Adenine incorporation in human and rat endothelium.

Adenine (ADE) reutilisation is an important pathway of adenylate pool regeneration. Data on the rate of this process in different types of cells, its regulation and the importance of species differences is limited. In this study we evaluated adenine incorporation rate and the effect of metabolic factors on this process in human and rat endothelium and compared it to adenine phosphoribosyltransferase (APRT) activity. Microvascular endothelial cells from human (HE) and rat (RE) hearts and a transformed human microvascular endothelial cell line (HMEC-1) were investigated. The rate of adenine incorporation into the adenine nucleotide pool under control conditions was 3.1+/-0.3, 82.8+/-11.1 and 115.1+/-11.2 pmol/min per mg protein for HE, RE and HMEC-1, respectively. In the presence of 2.5 mM ribose or elevated inorganic phosphate concentration in the medium (4.8 mM), few changes were observed in all types of cells. In the presence of both ribose and high inorganic phosphate, the rate of adenine incorporation for RE and HMEC-1 was not significantly different from control, while in HE the rate of adenine incorporation into adenine nucleotides was increased by 75%. Activities of APRT in RE and HMEC-1 were 237.7+/-23.2 and 262.0+/-30.6 pmol/min per mg protein respectively while the activity in HE was markedly lower 48.7+/-3.0 pmol/min per mg protein. In conclusion, nucleotide synthesis from adenine seems to be a slow process in human cardiac microvascular endothelium but it is fast and efficient in rat heart microvascular endothelial cells. Low APRT activity in normal human endothelial cells seems to be the most likely mechanism for this. However, adenine incorporation rate and APRT activity could be greatly enhanced in human endothelium, as demonstrated in transformed cells.

Purine metabolism in pigs and humans and its implications for xenotransplantation.

We compared concentrations of nucleotide substrates and activities of enzymes of nucleotide metabolism in pig and human blood, heart, and kidney. The most important difference was lower ecto-5-nucleotidase (ESN) activity in both pig hearts and kidney. Furthermore, higher hypoxanthine, inosine, adenine, and uracil, but lower uridine and uric acid concentrations were observed in pig blood as compared to human. A twofold increase in UTP concentration has been observed in pig hearts following 4 h perfusion with human blood. Purine metabolism is an important target for genetic and pharmacological manipulation during xenotransplantations.

Exposure to human blood inactivates swine endothelial ecto-5'-nucleotidase.

Ecto-5'-nucleotidase (E5'N) is an extracellular enzyme forming anti-inflammatory and immunosuppressive adenosine. We evaluated whether confrontation of pig heart and endothelial cells with human blood changes the activity of E5'N. Pig hearts were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were incubated in vitro with human plasma for 3 h. Ex vivo perfusion of pig heart with fresh human blood resulted in a decrease in E5'N activity to 62% and 61% of initial in wild-type and transgenic pig hearts, respectively. PAEC activity of E5'N decreased to 71% and 50% of initial after 3 h exposure to heat-inactivated and active complement human plasma, respectively, while it remained constant in controls. Pig heart activity of E5'N decreased following exposure to human blood, which may affect adenosine production and exacerbate hyperacute and vascular rejection.

Endothelial nucleotide catabolism and adenosine production.

