Effect of allylisopropylacetamide on Nuclear Ribonucleic Acid synthesis in rat liver

The porphyrogenic drug allylisopropylacetamide, a potent inducer of delta-aminolaevulinate synthetase, specifically increases nucleoplasmic RNA synthesis in rat liver. The drug-mediated increase in nucleoplasmic RNA synthesis is blocked by cycloheximide and haemin, which also inhibit the enzyme induction.

Genistein and 17β-estradiol fatty acid esters in blood and adipose tissue and the structure-related antioxidant activity of estrogens on lipoproteins

Soy-derived phytoestrogen genistein and 17β-estradiol (E2), the principal endogenous estrogen in women, are also potent antioxidants protecting LDL and HDL lipoproteins against oxidation. This protection is enhanced by esterification with fatty acids, resulting in lipophilic molecules that accumulate in lipoproteins or fatty tissues. The aims were to investigate, whether genistein becomes esterified with fatty acids in human plasma accumulating in lipoproteins, and to develop a method for their quantitation; to study the antioxidant activity of different natural and synthetic estrogens in LDL and HDL; and to determine the E2 esters in visceral and subcutaneous fat in late pregnancy and in pre- and postmenopause. Human plasma was incubated with [3H]genistein and its esters were analyzed from lipoprotein fractions. Time-resolved fluoroimmunoassay (TR-FIA) was used to quantitate genistein esters in monkey plasma after subcutaneous and oral administration. The E2 esters in women s serum and adipose tissue were also quantitated using TR-FIA. The antioxidant activity of estrogen derivatives (n=43) on LDL and HDL was assessed by monitoring the copper induced formation of conjugated dienes. Human plasma was shown to produce lipoprotein-bound genistein fatty acid esters, providing a possible explanation for the previously reported increased oxidation resistance of LDL particles during intake of soybean phytoestrogens. Genistein esters were introduced into blood by subcutaneous administration. The antioxidant effect of estrogens on lipoproteins is highly structure-dependent. LDL and HDL were protected against oxidation by many unesterified, yet lipophilic derivatives. The strongest antioxidants had an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups. E2 ester levels were high during late pregnancy. The median concentration of E2 esters in pregnancy serum was 0.42 nmol/l (n=13) and in pre- (n=8) and postmenopause (n=6) 0.07 and 0.06 nmol/l, respectively. In pregnancy visceral fat the concentration of E2 esters was 4.24 nmol/l and in pre- and postmenopause 0.82 and 0.74 nmol/l. The results from subcutaneous fat were similar. In serum and fat during pregnancy, E2 esters constituted about 0.5 and 10% of the free E2. In non-pregnant women most of the E2 in fat was esterified (the ester/free ratio 150 - 490%). In postmenopause, E2 levels in fat highly exceeded those in serum, the majority being esterified. The pathways for fatty acid esterification of steroid hormones are found in organisms ranging from invertebrates to vertebrates. The evolutionary preservation and relative abundance of E2 esters, especially in fat tissue, suggest a biological function, most likely in providing a readily available source of E2. The body s own estrogen reservoir could be used as a source of E2 by pharmacologically regulating the E2 esterification or hydrolysis.

Self-acylation properties of type II fatty acid biosynthesis acyl carrier protein

Acyl carrier protein (ACIP) plays a central role in many metabolic processes inside the cell, and almost 4% of the total enzymes inside the cell require it as a cofactor. Here, we report self-acylation properties in ACPs from Plasmodium falciparum and Brassica napus that are essential components of type II fatty acid biosynthesis (FAS II), disproving the existing notion that this phenomenon is restricted only to ACPs involved in polyketide biosynthesis. We also provide strong evidence to suggest that catalytic self-acylation is intrinsic to the individual ACP. Mutational analysis of these ACPs revealed the key residue(s) involved in this phenomenon. We also demonstrate that these FAS 11 ACPs exhibit a high degree of selectivity for self-acylation employing only dicarboxylic acids as substrates. A plausible mechanism for the self-acylation reaction is also proposed.

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C4), -octanoyl (C8), -decanoyl (C10), -dodecanoyl (C12) and -tetradecanoyl (C14)-ACPs display marked changes in backbone HN, Cα and Cβ chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.

