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.     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

The disposition of [24-3H]cerebrosterol in developing rat brain.

—The uptake into subcellular fractions of developing rat brain in vivo of intracerebrally injected [4-¹⁴C]cholesterol, [24-³H]cerebrosterol, and [24-³H]24-epicerebrosterol was measured for periods up to 30 days following administration. [4-¹⁴C]cholesterol was accumulated rapidly in nuclei, nerve endings, and microsomes, more slowly in myelin and mitochondria. [24-³H]cerebrosterol was accumulated rapidly in myelin, nerve endings, and microsomes, more slowly in nuclei and mitochondria. The uptake of [24-³H]24-epicerebrosterol was essentially the same as that of [24-³H]cerebrosterol. Ratios of radioactivities of [24-³H]cerebrosterol and [4-¹⁴C]cholesterol accentuated the early accumulation of [24-³H]cerebrosterol in myelin, nerve endings, and microsomes, and declining ³H:¹⁴C ratios disclosed the rapid elimination of [24-³H]cerebrosterol and [24-³H]24-epicerebrosterol relative to [4-¹⁴C]cholesterol in nerve endings and microsomes. The data suggest that the removal of [24-³H]cerebrosterol from brain results from an enzymic metabolism of the sterol, therefore that cerebrosterol exists in brain in a dynamic state of biosynthesis and catabolism.     

Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [¹⁴C]-mevalonate, [¹⁴C]isopentenyl- and [¹⁴C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [¹⁴C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg²⁺ ions in combination to the culture of a strain of Arthrobacter gave rise to ¹⁴C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.     

Obligately phototrophic Chloroflexus: primary production in anaerobic hot spring microbial mats

Microbial mats which lack cyanobacteria occur at 50° to 65° C in the sulfide-containing Mammoth Springs of Yellowstone National Park. The principal organisms within these mats are filamentous bacteria which resemble Chloroflexus aurantiacus. The incorporation of [¹⁴C]-HCO ₃ ^- into mat material depended upon both light and sulfide, and was not inhibited when complete natural light was replaced with far-red and infra-red radiation. [¹⁴C]-acetate was incorporated in a light-dependent reaction which was stimulated by, but did not require, sulfide. In situ experiments with microelectrodes demonstrated net sulfide uptake by the mat in the light, and net sulfide production by the mat in the dark, suggesting the operation of a sulfur cycle.Filamentous phototrophic bacteria isolated from the mat were incapable of sustained growth in the presence of O₂.Simultaneous exposure of cultures to light and O₂ caused degradation of bacteriochlorophyll c. The stimulation of light-dependent [¹⁴C]-HCO ₃ ^- -uptake by sulfide was more pronounced in these isolates than in strains of Chloroflexus aurantiacus.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Similar effects ofβ-alanine and taurine in cholesterol metabolism

β-Alanine, though producing a deficiency of taurine in the tissues, had a similar effect on cholesterol metabolism as taurine. Both caused increased activity of hepatic hydroxymethylglutaryl coenzyme A reductase and increased incorporation of 1, 2 of [¹⁴C]-acetate into liver cholesterol. Both caused increased concentration of biliary cholesterol and bile acids. There was increased activity of lipoprotein lipase in heart, but decreased activity in the adipose tissue in both cases. Release of lipoproteins into circulation was decreased in both cases.     

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.     

Early stages of ecdysteroid biosynthesis: The role of 7-dehydrocholesterol

The synthesis of [4-¹⁴C]cholesta-4,6-dien-3-one and [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one is described. Both [4-¹⁴C]cholest-4-en-3-one and [4-¹⁴C]cholesta-4,6-dien-3-one were not incorporated significantly into ecdysteroids compared to [1α,2α-³H]cholesterol in fifth instar and maturing adult female Schistocerca gregaria. Similarly, [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one was not incorporated significantly in the latter system. The results suggest that none of the three ¹⁴C-substrates are intermediates in ecdysteroid biosynthesis from cholesterol, although possible complications from permeability barriers cannot be discounted. [4-¹⁴C, 7-³H]7-dehydrocholesterol has been synthesized and incorporated into ecdysteroids in adult female Schistocerca gregaria and in Spodoptera littoralis pupae. Although approximately half the tritium was eliminated during ecdysteroid synthesis in S. gregaria, there was essentially complete retention of the tritium in Spodoptera. The results support the direct incorporation of 7-dehydrocholesterol into ecdysteroids and not via cholesterol. A possible explanation for the loss of appreciable tritium in S. gregaria is discussed.     

