The sensitivity of acetyl-coenzyme A carboxylase to citrate stimulation in a homogenate of rat liver containing subcellular particles

1. Although citrate is known to activate purified preparations of acetyl-CoA carboxylase, it had no stimulatory effect on the incorporation of [¹⁴C]acetate into long-chain fatty acids in a whole homogenate of rat liver (S_0.7) under conditions in which the activity of acetyl-CoA carboxylase was rate-limiting for fatty acid synthesis. 2. The rate of incorporation of acetyl carbon into fatty acids was estimated in S_0.7 preparations incubated with [¹⁴C]acetate, by measuring the specific radioactivity of the acetyl carbon of acetyl-CoA and the incorporation of ¹⁴C into fatty acids. These estimates were compared with estimates of acetyl-CoA carboxylase activity in the S_0.7 preparation obtained by direct assay in conditions in which the enzyme was in the fully activated state. 3. In the absence of citrate, incorporation of acetyl carbon into fatty acids was about 75% of the value expected if the acetyl-CoA carboxylase in the S_0.7 preparation were in the fully activated state. 4. Incorporation of acetyl carbon into fatty acids in the S_0.7 preparation was stimulated by citrate, but the effect was many times less than the stimulation of [¹⁴C]acetate incorporation by citrate in particle-free preparations. 5. When the mitochondria and microsomes were removed from the S_0.7 preparation, [¹⁴C]acetate incorporation into fatty acids fell to a negligible value and the preparation became highly sensitive to stimulation by citrate. 6. It is suggested that in the presence of mitochondria and microsomes, and in the intact liver cell, the degree of activation of acetyl-CoA carboxylase is such that citrate activation may not be of physiological significance.     

Cholesterogenesis from propionate: facts and speculations

Cholesterogenesis from [1-14C]acetate and [2-14C]propionate by the liver and adipose tissue has been studied in vitro. In all species tested including the rat, mouse, chicken, cow and pig, labelled propionate was recovered in cholesterol following the same trend as acetate, but at lower incorporation rates. Chicken liver was the most active in incorporating both substrates into cholesterol. In the cow and pig, the rates of cholesterol synthesis were higher in the adipose tissue than in the liver. Three alternative mechanisms are proposed to explain the recovery of propionate in cholesterol.     

Investigations on the mechanism of hypercholesterolemia observed in copper deficiency in rats

The mechanism of hypercholesterolemia effect of Cu²⁺ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased incorporation of labelled acetate into free cholesterol of liver in the Cu²⁺ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu²⁺ deficient rats.     

Turnover of14C-labelled oat residues and small molecular organic compounds in two soils under different levels of mineral nutrition

Mineralization and redistribution of carbon from¹⁴C-labelled oat shoots and [¹⁴C(U)] labelled glucose, leucine, acetate and phenylacetate were studied in light loamy sand and medium clay loam under different levels of mineral nutrition. Losses of mineralized¹⁴C as CO₂ were greater in the sandy soil than in the clay soil. NPK and NPK+Ca fertilization increased the rates of decay of the introduced plant organic matter. Among the small molecular organic compounds glucose was degraded fastest and phenylacetate slowest. Incorporation of radioactive carbon into humus fractions varied and depended on the nature of the compound introduced and on the soil type. Carbon of glucose, phenylacetate and acetate was mainly incorporated into fulvic acids, whereas¹⁴C of leucine was almost evenly distributed between humic and fulvic acids and¹⁴C of oat residues in fulvic acids and humin fractions. There was significantly higher incorporation of¹⁴C into humic acids and lower incorporation into humins in the sandy soil compared to the clay soil. NPK+Ca decreased the conversion of¹⁴C from phenylacetate and acetate to bitumens and increased its content in humic acids, particularly in the clay soil. The incorporation of¹⁴C from phenylacetate to humins benefitted from mineral fertilization during the first 30 days of the experiment in both soils.     