Effect of dietary cholesterol and ubiquinone on isoprene synthesis in rat liver

The effect of dietary cholesterol and ubiquinone on the synthesis of isoprene compounds in the liver, as tested by the incorporation of acetate-1-14C and mevalonate-2-14C, was studied in rats. In cholesterol feeding, there appears to be a second site of inhibition after squalene in addition to the previously known primary site of inhibition at the β-hydroxy-β-methyl glutaryl-CoA reductase. Feeding ubiquinone inhibited at some common step between acetate and mevalonate in the synthesis of both cholesterol and ubiquinone, without affecting the acetate activation or fatty acid synthesis, and also at a step in the synthesis of ubiquinone not common with the synthesis of cholesterol. These results are suggestive of a role for ubiquinone in the regulation of isoprene synthesis.

Stimulation by thyrotropin, long-acting thyroid stimulator, and dibutyryl 3′, 5′-adenosine monophosphate of protein and ribonucleic acid synthesis and ribonucleic acid polymerase activities in porcine thyroid in Vitro

The in vitro incorporation of [3H]uridine into RNA and [3H]leucine into protein in slices of porcine thyroid was studied. Thyrotropin (10-500 mU/ml of medium), when added with [3H]uridine, inhibited incorporation into RNA, but as little as 10 mU of thyrotropin per ml stimulated incorporation of [3H]orotic acid into RNA. Uridine kinase (EC 2.7.1.48) was found to be inhibited in slices incubated with thyrotropin whereas UMP 5′ nucleotidase (EC 2.1.3.5) was not. Preincubation of slices with thyrotropin (5-50 mU/ml) led to enhanced incorporation of subsequently added [3H]uridine and [3H]leucine. When slices were preincubated with long-acting thyroid stimulator-IgG (2.5 or 5 mg per ml of medium) incorporation of [3H]uridine and [3H]leucine was similarly enhanced, with the smaller concentration being more effective. Without preincubation these stimulatory effects were mimicked by 1 mM dibutyryl 3′,5′-AMP and, to a lesser extent, 1 mM 3′,5′-AMP. AMP and ATP also stimulated [3H]uridine incorporation in this system but only after more prolonged periods of incubation than were required for the other nucleotides. RNA polymerase (EC 2.7.7.6) activity measured in isolated thyroid nuclei had two components, one Mg2+-stimulated and the other requ ring Mn2+ and high salt content [0.4 M (NH4)2SO4]. These activities, and particularly the former, were enhanced if thyroid slices were incubated with thyrotropin (5-100 mU/ml of medium), 2.5 mg or 5.0 mg of long-acting thyroid stimulator-IgG per ml, or 1 mM dibutyryl 3′,5′-AMP, before isolatior of the nuclei and measurement of enzyme activities; 1 mM AMP, ADP, or 2′,3′-GMP had no influence. Added directly to the nuclei, thyrotropin, long-acting thyroid stimulator-IgG, and dibutyryl 3′,5′-AMP had no effect on RNA polymerase activities. These data are seen as affording evidence for mediation by 3′,5′-AMP of effects of thyrotropin and long-acting thyroid stimulator on thyroid RNA and protein synthesis, at least in part through an indirect stimulation of nuclear RNA polymerase activities.

Fatty acid specificity for the esterification of vitamin A and cholesterol by intestinal and pancreatic enzymes in rats

VITAMIN A and cholesterol esters have been shown to undergo extensive hydrolysis in the lumen of the small intestine during the process of absorption; they are re-esterified to appear in the lymph mostly as esters1,2. However, the vitamin A esters of the lymph, blood and liver of the rat are formed by long-chain fatty acids3 and in the normal rat liver, probably as palmitates4. On the other hand, cholesterol esters are usually made up of poly-unsaturated fatty acids in the lymph and blood of rats5. For the absorption of the two lipid materials, the enzymes of the pancreas have been largely implicated, while not much attention has been paid to the possible role of the mucosal enzymes. From the behaviour of the mucosal enzymes, as presented here, it appears that probably these enzymes play a more important part in the re-esterification of the two lipid materials during their absorption.

Fatty acid component of vitamin A ester in sheep liver

Vitamin A, when extracted along with other lipids from sheep liver, had an E1cm.1% value of 14.4, which was raised to 45.57 on removal of the phospholipids by cold acetone. Selective hydrolysis of triglycerides by an extract of acetone-dried sheep pancreas in the presence of HgCl2 as inhibitor of vitamin A esterase, followed by chromatography through alumina gave a product with E1cm.1% value of 276. This on chromatography through magnesium oxide raised the E1cm.1, value to 601.5, representing 64% pure vitamin A ester calculated as palmitate, and the total recovery was 23% of the starting oil. The purified ester preparation, when subjected to reverse-phase chromatography on silicone-impregnated paper, gave a single ultraviolet fluorescent band. The fluorescent band on hydrolysis gave only one fatty acid. This was conclusively identified to be palmitic acid.

