Incorporation Of Radioactive Acetate Research Articles

Effects of acute heat stress on lipid metabolism of bovine primary adipocytes

Heat stress (HS) affects numerous physiological processes including nutrient partitioning and lipid metabolism. Objectives of this study were to evaluate how acute HS affects lipid metabolism in subcutaneous adipose tissue of dairy cattle. Adipose tissue biopsies were performed on Holstein cows for bovine primary adipocyte isolation and cultured at either 42°C (HS) or 37°C (thermal neutral, TN). Adipocytes were incubated with increasing isoproterenol (ISO), and with increasing concentrations of insulin in the presence of ISO to evaluate changes in lipolysis. Incorporation of radioactive acetate into lipids was measured as an indicator of lipogenesis. Abundance and phosphorylation of several lipolytic and lipogenic proteins were also measured. Adipocytes exposed to HS had an elevated maximal response to ISO and were more sensitive to lipolytic stimulation by ISO compared with cells cultured at TN. Thermal treatment did not affect the antilipolytic effects of insulin in the presence of ISO. Lipogenesis measured as acetate incorporation was not altered by HS, but a temperature by insulin interaction was observed for the regulation of acetyl CoA carboxylase, such that the presence of insulin resulted in a reduction in phosphorylation of acetyl CoA carboxylase in adipocytes cultured at TN but not HS conditions. Results of this study demonstrate that acute HS has a direct effect on the regulation of lipolysis and the rate-limiting enzyme of lipogenesis in isolated bovine adipocytes.

Effects of dehydration and rehydration on the leaf lipids and lipid metabolism in Parkinsonia aculeata (Caesalpiniaceae)

Plants of Parkinsonia aculeata L. were dehydrated by withholding water for 13 days and then rehydrated. The leaves of control plants and stressed plants were analyzed every four days from the beginning of stress and 6, 24, and 48 h after stress relief. Analysis of leaf water status showed a decrease in relative water content of nearly 16% during dehydration and a return to its control value after 48 h of rehydration. The contents of chlorophylls, chloroplast lipids (monogalactosyldiacylglycerol and digalactosyldiacylglycerol), and neutral lipids increased by 38%, 20%, and 125% (in μg·mg dry mass−1), respectively, when the daily decrease of relative water content reached 1.4%, while the mass of extra-plastidial lipids (phosphatidylcholine and phosphatidylethanolamine) increased by 25% (in μg·mg dry mass−1) only when the daily water loss was 2.6%. In addition, incorporation of radioactive acetate in lipids showed a preferential synthesis of digalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The unsaturation degree of galactolipids remained stable, while a slight increase of unsaturation of phosphatidylcholine and phosphatidylethanolamine was observed. After rehydration, the mass of chlorophylls fell sharply, while the amount of total lipids decreased slowly as the level remained 17% higher than in the control 6 h after stress relief. Our results show that Parkinsonia plants are sensitive to a slight variation of the water potential and respond with an increased synthesis of membrane lipids during stress and after rehydration.

Comparative evaluation of the hypolipidemic effects of coconut water and lovastatin in rats fed fat–cholesterol enriched diet

The coconut water presents a series of nutritional and therapeutic properties, being a natural, acid and sterile solution, which contains several biologically active components, l-arginine, ascorbic acid, minerals such as calcium, magnesium and potassium, which have beneficial effects on lipid levels. Recent studies in our laboratory showed that both tender and mature coconut water feeding significantly ( P < 0.05) reduced hyperlipidemia in cholesterol fed rats [Sandhya, V.G., Rajamohan, T., 2006. Beneficial effects of coconut water feeding on lipid metabolism in cholesterol fed rats. J. Med. Food 9, 400–407]. The current study evaluated the hypolipidemic effect of coconut water (4 ml/100 g body weight) with a lipid lowering drug, lovastatin (0.1/100 g diet) in rats fed fat–cholesterol enriched diet ad libitum for 45 days. Coconut water or lovastatin supplementation lowered the levels of serum total cholesterol, VLDL + LDL cholesterol, triglycerides and increased HDL cholesterol in experimental rats ( P < 0.05). Coconut water feeding decreased activities of hepatic lipogenic enzymes and increased HMG CoA reductase and lipoprotein lipase activity ( P < 0.05). Incorporation of radioactive acetate into free and ester cholesterol in the liver were higher in coconut water treated rats. Coconut water supplementation increased hepatic bile acid and fecal bile acids and neutral sterols ( P < 0.05). Coconut water has lipid lowering effect similar to the drug lovastatin in rats fed fat–cholesterol enriched diet.