Evidence for negative feedback control of cholesterogenesis from mevalonate in liver: Absence in the intestine of guinea pigs fed A 0,5 % cholesterol diet

The in vitro rate of incorporation of [2-¹⁴C]-acetate and [2-¹⁴C]-mevalonate into cholesterol of liver, ileum and caecum was determined in guinea pigs. In control animals, contrary to the situation observed when acetate was used as precursor, the rate of conversion of mevalonate to cholesterol was higher in liver than in intestine. In this latter tissue, the cholesterogenesis varied depending on the portion tested. The distribution of radiolabel derived from mevalonate between esterified and unesterified cholesterol differed among the various tissues. In cholesterol-fed guinea pigs, the plasma, liver, intestine and aorta cholesterol contents increased significantly. In addition, a negative feedback control existed for hepatic cholesterol synthesis for mevalonate and acetate. This control was absent in intestinal tissues.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Pathways of Propionate Degradation by Enriched Methanogenic Cultures

A mixed methanogenic culture was highly enriched in a growth medium containing propionate as the sole organic carbon and energy source. With this culture, the pathways of propionate degradation were studied by use of ¹⁴C-radiotracers. Propionate was first metabolized to acetate, carbon dioxide, and hydrogen by nonmethanogenic organisms. Formate was not excreted. The carbon dioxide originated exclusively from the carboxyl group of propionate, whereas both [2-¹⁴C]- and [3-¹⁴C]propionate lead to the production of radioactive acetate. The methyl and carboxyl groups of the acetate produced were equally labeled, regardless of whether [2-¹⁴C]- or [3-¹⁴C]propionate was used. These observations suggest that in the culture, propionate was degraded through a randomizing pathway.     

Preincubation stimulates fatty acid synthesis by liver slices and isolated hypatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1-¹⁴C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10^?4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1-¹⁴C] acetate into fatty acids or cholesterol. The rate of ¹⁴CO₂ synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1-¹⁴C] acetate or [³H] H₂O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control     

Adriamycin treatment increases 3-hydroxy-3-methylglutaryl CoA reductase and isoprene biosynthesis in the rat liver

Treatment of rats with Adriamycin caused an increase in the incorporation into hepatic cholesterol of [1-¹⁴C] acetate, but not of [2-¹⁴C] mevalonate. The step affected was found to be 3-hydroxy-3-methylglutaryl CoA reductase whose activity in the liver microsomes increased in Adriamycin-treated animals, but was inhibited when the drug was added in the assay medium. Also, the concentration of ubiquinone in the liver and of cholesterol in the plasma increased.     

Biosynthesis of internally branched monomethylalkanes in the cockroach Periplaneta fuliginosa.

Sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]propionate, sodium [2-¹⁴C]propionate, sodium [3-¹⁴C]propionate and sodium [methyl-¹⁴C]methylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fuliginosa both in vivo and in vitro, whereas no incorporation of [methyl-¹⁴C]methionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3-methylalkanes, and 59+% internally branched monomethylalkanes, principally 13-methylpentacosane. Sodium [1-¹⁴C]acetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radio-gas-liquid chromatography showed that sodium [1-¹⁴C]propionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium [1-¹⁴C]acetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.     

Incorporation of Labeled Precursors into, and Accumulation of, Triglycerides and Phosphoglycerides at Selected Stages of Fungal Growth

