Effect of 3-deazaadenosine on methionine metabolism in isolated perfused livers

The effect of the purine analog 3-deazaadenosine (dzAdo) on the metabolism of sulfur-containing compounds was examined in hepatocytes. The uptake of exogenous methionine by the liver was not affected by the addition of dzAdo to the perfusate, while the intracellular concentrations of S-adenosyl-L-methionine (AdoMet) and S-adenosyl-L-homocysteine (AdoHcy) continued to increase as long as exogenous methionine was available. In addition, large amounts of 3-deazaadenosyl-L-homocysteine (dzAdoHcy) accumulated in the cell. The specific radioactivity of the carbon chain of dzAdoHcy was the same as that of AdoMet and AdoHcy. Consequently, an equivalent amount of homocysteine (Hcy) must have been generated via hydrolysis of AdoHcy. Free Hcy could not be detected either in the tissue or perfusate when dzAdo was present, while Hcy was excreted into the perfusate by control livers. Consequently, the AdoHcy and DzAdoHcy that accumulate in the cell not only function as inhibitors of methylation reactions, but serve as a trap for Hcy. This could result in methionine starvation and hence, inhibition of protein synthesis.     

Palmitic and erucic acid metabolism in isolated perfused hearts from weanling pigs

Hearts from 4 week-old weanling pigs were capable of continuous work output when perfused with Krebs-Henseleit buffer containing 11 mM glucose. Perfused hearts metabolized either glucose or fatty acids, but optimum work output was achieved by a combination of glucose plus physiological concentrations (0.1 mM) of either palmitate or erucate. Higher concentrations of free fatty acids increased their rate of oxidation but also resulted in a large accumulation of neutral lipids in the myocardium, as well as a tendency to increased acetylation and acylation of coenzyme A and carnitine. When hearts were perfused with 1 mM fatty acids, the work output declined below control values. Erucic acid is known to be poorly oxidized by isolated rat heart mitochondria and, to a lesser degree, by perfused rat hearts. In addition, it has been reported that erucic acid acts as an uncoupler of oxidative phosphorylation. In isolated perfused pig hearts used in the present study, erucic acid oxidation rates were as high as palmitate oxidation rates. When energy coupling was measured by 31P-NMR, the steady-state levels of ATP and phosphocreatine during erucic acid perfusion did not change noticeably from those during glucose perfusion. It was concluded that the severe decrease in oxidation rates and ATP production resulting from the exposure of isolated pig and heart mitochondria to erucic acid are not replicated in the intact pig heart.     

Influence of ethanol on the metabolism of perfused normal, fatty and cirrhotic rat livers

1. The influence of ethanol on the metabolism of perfused livers from normal rats and rats in various stages of development of dietary cirrhosis was studied. A choline-deficient, low-protein and high-fat diet was used. Results were obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in fatty and cirrhotic livers than in normal livers. Ethanol had no effect on the oxygen consumption of any of the various livers. After addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely in normal livers. Only a slight decrease in the carbon dioxide production occurred in fatty and cirrhotic livers. 3. With every type of liver glucose was released from the liver into the perfusion medium during the initial control period. This release continued after the addition of ethanol to the perfusion medium in experiments with normal and fatty livers, whereas with cirrhotic livers a marked uptake of glucose from the medium was found. A simultaneous release of the glycolytic end products lactate and pyruvate into the medium occurred. 4. The production of ketone bodies was equal in normal and early fatty livers (6 weeks on the fat diet). It was smaller in late fatty livers (3–4 months on the fatty diet) and in cirrhotic livers. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 11 to 67 with normal livers, from 12 to 16 with early fatty livers, from 13 to 26 with late fatty livers and from 21 to 55 with cirrhotic livers when the livers were perfused with a medium containing ethanol. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 1·2 to 8·4 in normal livers, from 2·0 to 2·8 in early fatty livers, from 1·2 to 2·4 in late fatty livers and from 2·1 to 4·0 in cirrhotic livers when ethanol was added to the medium. 6. The effects of ethanol on liver metabolism during the development of dietary cirrhosis are discussed and related to human fatty liver and cirrhosis during chronic ethanol consumption.     

