Formation of activated oxygen in the hypoxic rat liver

The biliary GSSG efflux rate of normoxic perfused rat liver was 1.5 +/- 0.2 nmol/min/g liver wet weight. The GSSG efflux rate as indicator for the flux through the glutathione peroxidase reaction and, therefore, for an oxidative loading increased with the extent of hypoxia. 2.6 +/- 0.5 nmol/min/g were released from the severely hypoxic liver. The hydroxyl radical scavenger formate as well as the xanthine oxidase inhibitor allopurinol reduced the efflux rate of GSSG. GSH was released from the perfused liver at a rate of 15.5 nmol/min/g which was nearly unchanged in severe hypoxia. The high rate of glucose liberation from the hypoxic liver declined to almost that of the normoxic organ in the presence of formate. There is an 'oxidative stress' during hypoxic liver perfusion which probably originates from increased generation of activated oxygen species in the degradation of purine nucleotides.     

The response of the rat liver to normobaric hypoxia stimulated in vivo and in vitro

The metabolic features of the rat liver were studied in artificial homeostasis conditions, using an isolated perfused organ as a model. The metabolism of the liver isolated from an intact rat and perfused with a normobaric hypoxic medium was compared with that of a liver that was isolated from a rat preliminarily kept in a chamber to simulate hypoxia of the total body and perfused using a medium with a normal oxygen content. The functional activity of the liver was assessed by portal pressure; oxygen consumption; and carbon dioxide gas, urea, glucose, and lactate contents in the perfusion medium. Metabolic changes in the perfused liver during oxygen deficiency became detectable at the same time point after exposure regardless of the method used to experimentally simulate hypoxia. This finding directly points to the metabolic autonomy of the liver.     

On the stimulation of respiration by alpha-adrenergic agonists in perfused rat liver

Interactions between phenylephrine-induced oxygen consumption, lactate and pyruvate output, and urea and glucose production were examined in perfused livers from fed or 48-h-fasted rats. Within 2 min of phenylephrine infusion, oxygen consumption in perfused livers was increased by approximately 40%. Increases in oxygen consumption induced by phenylephrine were essentially abolished in the presence of carboxyatractyloside, whereas those induced by dinitrophenol were still evident. Phenylephrine-induced increases in oxygen consumption were accompanied by enhanced rates of gluconeogenesis and ureogenesis in livers from fed or 48-h-fasted animals. These data indicate that phenylephrine-induced increases in respiration in perfused rat liver may result from an enhanced rate of mitochondrial oxidative phosphorylation in response to an increased cellular energy requirement.     

Limb preservation in replantation surgery

Five patients are presented in whom a traumatically amputated extremity or digit was perfused with oxygenated Fluosol DA-20%. Perfusion lasted between 16 and 46 hours. Microcirculatory oxygenation was monitored using pO2 measurements. Moreover, K?lactate, glucose, and ATP levels were measured and electron microscopic sections of the vessels were investigated at the end of the experiment. All above-mentioned parameters suggest that the tissue was sufficiently provided with oxygen and viable after many hours of perfusion. Conclusive evidence of the viability of a perfused digit was given by its successful replantation after 17 hours of Fluosol perfusion (at approximately 15 degrees C).     

Acetylcholine affects rat liver metabolism via type 3 muscarinic receptors in hepatocytes