Role for sterol regulatory element binding protein in the regulation of farnesyl diphosphate synthase and in the control of cellular levels of cholesterol and triglyceride: evidence from sterol regulation-defective cells.

In order to define the factors involved in the regulation of farnesyl diphosphate (FPP) synthase, we used sterol regulation-defective (SRD) cell lines that constitutively express either high (SRD-2) or low (SRD-6) levels of transcriptionally active sterol regulatory element binding protein (SREBP). FPP synthase mRNA levels were high in SRD-2 cells and low in SRD-6 cells and were unaffected by the addition or removal of sterols from the media. In contrast, the mRNA levels in parental CHO-7 cells were regulated by sterols. SRD-2, SRD-6, and CHO-7 cells were also transiently transfected with plasmids containing FPP synthase promoter-reporter genes. Reporter gene activity was significantly higher in SRD-2 cells than in either SRD-6 or CHO-7 cells, consistent with a higher rate of transcription of the reporter gene in SRD-2 cells. The high expression of the reporter gene in SRD-2 cells was not observed when the FPP synthase promoter contained a three base pair mutation within an SREBP binding site, termed sterol regulatory element-3 (SRE-3). These observations are consistent with the hypothesis that high levels of transcription of the FPP synthase gene are dependent on the availability of transcriptionally active SREBP. We also demonstrate that the incorporation of radioactive acetate into both cholesterol and fatty acids was enhanced in SRD-2 cells as compared to CHO-7 or SRD-6 cells. Finally, we demonstrate that the concentrations of cholesterol, cholesteryl ester, and triglyceride were all significantly elevated in SRD-2 cells. We conclude that SREBP is involved not only in the regulation of FPP synthase and cholesterogenesis but also in fatty acid and triglyceride synthesis.

Lipogenesis and Lipolysis in Fed and Fasted Chicks from High and Low Body Weight Lines

Lipogenic and lipolytic capacities were examined in fasted and nonfasted 28-day-old chicks from high-weight (HW) and low-weight (LW) selected lines. Lipogenesis was assessed in liver and bone (sternum) tissues through the activities of malic enzyme (EC 1.1.1.40), citrate cleavage enzyme (EC 4.1.3.8), and acetyl coenzyme A carboxylase (EC 6.4.1.2), as well as through the in vitro incorporation of acetate-1-14C into liver and bone lipid fractions. Lipolysis was estimated through the in vitro release of free fatty acids (FFA) from abdominal adipose tissue and through plasma FFA.Fasting depressed lipogenesis and increased lipolysis. Regardless of the feeding state, LW chicks exhibited higher lipogenic and lipolytic capacities than their HW counterparts, suggesting that lipid degradation may be relatively more important than synthesis in determining net fat deposition. In addition, the incorporation of radioactive acetate into bone lipid was associated with detectable activity of lipogenic enzymes, providing further evidence that the skeleton is an important site of lipogenesis in the chicken.

Increased hepatic dolichol and dolichyl phosphate-mediated glycosylation in rats fed cholesterol

The concentrations of dolichol and cholesterol in livers of rats maintained for 2 weeks on a diet enriched with cholesterol (1%) were significantly higher than those in animals on a normal diet. The incorporation of radioactive mevalonate into dolichol and into a dolichyl diphosphate oligosaccharide fraction by liver slices of the cholesterol-fed animals was increased over that of the control group. However, the incorporation of radioactive mevalonate into cholesterol was decreased, as was the incorporation of radioactive acetate into both dolichol and, more markedly, cholesterol. These results are consistent with cholesterol feeding causing partial inhibition of the cholesterol-biosynthetic pathway both at β-hydroxy-β-methylglutaryl coenzyme A reductase and at a step after farnesyl pyrophosphate formation, resulting in a greater flux of mevalonate to dolichol and an increase in pool sizes of precursors of β-hydroxy-β-methylglutaryl coenzyme A. Maximal activity of glycosyl transfer to dolichyl phosphate was greater in microsomal preparations from livers of cholesterol-fed animals compared with those of control animals. A corresponding higher degree of in vitro glycosylation of endogenous protein was also observed. It is concluded that the cholesterol-enriched diet caused an increase in the biosynthesis and concentration of dolichyl monophosphate which resulted in a higher level of N-glycosylation of protein. These effects were complicated by differences in the kinetics of glycosyl transfer and in its response to exogenous dolichyl monophosphate.