The transport of substances out of developing fruits in relation to the induction of apical senescence in Pisum sativum line G2

Apical sencscence in G2 peas occurs only in long days in the presence of fruit. The effect of fruits could be caused by the export of a senescence hormone from the fruits to the shoot tip. Export of radiolabeled material from developing fruits of G2 peas grown in long days was therefore examined following injection of the pods with [¹⁴C]-sucrose, [¹⁴C]-acetate, or [¹⁴C]-mevalonate or after allowing the pods to photosynthesize in ¹⁴CO₂ for 48 h. In all cases a small amount (<1%) of radioactivity was exported, primarily to the younger fruits on the same side of the plant and the to the shoot apex. After feeding ¹⁴CO₂ to the fruit, the radiolabeled material partitioned into acidic ethyl acctate and possessed a carboxyl group. While this radioactivity had chromatographic properties similar to abscisic acid (ABA) in a number of solvent systems, it was not identical to either ABA, phascic acid or dihydrophaseic acid. The nature of the labeled material found in the apex was different in short days, in which senescence does not occur, or when the leaves were the source of the radioactive compounds. The labeled material in the apex was similar after feeding ¹⁴CO₂, [¹⁴C]-acctatc, or [¹⁴C]-sucrose, but different if the fruits were injected with[¹⁴C]-mevalonate.Identification of the chemical nature of the labeled material in the apex was not possible due to the small amount present. Parallel purification of an extract from treated fruits led to the identification of N-benzoylaspartate and N-phenylacetyl-aspartate. The radiolabeled substance from the apex was run with these two chemically synthesized compounds on several gas chromatogtaphic columns, and was also recrystallized together several times. The label and the pure material did not have identical retention times; neither did they co-purify so that, while similar, the material exported to the apex is not the above compounds.     

The effect of different temperatures on fatty-acid synthesis and polyunsaturation in cell suspension cultures

Cell suspension cultures of Catharanthus roseus G. Don, Glycine max (L.) Merr. and Nicotiana tabacum L. were incubated with [¹⁴C]acetate, [¹⁴C]oleic acid and [¹⁴C]linoleic acid at five different temperatures ranging from 15 to 35° C. When the incubation temperature was increased, [¹⁴C]acetate was incorporated preferentially into [¹⁴C]palmitate, with a concomitant drop in [¹⁴C]oleate formation. Between 15 and 20° C, [¹⁴C]oleic acid accumulated in C. roseus cells. In all cultures, optimum desaturation of [¹⁴C]oleic acid to [¹⁴C]linoleic acid occurred between 20 and 25° C, and in G. max this was also the optimal range for desaturation of [¹⁴C]linoleic acid to [¹⁴C]linolenic acid. Elongation of [¹⁴C]palmitic acid was inhibited when cultures grown at 15° C for 25 h were subsequently incubated with [¹⁴C]acetate at 25° C. [¹⁴C]oleic acid accumulated in G. max and C. roseus cultures grown at 35° C for 25 h and subsequently incubated at 25° C. Desaturation of [¹⁴C]oleic acid increased up to 25° C, but then decreased or leveled off depending on the cell line and on the temperature prior to incubation.     

Incorporation of labeled small molecules into rubratoxin

A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28° C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1¹⁴C]acetate, [1,5¹⁴C]citrate, [2¹⁴C]malonate, [1¹⁴C]glucose, [U¹⁴C]glucose or [1¹⁴C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1¹⁴C]acetate and [2¹⁴C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.Abbreviations GMS glucose-mineral salts - RCM replacement culture medium - TCA tricarboxylic acid - PEP phosphoenolpyruvate - RIC relative isotopic content - PI percent incorporation     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Evidence for intra- and extra-protoplasmic feruloylation and cross-linking in wheat seedling roots

The sub-cellular feruloylation and oxidative coupling sites of cell wall polysaccharides were investigated in planta by monitoring the kinetics of appearance of arabinosyl- and feruloyl-radiolabelled polysaccharides in the protoplasmic compartment and their secretion in the wall either in the presence or absence of brefeldin A (BFA). By using root apical segments excised from wheat seedlings (Triticum durum Desf.), incubated with trans-[U-¹⁴C]cinnamic acid, we demonstrated that [¹⁴C]ferulate, likely [¹⁴C]diferulate, as well as trimers and larger products of ferulate are incorporated into the protoplasmic polysaccharides very rapidly within 1–3 min of [¹⁴C]cinnamate feeding. This agrees with the assumption that (glucurono)arabinoxylans [(G)AX] feruloylation and oxidative coupling occur intracellularly, likely in the Golgi apparatus. Simultaneously, polymer bound radioactive hydroxycinnamic acids appeared to be incorporated into the cell wall of root apical segments as early as 2 min after trans-[U-¹⁴C]cinnamic acid feeding. On the contrary, starting from l-[1-¹⁴C]arabinose as tracer, the secretion of the pentose-containing polymers into the wall was between 5 to 10 min. These results indicated that (G)AX feruloylation and oxidative coupling occur both intra-protoplasmically and in muro. The occurrence of in muro feruloylation and oxidative coupling was confirmed by the use of BFA a well known inhibitor of secretion. The drug caused a strong inhibition of the synthesis and secretion into the wall of the ¹⁴C-pentosyl-labelled polymers as well as of ¹⁴C-feruloyl-polymers. In spite of this, the total amount of ¹⁴C-feruloyl-polymers incorporated into the wall was only slightly affected by BFA. This indicates the existence of a mechanism involved into secretion of the activated hydroxycinnamoyl precursors to the wall, alternative to that involved in polysaccharide secretion. Lucia Ilenia Mastrangelo and Marcello Salvatore Lenucci equally contributed to this work.     

