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.     

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.     

In vivo study of cholesterol turnover in tissues of adult sows

The in vivo study of free and esterified cholesterol turnover was carried out in 15 tissues of adult Large White sows maintained at a constant weight for 10–12 weeks. They received a single intravenous injection either of [1-¹⁴C] acetate, or of an autologous red cell suspension or of plasma, previously labelled in vitro (for red cells) or in vivo (for plasma) with tritiated cholesterol.The tissues can be separated into four groups according to their relative rate of free cholesterol exchange between plasma and tissues. The liver and the lungs have a very fast exchange rate whereas the brain and the spinal cord have a very slow one. The whole lipoprotein particle transfer — an exclusive model for the esterified cholesterol transport from plasma to tissues — has been found in all sow tissues. When [1-¹⁴C] acetate is used as a substrate for cholesterol synthesis, lungs, adrenal glands and heart do not seem — or at an extremely low rate — to convert acetate into cholesterol whereas an intense cholesterol synthesis takes place in the small intestine. Its contribution to cholesterol synthesis in sows — taking into account the cholesterol transfer processes — reaches 70 per cent.     

Cholesterol synthesis in rat intestine: effect of fasting and of porphyrogenic chemical allylisopropylacetamide

(a) Administration of allylisopropylacetamide to fasting rats stimulates intestinal sterol synthesis as measured by incorporation of ¹⁴C from [1-¹⁴C]sodium acetate. Stimulatory effect of AIA is confined to the acetate to mevalonate segment of cholesterol biosynthetic pathway.(b) It is also shown that the suppression of sterol synthesis in the ileum of intact rats produced by fasting is of the same order of magnitude as that observed for liver sterol synthesis due to fasting.     

Incorporation of intravenously injected (2-(14)C) acetate into tissue lipids of hamsters during the last hour of 3-, 6-, and 24-hour periods of hypothermia.

The in vivo incorporation of total lipid ¹⁴C from [2-¹⁴C]acetate is decreased in kidney, liver, and small intestine tissue from 3-, 6-, and 24-hr hypothermic hamsters compared to tissues from normothermic animals. The length of time in hypothermia affects hamster tissues differently; thus, ¹⁴C activity: decreases with time in kidney; increases with time in liver; and increases at 3 and 6 hr but decreases from 6 to 24 hr of hypothermia in small intestine.Tissues from hypothermic hamsters incorporated a greater percentage of [2-¹⁴C] acetate into free sterols and diglycerides and a smaller percentage into phospholipid than did corresponding tissues from normothermic hamsters.The percentage of total fatty acid ¹⁴C activity found as polyunsaturated fatty acid ¹⁴C activity increases in hypothermic kidney, liver, and small intestine with a decrease in the percentage of ¹⁴C activity measured in the saturated fatty acids. Esterification of fatty acid was inhibited in all tissues taken from hypothermic hamsters.     

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.     

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of ¹⁴CO₂ from l-[1-¹⁴C]fucose and d-[1-¹⁴C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-¹⁴C]fucose or of d-[1-¹⁴C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more ¹⁴CO₂ from l-[1-¹⁴C]fucose or d-[1-¹⁴C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies.     

Cholesterol biosynthesis in human lymphocytes,monocytes, and granulocytes

Lymphocytes, monocytes and granulocytes were separated by counter-flow centrifugation from the blood of normal individuals and were incubated in full serum medium or lipid-depleted medium. The monocytes incorporated about five times more [2-¹⁴C]acetate into sterols than did the lymphocytes in full serum medium and approximately twenty times more than the lymphocytes in lipid-depleted medium. The granulocytes were unable to synthesize sterols from either [2-¹⁴C]acetate or [2-¹⁴C]mevalonate, but they were able to use these substrates for the synthesis of squalene and demonstrated approximately a two fold increase in the incorporation of [2-¹⁴C]acetate (but not [2-¹⁴C]mevalonate) into squalene when incubated in the lipid-depleted medium as compared to the full serum medium.     

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     

Measurement of uridine diphosphate glucuronic acid concentrations and synthesis in animal tissues

1. A method for the isolation from animal tissues of UDP-glucuronic acid by one-dimensional paper chromatography is described and its concentrations in some tissues of several species of vertebrates are reported; the incorporation of [³²P]-phosphate into UDP-glucuronic acid in vivo was also investigated. 2. The concentration of UDP-glucuronic acid was higher in the liver of rats, rabbits and guinea pigs than in the same tissue of some species of birds, amphibia and fishes; also, the concentration of UDP-glucuronic acid in rat liver, kidney and small intestine was several times lower than that of the same tissues of guinea pigs. 3. The rate of [³²P]-phosphate incorporation into UDP-glucuronic acid was very high in rat liver and kidney and almost reached equilibrium with the radioactivity of UDP-glucose 30min after the administration of the [³²P]phosphate.     