OBJECTIVE: The aim was to study pathways of nucleotide catabolism and adenosine production in cultured human umbilical vein endothelial cells (HUVEC) incubated under normal conditions and following inhibition of ATP synthesis. METHODS: Confluent cultures of HUVEC were incubated for 45 min in Hank's balanced salt solution in a 95% O2/5%CO2 atmosphere under the following experimental conditions: (1) in presence of the adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA); (2) with EHNA and the adenosine kinase inhibitor, 5'-iodotubercidin (ITu); (3) with iodoacetate and oligomycin (I+O), inhibitors of ATP production, and EHNA; (4) with I+O, EHNA, and an inhibitor of ecto 5'-nucleotidase, alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP). Nucleotide and catabolite contents in both cells and medium were analysed by HPLC. RESULTS: The initial contents of ATP, ADP, and AMP were 9.12(SEM 1.2), 0.73(0.08) and 0.11(0.02) nmol per culture flask respectively. These levels were maintained under experimental condition (1), with only a small increase in hypoxanthine in the medium of 0.9(0.2) nmol. Under experimental condition (2), adenosine accumulation in the medium was greatly enhanced [increase by 0.9(0.2) nmol], while the rise in hypoxanthine was similar to that in experimental condition (1). Under experimental condition (3), cellular ATP was totally depleted after 45 min and AMP increased to 4.3(0.9) nmol. Adenosine in the medium was increased by 5.4(0.2) nmol, the increase in the sum of hypoxanthine and inosine was 1.6(0.2) nmol, and the increases in uric acid and xanthine were less than 0.4 nmol. Under experimental condition (4), where AOPCP was present in addition to I+O and EHNA, adenosine production was markedly reduced and AMP accumulated in the medium [1.2(0.2) nmol]. CONCLUSIONS: Adenosine is generated continuously in cultured HUVEC under normal conditions, but is immediately recycled via adenosine kinase. In ATP depleted cells, nucleotide catabolism proceeds predominantly via intracellular dephosphorylation of AMP with a small contribution from the extracellular dephosphorylation pathway. The capacity of the AMP deamination pathway in endothelium is small and the flux through xanthine oxidoreductase is minimal.

Different regulatory properties of the cytosolic and mitochondrial forms of malic enzyme isolated from human brain.

The human brain contains a cytosolic and mitochondrial form of NADP(+)-dependent malic enzyme. To investigate their possible metabolic roles we compared the regulatory properties of these two iso-enzymes. The mitochondrial malic enzyme exhibited a sigmoid substrate saturation curve at low malate concentration which was shifted to the right at both higher pH values and in the presence of low concentration of Mn2+ or Mg2+. Succinate or fumarate increased the activity of the mitochondrial malic enzyme at low malate concentration. Both activators shifted the plot of reaction velocity versus malate concentration to the left, and removed sigmoidicity, but the maximum velocity was unaffected. The activation was associated with a decrease in Hill coefficient from 2.3 to 1.1. The human brain cytosolic malic enzyme displayed a hyperbolic substrate saturation kinetics and no sigmoidicity was detected even at high pH and low malate concentrations. Succinate or fumarate exerted no effect on the enzyme activity. Excess of malate inhibited the oxidative decarboxylation catalysed by cytosolic enzyme at pH 7.0 and below. In contrast, decarboxylation catalysed by mitochondrial malic enzyme, was unaffected by the substrate. These results suggest that under in vivo conditions, cytosolic malic enzyme catalyses both oxidative decarboxylation of malate and reductive carboxylation of pyruvate, whereas the role of mitochondrial enzyme is limited to decarboxylation of malate. One may speculate that in vivo the reaction catalysed by cytosolic malic enzyme supplies dicarboxylic acids (anaplerotic function) for the formation of neurotransmitters, while the mitochondrial enzyme regulates the flux rate via Krebs cycle by disposition of the tricarboxylic acid cycle intermediates (cataplerotic function).

Purification and properties of cytosolic and mitochondrial malic enzyme isolated from human brain.

