Mitochondrial and cytosolic ATP/ADP ratios in isolated hepatocytes. A comparison of the digitonin method and the non-aqueous fractionation procedure

Klevets

Cell Biochemistry & Function

2012

Mitochondria maintain numerous energy-consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl- and sucrose-based solutions using Clark oxygen electrode. Respiration of digitonin-treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate-stimulated respiration rate did not depend on solution composition. In sucrose-based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl-based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose-based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non-mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose-based solution.

Section: Discussionsupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Klevets

Cell Biochemistry & Function

2012

European Journal of Biochemistry

“…In their studies with perfused liver, ATPiADP ratios were lowered markedly in the presence of fatty acid, and this led them to consider the possibility that 'futile cycles' [67, 681 involving ATP cleavage (e. g. recycling between triacylglycerol and longchain fatty acids [69]) may have been involved. However, the low ATP/ADP ratios observed may have been an experimental phenomenon due to perfusion of the organ with an erythrocytefree medium [70]. In our experiments ATPiADP ratios were maintained within the normal range in the presence of added fatty acid.…”

Section: Discussionmentioning

confidence: 49%

The Calorigenic Nature of Hepatic Ketogenesis: An Explanation for the Stimulation of Respiration Induced by Fatty Acid Substrates

Berry

Clark

Grivell

et al. 1983

The rates of 0, consumption during fatty acid oxidation to acetyl-CoA and to COZ were determined for isolated liver cells from starved rats on the basis of measured rates of respiration and ketogenesis. About 60% of the endogenous 0, uptake was associated with acetyl-CoA formation. The remainder was assumed to represent total combustion of fatty acid to CO, and H,O through the Krebs cycle. In the absence of added fatty acid, I .90 pmol ' inin-' . g-' of acetyl-CoA was generated, of which 1.40pmol . min-' . g-' gave rise to ketone bodies. 0, consumption was stimulated about 30 ::<, by the addition of 2 mM palmitate or 4mM hexanoate. This increase was entirely due to stimulation of 0, consumption related to oxidation of fatty acid to acetyl-CoA. The extra acetyl-CoA produced was channelled into ketone body formation.The gluconeogenic substrate lactate, which increases hepatic demand for ATP, stimulated O2 consumption almost 4076, but in this case the increase was due to an enhancement of Krebs cycle activity, and acetyl-CoA generation was depressed 25 7;. In the absence of added fatty acid, 83 of the total acetyl-CoA produced by cells incubated with lactate was oxidized in the Krebs cycle. Addition of fatty acid caused a further stimulation of 0, uptake which reflected a promotion of acetyl-CoA production with an associated increase in ketone body formation.The uncoupling agent, 2,4-dinitrophenol, stimulated total 0, consumption of cells incubated without exogenous substrate or with added fatty acid. In each circumstance this stimulation of 0, uptake was related entirely to enhancement of Krebs cycle activity and depression of ketogenesis. The large increment in 0, consumption induced by the combination of lactate and 2,4-dinitrophenol in the presence of added fatty acid was likewise attributable solely to a marked stimulation of Krebs cycle flux. Added ethanol caused only a small inhibition of total acetyl-CoA production and had little effect on respiration, but considerably depressed Krebs cycle activity. This depression was relieved by 2.4-dinitrophenol.Oligomycin and antimycin had markedly different effects on fatty acid catabolism although both inhibitors depressed O2 consumption to the same extent. Oligomycin caused only a small diminution of rates of acetyl-CoA formation but induced a strong depression of cellular ATP concentrations and of acetyl-CoA oxidation to C0,. In contrast antimycin had no effect on acetyl-CoA oxidation and brought about only a small decline in cellular ATP levels, but considerably depressed formation of acetyl-CoA.These results are interpreted as indicating that addition of fatty acid to liver cells induces various energydependent processes, including ATP synthesis, reversed electron transfer and metabolite translocation, all of which contribute to the observed stimulation of O2 uptake. It is inferred that oxidation of acetyl-CoA to CO, is preferentially coupled to ATP synthesis whereas oxidation of fatty acid to acetyl-CoA is obligatorily associated with reversed electron transfer....

“…The ATP content in fresh isolated hepatocytes was the same in both HR and LR rats nonadapted and adapted to hypoxia and was around 2.5 pmol/g wet weight, which corresponds to reported mean values of ATP [13]. A steady-state ATP level was maintained in hepatocytes from nonadapted HR and LR rats during 2-h incubation in medium with 890-100 gM O v attesting to intact energy synthesis in them ( Table 1).…”

Section: Resoltsmentioning

confidence: 61%

Effect of various oxygen concentrations on the ATP content in isolated hepatocytes of rats adapted and nonadapted to hypoxia

1994

It is shown that isolated hepatocytes are capable of perceiving slight changes in the environmental oxygen concentration. A complicated phase dependence exists between adenosine triphosphate and partial oxygen pressure, which differs in cells from animals with high and low resistance to hypoxia, the former showing a more stable and resistant energy-synthesizing function than the latter. After long-term adaptation to periodic hypoxia, the resistance of the energy-synthesizing function rises in hepatocytes from high-resistant animals, and falls in low-resistant animals suggesting a fundamentally different organization of the emergency compensatory mechanisms of the energysynthesizing function in hepatocytes of animals of these two types.Key Words: hepatocytes; hypoxia; adenosine triphosphate; resistance; adaptation It is well known that aerobic organisms respond to a decrease of environmental oxygen by a change in respiration and adenosine triphosphate (ATP) synthesis. For oxygen-dependent enzyme systems, such as cytochrome oxidase, this relationship is described by the Michaelis-Menten equation: the process is governed by zero-order kinetics within a wide range of oxygen concentrations (respiration is independent of the partial oxygen pressurepO2), but within a very narrow range of low pO2 values it obeys ftrst-order kinetics (respiration linearly decreases as pO 2 declines) [10,12]. This relationship holds true for a mitochondrial suspension. However, cell systems exhibit some deviations from this law both due to the limited oxygen diffusion across the histohematic barriers [11,12] and due to specific features of cell metabolism and energetics [5,6,14]. Nonmitochondrial oxygen-deLaboratory of Bioenergetics, Research Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow pendent enzyme systems which have a lesser affinity to oxygen than cytochrome oxidase, as well as regulatory interactions between glycolysis and aerobic oxidation significantly distort this relationship [5,9]. A knowledge of how a cell or tissue responds to oxygen deficiency and of the mechanisms controlling energy synthesis in the intact cell in hypoxia is of fundamental importance, and yet the topic has hardly been addressed. Isolated cells are most suitable model systems for such study, for in then the diffusional limitations for oxygen are minimized, and energy synthesis and utilization, as well as other important metabolic processes, remain intact. In view of this, in the present investigation we studied the specifics of ATP formation in isolated hepatocytes under conditions of various oxygen concentrations in the incubation medium. The motivation for the study came from the high incidence of ischemic liver pathology, which is difficult to treat because the molecular and bioenergy mechanisms of this disorder are poorly uderstood.