There was greater incorporation of [2^?14C] acetate and of [6^?14C] glucose into phosphoglycerides than into triglycerides, of 1 1/2, 2, 3, 4 and 6 day old mycelial sample of the fungus Glomerella cingulata. Maximum incorporation into both classes of lipids occurred in young mycelial samples (2 to 3 days of age) which had a high content of total nitrogen. The five sets of mycelial samples all contained somewhat larger quantities of phosphoglycerides than triglycerides, and changes in content of both classes of lipids were similar in pattern to changes in content of total nitrogen. Incorporation, accumulation and total nitrogen of the mycelial samples, decreased at 4 days but increased again by 6 days. The apparent turnover of the triglycerides and phosphoglycerides was qualitatively similar although there was greater apparent turnover of phosphoglycerides than triglycerides; the similarity in patterns of apparent turnover was inferred to be a consequence of acyl exchange between labeled and unlabeled triglycerides and phosphoglycerides. There was greater incorporation of [2^?14C] acetate into the acyl than into the glyceryl moieties of both classes of lipids but greater incorporation of [6^?14C] glucose into the glyceryl than acyl moieties. With both precursors, the glyceryl and acyl moieties of the phosphoglycerides were more heavily labeled than corresponding moieties of the triglycerides.     

Partial characterization of the pathway from propionate to acetate in the cabbage looper,Trichoplusia ni

Experiments were performed to characterize the metabolism of propionate to acetate in the cabbage looper Trichoplusia ni and correlate the results with vitamin B₁₂ levels. Fourth and fifth instar larvae contain 2–4 pg vitamin B₁₂/mg dry wt whereas pupae and adults do not contain detectable amounts. In vivo studies as a function of time in larvae, pupae and adults gave evidence that [2-¹⁴C]propionate was converted to 3-hydroxypropionate and then to acetate, which subsequently labeled Krebs cycle intermediates. Radioactivity from [1-¹⁴C]propionate was recovered only in the propionate and 3-hydroxypropionate fractions, and not in acetate or Krebs cycle intermediates, suggesting that carbon 1 of propionate was lost as carbon dioxide and that carbons 2 and 3 of propionate were retained during conversion to acetate. The enzymes of this pathway were located entirely in the mitochondrial fraction. Cyanide inhibited the metabolism of propionate to 3-hydroxypropionate and acetate in mitochondrial preparations, whereas carbon monoxide did not. [2,3-¹⁴C]Acrylic acid was metabolized to 3-hydroxypropionate, which is consistent with a dehydrogenase converting propionate to acrylate which is then hydrated to 3-hydroxypropionate and then oxidized and decarboxylated to acetate.     

Conversion of propionate to acetate and methane by syntrophic consortia

Summary The anaerobic degradation of propionate to acetate and methane by a defined sulfidogenic syntrophic co-culture consisting of Syntrophobacter wolinii and Desulfovibrio G11, and a new thermophilic, methanogenic consortium T13 was studied. Tracer experiments using (¹⁴C) propionate produced evidence for the generally accepted biochemical pathway involving methylmalonyl-CoA as an intermediate in the degradation of propionate. The degradation of (1-¹⁴C) propionate led exclusively to the formation of ¹⁴CO₂ by S. wolinii/D. G11 and to the formation of ¹⁴CH₄ by the methanogenic consortium T13. The conversion of either (2-¹⁴) or (3-¹⁴) propionate by S. wolinii/D. G11 resulted in uniform labelled acetate as the endproduct. The methanogenic consortium formed (U-¹⁴C) acetate from (2-¹⁴) and (3-¹⁴) propionate as an intermediary product followed by aceticlastic splitting to yield equivalent amounts of ¹⁴CO₂ and ¹⁴CH₄.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using 13C labels detected by nuclear magnetic resonance and 14C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [${}^{14}\text{CH}{}_3$]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using C labels detected by nuclear magnetic resonance and C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [¹⁴CH₃]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

Degradation patterns and intermediates in the anaerobic digestion of glucose: Experiments with 14C-labeled substrates

A mineral salts medium containing 1% (w/v) glucose was subjected to anaerobic digestion in an upflow reactor. Performance with respect to utilization of glucose was monitored by collection of fermentation gases and calculation of carbon mass balances. Sub-samples of bacterial supennsions from the upflow reactor were incubated with (U-¹⁴C)-glucose, (U-¹⁴C)-acetate, (2-¹⁴C)-propionate, (1-¹⁴C)-butyrate or ¹⁴C-carbonate. Individual radioactive products in samples from incubation mixtures were analysed by radio gas chromatography.Quantitatively, acetate and propionate were the only important intermediates in glucose degradation by glucose-adapted sludge, with acetate accounting for the largest part of intermediary fatty acid flux.Abbreviations used ATP Adenosine Triphosphate - TIC Total Inorganic Carbon - TOC Total Organic Carbon - VFA Volatile Fatty Acids The research described in this publication was supported by the Dutch Ministry of Public Health and Environmental Protection.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