Three isoforms of malic enzyme have been described in mammalian tissues: a cytosolic NADP(+)-dependent enzyme, a NADP(+)-dependent mitochondrial isoform and a mitochondrial isozyme which can use both NAD+ and NADP+ but is more effective with NAD+. We purified mitochondrial and cytosolic malic enzyme from human brain extract to apparent homogeneity in order to compare properties of these isozymes and to verify whether mitochondria contain one or two malic enzyme. Specific activities of both isoforms are approx. 90 mumol/min/mg of protein, which corresponds to about 1900-fold purification. The two isozymes have identical native molecular mass (257 kDa) and are presumably tetramers composed of four identical subunits (M(r) = 64 kDa). The isoelectric point of cytosolic isozyme is 5.65, and that of mitochondrial one is 7.0. The isozymes show a substantial difference in their capability to catalyse the reductive carboxylation of pyruvate to malate: the maximal carboxylation rate approaches 80% that of decarboxylation velocity for the cytosolic enzyme, and only 17% for the mitochondrial isozyme. The coenzyme specificity of both isozymes is not stringent; NADP+ is the preferred and NAD+ can substitute it, although with much lower efficiency. The homogenous cytosolic malic enzyme catalysed decarboxylation of oxaloacetate and NADPH-dependent reduction of pyruvate at about 24 and 0.5% of the maximum rate of NADP-dependent oxidative decarboxylation of malate respectively. Decarboxylation of oxaloacetate catalysed by mitochondrial malic enzyme has not been detectable, while NADP-linked reduction of pyruvate approaches only 0.15% of the maximum rate of NADP-linked oxidative decarboxylation of malate.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of lipogenesis in rat brown adipose tissue by clofibrate.

The effect of clofibrate (Atromid S, ethyl-2-(4-chlorophenoxy)-2-methylpropionate) administration for 7 days to rats on lipogenesis and on some lipogenic enzyme activities in brown adipose tissue (BAT), liver and white adipose tissue (WAT) was examined. As compared to control rats the rate of lipogenesis in BAT in the clofibrate-treated animals was significantly decreased. The rate of liver lipogenesis increased slightly, whereas lipogenesis in the WAT was not affected by clofibrate. In BAT, the drug treatment resulted in depression of fatty acid synthase, ATP-citrate lyase, malic enzyme, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. The activity of liver fatty acid synthase did not change, ATP-citrate lyase activity slightly decreased, whereas the activity of malic enzyme significantly increased in this organ after clofibrate feeding. The ATP-citrate lyase activity in WAT decreased, while fatty acid synthase and other lipogenic enzymes were not changed after clofibrate feeding. Clofibrate treatment did not influence the activity of NADP-linked isocitrate dehydrogenase and malate dehydrogenase (enzymes not linked directly to lipogenesis), either in BAT, liver or WAT. The data presented suggest that the hypolipidaemic effect of clofibrate in the rat may be due (possibly among other mechanisms) to reduction of the rate of fatty acid synthesis in BAT but not in the liver and WAT.

Increase of lipogenic enzyme mRNA levels in rat white adipose tissue after multiple cycles of starvation-refeeding.

Recently, we have found that despite the significant reduction of body weight after multiple starvation-refeeding cycles, white adipose tissue (WAT) exhibits surprisingly high rates of lipogenesis and lipogenic enzyme activities. The purpose of this study was to determine the response of WAT lipogenic enzyme mRNAs of rats subjected to multiple cycles of 3 days fasting and 3 days of refeeding. Despite the body weight reduction, significant increase of lipogenic enzymes (ie, fatty acid synthase [FAS], acetyl-coenzyme A [CoA] carboxylase [ACC], adenosine triphosphate (ATP)-citrate lyase [ACL], NADP-linked malic enzyme [ME], and glucose 6-phosphate dehydrogenase [G6PDH]) mRNAs in WAT was found after multiple cycles of starvation-refeeding of rats on standard laboratory diet. These findings, together with the results published recently, indicate that multiple cycles of starvation-refeeding cause the increased lipogenesis in WAT by upregulation of the lipogenic enzymes gene expression.

Unususal increase of lipogenesis in rat white adipose tissue after multiple cycles of starvation-refeeding.