Influence of selenium deficiency on glutathione disulfide metabolism in isolated perfused rat heart

Selenium deficiency causes a fall in rat cardiac glutathione peroxidase activity. As a consequence, isolated perfused selenium-deficient heart does not release increased amounts of GSSG when hydroperoxide is infused. However, the total amount of glutathione measured as intracellular GSH, intracellular GSSG and GSSG released from the heart when hydroperoxide is infused does not equal the total glutathione measured in these pools in untreated hearts (Xia, Y., Hill, K.E. and Burk, R.F. (1985) J. Nutr. 115, 733-742). GSSG can react with protein sulfhydryl groups to form glutathione-protein mixed disulfides (PrS-SG). PrS-SG were measured in perfused selenium-deficient and control hearts infused with t-butylhydroperoxide and were found to account for the previously unmeasured glutathione. The ability of the selenium-deficient heart to transport GSSG was also examined. GSSG was produced non-enzymatically by infusing diamide. The diamide-treated selenium-deficient heart formed GSSG and released it at the same rate as similarly-treated control heart. Thus although selenium deficiency decreases GSSG formation by glutathione peroxidase, it does not affect cardiac GSSG transport.     

Amino acid release by isolated perfused cirrhotic livers

Livers of normal and cirrhotic rats were perfused in vitro with and without amino acid substrates (2.3 mM ornithine, 10 mM glutamine or 20 mM alanine) in order to assess urea formation and amino acid release. The rates of urea production were lower in the livers of cirrhotic rats when compared to those of controls only in perfusions with added substrates. The release of several amino acids by livers of cirrhotic rats was higher than that of controls although the pattern of amino acids in the perfusate was different from that reported in plasma during hepatic insufficiency.     

Metabolism of testosterone in the isolated perfused rat lungs

The metabolism of [4-¹⁴C]-testosterone in the isolated perfused rat lungs was investigated following the administration of the substrate eithervia the pulmonary artery orvia the trachea. After administration of testosterone in the circulating medium, 3.5% of the hormone was metabolized to various unconjugated metabolites during a single passage through the pulmonary circulation. It so seems that the lungs, receiving all the cardiac output, are one of the major sites of androgen catabolism in the rat organism. The major metabolites were 5α- and 5β-androstane-3α,17β-diols and various non-conjugated polar metabolites. After intratracheal instillation, testosterone was rapidly absorbed from rat lungs. Two minutes following installation, 62% of the dose was recovered from the lungs. Two thirds of this was present as metabolites.It is concluded that the lungs have an efficient metabolic capacity towards androgens. The availability of extractable substrate seems to be rate limiting for the pulmonary testosterone metabolism.     

The effect of purification of commercial bovine serum albumin on the performance of isolated rat livers perfused at 5 degrees C

Commercial bovine serum albumin was purified by gel filtration, in an attempt to extend the period during which isolated rat livers could be maintained in a viable condition while in a state of hypothermia, and to remove some of the variability of data obtained from livers perfused with different batches of BSA. A minor component, accounting for about 5% of the total protein, was removed and the remainder used to support isolated perfused rat livers.Perfusion of rat livers with purified albumin at a concentration of 62 g/liter achieved two results: one, an improvement in the biochemical performance of rat livers perfused at 35 °C; and two, an increase in the period of hypothermic perfusion from 12 to 24 hr during which the liver would remain viable, as indicated by the biochemical tests performed on rewarming to 35 °C.     