Inhibition of cholesterol synthesis in cultured cells by 25-azidonorcholesterol.

25-Azidonorcholesterol, a side chain analogue of cholesterol, inhibits cholesterol biosynthesis in BHK 21 cells in tissue culture; but 3-azido-5-cholestene, the azido analogue of cholesterol has no inhibitory effect. Cell growth was unimpaired by the azido analogue, which showed only a minimal uptake by the cell, compared with that of cholesterol or 25-azidonorcholesterol. 25-Azidonorcholesterol is incorporated into the membranes of BHK 21 cells in place of cholesterol as demonstrated by isotope experiments. The incorporation of radioactive acetate is strongly inhibited by the side chain azido substituted cholesterol derivative; the incorporation of radioactive mevalonate is, however, unimpaired. We conclude that the inhibition occurs at the hydroxymethylglutaryl-CoA-reductase step. Cell growth is not significantly altered.

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small. Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis. Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.

Incorporation of I-14C-acetate into the lipids of isolated epidermal cells.

Suspensions of epidermal cells were prepared by trypsinization of rat epidermis and incubated with I-14C-acetate in Eagle's minimum essential medium. The incorporation of radioactivity into the total lipids of the cells was increased by the addition of serum to the medium. The pattern of incorporation into the main lipid classes was affected by the addition of serum to the medium and by alterations in the density of the cell suspensions. The incorporation of radioactivity into the total lipids increased approximately linearly over a 24-h period of incubation and the pattern of incorporation into the phospholipids and glycolipids altered with time. However, at each time interval studied, reporducible patterns of incorporation were obtained under controlled conditions. The incorporation of radioactive acetate therefore provides the basis for a very sensitive method of lipid analysis.

Preovulatory changes of steroidogenesis in isolated rabbit follicles (author's transl)

In an attempt to investigate the effect of ovulating hormone on the steroidogenesis of mature follicles in the course of ovulation, transitory changes of steroidogenesis in isolated rabbit follicles have been studied at several intervals after injection of an ovulatory dose of human chorionic gonadotropin (hCG). Five to ten follicles of approximately 1-2 mm in diameter were isolated from ovaries of a mature rabbit (2.5-3.0 kg) under streomicroscope, before and at the 3rd, 6th, 9th and 12th hours after intravenous injection of of 100 IU/kg of hCG. Follicles were incubated with 100 muCi of acetate-1-14C in 2 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) at 37 degrees C for 3 hours under 95% oxygen plus 5% carbon dioxide. Each incubation was terminated by quick freezing and stored forzen at -20 degrees C until eighty follicles had been collected for each time period before commencement of analysis. Incorporation of radioactive acetate into pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone., 20 alpha-dihydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone and estradiol-17beta were analysed by the reverse dilution technique and identified in radiochemically pure form by recrystallization to constant specific activities. The steroidogenic activity of the follicles was evaluated by overall as well as fractionated incorporations. A peak in the overall incorporation of 14C- acetate into the ten steroids at the 3rd hour after hCG injection, followed by gradual decrease up to the 9th hour was observed. The incorporation decreased markedly to a minimum level at the 12th hour after hCG injection, which was below the level of preinjection control. Comparable quantitative fluctuations were found with the fractionated incorporation of 14C-acetate into the C21 and C18 steroids in the time sequence following hCG injection. However, the fractionated incorporation into C19 steroids reached to a maximum at the 6th hour after hCG injection. 5istribution patterns of incorporation among the individual steroids were varied at each interval of time. In the non-injected control, mature follicles synthesized predominantly estradiol-17beta, testosterone and androstenedione. Divergent steroids were formed from radioactive acetate at the 3rd hour after hCG injection. These included porgestogen, androgen and estrogen, but pregnenolone and 17hydroxyprogesterone were the two principal steroids produced. There was no essential difference in the steroidogenic patterns between the 6th and 9th hour, the major products being C21 and C19 steroids such as pregnenolone, 17hydroxyprogesterone, dehydroipiandrosterone and testosterone. The three androgens were the major steroids formed at the 12th hour after hCG injection. Thus the chages in the steroidogenic profile of the follicle was obvious in the course of ovulation. The basis of qualitative changes in follicular steroidogenesis during the process of ovulation have been discussed in connection with an accompanying effect of an ovulatory dose of hCG.

Effect of inhibition of protein synthesis on lipid metabolism in Lactobacillus plantarum.