Although the role of acetylcholine (Ach) in hepatic glucose metabolism is well elucidated, it is still unclear if it influences gluconeogenesis, glycogenolysis and high-energy phosphate metabolism, and if it does what the mechanisms of this influence are. Therefore, using isolated perfused rat liver as a model, we have studied the effect of Ach on oxygen consumption, synthesis of glucose from lactate and pyruvate, glycogen formation, mitochondrial oxidative phosphorylation and ATP-synthesis. We have established that effects of Ach on oxygen consumption depend on its concentration. When used at a concentration of 10(-7) M, Ach exerts maximum stimulatory effect, while its infusion at 10(-6) M causes a decrease of oxygen consumption by the liver. Moreover, when used at a concentration of 10(-6) M or 10(-7) M, Ach increases rates of glucose production from the gluconeogenic substrates lactate and pyruvate, leading to enhanced glycogen content in perfused liver. It was also shown that Ach possesses a stimulating effect on alanine and aspartate aminotransferases. As detected by 31P NMR spectroscopy, continuous liver perfusion with pyruvate and lactate in the presence of Ach leads to a significant decrease of ATP level, implying enhanced energy requirements for gluconeogenesis under these conditions. Elimination of the described effects of Ach by atropine, the antagonist of muscarinic receptors, and identification of the type 3 muscarinic receptors (m3) in isolated hepatocytes as well as in whole liver, imply that Ach may exert its effect on liver metabolism through m3 receptors.     

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent K_m=22 μM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 μM), dibutyryl cyclic AMP (0.1 mM) and the β-adrenergic agonist ritodrine (10 μM) also had no effect, however the α₁-adrenergic agonist, methoxamine (10 μM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the α-adrenergic agonist methoxamine.     

Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism

1. The metabolic integrity of a new isolated rat hindquarter preparation was studied. The hindquarter was perfused with a semi-synthetic medium containing aged human erythrocytes. More than 95% of the oxidative metabolism of the preparation was due to muscle, the remainder being due to bone, adipose tissue and, where present, skin. 2. Consumption of O(2), glucose utilization, glycerol release and lactate production were similar in the presence and in the absence of the skin, indicating that the latter contributed little to the overall metabolism of the preparation. 3. After 40min of perfusion, tissue concentrations of creatine phosphate, ATP and ADP were similar to those found in muscle taken directly from intact animals. The muscle also appeared normal under the electron microscope. 4. The hindquarter did not lose K(+) to the medium during a 30min perfusion. In the presence of insulin it had a net K(+) uptake. 5. Insulin caused a sixfold increase in glucose uptake, stimulated O(2) consumption by nearly 40% and depressed glycerol release to less than half the control value. 6. Bilateral sciatic-nerve stimulation caused severalfold increases in O(2) consumption and lactate production. In the absence of insulin nerve stimulation also enhanced glucose uptake; in the presence of insulin it did not further increase the already high rate of glucose uptake. 7. Rates of lactate production and O(2) consumption of the rat hindquarter in vivo and the isolated perfused hindquarter were very similar. 8. Ketone bodies were a major oxidative fuel in vivo of the hindquarter of a rat starved for 2 days. If the acetoacetate and 3-hydroxybutyrate removed by the tissue were completely oxidized, they would have accounted for 77% of the O(2) consumption. 9. Acetoacetate accounted for 84% of the ketone bodies removed by the hindquarter in vivo even though its arterial concentration was half that of 3-hydroxybutyrate. 10. Similar rates of acetoacetate and 3-hydroxybutyrate utilization were observed in the perfused hindquarter. 11. Acetoacetate utilization by the perfused hindquarter was not diminished by the addition of either oleate or insulin to the perfusate. 12. Oxidation of glucose to CO(2) accounted for less than 4% of the O(2) consumed by the perfused hindquarter in both the presence and the absence of insulin. 13. The results indicate that the isolated perfused hindquarter is a useful tool for studying muscle metabolism. They also suggest that ketone bodies, if present in sufficient concentration, are the preferred oxidative fuel of resting muscle.     

Effects of added adenine nucleotides on renal carbohydrate metabolism

1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or P(i) or the formation of complexes with Mg(2+) ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30-50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C(3) phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of alpha-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes.     

Effects of ranolazine on fatty acid transformation in the isolated perfused rat liver

It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-¹⁴C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-¹⁴C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD⁺ ratio, but stimulated ¹⁴CO₂ production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.     

Liver parenchyma heterogeneity in the response to extracellular NAD+

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.     

Stimulation of hepatic glycogenolysis by phorbol 12-myristate 13-acetate.

