Effect of testosterone on purine synthesis de novo in rat perineal muscle in vivo

We examined in vivo the influence of testosterone on purine synthetis de nov, in the levator ani and gastrocnemius muscles of the rat. The hypoxanthine, adenine and guanine contents and the rate of incorporation of [¹⁴C]formate into these purine bases were determined in castrated adult and prepubertal rats (groups 1 and 2) both before and after orchiectomy and, in the second case, at different times after testosterone treatment. Substantially similar behavior was found in both groups, with some specific differences. The results showed an increase in the basal levels after castration (except for a dramatic decrease in adenine and a rise in the Gua/Ade molar ratio in prepubertal rats) and a return to basal levels after hormone administration, which was also accompanied by variations in the Gua/Ade molar ratio. The kinetics of purine nucleotide synthesis de novo and, spefically, of the overall reactions: IMP formation from PRib-PP, IMP → AMP and IMP → GMP, were followed by evaluating the incorporation curves of [¹⁴C]formate into hypoxanthine, adenine and guanine. Our results show that testosterone administration enhanced the incorporation rate and gave characteristic patterns: a diphasic cyclic oscillation of the Ade values in adult castrated rats, and single peaks having a specific shape in the other cases. The Gua/Ade labeling ratio was unchaned in castrated rats and increased in both groups during ther first 5 days after testosterone treatment, after which values even fell below normal; in most cases, values overlapped the pattern of the Gua/Ade molar ratio. The specific profile of the curves indicated that testosterone initially accelerated the turnover of guanylic acid and in the second phase re-established the normal behavior and ratio of AMP and GMP formation. These results indicate that the ‘inosinic branch point’ was subject to regulation by testosterone. The profiles of the incorporation curves and of the Gua/Ade ratio were indicative of a primary and secondary response to hormone action.     

Lysolecithin as regulator of de novo lecithin synthesis in rat liver microsomes.

1-Acyl-sn-glycero-3-phosphocholine (lysolecithin) was found to affect 1,2-diacyl-sn-glycerol:CDPcholine cholinephosphotransferase (CPT; EC 2.7.8.2) activity of rat liver microsomes in a concentration dependent, characteristic manner. Cholinephosphate transfer was activated at lysolecithin concentrations below 0.5 mM with a maximum stimulation occurring at 75–100 μM lysolecithin levels. At concentrations above 0.5 mM, CPT activity was inhibited by lysolecithin. It was shown that CPT inhibition by lysolecithin is competitive (Ki ≈ 0.6 mM) with respect to CDPcholine. The possible role of lysolecithin as regulator of de novo lecithin synthesis in vivo is outlined.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

Inhibition of carnitine palmitoyltransferase leads to induction of 3-hydroxymethylglutaryl coenzyme A reductase activity in rat liver

The relation between carnitine palmitoyltransferase (CPT) activity and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated. Rats were treated with aminocarnitine or 1-carnitine overnight. In rats, in which CPT activity was inhibited by aminocarnitine, plasma and hepatic triacylglycerol contents were increased 5- to 6-fold. The plasma cholesterol concentration was unchanged, while the hepatic cholesterol content was lowered (-16%). Hepatic cholesterol synthesis, determined by following the incorporation of 14C-acetate and 3H2O into digitonin-precipitable sterols, in liver slices was increased 5- to 7-fold. HMG-CoA reductase activity in liver microsomes was increased to the same extent.     

Ethanol enhances de novo synthesis of high density lipoprotein cholesterol

Male squirrel monkeys fed ethanol at variable doses were used to assess whether alcohol enhances de novo synthesis of high density lipoprotein (HDL) cholesterol in vivo. Monkeys were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys fed liquid diet with vodka substituted isocalorically for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. High Ethanol primates had significantly higher levels of HDL nonesterified cholesterol than Control and Low Ethanol animals while serum glutamate oxaloacetate transaminase was similar for the three treatments. There were no significant differences between the groups in HDL cholesteryl ester mass or specific activity following intravenous injection of labeled mevalonolactone. By contrast, High Ethanol monkeys had significantly greater HDL nonesterified cholesterol specific activity with approximately 60% of the radioactivity distributed in the HDL3 subfraction. This report provides the first experimental evidence that ethanol at 24% of calories induces elevations in HDL cholesterol in primates through enhanced de novo synthesis without adverse effects on liver function.     

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.     

Inhibition of in vivo DNA synthesis in regenerating rat liver following thermal injury

Hepatic repair and regeneration which is extremely important after thermal injuries can be inhibited by the acute inflammatory reaction. Since thermal injury initiates this acute inflammatory reaction, DNA synthesis was studied in the regenerating liver following this injury. In vivo incorporation of [3H]-thymidine into hepatic DNA, autoradiographic determination of a labeling index, and thymidine kinase activity were determined. Incorporation of [3H]-thymidine into hepatic DNA and labeling indices were markedly diminished at 24 hours if partial hepatectomy and thermal injury were carried out concurrently. After partial hepatectomy, the expected elevations in thymidine kinase activity were inhibited by the thermal injury (p less than 0.01) and elevation of serum fibrinogen, a marker of the acute phase reaction that normally follows thermal injury, was blunted by the partial hepatectomy (p less than 0.05). The combination of thermal injury and partial hepatectomy resulted in a greatly diminished DNA replicative response as compared to partial hepatectomy alone and suggests that multiplicative injury is more likely to result in multi-system failure.     