The purpose of the study was to determine the response of liver and brown (BAT) and white (WAT) adipose tissue lipogenesis and total body weight in rats subjected to multiple cycles of 3 days of fasting and 3 days of refeeding. Rats fasted for 3 days showed significant reduction in body weight. These changes were reversed on 3 days' refeeding. Body weight was much higher in rats fed ad libitum than in animals experiencing more than one cycle of 3 days of fasting followed by 3 days of refeeding. Despite the significant body weight reduction, an unusual increase of lipogenesis in WAT was found after multiple cycles of starvation-refeeding of rats on standard laboratory diet. The rate of lipogenesis in the liver and BAT was also elevated but to a much smaller extent. A parallel increase in enzymatic activities related to fatty acid synthesis, ie, fatty acid synthase, acetyl-coenzyme A carboxylase, adenosine triphosphate (ATP)-citrate lyase, NADP-linked malic enzyme, and hexose monophosphate shunt dehydrogenases, suggests that the increased rate of lipogenesis in WAT is a consequence of increased lipogenic enzyme activities. These data suggest that upregulation of WAT lipogenesis occurs after the multiple cycles of the starvation-refeeding protocol. An unusual increase of lipogenesis in rat WAT may have a survival advantage, because starved-refed rats must develop the ability to ingest large amounts of food during a refeeding period to store it in a convenient form than can be used as an oxidizable substrate during a period of starvation. Moreover, these results suggest that it is possible to develop appropriate starvation-refeeding conditions that may inhibit body weight gain.

Comparative study of the lipogenic potential of human and rat adipose tissue.

The reported low activity of lipogenic enzymes (especially adenosine triphosphate [ATP]-citrate lyase) in human adipose tissue led to the general conclusion that in humans lipogenesis occurs primarily in the liver. However, recent studies indicate that the liver plays a minor role in de novo lipogenesis and suggest that adipose tissue may be the principal lipogenic human tissue. In an attempt to resolve these contradictions we reinvestigated the lipogenic potential of human adipose tissue and compared with adipose tissue of rats fed a high-fat diet for 2 weeks and fasted overnight before death. These conditions mimic the nutritional state of patients at the moment of tissue sampling. We found that overnight fasting of the rats maintained previously for 12 days on a high-fat diet caused a decrease of ATP-citrate lyase of about 7-fold. Thus, in human adipose tissue, the mean activity of ATP-citrate lyase was approximately 8 times lower than in rats fed a high-fat diet and fasted overnight, and about 50 times lower than in rats maintained on normal laboratory diet. Unlike ATP-citrate lyase, fatty acid synthase (FAS) activity was only slightly lower in human adipose tissue than in rats maintained on a normal laboratory diet. Comparable FAS activity was found when rats were fed a high-fat diet and fasted overnight. The average activities of human adipose tissue acetyl-coenzyme A carboxylase, malic enzyme, and glucose-6-phosphate dehydrogenase were approximately 3-, 4-, and 6-fold lower than in adipose tissue from rats fed a high-fat diet and fasted overnight before tissue sampling, while the activity of 6-phosphogluconate dehydrogenase in humans was higher than in rat adipose tissue. No significant differences in lipogenic enzyme activities were found between male and female and between lean and obese patients. The rate of fatty acid synthesis in intact pieces of human adipose tissue was approximately 5 times lower than in adipose tissue pieces of rats fed a high-fat diet and fasted overnight before tissue samples were taken. The comparison of the lipogenic potential of humans and rats (maintained on the diet to mimic the nutritional state of patients at the time of tissue sampling) suggests that human adipose tissue is an important site of fatty acid synthesis.

Tissue-specific effect of clofibrate on rat lipogenic enzyme gene expression.

Fibrate derivatives are commonly used to treat hyperlipidaemia; however, the mechanism of the antilipidaemic action of these drugs is still unknown. The effect of clofibrate (fibrate derivative) administration for 14 days on lipogenesis and on malic enzyme (EC 1.1.1.40) and fatty acid synthase (EC 2.3.1.85) gene expression in brown and white adipose tissues and in the liver was examined in rats. The rate of brown adipose tissue lipogenesis in the clofibrate-treated animals was significantly lower than that of the control rats. The rate of liver and white adipose tissue lipogenesis was not affected significantly by clofibrate. In brown adipose tissue, the drug treatment resulted in a depression of fatty acid synthase and malic enzyme mRNA levels. The fatty acid synthase mRNA level did not change significantly in the liver, whereas the malic enzyme mRNA level increased approximately 6-fold in this organ after clofibrate treatment. The malic enzyme mRNA level in white adipose tissue increased about 2-fold, while the fatty acid synthase mRNA level was unchanged after clofibrate feeding. The results presented in this paper provide further evidence that the hypolipidaemia caused by treatment of rats with clofibrate cannot be related to the inhibition of fatty acid synthesis in the liver and white adipose tissue. These data also indicate that clofibrate exhibits tissue specificity.