Propranolol metabolism by isolated hepatocytes from normal and cirrhotic rat livers: the effect of albumin

Several recent reports have shown that the hepatic uptake and subsequent elimination of some substrates is faster in the presence of albumin than in its absence, as if some of the substrate bound to albumin was also available for uptake. In the present study, we examined the effect of albumin on the clearance of propranolol by isolated rat hepatocyte suspensions. The clearance of total drug decreased progressively as albumin concentration increased. There was also a progressive decrease in the free fraction of propranolol and the net result was an increase in the clearance of unbound drug (+50% at 40 g/L albumin). This increase was not due to an oncotic pressure effect of albumin, nor to the presence of fatty acids bound to albumin. The clearance of propranolol by isolated hepatocytes from cirrhotic rats was decreased compared with controls (-50%), and albumin also increased propranolol free clearance, albeit to a lesser extent than in control animals. Our results indicate that albumin facilitates the elimination of propranolol by hepatocytes, possibly because of surface-mediated catalysis of the albumin-propranolol complexes.     

Comparison of metabolism of free fatty acid by isolated perfused livers from male and female rats.

Livers from normal, fed male and female rats were perfused with different amounts of [1-14C]oleate under steady state conditions, and the rates of uptake and utilization of free fatty acid (FFA) were measured. The uptake of FFA by livers from either male or female rats was proportional to the concentration of FFA in the medium. The rate of uptake of FFA, per g of liver, by livers from female rats exceeded that of the males for the same amount of FFA infused. The incorporation by the liver of exogenous oleic acid into triglyceride, phospholipid, and oxidation products was proportional to the uptake of FFA. Livers from female rats incorporated more oleate into triglyceride (TG) and less into phospholipid (PL) and oxidation products than did livers from male animals. Livers from female rats secreted more TG than did livers from male animals when infused with equal quantities of oleate. The incorporation of endogenous fatty acid into TG of the perfusate was inhibite) by exogenous oleate. At low concentrations of perfusate FFA, however, endogenous fatty acids contributed substantially to the increased output of TG by livers from female animals. Production of 14CO2 and radioactive ketone bodies increased with increasing uptake of FFA. The partition of oleate between oxidative pathways (CO2 production and ketogenesis) was modified by the availability of the fatty acid substrate with livers from either sex. The percent incorporation of radioactivity into CO2 reached a maximum, whereas incorporation into ketone bodies continued to increase. The output of ketone bodies was dependent on the uptake of FFA, and output by livers from female animals was less than by livers from male rats. The increase in rate of ketogenesis was dependent on the influx of exogenous FFA, while ketogenesis from endogenous sources remained relatively stable. The output of glucose by the liver increased with the uptake of FFA, but no difference due to sex was observed. The output of urea by livers from male rats was unaffected by oleate, while the output of urea by livers from females decreased as the uptake of FFA increased. A major conclusion to be derived from this work is that oleate is not metabolized identically by livers from the two sexes, but rather, per gram of liver, livers from female rats take up and esterify more fatty acid to TG and oxidize less than do livers from male animals; livers from female animals synthesize and secrete more triglyceride than do livers from male animals when provided with equal quantities of free fatty acid.     

Regulation of carbohydrate metabolism in mouse liver. Effect of glucagon on gluconeogenic/glycolytic flux in isolated perfused livers.

1. Glucagon stimulated gluconeogenesis from both [U-14C]lactate and [14C]xylitol in isolated perfused mouse liver. 2. Addition of cyclic AMP also stimulated gluconeogenesis from [U-14C]lactate. 3. Glucagon caused a rapid (2.5 min) 12-fold increase in hepatic cyclic AMP but not cyclic GMP concentration. 4. Glucagon caused a rapid and stable decrease in hepatic fructose 1,6-diphosphatase activity measured in vitro. 5. The results are interpreted to indicate that glucagon stimulates hepatic gluconeogenesis in mice via cyclic AMP by two different mechanisms: (a) increased substrate uptake (i.e. utilization) and (b) increased gluconeogenic efficiency (i.e. inhibition of alternate substrate fates).     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

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.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.