The role of kidneys in mevalonate metabolism: fact and artifact

This laboratory has previously demonstrated that circulating mevalonate is metabolized primarily by the kidneys by both the sterol and nonsterol pathways of mevalonate metabolism. Bardenheier and Popják recently challenged this conclusion, claiming that a contaminant in commercially obtained [5-¹⁴C]mevalonate can artifactually account for much of the ¹⁴C reported as ¹⁴CO₂ in such experiments.In the present study, this contaminant was shown to occur at levels averaging only 0.08% and even if not corrected for, would cause less than a 4% error in calculating renal mevalonate oxidation. Further, regardless of the minor degree of contamination of commercial ¹⁴C-mevalonate, the kidney oxidized mevalonate to CO₂ at rates averaging 24 times that of the liver. Finally, purified (R)-[5-¹⁴C]mevalonate was shown to yield results virtually identical, both in vitro and in vivo, to those obtained with commercial [5-¹⁴C]mevalonate. These data, therefore, fully confirm our previous conclusion that the kidney represents the primary organ site of mevalonate metabolism by the nonsterol, or shunt, pathway.     

The chemical synthesis and biological oxidation of 7α-hydroxy[26-14C]cholesterol, 7-dehydro[26-14C]cholesterol and 26-hydroxy[26-14C]cholesterol

1. 26-Hydroxycholesterol was obtained by reducing the methyl ester of (±)-3β-hydroxycholest-5-en-26-oic acid, which was synthesized from 25-oxonorcholesterol. 2. Methods for preparing 7α-hydroxycholesterol and 7-dehydrocholesterol were modified to allow the micro-scale preparation of these [¹⁴C]sterols from [26-¹⁴C]-cholesterol. 3. 26-Hydroxycholesterol was oxidized more readily than 7α-hydroxycholesterol, 7-dehydrocholesterol or cholesterol by mitochondrial preparations from livers of mice, rats, guinea pigs, common toads (Bufo vulgaris) and Caiman crocodylus. 4. (±)-3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was oxidized very rapidly to ¹⁴CO₂ by mouse and guinea-pig mitochondria without evident discrimination between the two optical isomers. 5. An enzyme system that oxidizes 26-hydroxycholesterol to 3β-hydroxycholest-5-en-26-oic acid was identified in the soluble extract of rat-liver mitochondria. This enzyme could use NADP in place of NAD but was not identical with liver alcohol dehydrogenase (EC 1.1.1.1). 6. [26-¹⁴C]Cholesteryl 3β-sulphate was not oxidized by fortified mouse-liver preparations that oxidized [26-¹⁴C]cholesterol to ¹⁴CO₂.     

Sesquiterpene biosynthesis in maritime pine needles

The biosynthesis of sesquiterpene hydrocarbons was studied in maritime pine (Pinus pinaster) needles by incorporation of ¹⁴CO₂, [1-¹⁴C] acetate and [2-¹⁴C] mevalonate. It was shown that the mechanisms of sesquiterpene biosynthesis are different according to the applied tracer. The important role of the acyclic compound, trans-β-farnesene, before cyclisation processes is discussed.     

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. ¹⁴C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [¹⁴C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.     

Ontogeny of mitochondrial steroidogenesis in the guinea pig gonad

To determine whether steroidogenesis in the developing guinea pig may be limited by the formation of pregnenolone, cholesterol side chain cleavage activity was ascertained at various stages of development. The conversion of [1,2-³H]cholesterol to [1,2-³H]pregnenolone was detected in mitochondria isolated from fetal guinea pig ovaries and testes as early as day 35 of gestation, while no metabolism was noted in day 30 animals. Moreover, no [l,2-³H]progesterone was formed during the 60 minute incubation. From day 35 of gestation to the day of birth, the percentage of pregnenolone formed per testis (total activity) increased, while total activity in the ovary declined. In contrast, gonadal mitochondria from adult guinea pigs converted cholesterol to both pregnenolone and progesterone and total activity in these animals was substantially higher than in their fetal counterparts. In the three females examined, the rate of pregnenolone and progesterone synthesis varied according to the stage of the estrous cycle during which these animals were sacrificed. Conversion of pregnenolone to progesterone was most rapid in the early luteal phase animal, while conversion of cholesterol to pregnenolone occurred more rapidly in the periovulatory animals than in ovarian mitochondria from the late luteal phase of the cycle. The results indicate that during prenatal and postnatal development of the gonad, cholesterol side chain cleavage activity changes and that mitochondria may acquire a Δ⁵-3β-hydroxysteroid dehydrogenase.     