Differential effect of clofibrate on acetyl-CoA carboxylase mRNA level in rat white and brown adipose tissue.

Regulation of some lipogenic enzyme gene expression by clofibrate was studied in rat white and brown adipose tissue. In white adipose tissue the drug administration for 14 days to rats resulted in the increase in acetyl-CoA carboxylase, ATP-citrate lyase, and glucose 6-phosphate dehydrogenase mRNA levels. Opposing effect of clofibrate on the acetyl-CoA carboxylase, ATP-citrate lyase, and glucose 6-phosphate dehydrogenase mRNA levels was found in brown adipose tissue. These data indicate a tissue specificity of clofibrate action on lipogenic enzyme gene expression. The results presented in this paper provide further evidence that hypolipidaemia caused by the treatment with clofibrate cannot be related to the inhibition of fatty acid synthesis in white adipose tissue in rat.

Dietary alpha-tocopherol prevents dehydroepiandrosterone-induced lipid peroxidation in rat liver microsomes and mitochondria.

Dehydroepiandrosterone (DHEA), an adrenal steroid, causes lipid peroxidation in rat liver microsomes and mitochondria and induces hepatocarcinogenesis. It was investigated whether alpha-tocopherol, a naturally occurring free radical chain terminator, could decrease lipid peroxidation. When DHEA-free diet supplemented with increasing concentrations of alpha-tocopherol (25, 50, 100, 200, 400 and 1000 mg/kg diet) was fed to rats for 7 days, a marked lipid peroxidation (measured as thiobarbituric acid reactive substances formation) was observed at concentrations 25 and 50 mg/kg in liver microsomes and mitochondria isolated from these animals. Lipid peroxidation was significantly reduced at concentrations > or = 100 mg/kg. When DHEA (500 mg/kg diet) was fed to rats simultaneously with increasing concentrations of alpha-tocopherol, strong lipid peroxidation was observed at alpha-tocopherol concentrations < or = 200 mg/kg diet. However, microsomes and mitochondria isolated from livers of rats fed alpha-tocopherol at doses of 400 and 1000 mg/kg diet produced only negligible amounts of thiobarbituric acid reactive substances. The data show that high concentrations of alpha-tocopherol in the diet decrease DHEA-induced microsomal and mitochondrial lipid peroxidation. Our results support the concept that alpha-tocopherol can protect against DHEA-induced lipid peroxidation and consequently against steroid-induced liver cell damage and, perhaps, also tumour development.

Comparative studies on NADP(+)-linked malic enzyme in the central nervous system of ectothermic and endothermic animals.

The maximum activity and intracellular distribution of NADP(+)-linked malic enzyme in brain of Mammalia, Aves, Reptilia, Amphibia and Pisces are reported. Malic enzyme activity was present in all animals brains investigated. Most of the enzyme activity was located in the mitochondrial fraction. In brain of endothermic animals the activity of malic enzyme was several-fold higher than in ectothermic animals. Other NADPH-producing enzymes (i.e. NADP(+)-linked isocitrate dehydrogenase and hexosemonophosphate shunt dehydrogenase) activities were essentially similar in all animals brains tested. However, the total potential capability of NADPH production was lower in ectothermic animals (due mainly to lower malic enzyme activity). It is suggested that the presence of NADP(+)-linked malic enzyme in the brain may be related mainly to mitochondrial metabolism, especially to maintain the mitochondrial pool of NADP+ in reduced form.