In isolated perfused rat livers, infusion of phorbol 12-myristate 13-acetate (PMA) (150 nM) resulted in a 3-fold stimulation of the rate of glucose production. This response was maximal at a perfusate PMA concentration of 150 nM, and was significantly diminished at higher concentrations of PMA (e.g. 300 nM). Stimulation of glycogenolysis by PMA was greatly decreased in livers perfused with Ca2+-free medium. PMA infusion into livers perfused in the absence of Ca2+ did not result in Ca2+ efflux from the livers. Additionally, in hepatocytes isolated from livers of fed rats, neither PMA nor 1-oleoyl-2-acetyl-rac-glycerol stimulated the rate of glucose production. Although indomethacin has been demonstrated to block PMA-stimulated hepatic glycogenolysis [Garcia-Sainz & Hernandez-Sotomayor (1985) Biochem. Biophys. Res. Commun. 132, 204-209], infusion of PMA into perfused rat livers did not alter the rates of production of either prostaglandin E2 or 6-oxo-prostaglandin F1 alpha in the livers. These data, along with the observed increases in the perfusion pressure and decrease in O2 consumption in isolated perfused livers suggest that phorbol-ester-stimulated glycogenolysis is not a consequence of a direct effect of phorbol ester on liver parenchymal cells.     

An improved apparatus for blood perfusion of the canine cerebral vasculature

An improved perfusion apparatus is described which consists of a membrane oxygenator, roller pump, reservoir, heat exchanger, blood filter, and inert tubing. Heparinized blood may be used and is delivered at flow rates from 10 to 250 ml/min. Dogs are anesthetized with halothane and their cerebral arterial blood supply isolated by the method of Gilboe et al. (8). When the canine brain is perfused for 5 hr using the described apparatus, the rates of cerebral oxygen and glucose consumption are 5.19±0.12 ml/100 g/min and 39.9±6.5 mol/100 g/min, respectively. Of the total glucose consumed by the brain, about 1/4 is contributed by the erythrocytes. An equivalent of about 9% of the consumed glucose is returned to the blood as lactate. Electron microscopic examination of cerebral cortex samples reveals no differences between 5-hr perfused brain and appropriate nonperfused controls. It is concluded that the apparatus is a useful system for organ perfusion and that the canine brain perfused by this method remains physiologically and metabolically active for at least 5 hr.With the technical assistance of Evelyn Townsend.     

Impact of altered substrate utilization on cardiac function in isolated hearts from Zucker diabetic fatty rats

The goal of this study was to determine whether changes in cardiac metabolism in Type 2 diabetes are associated with contractile dysfunction or impaired response to ischemia. Hearts from Zucker diabetic fatty (ZDF) and lean control rats were isolated and perfused with glucose, lactate, pyruvate, and palmitate. The rates of glucose, lactate, pyruvate, and palmitate oxidation rates and glycolysis were determined during baseline perfusion and low-flow ischemia (LFI; 0.3 ml/min for 30 min) and after LFI and reperfusion. Under all conditions, ATP synthesis from palmitate was increased and synthesis from lactate was decreased in the ZDF group, whereas the contribution from glucose was unchanged. During baseline perfusion, the rate of glycolysis was lower in the ZDF group; however, during LFI and reperfusion, there were no differences between groups. Despite these metabolic shifts, there were no differences in oxygen consumption or ATP production rates between the groups under any perfusion conditions. Cardiac function was slightly depressed before LFI in the ZDF group, but during reperfusion, function was improved relative to the control group despite the increased dependence on fatty acids for energy production. These data suggest that in this model of diabetes, the shift from carbohydrates to fatty acids for oxidative energy production did not increase myocardial oxygen consumption and was not associated with impaired response to ischemia and reperfusion.     

