Effect of experimental diabetes and insulin on lipid metabolism in the isolated perfused rat lung.

The isolated perfused rat lung was used as a model to study the possible hormonal regulation of lipid metabolism in the mammalian adult lung. Experimental diabetes, whether induced by alloxan or streptozotocin, decreased the incorporation of [U-14C]glucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60-80%. Glucose incorporation into phosphatidylcholine and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the incorporation of glucose into the phosphatidylcholine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidylcholine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mammalian pulmonary surfactant complex.     

Use of the isolated perfused rat lung in studies on lung lipid metabolism

A procedure for the use of the isolated perfused rat lung in studies on metabolic regulation has been developed. The procedure, reasonably uncomplicated, yet physiological, maintains the lung so that edema is not observed. The phospholipid content remains normal, and incorporation of [1-(14)C]-palmitate, [2-(14)C]acetate, and [U-(14)C]glucose is linear with time for a minimum of 2 hr. The incorporation of [1-(14)C]-palmitate and [2-(14)C]acetate into the total lung phospholipid fraction and into the phosphatidylcholine and phospatidylethanolamine fractions has been studied. Increasing the concentration of palmitate in the medium from 0.14 to 0.51 mm increased by 60% the incorporation of [1-(14)C]palmitate into the total lung phospholipid fraction at 2 hr. When the palmitate concentration of the medium was 0.14 mm, addition of 0.11 and 0.79 mm oleate to the medium decreased [1-(14)C]palmitate incorporation into the total lung phospholipid fraction at 2 hr by 37 and 49%, respectively. The results suggest that the incorporation of exogenous fatty acids, present in the medium perfusing the lung, into lung phospholipids may depend upon the fatty acid composition of the medium. Known specific acyltransferase activities may be responsible for the ordered incorporation of available fatty acids into lung phospholipids.     

Insulin responsiveness of isolated perfused livers from rats with streptozotocin induced diabetes.

The ability of insulin to inhibit efflux of potassium (K) and amino acid nitrogen (AAN) from perfused livers of normal and insulin deficient rats was studied. Two groups of rats with different degrees of insulin deficiency were produced by injecting varying amounts of streptozotocin. One group, classified as being moderately diabetic (MD), had fasting plasma glucose levels between 235--425 mg%, while the other group, whose plasma glucose levels greater than 425 mg%, were considered to have severe diabetes (SD). Two other groups of rats were food restricted in order to attain body weights comparable to the two groups of diabetic rats, and livers from these animals were used for control perfusions. The results indicated that the ability of insulin to suppress efflux of K and AAN from perfused livers of rats with MD was comparable to that seen in control perfusions. On the other hand, insulin could not suppress the efflux of either K or AAN from perfused livers of rats with SD. These results indicate that normal hepatic responsiveness to insulin can be lost secondary to the production of insulin deficiency.     

Surfactant cholesterol metabolism of the isolated perfused rat lung

The cuticle (0.15 to 0.5 microns thick) of the microscopic free-living nematode Panagrellus silusiae was isolated intact by incubating worms with 1% sodium dodecyl sulfate at 37 degrees C overnight. After shearing and further treatment with detergent, electron microscopy revealed that the cuticular pieces were free of contaminating material and retained their characteristic in situ ultrastructure. From amino acid determinations, the cuticle is collagen-like with high levels of glycine (approximately equal to 31 residue %), proline (approximately equal to 20 residue %) and alanine (approximately equal to 21 residue %) although the hydroxyproline (2.6 residue %) content is low. Half-cystine (approximately equal to 1 residue %) is present in purified cuticles. Treatment with 8 M guanidine hydrochloride-2% beta-mercaptoethanol can solubilize more than 85% of the cuticular preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized cuticles from juvenile, adult and old dead worms revealed, at least, 18 discrete components. Estimated molecular weights ranged from about 26 000 (peak 1) to 250 000 (peak 18).     

The effects of cigarette smoke exposure on testosterone metabolism in the isolated perfused rat lung

Rats were exposed daily to cigarette smoke for 1 h for five consecutive days. The metabolism of [4-¹⁴C]-testosterone by the isolated perfused lungs was studied on the sixth day. Slightly diluted rat blood. containing the radioactive substrate, was used as the perfusion medium. Compared to sham-exposure rats, the formation of reduced metabolites (dihydroxysteroids) was less and the metabolism of testosterone diminished after cigarette smoke exposure. After the perfusion the amount of polar non-conjugated metabolites was significantly higher in the smoke-exposed lung tissue than in the control lungs. No conjugate formation was observed.The decrease in the formation of reduced metabolites seems to be due to an inhibition of the 4-ene-5α-reductase activity.     

