusate. Each group was divided into two subgroups: static cold storage (SCS) and HMP (n = 6 per subgroup). The liver graft was retrieved according to the method described by Kamada. For the SCS group, the graft was directly placed into cold perfusate (0-4 °C) for 6 h after liver isolation while the portal vein of the graft was connected to the perfusion machine for the HMP group. Then the perfusates were collected at different time points for analysis of aspartate aminotransferase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) levels. Liver tissues were obtained for evaluation of histology, dry/wet weight (D/W) ratio, and malondialdehyde (MDA) and adenosine-triphosphate (ATP) levels. The portal vein pressure and velocity were monitored in real time in all HMP subgroups. Comparison of HMP and SCS: Regardless of the perfusate, HMP improved the architecture of donor graft in reducing the congestion around sinusoids and central vein and maintaining sinusoid lining in morphology; HMP improved liver function in terms of ALT, AST and LDH, especially during the 3-6 h period (SCS vs HMP using saline: ALT3, 225.00 ± 105.62 vs 49.50 ± 18.50, P = 0.047; LDH3, 1362.17 ± 563.30 vs 325.75 ± 147.43, P = 0.041; UW: LDH6, 2880.14 ± 948.46 vs 2135.00 ± 174.27, P = 0.049; HTK, AST6, 307.50 ± 52.95 vs 185.20 ± 20.46, P = 0.041); HMP decreased MDA level (saline, 2.79 ± 0.30 vs 1.09 ± 0.09, P = 0.008; UW, 3.01 ± 0.77 vs 1.23 ± 0.68, P = 0.005; HTK, 3.30 ± 0.52 vs 1.56 ± 0.22, P = 0.006). Comparison among HMP subgroups: HTK showed less portal vein resistance than UW and saline (vs saline, 3.41 ± 0.49 vs 5.00 ± 0.38, P < 0.001; vs UW, 3.41 ± 0.49 vs 4.52 ± 0.63, P = 0.007); UW reduced edema most efficiently (vs saline, 0.68 ± 0.02 vs 0.79 ± 0.05, P = 0.013), while HTK maintained ATP levels best (vs saline, 622.60 ± 29.11 vs 327.43 ± 44.66, P < 0.001; vs UW, 622.60 ± 29.11 vs 301.80 ± 37.68, P < 0.001). HMP is superior to SCS in maintaining both architecture and function of liver grafts. Further, HTK was found to be the optimal perfusate for HMP.

Effects of fructose-1,6-bisphosphate on morphological and functional neuronal integrity in rat hippocampal slices during energy deprivation

d-Fructose-1,6-bisphosphate, a high energy glycolytic intermediate, attenuates ischemic damage in a variety of tissues, including brain. To determine whether d-fructose-1,6-bisphosphate serves as an alternate energy substrate in the CNS, rat hippocampal slices were treated with d-fructose-1,6-bisphosphate during glucose deprivation. Unlike pyruvate, an endproduct of glycolysis, 10 mM d-fructose-1,6-bisphosphate did not preserve synaptic transmission or morphological integrity of CA1 pyramidal neurons during glucose deprivation. Moreover, during glucose deprivation, 10-mM d-fructose-1,6-bisphosphate failed to maintain adenosine triphosphate levels in slices. d-Fructose-1,6-bisphosphate, however, attenuated acute neuronal degeneration produced by 200 μM iodoacetate, an inhibitor of glycolysis downstream of d-fructose-1,6-bisphosphate. Because (5 S, 10 R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, an antagonist of N-methyl- d-aspartate receptors, exhibited similar protection against iodoacetate damage, we examined whether (5 S, 10 R)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine and d-fructose-1,6-bisphosphate share a common neuroprotective mechanism. Indeed, d-fructose-1,6-bisphosphate diminished N-methyl- d-aspartate receptor-mediated synaptic responses and partially attenuated neuronal degeneration induced by 100-μM N-methyl- d-aspartate. Taken together, these results indicate that d-fructose-1,6-bisphosphate is unlikely to serve as an energy substrate in the hippocampus, and that neuroprotective effects of d-fructose-1,6-bisphosphate are mediated by mechanisms other than anaerobic energy supply.

Heat-shock preconditioning reduces oxidative protein denaturation and ameliorates liver injury by carbon tetrachloride in rats