Xylitol metabolism in the isolated perfused rat liver

1. Loading the isolated perfused liver from well-fed rats with xylitol (20mm) caused a depletion of adenine nucleotides and P_i and an accumulation of α-glycerophosphate. The ATP content fell to 66% of the control value after 10min and to 32% after 80min. The ADP and AMP contents also fell. After 80min 63% of the total adenine nucleotides and 59% of the P_i had been lost. 2. The α-glycerophosphate content rose from 0.13 to 4.74μmol/g at 10min and reached 8.02μmol/g at 40min. 3. Xylitol was rapidly metabolized, the main products being glucose, lactate and pyruvate. 4. The [lactate]/[pyruvate] ratio in the presence of xylitol rose to 30–40. 5. On perfusion of livers from starved animals the main product of xylitol metabolism was glucose and the mean ratio xylitol removed/glucose formed was 1.29 (corrected for endogenous glucose and lactate production). This is close to the predicted value of 1.2. 6. Evidence is presented indicating that the loss of adenine nucleotides caused by xylitol is not due to the increased ATP consumption but to the accumulation of α-glycerophosphate and depletion of P_i. 7. The loss of adenine nucleotides accounts for the hyperuricaemia which can occur after xylitol infusion in man. 8. The relevance of the findings to the clinical use of xylitol as an energy source is discussed.     

The metabolism of fructose in the bivascularly perfused rat liver.

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 mumols min-1 g-1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained. (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion; oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 +/- 0.14 mumols min-1 ml-1 in the retrograde mode; and, 2.05 +/- 0.19 mumols min-1 ml-1 in the anterograde mode (P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 +/- 0.16 mumols min-1 ml-1 in the retrograde mode; and, 0.51 +/- 0.08 mumols min-1 ml-1 in the anterograde mode (P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 +/- 0.18 mumols min-1 ml-1 in the retrograde mode; and, 1.83 +/- 0.16 mumols min-1 ml-1 in the anterograde mode (P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.     

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.     

Glucose oxidation and oxygen consumption of isolated guinea pig and muskrat hearts

Glucose in Krebs-Henseleit buffer was presented to isolated Langendorff perfused muskrat and guinea pig hearts that were paced at 240 beats/min. Glucose uptake (amount removed from the perfusion fluid) was 3 times greater in the muskrat hearts than in the guinea pig heart. Glucose oxidation (amount converted to CO2) and oxygen consumption did not differ in the hearts of the two species. When glucose is the only exogenous substrate, isolated muskrat hearts extract more glucose than guinea pig hearts but oxidize similar amounts of glucose and have a similar myocardial oxygen consumption.     

Effects of increased mechanical work by isolated perfused rat heart during production or uptake of ketone bodies. Assessment of mitochondrial oxidized to reduced free nicotinamide-adenine dinucleotide ratios and oxaloacetate concentrations.

Metabolic effects of increased mechanical work were studied by comparing isolated pumping rat hearts perfused by the atrial-filling technique with aortic-perfused non-pumping hearts perfused by the technique of Langendorff. The initial medium usually contained glucose (11 mm) and palmitate (0.6 mm bound to 0.1 mm albumin). During increased heart work (comparing pumping with non-pumping hearts) the uptake of oxygen and glucose increased threefold, but that of free fatty acids was unchanged. Tissue contents of alpha-oxoglutarate, NH4+, malate, lactate, pyruvate and Pi rose with increased heart work, but contents of ATP, phosphocreatine and citrate fell. Ketone bodies were produced with a ratio of beta-hydroxybutyrate/acetoacetate of about 3:1 in both pumping and non-pumping hearts but with higher net production rates in non-pumping hearts. When ketone bodies were added in relatively high concentrations (total 4 mm) to a glucose (11 mm) medium the medium, ratios of beta-hydroxybutyrate/acetoacetate were not steady even after 60 min of perfusion. The validity of calculating mitochondrial free NAD+/NADH ratios from the tissue contents of the reactants of the glutamate dehydrogenase system or the beta-hydroxybutyrate dehydrogenase system is assessed. The activities of these enzymes are considerably less in the rat heart than in the rat liver, introducing reservations into the application to the heart of the principles used by Williamson et al. (1967) for calculation of mitochondrial free NAD+/NADH ratios of liver mitochondria...