Pharmacokinetics of oltipraz in rat models of diabetes mellitus induced by alloxan or streptozotocin

Pharmacokinetic parameters of oltipraz were compared after intravenous (10 mg/kg) and oral (30 mg/kg) administration in rat model of diabetes mellitus induced by alloxan (rat model of DMIA) or streptozotocin (rat model of DMIS) and their respective control male Sprague-Dawley rats. In rat models of DMIA and DMIS, the expressions and mRNA levels of CYP1A2, 2B1/2, and 3A1(23) increased, and oltipraz was metabolized mainly via CYP1A1/2, 2B1/2, 2C11, 2D1, and 3A1/2 in male Sprague-Dawley rats. Hence, it would be expected that the AUC and CL values of oltipraz would be significantly smaller and faster, respectively, in rat models of diabetes. This was proven by the following results. After intravenous administration, the AUC values were significantly smaller in rat models of DMIA (40.1% decrease) and DMIS (26.0% decrease) than those in respective control rats, and this could be due to significantly faster CL values in rat models of DMIA (40.1% increase) and DMIS (26.0% increase). The faster CL could be due to increase in hepatic blood flow rate and significantly faster CL(int) in rat models of diabetes, since oltipraz is an intermediate hepatic extraction ratio drug in male Sprague-Dawley rats. After oral administration, the AUC values of oltipraz were also significantly smaller in rat models of DMIA (54.0% decrease) and DMIS (63.2% decrease). This could be due to increase in hepatic blood flow rate, significantly faster CL(int), and changes in the intestinal first-pass effect in rat models of diabetes. However, this was not due to decrease in absorption in rat models of diabetes.     

Prostaglandin removal and metabolism by isolated perfused rat lung

We have investigated the mechanism(s) involved in the removal of prostaglandins (PG) from the pulmonary circulation by the lung. Unidirectional fluxes of PG from the circulation into the lung are measured in an isolated perfused rat lung preparation. Evidence is presented which suggests that a transport system for PG exists in lung tissue. This transport system is responsible for the removal of some PG from the circulation by the lung. PGE₁ and PGF_2α are substrates for this system, whereas PGB₁, PGA₁, and 15-keto-PGF_2α are not. Since PGA₁ is a substrate for the intracellular PG dehydrogenase, the selectivity of the lung's metabolism system for circulating PG is probably due to the selectivity of the transport system for PG. It is shown that the percentage of the pulmonary arterial concentration (C_A) of PGE₁ or PGF_2α that is metabolized on passage through the pulmonary circulation decreases rapidly as C_A increases. When the lungs were perfused with PGE₁ (PGF_2α), the metabolites detected in the venous effluent were 15-keto-PGE₁ (PGF_2α) and 15-keto-13,14-dihydro-PGE₁ (PGF_2α). The time course pattern of the appearance of metabolites in the venous effluent after the initiation of a constant C_A, and the relative concentrations of the metabolites in the venous effluent, were examined as a function of C_A.     

Metabolic effects of acetaminophen. Studies in the isolated perfused rat liver

The effects of acetaminophen on the metabolism of the isolated perfused rat liver were investigated. The following results were obtained: (1) Acetaminophen increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). (2) Oxygen uptake, gluconeogenesis from either pyruvate or fructose and glycogen synthesis were inhibited. (3) In isolated rat liver mitochondria acetaminophen decreased state III and state IV respiration; it also decreased the ADP/O ratio and the respiratory control ratio. (4) The action of acetaminophen on glycogenolysis was not affected by N-acetylcysteine; this compound, however, increased glycogen synthesis. (5) The effects of acetaminophen are reversible. It was concluded that glycogen depletion by acetaminophen can be produced by two mechanisms. The first, as previously demonstrated by several workers, depends on irreversible binding of a reactive metabolite. The second, however, is reversible and depends primarily on an inhibition of mitochondrial energy metabolism.     

Effect of salvipholin on the carbohydrate and lipid metabolism in the rat liver in alloxan diabetes]

Peroral administration of salvipholin in a dose of 50 mg/kg to intact male rats (the body weight 180-220 g) had a positive effect on the carbohydrate-lipid metabolism in the liver and blood serum of animals. Administration of the same dose to rats with alloxan diabetes induced a significant decrease in the content of glucose, free fat acids, triglycerides and lysophospholipids of the liver and blood serum. The level of these components has sharply increased after subcutaneous alloxan administration in a dose of 150 mg/kg, the content of glycogen and pyruvic acid being normalized and insulin deficiency removed. These changes are closely related to salvipholin-promoted restoration of phospholipid spectra of the blood serum and liver of experimental animals disturbed under conditions of insulin deficiency. The possible mechanism of the salvipholin action is analyzed.     