In Lactobacillus plantarum 17-5, lipid synthesis appears to be correlated with protein synthesis. Inhibition of protein synthesis by chloramphenicol (50 mug/ml) caused the nearly simultaneous inhibition of incorporation of radioactive oleic acid into polar lipids before the cessation of growth. In addition, de novo fatty acid synthesis, as determined by the incorporation of radioactive acetate into cellular lipids, was also inhibited. Removal of the antibiotic resulted in the resumption of growth, protein synthesis, and polar lipid synthesis. Inhibition of protein synthesis by leucine deprivation also produced a marked reduction in the incorporation of radioactive oleic acid into the total polar lipids at about the same time that growth stopped (30 to 60 min after the removal of leucine). However, the different classes of lipids behaved differently. For example, the incorporation of oleic acid into cardiolipin was inhibited immediately upon removal of leucine from the cultures, whereas incorporation into phosphatidyl-glycerol was maintained at near normal rates for 60 min after the removal of leucine and then ceased. In contrast, the accumulation of radioactive oleic acid in a neutral lipid identified as diglyceride occurred to a much greater extent in leucine-deprived cultures than in control (+ leucine) cultures. Upon addition of leucine to leucine-deprived cultures, the rates of synthesis of phosphatidyl-glycerol and cardiolipin returned to normal; the amount of radioactivity in the diglyceride fraction decreased to normal levels concomitantly with increased phospholipid synthesis.

Diagnosis of familial hypercholesterolemia by measurement of sterol synthesis in cultured skin fibroblasts

The incorporation of radioactive acetate into the digitonin precipitable fraction (cholesterol) was measured in monolayers of primary cultures of skin fibroblasts. Mean incorporation was increased approximately 20-fold in 4 subjects homozygous for familial hypercholesterolemia (FH) and 4-fold in 6 heterozygotes derived from the immediate family of homozygotes. Incorporation was normal in 4 subjects with Type IV and V hyperlipoproteinemia. In cells that had been preincubated in lipid free medium, incorporation by cells from homozygotes was equal to controls, denoting a derangement in the feedback inhibition of cholesterol synthesis by medium lipids in FH. The activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase paralleled the values obtained for sterol synthesis. The assay described could be useful in making an “etiologic” diagnosis of familial hypercholesterolemia and could possibly identify variants of monogenic hyperbetalipoproteinemia.

Steroid hormone formation in bovine ovarian follicles.

In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions.

Incorporation of radioactive acetate into diacetyl by Streptococcus diacetilactis.

Streptococcus diacetilactis was grown in a partially defined, lipoic acid-free medium containing radioactive acetate with and without addition of 0.1% unlabeled sodium pyruvate. Labeled carbon was incorporated into diacetyl, but neither the amount of diacetyl produced nor its specific activity was influenced by addition of pyruvate. Acetoin had low specific activity, indicating that it was a mixture of radioactive and nonradioactive acetoin. The specific activity of acetoin was lower when pyruvate, a precursor of unlabeled acetoin, was added to the medium, which indicated that the radioactive acetoin was produced from radioactive diacetyl by diacetyl reductase. Results substantiate condensation of acetyl-coenzyme A with hydroxyethylthiamine pyrophosphate as the in vivo mechanism for synthesis of diacetyl.

STUDIES ON HISTONE FRACTION F2A1 IN MACRO- AND MICRONUCLEI OF TETRAHYMENA PYRIFORMIS

Histone fraction F2A1 has been isolated and purified from macronuclei of the ciliate Tetrahymena pyriformis. It migrates as a single species on sodium dodecyl sulphate-acrylamide gel electrophoresis, with a molecular weight indistinguishable from that of calf thymus F2A1. The solubility properties of Tetrahymena F2A1 are also similar to those of calf thymus F2A1. Electrophoretic analyses on urea-acrylamide gels indicate that Tetrahymena F2A1 consists of four or five subspecies, the two fastest having electrophoretic mobilities identical with those of the two major electrophoretically separable forms of calf thymus F2A1. High resolution (long gel) electrophoresis coupled with incorporation of radioactive acetate both in vivo and in vitro suggest that, as in the case of calf thymus F2A1, differentical acetylation of a parent molecule can explain the observed electrophoretic heterogeneity of Tetrahymena F2A1. Electrophoretic analysis of histones isolated from the micronucleus, which is genetically less active than the macronucleus, indicates that it contains largely the relatively unacetylated (parent) form of histone F2A1.