Membrane lipids and cytosolic proteins are major targets of oxidative injury. This study examined the effect of heat-shock preconditioning associated with the induction of heat-shock protein 72 on liver injury, from the aspect of lipid peroxidation and protein denaturation after carbon tetrachloride (CCl4) administration in rats--one of the representative oxidative injuries. Male Wistar rats were divided into two groups, group HS (preconditioned by heat exposure) and group C (not preconditioned). Expression of HSP72 in the liver tissue was confirmed by Western blot analysis. After a 48-h recovery period, all rats were given CCl4 intragastrically. Liver damage was assessed by measuring serum liver-related enzyme levels and adenine nucleotide concentration in the liver tissue. Lipid peroxidation and protein denaturation were evaluated by measuring tiobarbituric acid reactive substances (TBARS) and by immunohistochemical staining of 4-hydroxy-2-nonenal(HNE)-modified proteins in the liver. Survival rates of the rats after CCl4 administration were also compared. Expression of HSP72 was clearly detected in group HS, but not in group C. Heat-shock preconditioning significantly improved the survival rate, suppressed the increase in liver-related enzyme levels and maintained adenosine triphosphate levels (P<0.01 each). HNE-modified proteins--denatured proteins by free radical attack--were significantly less stained in group HS than in group C (P<0.05). However, TBARS levels did not differ between groups. Because heat-shock preconditioning did not alter TBARS levels but reduced HNE-modified proteins in association with the expression of HSP72, it is suggested that HSP72 did not prevent lipid peroxidation but decreased the lipid peroxidation-induced denaturation of proteins. This seemed to be a mechanism of heat-shock preconditioning to ameliorate oxidative liver injury.

In a canine model, lung preservation at 10° C is superior to that at 4° C

Techniques for organ preservation generally use hypothermia to retard metabolic requirements. However, excessive hypothermia may also produce injury. Using a canine left lung allotransplantation procedure, we compared two preservation temperatures (4 degrees and 10 degrees C) in terms of subsequent lung function measured by temporary occlusion of the right pulmonary artery after implantation of the preserved left donor lung. The lungs were flushed with low-potassium dextran electrolyte solution, inflated with 100% oxygen, and preserved for 18 hours. To investigate possible changes of energy stores at different temperatures, we performed phosphorus 31-nuclear magnetic resonance analyses of lung samples. Sequential determinations of adenosine triphosphate levels in lung tissue preserved at 4 degrees, 10 degrees, and 22 degrees C were studied. After transplantation, lungs preserved at 10 degrees C (n = 6) provided significantly better arterial oxygen tension than those preserved at 4 degrees C (n = 6), 451 +/- 46 mm Hg versus 243 +/- 86 mm Hg (p less than 0.05), and lower pulmonary vascular resistance, 581 +/- 68 dynes.sec.cm-5 versus 1006 +/- 157 dynes.sec.cm-5 (p less than 0.05). Adenosine triphosphate levels at 4 degrees and 10 degrees C were stable and did not differ from each other at the end of the 18-hour preservation period: 0.86 +/- 0.04 mumol/gm wet weight for control versus 0.86 +/- 0.07 mumol/gm wet weight for 4 degrees C and 0.93 +/- 0.06 mumol/gm wet weight for 10 degrees C after 18 hours of preservation. Preservation at 22 degrees C caused a 28% depression of adenosine triphosphate after 18 hours of preservation. These results lead us to conclude the following: (1) Optimal temperature for lung preservation is in the vicinity of 10 degrees C, and (2) lung dysfunction caused by excessive hypothermia is not due to a failure to maintain adenosine triphosphate levels. We suspect that adenosine triphosphate is generated by oxidative phosphorylation during lung preservation.

Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans

Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.

Preservation of high-energy phosphates in human myocardium A phosphorus 31-nuclear magnetic resonance study of the effect of temperature on atrial appendages

After prolonged exposure to low temperatures (1 degree and 4 degrees C), human atrial trabeculae show poor recovery of contraction. At somewhat higher temperatures (12 degrees and 20 degrees C), recovery is much better (Keon and associates. Ann Thorac Surg 1988;46:337-41). Although better preservation of adenosine triphosphate and therefore improved contractile recovery might be expected after exposure to lower temperatures, it remained possible that, below a certain temperature, adenosine triphosphate-generating mechanisms could be slowed more than adenosine triphosphate utilization. To investigate this phenomenon further, we followed the time course of metabolic changes in human atrial appendages, harvested during cardiac bypass operations, at 1 degree, 4 degrees, 12 degrees, and 20 degrees C using high-resolution 31P and 1H nuclear magnetic resonance spectroscopy. The results are quantitated by correlation with data obtained from biochemical assays on quick-frozen tissues. Initial adenosine triphosphate levels in myocytes of human atrial appendages are 3.3 to 4.3 mumol.gm-1 tissue wet weight. At 20 degrees C, adenosine triphosphate disappears after 6 hours; at 12 degrees C, about half the initial adenosine triphosphate is still observable at this time; at 4 degrees C or 1 degree C, the decline is still slower. Only a small contribution toward adenosine triphosphate maintenance comes from creatine phosphate, since creatine phosphate, inorganic phosphate, and total creatine levels in the appendage are low (less than 2 mumol.gm-1 tissue wet weight). Glycolysis is active at all temperatures; the rate of glycolysis correlates positively with increasing temperature. Adenosine triphosphate generated by glycolysis falls just short of demand at all temperatures, but the difference is small at 1 degree and 4 degrees C. These studies lead us to conclude that the relatively poor recovery of contractile response of human atrial trabeculae, together with contracture reported previously at lower temperatures (1 degree and 4 degrees C), is not due to a failure to maintain adenosine triphosphate levels.