Effect of diabetes on the metabolism of chenodeoxycholic acid in isolated perfused rat liver

The formation of alpha-muricholic acid and beta-muricholic acid from chenodeoxycholic acid was comparatively investigated in livers isolated from normal, streptozotocin-diabetic, and insulin-treated diabetic rats. [24-14C]Chenodeoxycholic acid or [24-14C]alpha-muricholic acid was infused into the perfused livers. There was no difference in biliary excretion of 14C among the different groups of rats after the infusion of each 14C-labelled bile acid. Biliary [14C]bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. In the diabetic group, the formation ratio of alpha-muricholic acid and beta-muricholic acid from [24-14C]chenodeoxycholic acid and also that of beta-muricholic acid from [24-14C]alpha-muricholic acid were much smaller than in the normal group. Treatment of the diabetic group with insulin cancelled the difference in the infusion of each [24-14C]bile acid. The results indicate that not only 6 beta-hydroxylation of chenodeoxycholic acid to alpha-muricholic acid but also 7-epimerization of the latter acid to beta-muricholic acid is suppressed in an insulin-deficient state in rats.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.     

ADPase activity of isolated perfused rat lung.

More than 90% of 3H-ADP was metabolized following bolus injection into rat isolated perfused lungs. The major metabolite was inosine, with lesser amounts of adenosine and AMP. The mean pulmonary transit time for the radioactivity associated with ADP and its metabolites was the same as that for the vascular marker 14C-dextran, indicating that ADP is metabolized by enzymes in the pulmonary vessel walls. The metabolism of 3H-ADP was apparently unaffected by the simultaneous injection of prostacyclin or by continuous infusion of indomethacin or aspirin. 3H-ADP was similarly metabolized by the lung following continuous infusion, although relatively higher amounts of adenosine were observed. The metabolism of ADP by the lung represents biological inactivation since over 95% of the platelet-aggregatory activity of ADP was lost on passage through the lung.     

Acute effects of ethanol on the perfused rat liver. Studies on lipid and carbohydrate metabolism, substrate cycling and perfusate amino acids

1. Livers from fed rats were perfused in situ with whole rat blood containing glucose labelled uniformly with (14)C and specifically with (3)H at positions 2, 3 or 6. 2. When ethanol was infused at a concentration of 24mumol/ml of blood the rate of utilization was 2.8mumol/min per g of liver. 3. Ethanol infusion raised perfusate glucose concentrations and caused a 2.5-fold increase in hepatic glucose output. 4. Final blood lactate concentrations were decreased in ethanol-infused livers, but the mean uptake of lactate from erythrocyte glycolysis was unaffected. 5. Production of ketone bodies (3-hydroxybutyrate+3-oxobutyrate) and the ratio [3-hydroxybutyrate]/[3-oxobutyrate] were raised by ethanol. 6. Formation of (3)H(2)O from specifically (3)H-labelled glucoses increased in the order [6-(3)H]<[3-(3)H]<[2-(3)H]. Production of (3)H(2)O from [2-(3)H]glucose was significantly greater than that from [3-(3)H]glucose in both control and ethanol-infused livers. Ethanol significantly decreased (3)H(2)O formation from all [(3)H]glucoses. 7. Liver glycogen content was unaffected by ethanol infusion. 8. Production of very-low-density lipoprotein triacylglycerols was inhibited by ethanol and there was a small increase in liver triacylglycerols. Very-low-density-lipoprotein secretion was negatively correlated with the ratio [3-hydroxybutyrate]/[3-oxobutyrate]. Perfusate fatty acid concentrations and molar composition were unaffected by perfusion with ethanol. 9. Ethanol decreased the incorporation of [U-(14)C]glucose into fatty acids and cholesterol. 10. The concentration of total plasma amino acids was unchanged by ethanol, but the concentrations of alanine and glycine were decreased and ([glutamate]+[glutamine]) was raised. 11. It is proposed that the observed effects of ethanol on carbohydrate metabolism are due to an increased conversion of lactate into glucose, possibly by inhibition of pyruvate dehydrogenase. The increase in gluconeogenesis is accompanied by diminished substrate cycling at glucose-glucose 6-phosphate and at fructose 6-phosphate-fructose 1,6-bisphosphate.     

Hydroperoxide and cigarette smoke induced effects on lung mechanics and glutathione status in rat isolated perfused and ventilated lungs

The effects of t-butylhydroperoxide (TBH) and cigarette smoke on lung mechanics (CDYN and RL) and glutathione status (GSH) were studied using an isolated perfused and ventilated rat lung preparation. TBH (200, 400, 1000 microM) infused via the pulmonary circulation caused a dose-related bronchoconstriction. The lung GSH-levels were also significantly reduced. Pretreatment of rats with diethylmaleate (DEM) potentiated the TBH elicited bronchoconstriction. DEM (1 mM) infused into the pulmonary circulation caused an almost complete depletion of GSH-content but no effects on lung mechanics were seen. Indomethacin (2.8 and 28 microM) infusion attenuated TBH (400 microM) induced bronchoconstriction. These findings suggest that the TBH induced bronchoconstriction is at least partly mediated via arachidonic acid metabolites. When TBH was administered intratracheally, weak and not dose-related bronchoconstriction was observed even though doses higher than those given intravascularly were used. However, the GSH-content of the lungs was markedly decreased. Cigarette smoke caused weak if any effects on lung mechanics but significantly decreased lung GSH-content.     

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.