Pioglitazone induces de novo ceramide synthesis in the rat heart

Ceramide (CER) is an important mediator of lipotoxicity in the heart. It was found that Zucker diabetic fatty rats develop an age-dependent accumulation of myocardial CER leading to cardiomyocyte apoptosis. However, administration of peroxisome proliferator-activated receptor (PPAR) gamma agonist decreased the content of CER and prevented cardiomyocyte apoptosis [Zhou et al. Proc Natl Acad Sci USA 2000;97:1784-9]. These data suggest that PPARgamma activators affect myocardial CER metabolism. Therefore, the aim of our study was to examine the effects of pioglitazone, a selective PPARgamma agonist, on the content of CER and its metabolites and on the activity of key enzymes of CER metabolism in the heart. The experiments were conducted on rats fed either a standard chow (STD) or a high-fat diet (HFD) for 21 days. Each group was divided into two subgroups: control and treated with pioglitazone for 14 days. Surprisingly, administration of PPARgamma agonist significantly increased myocardial CER content in both STD and HFD rats. In the latter group an elevation in the amount of sphingomyelin was also observed. In STD rats pioglitazone treatment increased the activity of neutral sphingomyelinase and acid ceramidase. However, in HFD group the compound did not affect the activity of the aforementioned enzymes. Interestingly, the activity of serine palmitoyltransferase in both STD and HFD rats increased two-fold after pioglitazone treatment. We conclude that pioglitazone induced accumulation of CER in rat myocardium as a result of augmented CER synthesis de novo. However, in the STD group increased activity of neutral sphingomyelinase could also contributed to this effect.     

Adenine nucleotide synthesis de novo in mature rat cardiac myocytes

Inadequate oxygenation of cardiac muscle leads to rapid loss of high energy compounds essential for contractile function. ATP can be regenerated by synthesis de novo, a route operating at a relatively slow rate in the heart. Myocytes isolated from mature rat heart have been used to measure the rate of ATP synthesis de novo from both [14C]glycine and [14C]ribose. Incorporation of glycine into ATP is accelerated 10-fold in the presence of 1 mM ribose. Myocytes also accumulate both precursors into IMP and four other metabolites on the de novo synthesis pathway. These metabolites represent 80% of the glycine entering the pathway. The potential of de novo synthesis for restoration of adenine nucleotides appears to be limited by the rates of early reactions, adenylosuccinate synthetase being only one of the enzymes operating at a sufficiently slow rate to make this pathway an inherently weak route for the restoration of normal energy status in post-ischemic myocardium. Interventions are being sought to alleviate these apparent metabolic delays.     

Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver

Our previous morphological studies illustrated the association of sterols with Plasmodium infecting hepatocytes. Because malaria parasites cannot synthesize sterols, they must scavenge these lipids from the host. In this paper, we have examined the source/s of sterols for intrahepatic Plasmodium and evaluated the importance of sterols for liver stage development. We show that Plasmodium continuously diverts cholesterol from hepatocytes until release of merozoites. Removal of plasma lipoproteins from the medium results in a 70% reduction of cholesterol content in hepatic merozoites but these parasites remain infectious in animals. Plasmodium salvages cholesterol that has been internalized by low-density lipoprotein but reduced expression of host low-density lipoprotein receptors by 70% does not influence liver stage burden. Plasmodium is also able to intercept cholesterol synthesized by hepatocytes. Pharmacological blockade of host squalene synthase or downregulation of the expression of this enzyme by 80% decreases by twofold the cholesterol content of merozoites without further impacting parasite development. These data enlighten that, on one hand, malaria parasites have moderate need of sterols for optimal development in hepatocytes and, on the other hand, they can adapt to survive in cholesterol-restrictive conditions by exploitation of accessible sterols derived from alternative sources in hepatocytes to maintain proper infectivity.     

Inhibition of protein synthesis and induction of helical polysomes in rat liver by N-hydroxy-2-fluorenylacetamide

After a single intraperitoneal injection of the hepatocarcinogen N-hydroxy-2-fluorenylacetamide (OH-FAA), numerous helical polysomes were found in the hepatocytic cytoplasm at 2 and 6 but not 24 h after treatment. Electron microscopy also demonstrated nucleolar segregation, disarray of endoplasmic reticuium (ER), and disaggregation of polyribosomes at the times when helical polysomes were present. Polyribosome disaggregation was confirmed and quantified by determining size distribution of polyribosomes at 2 h after OH-FAA treatment. Protein synthesis was inhibited at the time of helical polysome induction but the degree of inhibition did not noticeably alter the number of helical polysomes found electron microscopically.     

Effects of glycerol and fructose on purine synthesis de novo and on PP-ribose-P availability in rat liver cells

Incubation of freshly isolated rat liver cells with glycerol resulted in an initial decrease, followed by an increase in purine synthesis de novo and in PP-ribose-P availability. The magnitude of these effects was dependent on the concentration of glycerol; as it increased, the initial period of latency or inhibition was prolonged, and the extent of the subsequent stimulation was greater. The intracellular Pi concentration and the [14C]ATP/[14C]ADP ratio were also initially decreased in these cells, and they too returned subsequently to normal values. All these changes were similar to those induced by fructose under the same conditions. The increase in PP-ribose-P availability always preceded that in purine synthesis de novo, indicating that, under most circumstances, PP-ribose-P availability is limiting for purine synthesis de novo. Finally, PP-ribose-P synthesis in these cells varied in parallel with the intracellular Pi concentration and with the ATP/ADP and ATP/AMP ratios.