Lipid synthesis in the laticifers of Euphorbia lathyris L. seedlings

Various solutions of labeled precursors were absorbed by the cotyledons of etiolated Euphorbia lathyris L. seedlings. Incorporation of ¹⁴C into triterpenes from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]glyoxylate, [U-¹⁴C]glycerol, [U-¹⁴C]serine, [U-¹⁴C]xylose, [U-¹⁴C]glucose, and [U-¹⁴C]sucrose was obtained. The [¹⁴] triterpenes synthesized from [¹⁴C] sugars were mainly of latex origin. [¹⁴C]mevalonic acid was only involved in terpenoid synthesis outside the laticifers. Exogenously supplied glyoxylate, serine, and glycerol were hardly involved in lipid synthesis at all. The ¹⁴C-distribution over the various triterpenols was consistent with the mass distribution of these constituents in gas liquid chromatography when [¹⁴C]sugars, [¹⁴C]acetate, and [¹⁴C]pyruvate were used. These precursors were supplied to the seedlings in the presence of increasing amounts of unlabeled substrates. The amount of substrate directly involved in lipid synthesis as well as the absolute triterpenol yield was calculated from the obtained [¹⁴C]triterpenols. The highest yield was obtained in the sucrose incorporated seedlings, being 25% of the daily increase of latex triterpenes in growing seedlings.     

Production of [14C]-Labeled 3-Hydroxy-L-Kynurenine in a Butterfly,Heliconius charitonia L. (Heliconidae), and Precursor Studies in Butterfly Wing Ommatins

A method was developed to produce radiolabeled 3-hydroxy-L-kynurenine by injection of [¹⁴C]-L-tryptophan into pupae of the heliconid butterfly, Heliconius charitonia, which was converted into [¹⁴C]-3-hydroxy-L-kynurenine and deposited as a wing pigment. Extractions of 3-hydroxykynurenine (3-OHK) with 60% methanol from wings yielded in 14.4 μg per mg dry weight. In extracts from yellow wing areas, 3-OHK represented 100% of detectable amino acids. Resulting specific radioactivity of [¹⁴C]-3-OHK was between 0.05 and 0.07 mCi/mmol when 0.5 μCi [¹⁴C]-tryptophan was injected into pupae 1 or 2 days before emergence of the butterfly. Incorporation of [¹⁴C]-3-OHK into wing ommochromes was studied in nymphalid butterflies, Araschnia levana and Precis coenia. After injection into pupae [^I4C]-3-OHK as well as [¹⁴C]-tryptophan were specifically incorporated into red and red-brown wing scales as shown by autoradiography. The same incorporation occurred in isolated wings after incubation in Grace's medium containing [¹⁴C]-3-OHK. In Araschnia levana, [¹⁴C]-3-OHK offered to left wing pairs was incorporated into dihydroxanthommatin six times more effectively than [¹⁴C]-tryptophan offered to right wing pairs from the same specimen. Therefore, 3-OHK seems to be the ultimate precursor of wing ommatins.     

Metabolism of long-chain alcohols in cell suspension cultures of soya and rape

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-¹⁴C]octadecenol for 3–48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-¹⁴C]oleic acid, or [1-¹⁴C]stearoyl-CoA, or di[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.     

The stereochemistry of cyclization in abscisic acid

The hydroxylation of the pro-6′-(R)-methyl of (+)-abscisic acid, which then cyclises to phaseic acid, was used to define the origin in mevalonate of the 6′-methyl groups. Abscisic acid (ABA), biosynthesised from [2-¹⁴C, 2-³H₂]-mevalonate, was metabolized to phaseic acid by tomato shoots. The slight loss of [³H] from the phaseate, and to a lesser extent from the ABA, suggested that the unlabelled 6′-methyl was hydroxylated. This was confirmed by Kuhn-Roth oxidation of methyl phaseate to give [¹⁴C, ³H]-acetate. The data also suggest that ABA is converted to dihydrophaseate via free phaseate, the conjugates being formed from each free acid.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.