Regulation of dolichol and of cholesterol biosynthesis in cholesterol-fed rabbits

The feeding of rabbits with a diet supplemented with 2% cholesterol caused a significant increase in the concentration of serum and hepatic microsomal cholesterol while not affecting serum high-density lipoprotein cholesterol concentration. The concentration of cytochrome b₅ was also increased in the cholesterol-fed rabbits but no change in the concentration of cytochrome P-450 was apparent. The increase in microsomal cholesterol was accompanied by an inhibition of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and a marked stimulation of acyl-coenzyme A:cholesterol acyltransferase activity. The incorporation of [1-¹⁴C]acetate into cholesterol and dolichol was strongly inhibited in liver slices of cholesterol-fed animals. In contrast, while incorporation of [2-¹⁴C]mevalonate into cholesterol was also inhibited by approximately 90%, incorporation of this precursor into dolichol was stimulated fourfold. The increased incorporation of mevalonate into dolichol was consistent with a threefold increase in the activity of the dolichol phosphate-dependent mannosyl transferase. The possible significance of these differences is discussed.     

Incorporation of 14C from carboxyl-labeled oleoyl-, linoleoyl-, and arachidonoyl-CoA into water-soluble and insoluble fractions of rat liver slices: Methodology for in vitro experiments

¹⁴C incorporation into water soluble (WS) and insoluble (IS) liver fractions was studied in vitro by incubation of rat liver slices with [1-¹⁴C]oleoyl (OL)-, [1-¹⁴C]linoleoyl (LI)-, and [1-¹⁴C]arachidonoyl (AR)-CoAs. Livers (200–300 mg) from 6-day-old rats were cut into pieces and incubated for 1 h at 37°C in 4 ml Eagle's amino acid basal medium, supplemented with fetal calf serum. OL, LI, and AR were added to the medium at a concentration of 0.10–0.15 mm (1.2–1.8 μCi per flask), except in one experiment where the molar concentration was higher (0.58 mm) and the radioactivity more dilute (0.7 μCi per flask). Two groups of liver slices were incubated in serum-free Eagle's amino acid basal medium alone. After incubation and repeated washings, liver slices were extracted using a chloroform-methanol-water system which separated into three layers: an upper-phase WS containing water-methanol soluble compounds, a lower-phase FL containing substances freely soluble in the solvents, and an intermediary fluff (IS phase) of insoluble macromolecules. The WS, IS, and FL phases were washed until no further radioactivity could be removed. Distribution of radioactivity among the three WS, IS, and FL phases was determined in relation to the radioactive precursor used and the different compositions of the nutritional media. Radioactivity measurements indicated: (1) Incorporation of ¹⁴C from OL (oleoyl-CoA) into liver slices was much higher than that from free oleic acid; (2) incorporation of ¹⁴C into WS and IS phases was higher from LI than from OL and from AR when the acyl-CoA concentration did not exceed 0.15 mm (1.2–1.8 μCi per flask); (3) incorporation of ¹⁴C into polar phases was highly dependent on the presence of fetal calf serum (FCS), and the total ¹⁴C uptake into liver slices was, for example, much higher for AR when FCS was omitted from the medium; (4) thin-layer chromatography separation of lipid compounds bound to WS and IS proteins released by hydrolysis indicates differences in the distribution of the radioactivity among the (OL, LI, and AR) groups. The technique can possibly be extended to other studies concerning synthesis of lipids and coenzymes covalently bound to multienzyme complexes.