Effect of lipopolysaccharide on alteration of phospholipids and their fatty acid composition in spleen and thymus by in vitro metabolic labeling

Lipopolysaccharide (LPS) is an endotoxin, a potent stimulator of immune response and induction of LPS leads to acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). ARDS is a life-threatening disease worldwide with a high mortality rate. The immunological effect of LPS with spleen and thymus is well documented; however the impact on membrane phospholipid during endotoxemia has not yet been studied. Hence we aimed to investigate the influence of LPS on spleen and thymus phospholipid and fatty acid composition by 32P]orthophosphate labeling in rats. The in vitro labeling was carried out with phosphate-free medium (saline). Time course, LPS concentration-dependent, pre- and post-labeling with LPS and fatty acid analysis of phospholipid were performed. Labeling studies showed that 50 mu g LPS specifically altered the major phospholipids, phosphatidylcholine and phosphatidylglycerol in spleen and phosphatidylcholine in thymus. Fatty acid analysis showed a marked alteration of unsaturated fatty acids/saturated fatty acids in spleen and thymus leading to immune impairment via the fatty acid remodeling pathway. Our present in vitro lipid metabolic labeling study could open up new vistas for exploring LPS-induced immune impairment in spleen and thymus, as well as the underlying mechanism.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Terminalia paniculata bark extract attenuates non-alcoholic fatty liver via down regulation of fatty acid synthase in high fat diet-fed obese rats

Background: This study was performed to understand the possible therapeutic activity of Terminalia paniculata ethanolic extract (TPEE) on non alcoholic fatty liver in rats fed with high fat diet. Methods: Thirty six SD rats were divided into 6 groups (n = 6): Normal control (NC), high fat diet (HFD), remaining four groups were fed on HFD along with different doses of TPEE (100,150 and 200 mg/kg b.wt) or orlistat, for ten weeks. Liver tissue was homogenized and analyzed for lipid profiles, activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content. Further, the expression levels of FAS and AMPK-1 alpha were also studied in addition to histopathology examination of liver tissue in all the groups. Results: HFD significantly increased hepatic liver total cholesterol (TC), triglycerides (TG), free fatty acids (FFA) and MDA but decreased the activities of SOD and CAT which were subsequently reversed by supplementation with TPEE in a dose-dependent manner. In addition, TPEE administration significantly down regulated hepatic mRNA expression of FAS but up regulated AMPK-1 alpha compared to HFD alone fed group. Furthermore, western blot analysis of FAS has clearly demonstrated decreased expression of FAS in HFD + TPEE (200 mg/kg b. wt) treated group when compared to HFD group at protein level. Conclusions: Our biochemical studies on hepatic lipid profiles and antioxidant enzyme activities supported by histological and expression studies suggest a potential therapeutic role for TPEE in regulating obesity through FAS.

Survey of the fatty acid composition of Canadian beef: Backfat and longissimus lumborum muscle

[EN]A survey of Canadian retail beef was undertaken with emphasis on the trans fatty acid (TFA) and conjugated linoleic acid (CLA) isomers, and compared with current health recommendations. Thirty striploin steaks were collected in the winter and summer from major grocery stores in Calgary (Alberta, Canada). Steak fatty acid compositions (backfat and longissimus lumborum muscle analysed separately) showed minor seasonal differences with lower total saturates (PB0.05) and higher total monounsaturates (PB 0.01) in winter, but no differences in total polyunsaturated fatty acids. The ratio of n-6 and n-3 polyunsaturated fatty acid in longissimus lumborum averaged 5.8. The average TFA content in longissimus lumborum was 0.128 g 100 g_1 serving size, and 10t-18:1 was found to be the predominant isomer (32% of total trans), while vaccenic acid was second most abundant (15% of total trans). The CLA content in longissimus lumborum was similar to that of backfat, ranging from 0.43 to 0.60% of total fatty acids and rumenic acid represented 60% of total isomers. Overall, there is still room for improvement in the saturated, mono- and polyunsaturated fatty acid composition of Canadian beef to meet general dietary guidelines for human consumption and additional targets should include reducing 10t-18:1 while increasing both rumenic and vaccenic acids.