The Formation of Fungal Sporopollenin in the Zygospore Wall of Mucor mucedo: a Role for the Sexual Carotenogenesis in the Mucorales

SUMMARY: Exhaustive alkali and acid extraction of Mucor mucedo zygospores leaves a residue that is chemically very similar to higher plant and synthetic sporopollenins. This Mucor sporopollenin is probably a component of the outer part of the zygospore wall. The incorporation of radioactive acetate, mevalonate and β-carotene suggests that it is biosynthesized by an oxidative polymerization of β-carotene. Thus sporopollenin formation is probably a role for the increased carotenogenesis observed during sexual reproduction of many members of the Mucorales.

Incorporation of Radioactive Acetate into Diacetyl by Streptococcus diacetilactis

Streptococcus diacetilactis was grown in a partially defined, lipoic acid-free medium containing radioactive acetate with and without addition of 0.1% unlabeled sodium pyruvate. Labeled carbon was incorporated into diacetyl, but neither the amount of diacetyl produced nor its specific activity was influenced by addition of pyruvate. Acetoin had low specific activity, indicating that it was a mixture of radioactive and nonradioactive acetoin. The specific activity of acetoin was lower when pyruvate, a precursor of unlabeled acetoin, was added to the medium, which indicated that the radioactive acetoin was produced from radioactive diacetyl by diacetyl reductase. Results substantiate condensation of acetyl-coenzyme A with hydroxyethylthiamine pyrophosphate as the in vivo mechanism for synthesis of diacetyl.

Biopharmaceutical Studies on Guaiacol Glyceryl Ether and Related Compounds V

Filtration using a crosslinked dextran gel was performed to determine the mechanism by which guaiacol glyceryl ether mononicotinate causes an increase in the solubility of cholesterol. The effects of guaiacol glyceryl ether mononicotinate and its related compounds on the incorporation of radioactive acetate into cholesterol also were investigated to obtain information concerning their hypocholesteremic behavior. Blood levels and urinary metabolites of guaiacol glyceryl ether mononicotinate in man were determined. Although the main purpose of the present study was not to investigate pharmacological effects of guaiacol glyceryl ether mononicotinate, it was noted that this compound displayed some analgesic but no antihistamine or anti-inflammatory activity.

The synthesis of prostaglandins in human platelets.

AbstractHuman blood platelets were found to possess the whole biological system for synthesis of prostaglandins from C14‐labeled acetate and inherent derivatives of essential fatty acids. The incorporation of radioactive acetate into prostaglandins, polar lipids and neutral lipids was followed as function of acetate concentration and time. Of the fractions mentioned, ca. 10% of the label was found in PGEs. Among the PGEs, PGE1 and PGE2 account for 48.6 and 43.4% of the radioactive label. The results obtained were related to the theory of the inhibiting function of prostaglandins on the platelet agglutination.

The binding of radioactive label from labelled phenacetin and related compounds to rat tissues in vivo and to nucleic acids and bovine plasma albumin in vitro.

1. acetyl-(3)H- and ethyl-(14)C-labelled derivatives of phenacetin and related compounds are described. 2. Radioactive label from the ethyl-(14)C-labelled derivatives of 4-nitrophenetole, 4-phenetidine and phenacetin binds in vitro to various extents to bovine plasma albumin, salmon sperm DNA and yeast RNA; the extent of binding is increased in the presence of a rat liver microsomal hydroxylating system and further increased when the microsomal enymes are induced by prior treatment of rats with 3-methylcholanthrene. 3. The ratios of the bound radioactive labels in vitro from [ethyl-(14)C]phenacetin, N-acetoxy[ethyl-(14)C]phenacetin, [acetyl-(3)H]phenacetin and [diacetyl-(3)H]N-acetoxyphenacetin per g-atom of DNA P, RNA P and per mol of protein in the absence of the microsomal system are approximately 1:60:11:863, 1:68:41:1835 and 1:88:713:2399 respectively. 4. Radioactive label from labelled phenacetin binds in vitro to all tissues examined, including the spleen, intestines, kidney and bladder; about 80% of the radioactivity bound to the liver is concentrated in the RNA and proteins. 5. Comparison of the relative extents of binding of radioactive label derived from equimolar amounts of labelled phenacetin, ethanol or acetate shows that the incorporation of labelled C(2) units into tissues and biological macromolecules in vivo and in vitro may account for only a part of the total bound radioactive label derived from phenacetin and not at all from the incorporation of radioactive acetate into nucleic acids. 6. Some implications of these findings are discussed.