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

1. [¹⁴C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO₃⁻. In a cell-free yeast preparation addition of HCO₃⁻ stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-¹⁴C]- and [1,3-¹⁴C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-¹⁴C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-¹⁴C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.     

Lipid composition and lipogenic activities in the livers of normo- and hereditary hyperlipidemic chickens

Lipid content of the liver and its lipogenic activities were compared among year-old laying hens and hereditary non-layers and roosters fed a low-fat and cholesterol-free grain diet. In contents of free cholesterol, cholesteryl ester and phospholipid, the differences between groups were relatively minor, but striking differences were noted in triglyceride with a marked accumulation in non-layers. Incorporation studies of [14C]acetate and [14C]mevalonate by liver slices revealed that in layers, acetate was preferentially incorporated into saponifiable lipids (SF), whereas mevalonate incorporated actively into both saponifiable and non-saponifiable (NSF) lipids. Lipid synthesis with acetate and mevalonate was extremely low in non-layers; in roosters, utilization of acetate was very poor but mevalonate was actively incorporated into SF and NSF lipids. During incubation of tissue slices, acetate was extensively oxidized to CO2, whereas mevalonate was poorly oxidized in all chickens. The level of substrate oxidation was similar between layers and roosters, but their level of oxidation was much greater than that of non-layers.     

Oestrogen-induced cholesterol and fatty acid biosynthesis in Xenopus laevis liver during vitellogenic response

1. Oestradiol-17β induces livers of Xenopus laevis (South African clawed toad) to synthesize and secrete into the serum large quantities of the egg-yolk-protein precursor, vitellogenin. The peak of this response occurs 9–16 days after hormone treatment [Dolphin, Ansari, Lazier, Munday & Akhtar (1971) Biochem. J. 124, 751–758]. It is now shown that 6 days after hormone treatment a 120–160-fold stimulation of the synthesis of cholesterol and fatty acid compared with control values occurred. 2. A cell-free system, derived from Xenopus liver, which synthesizes squalene and fatty acid is described. By using this system, several hundredfold stimulation of incorporation of [¹⁴C]acetate into squalene was recorded 6 days after the administration of oestradiol-17β, compared with a 3–4-fold stimulation of incorporation of [³H]mevalonate compared with control values. It is argued that oestradiol-17β must affect enzyme(s) catalysing step(s) between acetate and mevalonate in the biosynthetic pathway to cholesterol. 3. In incubation of liver slices in vitro, most of the lipid and cholesterol synthesized in response to the steroid hormone was associated with those subcellular fractions that contained membranes. Moreover, pulse-labelling experiments in vivo showed that 70% of this lipid and cholesterol was retained in the liver. The remainder appeared in the serum, where it was equally distributed between vitellogenin and vitellogenin-free serum. 4. G.l.c. analyses of the cholesterol content of liver microsomal fractions of Xenopus laevis indicated that the cholesterol content was at least 50% higher in microsomal fractions obtained from livers that had been exposed to oestradiol-17β. Meanwhile, g.l.c. analysis of the lipid moiety of secreted vitellogenin showed that up to 35% of its lipid was cholesterol.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Reduced synthesis and rapid esterification of cholesterol in the liver of hamsters with spontaneous hypercholesterolemia

The rates of in vitro incorporation of [2-14C]mevalonate and of [2-14C]acetate were determined in the liver and in the ileum obtained from two groups of hamsters. The first (N-H) were conventional hamsters with normal cholesterolemia, the second (FEC-H) were hamsters which develop high levels of cholesterol in plasma and in the liver with age. In FEC-H, the levels of cholesterol reached 130 to 220 mg/100 ml in plasma and 600-2400 mg/100 g fresh tissue in the liver in contrast to about 100 mg and 350 mg respectively in the N-H group. The accumulation of cholesterol in the tissues of FEC-H was found to be associated with a strong decrease in the incorporation rate of the precursors into cholesterol, but above all, the distribution of the radioactivity derived from [2-14C]mevalonate between free cholesterol and esterified cholesterol was markedly different in the two groups of hamsters. In FEC-H, 78% of the radioactivity was found in the esters, as opposed to 10% found in the N-H group. Thus, the development of hypercholesterolemia with age in FEC-H might be related to alterations in the enzyme systems (ACAT, CEH) which modulate the level of cholesterol esters in the liver. No significant difference was observed between the two groups of hamsters either in the concentration of cholesterol or in the rate of cholesterogenesis in the ileum.